66 SeCtIoN II • the hematopoIetIC SyStem
RBC
Skeletal protein
Red cell membrane organization lattice
Band 3 Glycophorin C
Spectrin Band 4.2 Band 4.1
Band
4.9 Vertical inrteractions
Ankyrin
Adducin
Spectrin tetramer
Spectrin dimer
Actin oligomer Tropomyosin
Tropomodulin
Horizontal interactions
■ Figure 5-5 Model of the organization of the erythrocyte membrane showing the peripheral and integral
proteins and lipids. Spectrin is the predominant protein of the skeletal protein lattice. Spectrin dimers join
head to head to form spectrin tetramers. At the tail end, spectrin tetramers come together at the junctional
complex. This complex is composed of actin oligomer and stabilized by tropomyosin, which sits in the groove
of the actin filaments. The actin oligomer is capped on one end by tropomodulin and on the other by adducin.
Band 4.9 (dematin) binds to actin and bundles actin filaments. Spectrin is attached to actin by band 4.1, which
also attaches the skeletal lattice to the lipid membrane via its interaction with glycophorin C (minor attachment
site). Ankyrin links the skeletal protein network to the inner side of the lipid bilayer via band 3. band 4.2
interacts with ankyrin and band 3 (major attachment site).
Band 3, also known as the anion exchange protein 1 (AE1), senescence. Band 3 is also important in attaching the skeletal protein
is the major integral protein of the red cell with 71 million copies network to the lipid bilayer by binding to the skeletal proteins ankyrin
10
per cell. Band 3 is the transport channel for the chloride- bicarbonate and band 4.2. The Ii blood group antigens are carried on the carbo-
exchange, which occurs during the transport of CO 2 from the tissues hydrate component of the red cell band 3 protein. 11,12
back to the lungs (Chapter 6). Like most transport channels, band 3 The red cell membrane contains 7100 additional inte-
8
spans the membrane multiple times (12–14). Anion exchange is gral proteins. These include all of the various transporter pro-
+
+
thought to occur by a “ping-pong” mechanism. An intracellular anion teins (glucose transporter, urea transporter, Na /K @ATPase,
++
++
enters the transport channel and is translocated outward and released. Ca @ATPase, Mg @ATPase), some red cell antigens (Rh, Kell), vari-
The channel remains in the outward conformation until an extracel- ous receptors (transferrin, insulin, etc.), and decay-accelerating factor
lular anion enters and triggers the reverse cycle. 8 (DAF; Chapter 17).
In addition to its role as an anion exchanger, band 3 is a major Peripheral proteins are found primarily on the cytoplasmic
binding site for a variety of enzymes and cytoplasmic membrane face of the erythrocyte membrane and include enzymes and structural
8
components. The, cytoplasmic domain of band 3 binds glycolytic proteins (Table 5.2). The structural proteins are organized into a two-
enzymes, regulating their activity; thus, band 3 serves as a regulator dimensional lattice network directly laminating the inner side of the
13
of red cell glycolysis. Band 3 also binds hemoglobin at its cytoplas- membrane lipid bilayer called the red cell membrane skeleton. The
mic domain with intact hemoglobin binding weakly but partially horizontal interactions of this lattice are parallel to the membrane’s
denatured hemoglobin (Heinz bodies) binding more avidly. Bind- plane and serve as a skeletal support for the membrane lipid layer. The
ing of hemoglobin to band 3 is thought to play a role in erythrocyte vertical interactions of the lattice are perpendicular to the membrane’s
M05_MCKE6011_03_SE_C05.indd 66 01/08/14 11:22 am
Chapter 5 • the erythroCyte 67
plane and serve to attach the skeletal lattice network to the lipid layer in the splenic vasculature (∙3 mcM). At the same time, cells must be
of the membrane. The skeletal proteins give red cell membranes their able to withstand the rigors of the turbulent circulation as they travel
viscoelastic properties and contribute to cell shape, deformability, throughout the body. Deformability of the red cell is due to three
and membrane stability. Defects in this cytoskeleton are associated unique cellular characteristics: 15,18
with abnormal cell shape, decreased stability, and hemolytic anemia • Its biconcave shape (large surface area-to-volume ratio)
(Chapter 17).
Spectrin, the predominant skeletal protein, exists as a heterodi- • The viscosity of its internal contents (the “solution” of hemoglobin)
mer of two large chains (a and b). The two chains associate in a side- • The unique viscoelastic properties of the erythrocyte membrane
by-side, antiparallel arrangement (N-terminal of a chain associated
with C-terminal of b chain). The ab heterodimers form a slender, Red cells have an “elastic extension” ability, primarily due to the
twisted, highly flexible molecule ∙100 nm in length. Spectrin het- elasticity of coiled spectrin tetramers and association–dissociation of
erodimers in turn self-associate head to head to form tetramers and skeletal proteins. As a result, the cells can resume a normal shape after
8
some larger oligomers. Spectrin is described as functioning like a being distorted by an external applied force. However, application of
spring. The spectrin tetramers are tightly coiled in vivo with an end- large or prolonged forces can result in reorganization of the cytoskel-
to-end distance of only ∙76 nm. (these could have an extended length etal proteins, producing a permanent deformation (e.g., dacryocytes)
18
14
of ∙200 nm). These coiled tetramers can extend reversibly when the or, if the force is excessive, fragmentation (e.g., schistocytes). In addi-
membrane is stretched but cannot exceed their maximum extended tion to being a major component of erythrocyte deformability, the
length (200 nm) without rupturing. membrane skeleton is the principal determinant of erythrocyte sta-
Ankyrin is a large protein that serves as the high-affinity bind- bility. The proportion of spectrin dimers versus tetramers (or higher
19
ing site for the attachment of spectrin to the inner membrane surface. oligomers) is a key factor influencing membrane stability. Higher
Ankyrin binds spectrin near the region involved in dimer–tetramer proportions of dimers result in increased fragility, and higher propor-
associations. In turn, ankyrin is bound with high affinity to the cyto- tions of tetramers and oligomers result in stabilization. Also, interac-
15
plasmic portion of band 3 (the anchor for the membrane skeleton). tion of the cytoskeleton with the lipid bilayer and integral membrane
Band 4.2 binds to ankyrin and band 3, strengthening their interaction proteins stabilizes the cell membrane. If the bilayer uncouples from the
and helping to bind the skeleton to the lipid bilayer at its major attach- skeleton, portions of lipid-rich membrane will be released in the form
ment point. 10,16 of microvesicles, resulting in a decrease in the surface area-to-volume
20
Red cell actin is functionally similar to actin in other cells. ratio and the formation of spherocytes (Chapter 17).
Red cell actin is organized into short, double-helical protofilaments
of 12–14 actin monomers. These short filaments are stabilized by CheCkpoint 5-4
their interactions with other proteins of the red cell skeleton includ-
ing tropomodulin, adducin, tropomyosin, and band 4.9. Spectrin Compare placement in the membrane and function of
dimers bind to actin filaments near the tail end of the spectrin dimer. peripheral and integral erythrocyte membrane proteins.
Band 4.1 interacts with spectrin and actin and with GPC in the over-
lying lipid bilayer. It serves to stabilize the otherwise weak interaction
between spectrin and actin and is necessary for normal membrane Membrane Permeability
8
stability. This complex of spectrin, actin, tropomodulin, tropomyosin,
adducin, band 4.9, and band 4.1 serves as the secondary attachment The red cell membrane is freely permeable to water (exchanged by
21
point for the red cell skeleton, binding to GPC of the membrane. a water channel protein) and to anions (exchanged by the anion
The skeletal proteins are in a continuous disassociation–association transport protein, band 3). In contrast, the red cell membrane is nearly
equilibrium with each other (e.g., spectrin dimer-tetramer interconver- impermeable to monovalent and divalent cations. Glucose is taken
sions) and with their attachment sites. This equilibrium occurs in response up by a glucose transporter in a process that does not require ATP
22
to various physical and chemical stimuli that affect the erythrocytes as nor insulin. + + ++ ++
they journey throughout the body. Calcium also influences the red cell The cations Na , K , Ca and Mg are maintained in the eryth-
cytoskeleton. Most intracellular calcium (80%) is found in association rocyte at levels much different than those in plasma (Table 5-3 ★).
with the erythrocyte membrane. Calcium is normally maintained at a Erythrocyte osmotic equilibrium is normally maintained by both the
++
low intracellular concentration by the activity of an ATP-fueled Ca selective (low) permeability of the membrane to cations and cation
++
++
pump (Ca @ATPase). Elevated Ca levels induce membrane protein
17
cross-linking. This cross-linking essentially acts as a fixative, stabiliz- ★ table 5-3 Concentration of Cations in the
ing red cell shape and reducing the cell’s deformability. For example, the Erythrocyte versus Plasma
abnormal erythrocyte shape of irreversibly sickled cells (Chapter 13) can
be produced by calcium-induced irreversible cross-linking and alteration cation erythrocyte (mmol/l) Plasma (mmol/l)
+
of the cytoskeletal proteins. Sodium (Na ) 5.4–7.0 135–145
+
The erythrocyte membrane together with the membrane skel- Potassium (K ) 98–106 3.6–5.0
eton is responsible for the dual cellular characteristics of structural Calcium (Ca ) 0.0059–0.019 2.1–2.6
++
integrity and deformability. The 7 mcM erythrocyte must be flexible Magnesium (Mg ) 3.06 0.65–1.05
++
enough to squeeze through the tiny capillary openings, particularly
M05_MCKE6011_03_SE_C05.indd 67 01/08/14 11:22 am
68 SeCtIoN II • the hematopoIetIC SyStem
pumps located in the cell membrane. To maintain low intracellular 3. Reduced sulfhydryl groups in hemoglobin and other proteins
++
+
+
Na and Ca and high K concentrations (relative to plasma con- 4. Red cell membrane integrity and deformability
centrations), the red cell utilizes two cation pumps, both of which use
intracellular ATP as an energy source. The most important metabolic pathways in the mature erythro-
+
+
The Na /K cation pump hydrolyzes one mole of ATP in the cyte are linked to glucose metabolism (Table 5-4 ★).Because the red
+
+
expulsion of 3Na and the uptake of 2K . This normally balances cell lacks a citric acid cycle (due to the lack of mitochondria), it is lim-
the passive “leaks” of each cation along its respective concentration ited to obtaining energy (ATP) solely by anaerobic glycolysis. Glucose
gradient between plasma and cytoplasm. Calcium plays a role in enters the red cell through a membrane-associated glucose carrier in
+
+
maintaining low membrane permeability to Na and K . An increase a process that does not require ATP or insulin.
+
+
++
in intracellular Ca allows Na and K to move in the direction
8
++
of their concentration gradients. Increased intracellular Ca also Glycolytic Pathway
+
activates the Gárdos channel, which causes selective loss of K and, The erythrocyte obtains its energy in the form of ATP from glucose
++
consequently, water, resulting in dehydration. Low intracellular Ca breakdown in the glycolytic pathway, formerly known as the Embden-
++
++
is maintained by a Ca @ATPase pump. The Ca pump depends Meyerhof pathway (Figure 5-6 ■). About 90–95% of the cell’s glucose
++
on magnesium to maintain its transport function. Although Mg consumption is metabolized by this pathway. Normal erythrocytes
++
++
is necessary for active extrusion of Ca from the cell, Mg is not do not store glycogen and depend entirely on plasma glucose for
transported across the cell membrane in the process. glycolysis. Glucose is metabolized by this pathway to lactate or pyru-
If erythrocyte membrane permeability to cations increases or the vate, producing a net gain of 2 moles of ATP per mole of glucose. If
cation pumps fail (either due to decreased glucose for generation of reduced nicotinamide-adenine dinucleotide (NADH) is available in
+
ATP via glycolysis or decreased ATP), Na accumulates in the cells in the cell, pyruvate is reduced to lactate. The lactate or pyruvate formed
+
excess of K loss. The result is an increase in intracellular monovalent is transported from the cell to the plasma and metabolized elsewhere
cations and water, cell swelling, and, ultimately, osmotic hemolysis. in the body.
ATP is necessary to maintain erythrocyte shape, flexibility, and
membrane integrity and to regulate intracellular cation concentration
CheCkpoint 5-5 (see previous discussion in the section “Membrane Permeability”).
How would an increase in RBC membrane permeability Increased osmotic fragility is noted in cells with abnormal cation per-
affect intracellular sodium balance? meability and/or decreased ATP production. Upon the exhaustion
of glucose, ATP for the cation pumps is no longer available, and cells
cannot maintain normal intracellular cation concentrations. The cells
become sodium and calcium loaded and potassium depleted. The cell
erytHrocyte MetaboliSM accumulates water and changes from a biconcave disc to a sphere and
Although the binding, transport, and release of O and CO are pas- is removed from the circulation.
2
2
sive processes not requiring energy, various energy-dependent meta-
bolic processes that are essential to erythrocyte viability occur. Energy Hexose Monophosphate (HMP) Shunt
is required by the erythrocyte to maintain:
Not all of the glucose metabolized by the red cell go through the
1. The cation pumps, moving cations against electrochemical gradi- direct glycolytic pathway. Of cellular glucose, 5 to 10% enters the oxi-
ents dative HMP shunt, an ancillary system for producing reducing sub-
2. Hemoglobin iron in the reduced state stances (Figure 5-6). Glucose-6-phosphate is oxidized by the enzyme
★ table 5-4 Role of Metabolic Pathways in the Erythrocyte
Metabolic Pathway Key enzymes Function Hematopathology
Glycolytic pathway Phosphofructokinase (PFK) Produces ATP accounting for 90% of glu- Hemolytic anemia
Pyruvate kinase (PK) cose consumption in RBC Hereditary PK deficiency
Hexose-monophosphate shunt Glutathione reductase (GR) Provides NADPH and glutathione to Hemolytic anemia
Glucose-6-phosphate reduce oxidants that would shift Hereditary G6PD deficiency
dehydrogenase (G6PD) the balance of oxyhemoglobin to Glutathione reductase deficiency
methemoglobin
Hemoglobinopathies
Rapoport-Luebering BPG-synthase Controls the amount of 2,3-BPG produced, Hypoxia
which in turn affects the oxygen affinity
of hemoglobin
Methemoglobin reductase Methemoglobin reductase Protects hemoglobin from oxidation via Hemolytic anemia
NADH (from glycolytic pathway) and Hypoxia
methemoglobin reductase
M05_MCKE6011_03_SE_C05.indd 68 01/08/14 11:22 am
Chapter 5 • the erythroCyte 69
Glycolytic pathway Hexose monophosphate pathway
H 2 O 2 H O
2
GP
Glucose
ATP GSH GSSG
Hexokinase GR
ADP
NADP NADPH
G6P 6–PG
G6PD
PI 6PDG
F6P PP
ATP
PFK
ADP
Fructose 1, 6-biP
Aldolase
Glyceraldehyde 3P
NAD Methemoglobin reductase + H Methemoglobin Methemoglobin
G3PD reductase
NADH Methemoglobin reductase Hemoglobin pathway
Rapoport-Luebering shunt
1,3 BPG
Mutase ADP
2,3 BPG BPG synthase PGK
ATP
Phosphatase 3 PG
2 PG
Enolase
PEP
ADP
PK
ATP
Pyruvate
LD
Lactate
■ Figure 5-6 Erythrocyte metabolic pathways. The glycolytic pathway is the major source of energy for
the erythrocyte through production of ATP. The hexose-monophosphate pathway is important for reducing
oxidants by coupling oxidative metabolism with pyridine nucleotide (NADP) and glutathione (GSSG) reduction.
The methemoglobin reductase pathway supports methemoglobin reduction. The Rapoport-Luebering Shunt
produces 2,3-BPG, which alters hemoglobin-oxygen affinity.
G6P = glucose- 6-phosphate; PI = glucose-6-phosphate isomerase; F6P = fructose-6-phosphate; PFK = 6-phosphofructokinase; fructose
1,6-biP = fructose 1,6-bisphosphate; Glyceraldehyde 3P = glyceraldehyde 3-phosphate; G3PD = glyceraldehyde 3-phosphate dehydrogenase;
1,3 BPG = 1, 3-bisphosphoglycerate; PGK = phosphoglycerate kinase; 3PG = 3-phosphoglycerate; 2PG = 2-phosphoglycerate; PEP =
phosphoenolpyruvate; PK = pyruvate kinase; LD = lactate dehydrogenase; GP = glutathione peroxidase; GR = glutathione reductase; GSH
= glutathione reduced; GSSG = glutathione oxidized; G6PD = glucose-6-phosphate dehydrogenase; 6-PG = 6-phosphogluconate; 6PDG =
6-phosphodehydrogenase gluconate; PP = pentose phosphate
M05_MCKE6011_03_SE_C05.indd 69 01/08/14 11:22 am
70 SeCtIoN II • the hematopoIetIC SyStem
glucose-6-phosphate dehydrogenase (G6PD) in the first step of the Rapoport-Luebering Shunt
+
HMP shunt. In the process, NADP is reduced to nicotinaminde- The Rapoport-Luebering shunt is a part of the glycolytic pathway
adenine dinucleotide phosphate NADPH. (Figure 5-6), which bypasses the formation of 3-phosphoglycerate
Glutathione is highly concentrated in the erythrocyte and is and ATP from 1,3-bisphosphoglycerate (1,3-BPG). Instead, 1,3-BPG
important in protecting the cell from oxidant damage by reactive oxy- forms 2,3-BPG (also known as 2,3-diphosphoglycerate, 2,3-DPG)
gen species (ROSs) produced during oxygen transport and by other catalyzed by BPG mutase. Therefore, the erythrocyte sacrifices one
oxidants such as chemicals and drugs. In the process of reducing oxi- of its two ATP-producing steps in order to form 2,3-BPG. When
dants, glutathione itself is oxidized. (Reduced glutathione is referred hemoglobin binds 2,3-BPG, oxygen release is facilitated (i.e., binding
to as GSH, and the oxidized form is referred to as GSSG.) NADPH 2,3-BPG causes a decrease in hemoglobin affinity for oxygen). Thus,
produced by the HMP shunt converts GSSG back to GSH, the form 2,3-BPG plays an important role in regulating oxygen delivery to the
necessary to maintain hemoglobin in the reduced functional state. The tissues (Chapter 6).
+
erythrocyte normally maintains a large ratio of NADPH to NADP .
When the HMP shunt is defective, hemoglobin sulfhydryl groups
(-SH) are oxidized, which leads to denaturation and precipitation of caSe Study (continued from page 63)
hemoglobin in the form of a Heinz body. Heinz bodies attach to the
inner surface of the cell membrane, decreasing cell flexibility. Macro- Stephen was admitted for identification and treatment
phages in the spleen remove them from the cell together with a por- of the anemia. More lab tests were ordered with the
tion of the membrane. These bodies can be visualized with supravital following results:
stains (Chapter 37). If large portions of the membrane are damaged in
this manner, the whole cell can be removed. This commonly occurs in Patient reference interval
patients with G6PD deficiency (Chapter 18). Total bilirubin 4.8 mg/dL 0.1–1.2
GSH also is responsible for maintaining reduced -SH groups of Direct bilirubin 1.6 mg/dL 0.1–1.0
cytoskeletal proteins and membrane lipids. Decreased GSH leads to Haptoglobin 25 mg/dL 35–165
oxidative injury of membrane protein -SH groups, compromising pro- Hemoglobin
tein function and resulting in “leaky” cell membranes. Cellular deple- electrophoresis
tion of ATP can then occur due to increased consumption of energy HbA 98% 795%
by the cation pumps. Hb-F 1% 62%
Ascorbic acid, or vitamin C, is also an important antioxidant in Hb-A2 1% 1.5–3.7%
the erythrocyte as it consumes oxygen free radicals and helps preserve Heinz body stain Positive Negative
alpha-tocopherol (vitamin E, another important antioxidant) in mem- Fluorescent spot test for Positive Negative
brane lipoproteins. 23 G6PD deficiency
2. What cellular mechanism results in hemolysis due to a
deficiency in G6PD?
Methemoglobin Reductase Pathway
3. Explain how Heinz body inclusions cause damage to
The methemoglobin reductase pathway, an offshoot of the glycolytic the erythrocyte membrane.
pathway, is essential to maintain heme iron in the reduced (ferrous)
++
state, Fe (Figure 5-6). Methemoglobin (hemoglobin with iron in the
oxidized ferric state, Fe +++ ) is generated simultaneously with the oxi-
dative compounds discussed earlier as O dissociates from the heme CheCkpoint 5-7
2
iron. Methemoglobin cannot bind oxygen. The enzyme methemoglo- Which erythrocyte metabolic pathway is responsible for
bin reductase (also known as NADH diaphorase, or cytochrome b ) with providing the majority of cellular energy? For regulating
5
NADH produced by the glycolytic pathway functions to reduce the oxygen affinity? For maintaining hemoglobin iron in the
ferric iron in methemoglobin, converting it back to ferrous hemoglo- reduced state?
bin. In the absence of this system, the 2% of methemoglobin formed
daily eventually builds up to 20–40%, severely limiting the blood’s
oxygen-carrying capacity. Certain oxidant drugs can interfere with erytHrocyte KineticS
methemoglobin reductase and cause even higher levels of methemo-
globin. This results in cyanosis (a bluish discoloration of the skin In the late 1800s, it was observed that individuals living at high alti-
due to an increased concentration of deoxyhemoglobin in the blood). tudes had an increase in erythrocytes, which was attributed to an
acquired adjustment to the reduced atmospheric pressure of oxy-
24
gen. Over the following decades, it was discovered that the stimula-
CheCkpoint 5-6 tion of erythropoiesis in the bone marrow in response to decreased
oxygen levels was the result of a hormone, erythropoietin (EPO), that
Uncontrolled oxidation of hemoglobin results in what is released into the peripheral blood by renal tissue in response to
RBC intracellular inclusion? hypoxia. Hormonal control of red blood cell mass is closely regulated
and is normally maintained in a steady state within narrow limits.
M05_MCKE6011_03_SE_C05.indd 70 01/08/14 11:22 am
Chapter 5 • the erythroCyte 71
Erythrocyte Concentration EPO is a thermostable renal glycoprotein hormone with a molec-
ular weight of about 34,000 daltons. Renal cortical interstitial cells
The normal erythrocyte concentration varies with sex, age, and geo- 29
12
graphic location. A high erythrocyte count 3.9–5.9 * 10 L) and secrete EPO in response to cellular hypoxia. This feedback control
hemoglobin concentration (13.5–20 g/dL) at birth are followed by a of erythropoiesis is the mechanism by which the body maintains opti-
gradual decrease that continues until about the second or third month mal erythrocyte mass for tissue oxygenation. Plasma levels of EPO are
of extrauterine life. At this time, red blood cell and hemoglobin values constant when the hemoglobin concentration is within the normal
25
12
fall to 3.1–4.3 * 10 /L and 9.0–13 g/dL, respectively. This eryth- range but increase steeply when the hemoglobin decreases below 12
30
rocyte decrease in infancy is sometimes called physiologic anemia g/dL. EPO is also produced by extrarenal sources, including marrow
of the newborn, the result of a temporary cessation of bone marrow macrophages and stromal cells, which likely contribute to steady-state
2
erythropoiesis after birth due to a low concentration of EPO. EPO erythropoiesis. However, under conditions of tissue hypoxia, oxygen
levels are high in the fetus due to the relatively hypoxic environment sensors in the kidneys trigger the release of renal EPO, resulting in an
in utero and the high oxygen affinity of hemoglobin F (fetal hemoglo- increased stimulus for erythropoiesis.
bin). After birth, however, when the lungs replace the placenta as the EPO has been defined in biologic terms to have an activity of
31
means of providing oxygen, the arterial blood oxygen saturation rises ∙130,000 IU/mg of protein. Normal plasma contains from 3 to 16
from ∙45% to ∙95%. EPO cannot be detected in the infant’s plasma IU of EPO per L of plasma. EPO can also be found in the urine at con-
32
from about the first week of extrauterine life until the second or third centrations proportional to that found in the plasma (Table 5-5 ★). In
month. Reticulocytes reflect the bone marrow activity during this anemia, EPO plasma levels are related to both hemoglobin concentra-
time. At birth and for the next few days, the mean reticulocyte count tion and the pathophysiology of the anemia. For example, patients with
is high (1.8–8.0%). Within a week, the count drops and remains low pure erythrocyte aplasia (Chapter 16) have plasma EPO levels signifi-
(61%) until about the second month of life, at which time EPO levels cantly higher than patients with iron deficiency anemia or megaloblas-
rise again (when the hemoglobin levels fall to ∙12 g/dL). This cor- tic anemia even though hemoglobin concentration in all three types of
responds to the recovery from “physiologic anemia of the newborn.” anemia can be similar. Plasma EPO levels reflect not only EPO produc-
Males have a higher erythrocyte concentration than females after tion but also its disappearance from the blood and/or utilization by the
puberty due to the presence of testosterone. Before puberty and after bone marrow (i.e., uptake by EPO-receptor-bearing cells in the marrow).
“male menopause,” males and females have comparable erythrocyte Patients with renal disease and nephrectomized patients are usu-
levels. 26,27 Testosterone stimulates renal and extrarenal EPO produc- ally severely anemic, but they continue to make some erythrocytes and
tion and directly enhances differentiation of marrow stem and pro- produce limited amounts of EPO in response to hypoxia. In addition
genitor cells. 28 to the production of EPO by marrow macrophages and stromal cells,
Individuals living at high altitudes have a higher mean eryth- hepatocytes act as an extrarenal source of EPO, but normally account
33
rocyte concentration than those living at sea level. Decreases in the for 615% of the total EPO production in humans. The adult liver
partial pressure of atmospheric oxygen at high altitudes result in a appears to require a more severe hypoxic stimulus for EPO production
physiologic increase in erythrocytes in the body’s attempt to provide than the kidney. The liver is the major site of EPO production during
adequate tissue oxygenation. fetal development, but at birth, a gradual shift from hepatic to renal
production of EPO occurs. 34
Increased EPO secretion is due to de novo synthesis of EPO
CheCkpoint 5-8 rather than release of preformed stores. The hypoxia-induced increase
Why are there different reference intervals for hemoglobin
concentration in male and female adults but not in male ★ table 5-5 Characteristics of Erythropoietin
and female children?
general characteristics
Composition Glycoprotein
Regulation of Erythrocyte Production Stimulus for synthesis Cellular hypoxia
The body can regulate the number of circulating erythrocytes by Origin Kidneys 80–90%
changing the rate of cell production in the marrow and/or the rate Liver 615%
of cell release from the marrow. Delivery of erythrocytes to the cir- Reference interval Plasma 5–30 U/L
culation is normally well balanced to match the rate of erythrocyte Functions
destruction, which does not vary significantly under steady-state con- Stimulates BFU-E and CFU-E to divide and mature
ditions. Impaired oxygen delivery to the tissues and low intracellular Increases rate of mRNA and protein (hemoglobin) synthesis
oxygen tension (PO ) trigger increased EPO release and increased Decreases normoblast maturation time
2
erythrocyte production by the marrow. Conditions that stimulate Increases rate of enucleation (extrusion of nucleus)
erythropoiesis include anemia, cardiac or pulmonary disorders, Stimulates early release of bone marrow reticulocytes (shift
abnormal hemoglobins, and high altitude. Erythropoiesis is influ- reticulocytes)
enced by a number of cytokines including SCF, IL-3, GM-CSF, and response to anemia
EPO (Chapter 4). However, EPO is the principal cytokine essential for Generally increased except in anemia of renal disease
terminal erythrocyte maturation.
M05_MCKE6011_03_SE_C05.indd 71 01/08/14 11:22 am
72 SeCtIoN II • the hematopoIetIC SyStem
of EPO is due to both increased gene transcription mediated by the A number of tumors have been reported to cause an increase
transcription factor hypoxia-inducible factor-1 (HIF-1), and stabiliza- in erythropoietin production. Stimulation of the hypothalamus can
tion of EPO messenger RNA. 35,36 Under hypoxic conditions, HIF-1 cause an increase in release of EPO from the kidneys, explaining the
binds to DNA regulatory sequences (hypoxia-responsive element association of polycythemia and cerebellar tumors. The serum EPO
[HRE]) in the EPO gene, activating transcription. Under conditions level increases dramatically in patients undergoing chemotherapy for
of normal oxygen concentration, HIF-1 is degraded by a hydroxylase leukemia as well as other cancers in response to marrow suppression
enzyme that requires oxygen for activity, resulting in a decreased pro- by chemotherapeutic agents. 39
duction of EPO mRNA. 33 The production of synthetic hematopoietic growth factors using
EPO exerts its action by binding to specific receptors (EPO-R) on recombinant DNA technology has revolutionized the management
erythropoietin-responsive cells. EPO’s major action is stimulation of of patients with some anemias. Several recombinant forms of human
committed progenitor cells, primarily the CFU-E, to survive, prolifer- EPO (rHuEPO) are available and are commonly used for treatment of
ate, and differentiate (see the section “Erythropoiesis and Red Blood the anemia associated with end-stage renal disease and chemotherapy
Cell Maturation” earlier in this chapter). A small subset of BFU-E has as well as HIV-related anemia. 31,40
EPO-R but in low number, and BFU-Es are largely unresponsive to
the effects of EPO. Thus, under conditions of EPO stimulation, the
primary elements of the erythroid precursor cells that respond are
the CFU-Es and early normoblasts. However, acute demands for caSe Study (continued from page 70)
erythropoiesis with extremely high EPO levels can stimulate the
BFU-E. When this occurs, the characteristics of the resulting eryth- 4. Would you predict Stephen’s serum erythropoietin
rocytes include an increase in mean corpuscular volume (MCV) and levels to be low, normal, or increased? Why?
2
an increase in i antigen and HbF concentration. EPO-Rs on the cell
membrane increase as the BFU-E matures to the CFU-E and gradually
decrease as the normoblasts mature. The EPO-R is absent on reticulo-
cytes. Other effects of EPO are described in Table 5-5. 37 CheCkpoint 5-9
A major way by which EPO increases RBC production is by
preventing apoptosis. Erythropoiesis is maintained by a finely tuned What would the predicted serum EPO levels be in a
balance between the positive signals generated by EPO and nega- patient with an anemia due to end-stage kidney disease?
tive signals from death receptor ligands and inhibitory cytokines
(Chapters 2, 4). Erythroid progenitors differ in their sensitivity to
EPO; some progenitors require much less EPO than others to survive
38
and mature to reticulocytes. Progenitors with increased sensitivity erytHrocyte deStruction
to EPO are thought to provide RBC production when EPO levels are Red blood cell destruction is normally the result of senescence. Several
normal or decreased. Progenitors that require high concentrations of theories have been proposed to explain the underlying pathology of
EPO die of apoptosis under these conditions. Progenitors requiring senescent red cells. Erythrocyte aging is characterized by a decline in
high concentrations of EPO, however, will be rescued from apoptosis certain cellular enzyme systems, including glycolytic enzymes and
when EPO concentrations are elevated as occurs in anemia, thus pro- enzymes needed for maintenance of redox status. This in turn leads
viding increased erythrocytes under these conditions. to decreased ATP production and loss of adequate reducing systems,
The EPO-R exists in the membrane as a homodimer and lacks resulting in oxidation of critical membrane proteins, lipids, and hemo-
intrinsic kinase activity. However, the cytoplasmic tail of the receptor globin, loss of the ability to maintain cell shape and deformability,
recruits and binds cytoplasmic kinases, Janus kinases 2 (JAK-2), which and loss of membrane integrity, all of which contribute to the cell’s
are activated when EPO binds to the EPO-R (see Figure 4-7). At least removal. 41,42,43 Oxidative damage also causes clustering of band
four different signaling pathways are activated by this EPO/EPO-R/ 3 molecules, which can be a senescence-identifying feature. The glu-
JAK-2 interaction. Abnormal interactions and/or function of these cose supply in the spleen is low, limiting the energy-producing process
components have been linked to familial forms of erythrocytosis and of glycolysis within the erythrocyte. Aged erythrocytes can quickly
certain myeloproliferative disorders (Chapter 24). deplete their cellular level of ATP, resulting in limited ability to main-
The normal bone marrow can increase erythropoiesis 5- to tain osmotic equilibrium via the energy-dependent cation pumps.
10-fold in response to increased EPO stimulation if sufficient iron Additionally, aged erythrocytes accumulate IgG (an immunoglobulin)
is available. Erythropoiesis is affected (and limited) by serum iron on their membrane. Splenic macrophages have receptors for this IgG,
39
levels and by transferrin saturation (Chapter 6). In hemolytic ane- which can enhance recognition of aged cells. The exposure of phospha-
mia, a readily available supply of iron is recycled from erythrocytes tidylserine (PS) on the outer leaflet of the erythrocyte membrane (nor-
destroyed in vivo that results in a sustained an ∙6@fold increase in mally concentrated on the inner leaflet of the membrane) is another
44
erythropoiesis. The rate of erythropoiesis in blood loss anemia during signal that allows macrophages to recognize senescent erythrocytes.
which iron is lost from the body, however, depends more on preexist- This is the only major difference between senescent and nonsenescent
45
ing iron stores. In this case, the rate of erythropoiesis usually does not erythrocytes that has been clearly documented. Any combination of
exceed 2.5 times normal unless large parenteral or oral doses of iron these events could contribute to the trapping of erythrocytes in the
are administered. vasculature of the spleen and their removal by splenic macrophages.
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Chapter 5 • the erythroCyte 73
The chromatin condensation and mitochondrial destruction Severe trauma to the RBC that damages the cell’s structural integrity
that occurs during erythrocyte production parallel changes seen in and leads to a breach in the cell membrane results in intravascular cell
apoptotic cells, as does the PS externalization seen in erythrocyte lysis and release of hemoglobin directly into the blood (intravascular
senescence. The parallels with apoptosis have led some researchers destruction). Only about 10% of erythrocyte destruction occurs in this
to speculate that erythrocyte maturation and senescence represent manner. Released hemoglobin is bound by plasma proteins, and hap-
“apoptosis in slow motion.” 46 toglobin and hemopexin are transported to the liver where the hemo-
Erythrocyte removal by the spleen, bone marrow, and liver is globin is catabolized similar to the process in extravascular hemolysis.
referred to as extravascular destruction. This pathway accounts for
about 90% of aged erythrocyte destruction. It is the most efficient
method of cell removal, conserving and recycling essential erythrocyte CheCkpoint 5-10
components such as amino acids and iron. (Hemoglobin catabolism is Explain how oxidation of RBC cellular components can
covered in detail in Chapter 6.) Most extravascular destruction of eryth- lead to extravascular hemolysis.
rocytes takes place in the macrophages of the spleen. The spleen’s archi-
tecture with its torturous circulation, sluggish blood flow, and relative
hypoxic and hypoglycemic environment makes it well suited for culling
aged erythrocytes and trapping those cells that have minimal defects caSe Study (continued from page 72)
(Chapter 3). In contrast to the macrophages in the spleen, the liver mac-
rophages lack the ability to detect cells with minimal defects. However, 5. Stephen’s haptoglobin level is 25 mg/dL. Explain why
because the liver receives 35% of the cardiac output (the spleen receives he has a low haptoglobin value.
5%) it can be more efficient in removing cells it recognizes as abnormal.
Summary
Erythrocytes are derived from the unipotent committed progen- becomes more rigid and is culled in the spleen. The normal
itor cells BFU-E and CFU-E. Morphologic developmental stages erythrocyte life span is 100–120 days.
of the erythroid cell include (in order of increasing maturity) the The erythrocyte derives its energy and reducing power
pronormoblast, basophilic normoblast, polychromatophilic nor- from glycolysis and ancillary pathways. The glycolytic pathway
moblast, orthochromatic normoblast, reticulocyte, and eryth- provides ATP to help the cell maintain erythrocyte shape, flex-
rocyte. Erythropoietin, a hormone produced in renal tissues, ibility, and membrane integrity through regulation of intracel-
stimulates erythropoiesis and is responsible for maintaining a lular cation permeability. The HMP shunt provides reducing
steady-state erythrocyte mass. Erythropoietin stimulates survival power to protect the cell from permanent oxidative injury. The
and differentiation of erythroid progenitor cells, increases the methemoglobin reductase pathway helps reduce heme from
++
rate of erythropoiesis, and induces early release of reticulocytes the ferric (Fe +++ ) back to the ferrous (Fe ) state. The Rapoport-
from the marrow. Luebering shunt facilitates oxygen delivery to the tissue by pro-
The erythrocyte concentration varies with sex, age, and ducing 2,3-BPG.
geographic location. Higher concentrations are found in males Destruction of aged erythrocytes occurs primarily in the
(after puberty) and newborns and at high altitudes. Decreases macrophages of the spleen and liver through processes known
below the reference interval result in a condition called anemia. as extravascular and intravascular destruction. Extravascular
The erythrocyte membrane is a lipid-protein bilayer com- destruction of the erythrocyte is the major physiological path-
plex that is important for maintaining cellular deformability way for aged or abnormal erythrocyte removal (splenic and
and selective permeability. As the cell ages, the membrane is hepatic macrophages).
reduced in surface area relative to cell volume, and the cell
Review Questions
Level i 2. This renal hormone stimulates erythropoiesis in the bone
marrow: (Objective 4)
1. The earliest recognizable erythroid precursor on a Wright-
stained smear of the bone marrow is: (Objective 1) A. IL-1
A. pronormoblast B. erythropoietin
B. basophilic normoblast C. granulopoietin
C. CFU-E D. thrombopoietin
D. BFU-E
M05_MCKE6011_03_SE_C05.indd 73 01/08/14 11:22 am
74 SeCtIoN II • the hematopoIetIC SyStem
3. An increase in 2,3-BPG occurs at high altitude in an effort 9. An increase of erythrocyte membrane rigidity would be
to: (Objective 9) predicted to have what effect? (Objective 5)
A. increase oxygen affinity of hemoglobin A. increase in erythropoietin production
B. decrease oxygen affinity of hemoglobin
B. increase in cell volume
C. decrease the concentration of methemoglobin
C. decrease in cell life span
D. protect the cell from oxidant damage
D. decrease in reticulocytosis
4. The erythrocyte life span is most directly determined by:
(Objective 5) 10. Extravascular erythrocyte destruction occurs in: (Objective 7)
A. spleen size A. the bloodstream
B. serum haptoglobin level
B. macrophages in the spleen
C. membrane deformability
C. the lymph nodes
D. cell size and shape
D. bone marrow sinuses
5. Which of the following depicts the normal sequence of
erythroid maturation? (Objective 1)
Level ii
A. pronormoblast S basophilic normoblast S poly-
chromatic normoblast S orthochromic normoblast 1. Results of a CBC revealed a MCHC of 40 g/dL. What char-
S reticulocyte acteristic of the RBC will this affect? (Objective 2)
B. pronormoblast S polychromatic normoblast S A. oxygen affinity
orthochromic normoblast S basophilic normoblast
S reticulocyte B. cell metabolism
C. basophilic normoblast S polychromatic normoblast C. membrane permeability
S reticulocyte S orthochromic normoblast S D. cell deformability
pronormoblast
D. orthochromic normoblast S basophilic normoblast 2. If the erythrocyte cation pump fails because of inadequate
S reticulocyte S polychromatic normoblast S generation of ATP, the result is: (Objective 3)
pronormoblast A. decreased osmotic fragility due to formation of target
6. The primary effector (cause) of increased erythrocyte pro- cell
duction, or erythropoiesis, is: (Objective 4)
B. formation of echinocytes due to influx of potassium
A. supply of iron
C. cell crenation due to efflux of water and sodium
B. rate of bilirubin production
D. cell swelling due to influx of water and cations
C. tissue hypoxia
D. rate of EPO secretion 3. As a person ascends to high altitudes, the increased activ-
ity of the Rapoport-Luebering pathway: (Objective 4)
7. An increase in the reticulocyte count should be accompa-
nied by: (Objective 2) A. causes precipitation of hemoglobin as Heinz bodies
A. a shift to the left in the Hb@O 2 dissociation curve B. has no effect on oxygen delivery to tissues
B. abnormal maturation of normoblasts in the bone C. causes increased release of oxygen to tissues
marrow
D. causes decreased release of oxygen to tissues
C. an increase in total and direct serum bilirubin
D. polychromasia on the Wright's-stained blood smear 4. A newborn has a hemoglobin level of 16.0 g/dL at birth.
Two months later, a CBC indicates a hemoglobin concen-
8. What property of the normal erythrocyte membrane allows tration of 11.0 g/dL. The difference in hemoglobin concen-
the 7@mcM cell to squeeze through 3@mcM fenestrations in tration is most likely due to: (Objective 1)
the spleen? (Objective 5)
A. chronic blood loss
A. fluidity
B. inherited anemia
B. elasticity
C. permeability C. increased intravascular hemolysis
D. deformability D. physiologic anemia of the newborn
M05_MCKE6011_03_SE_C05.indd 74 01/08/14 11:22 am
Chapter 5 • the erythroCyte 75
5. A 50-year-old patient had a splenectomy after a car acci- 8. A patient with kidney disease has a hemoglobin of 8 g/dL.
dent that damaged her spleen. She had a CBC performed This is most likely associated with: (Objective 6)
at her 6-week postsurgical checkup. Many target cells were
identified on the blood smear. This finding is most likely: A. decreased EPO production
(Objective 2) B. increased intravascular hemolysis
A. an indication of liver disease C. abnormal RBC membrane permeability
B. a loss of RBC membrane peripheral proteins D. RBC fragility due to accumulation of intracellular
C. an abnormal protein to phospholipid ratio of the RBC calcium
membrane
9. A laboratory professional finds evidence of Heinz bodies
D. an accumulation of cholesterol and phospholipid in in the erythrocytes of a 30-year-old male. This is evidence
the RBC membrane of: (Objective 4)
6. Which of the following is necessary to maintain reduced A. increased oxidant concentration in the cell
levels of methemoglobin in the erythrocyte? (Objective 4)
B. decreased hemoglobin-oxygen affinity
A. vitamin B 6
C. decreased production of ATP
B. NADH
D. decreased stability of the cell membrane
C. 2,3-BPG
10. A 65-year-old female presents with an anemia of 3 weeks’
D. lactate duration. In addition to a decrease in her hemoglobin
7. A patient lost about 1500 mL of blood during surgery but and hematocrit, she has a reticulocyte count of 6% and
was not given blood transfusions. His hemoglobin before 3+ polychromasia on her blood smear. Based on these
surgery was in the reference range. The most likely finding preliminary findings, what serum erythropoietin result is
3 days later would be: (Objective 1, 6) expected? (Objective 6)
A. increase in total bilirubin A. decreased
B. increase in indirect bilirubin B. normal
C. increase in erythropoietin C. increased
D. increased haptoglobin D. no correlation
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. Find additional information to help
organize information and figures to help understand concepts.
Disclaimer
The views expressed in this chapter are those of the author and do not necessarily reflect the official policy or position of the Depart-
ment of the Army, the Department of Defense, or the U.S. government.
References
1. Beutler E. The red cell: a tiny dynamo. In: Wintrobe, MM, ed. Blood, Pure 4. Bull BS, Herrmann PC. Morphology of the erythron. In: Kaushansky K, Licht-
and Eloquent. New york: mcGraw-hill; 1980:141–68. man MA, Beutler E, Kipps TJ, Seligsohn U, Prchal JT, eds. Williams Hema-
2. Papayannopoulou T, Migliaccio AR, Abkowitz JL et al. Biology of erythro- tology, 8th ed. New york: mcGraw-hill; 2010;409–27.
poiesis, erythroid differentiation and maturation. In: Hoffman R, Benz EJ 5. Jing L, Guo X, Mohandas N, Chasis JA, An X. Membrane remodeling dur-
Jr, Shattil SJ et al., eds. Hematology: Basic Principles and Practice, 5th ed. ing reticulocyte maturation. Blood. 2010;115(10);2021–27.
Philadelphia: Elsevier Churchill Livingstone; 2009:276–94. 6. Chasis JA, Mohandas N. Erythroblastic Islands: niches for erythropoiesis.
3. Gregg XT, Prchal JT. Anemia of endocrine disorders. In: Kaushansky K, Blood. 2008;112(3);470–78.
Lichtman MA, Beutler E et al., eds. Williams Hematology, 8th ed. New york: 7. Mohandas N. The red cell membrane. In: Hoffman R, Benz EJ Jr, Shattil SJ
McGraw-Hill; 2010;509–11. et al., eds. Hematology: Basic Principles and Practice. Philadelphia: Elsevier
Churchill Livingstone; 1995:264–69.
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8. Lux SE, Palek J. Disorders of the red cell membrane. In: Handin RI, Lux SE, 28. Besa EC. Hematologic effects of androgens revisited: an alternative therapy
Stossel TP , eds. Blood Principles and Practice of Hematology. Philadelphia: in various hematologic conditions. Semin Hematol. 1994;31:134–45.
J. B. Lippincott; 1995:1701–1818. 29. Bachmann S, Le Hir M, Eckardt KU. Co-localization of erythropoietin mRNA
9. Devaux PF. Protein involvement in transmembrane lipid asymmetry. Annu and ecto-5’-nucleotidase immunoreactivity in peritubular cells of rat renal
Rev Biophys Biomol Struct. 1992;21:417–39. cortex indicates that fibroblasts produce erythropoietin. J Histochem
10. Butler J, Mohandas N, Waugh RE. Integral protein linkage and the Cytochem. 1993;41:335–41.
bilayer-skeletal separation energy in red blood cells. Biophys J. 30. Gabrilove J. Overview: Erythropoiesis, anemia, and the impact of erythro-
2008;95(4);1826–36. poietin. Sem Hematol. 2000;l37(4)(supp):1–3.
11. Fukuda M, Dell A, Fukuda MN. Structure of fetal lactosaminoglycan: The 31. Jelkmann W. Erythropoietin after a century of research: younger than ever.
carbohydrate moiety of band 3 isolated from human umbilical cord erythro- Eur J Haematol. 2007;78:183–205.
cytes. J Biol Chem. 1984;259:4782–91. 32. Marsden JT. Erythropoietin measurement and clinical application. Ann Clin
12. Fukuda M, Dell A, Oates JE et al. Structure of branched lactosaminoglycan, Biochem. 2006;43:97–104.
the carbohydrate moiety of band 3 isolated from adult erythrocytes. J Biol 33. Hodges VM, Rainey S, Lappin TR et al. Pathophysiology of anemia and
Chem. 1984;259:8260–73. erythrocytosis. Crit Rev Oncol Hematol. 2007;64:139–58.
13. Platt OS. Inherited disorders of red cell membrane proteins. In: Nagel RL, 34. Zanjani ED, Ascensao JL, McGlave PB et al. Studies on the liver to kidney
ed. Genetically Abnormal Red Cells. Boca Raton, FL: CRC Press; 1988. switch of erythropoietin production. J Clin Invest. 1981;67:1183–88.
14. Vertessy BG, Steck TL. Elasticity of the human red cell membrane skeleton: 35. Nangaku M, Eckardt KU. Hypoxia and the HIF system in kidney disease.
Effects of temperature and denaturants. Biophys J. 1989;55:255–62. J Mol Med. 2007;85:1325–30.
15. Bennett V, Stenbuck PJ. The membrane attachment protein for spectrin 36. Wang GL, Semenza GL. A nuclear factor induced by hypoxia via de novo
is associated with band 3 in human erythrocyte membranes. Nature. protein synthesis binds to the human erythropoietin gene enhancer at a site
1979;280:468–73. required for transcriptional activation. Mol Cell Biol. 1992;12:5447–54.
16. Cohen CM, Dotimas E, Korsgren C. Human erythrocyte membrane protein 37. Unger EF, Thompson AM, Blank MJ et al. Erythropoiesis-stimulating agents:
band 4.2 (pallidin). Semin Hematol. 1993;30:119–37. time for a reevaluation. N Engl J Med. 2010;362;189–92.
17. Lorand L, Siefring GE Jr, Lowe-Krentz L. Enzymatic basis of membrane stiff- 38. Kelley LL, Koury MJ, Bondurant MC et al. Survival or death of indi-
ening in human erythrocytes. Semin Hematol. 1979;16:65–74. vidual proerythroblasts results from differing erythropoietin sensitivi-
18. Mohandas N, Chasis JA. Red cell deformability, membrane material proper- ties: a mechanism for controlled rates of erythrocyte production. Blood.
ties, and shape: regulation by transmembrane, skeletal, and cytosolic pro- 1993;82:2340–52.
teins and lipids. Semin Hematol. 1993;30:171–92. 39. Swabe y, takiguchi y, Kikuno K et al. Changes in levels of serum erythro-
19. Liu SC, Pallek J. Spectrin tetramer-dimer equilibrium and the stability of poietin, serum iron, and unsaturated iron binding capacity during chemo-
erythrocyte membrane skeletons. Nature. 1980;285:586–88. therapy for lung cancer. J Clin Onc. 1998;28(3):182–86.
20. Liu S-C, Derick LH. Molecular anatomy of the red blood cell membrane 40. Kimmel PL, Greer JW, Milam RA et al. Trends in erythropoietin therapy in
skeleton: structure-function relationships. Semin Hematol. 1992;29: the U.S. dialysis population. Semin Nephrol. 2000;20(4):335–44.
231–43. 41. Suzuki T, Dale GI. Senescent erythrocytes: isolation of in vivo aged cells and
21. Zeidel ML, Ambudkar SV, Smith BL et al. Reconstitution of functional water their biochemical characteristics. Proc Natl Acad Sci USA. 1998;85:1647–51.
channels in liposomes containing purified red cell CHIP28 protein. Bio- 42. Zimran A, Forman L, Suzuki T et al. In vivo aging of red cell enzymes: study
chemistry. 1992;31:7436–40. of biotinylated red cells in rabbits. Am J Haematol. 1990;33:249–54.
22. Mueckler M, Caruso C, Baldwin SA et al. Sequence and structure of a 43. Steinberg MH, Benz EJ Jr, Adewoye AH et al. Pathobiology of the human
human glucose transporter. Science. 1983;229:941–45. erythrocyte and its hemoglobins. In: Hoffman R, Benz EJ Jr, Shattil SJ et al.,
23. May JM. Ascorbate function and metabolism in the human erythrocyte. eds. Hematology: Basic Principles and Practice, 5th ed. Philadelphia: Else-
Frontiers in Bio. 1998;3(1):1–10. vier Churchill Livingstone; 2009:427–38.
24. Viault F. Sur l’augmentation considerable du nombre des globules ranges 44. Boas FE, Forman L, Beutler E. Phosphatidylserine exposure and red cell
dans le sang chez les habitants des haute plateaux de l’amerique du sud. viability in red cell aging and in hemolytic anemia. Proc Natl Acad Sci USA.
CR Acad Sci (Paris). 1890;119:917–18. 1998;95:3077–81.
25. Bao W, Dalferes ER Jr, Srinivasan SR. Normative distribution of complete 45. Beutler E. Destruction of erythrocytes. In: Kaushansky K, Lichtman MA,
blood count from early childhood through adolescence: the Bogalusa Heart Beutler E et al., eds. Williams Hematology, 8th ed. New york: mcGraw-hill;
Study. Prev Med. 1993;22:825–37. 2010;449–54.
26. Cavalieri TA, Chopra A, Bryman PN. When outside the norm is normal: 46. Gottlieb RA. Apoptosis. In: Kaushansky K, Lichtman MA, Beutler E, Kipps
interpreting lab data in the aged. Geriat. 1992;47:66–70. TJ, Seligsohn U, Prchal JT, eds. Williams Hematology, 8th ed. New york:
27. Kosower NS. Altered properties of erythrocytes in the aged. Am J Hematol. McGraw-Hill; 2010;161–67. http://accessmedicine.mhmedical.com.libproxy
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6 Hemoglobin
Shirlyn B. McKenzie, PhD
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Diagram the quaternary structure of a hemoglobin molecule, Objectives—Level I and Level II 77
identifying the heme ring, globin chains, and iron.
2. Assemble fetal and adult hemoglobin molecules with appropriate Key Terms 78
globin chains. Background Basics 78
3. Explain how pH, temperature, 2,3-BPG, and PO 2 affect the oxygen
dissociation curve (ODC). Case Study 78
4. List the types of hemoglobin normally found in adults and newborns Overview 78
and give their approximate concentration.
5. Summarize hemoglobin’s function in gaseous transport. Introduction 78
6. Define hemoglobin reference intervals Hemoglobin Structure 79
7. Explain how the fine balance of hemoglobin concentration is
maintained. Hemoglobin Synthesis 81
8. Compare HbA with HbA1c and explain what an increased Regulation of Hemoglobin Synthesis 83
concentration of HbA1c means.
9. Diagram and describe the mechanism of extravascular erythrocyte Ontogeny of Hemoglobin 84
destruction and hemoglobin catabolism and name laboratory tests that Hemoglobin Function 85
can be used to evaluate it.
10. Diagram and describe the mechanism of intravascular erythrocyte Hemoglobin Catabolism 90
destruction and hemoglobin catabolism and name laboratory tests that Acquired Nonfunctional
can be used to evaluate it. Hemoglobins 92
Objectives—Level II Summary 94
At the end of this unit of study, the student should be able to: Review Questions 94
1. Construct a diagram to show the synthesis of a hemoglobin molecule. Companion Resources 96
2. Describe the ontogeny of hemoglobin types; contrast differences in
oxygen affinity of HbF and HbA and relate them to the structure of the References 96
molecule.
3. Explain the molecular control of heme synthesis.
4. Given information on pH, 2,3-BPG, CO 2 , temperature, and HbF
concentration; interpret the ODC, and translate it into the physiologic
effect on oxygen delivery.
(continued)
M06_MCKE6011_03_SE_C06.indd 77 01/08/14 11:25 pm
78 SECTION II • ThE hEmaTOpOIETIC SySTEm
+
8. Compare and contrast the exchange of O 2 , CO 2 , H , and
Objectives—Level II (continued) Cl at the level of capillaries and the lungs.
-
5. Contrast the structures and functions of relaxed and 9. Explain the role of hemoglobin in the NO-control of
tense hemoglobin and propose how these structures blood flow and vessel homeostasis.
affect gaseous transport.
6. Describe how abnormal hemoglobins are acquired, and 10. Compare and contrast erythrocyte extravascular destruc-
tion with intravascular destruction and identify which pro-
select a method by which they can be detected in the cess is dominant given laboratory results.
laboratory.
7. Assess the oxygen affinity of abnormal, acquired hemo- 11. Identify the breakdown products of hemoglobin and
determine how the body conserves and recycles essential
globins and reason how this affects oxygen transport.
components.
Key Terms Background Basics
Artificial oxygen carrier (AOC) Hemopexin The information in this chapter builds on the concepts learned
Bilirubin Hemosiderin in previous chapters. To maximize your learning experience, you
Bohr effect Hemosiderinuria should review these concepts before starting this unit of study:
Carboxyhemoglobin Hypoxia
Chloride shift Oxygen affinity level i
Cyanosis Oxyhemoglobin • List and describe the stages of erythrocyte maturation
Deoxyhemoglobin methemoglobin (Chapter 5).
Ferritin Relaxed (R) structure • Summarize the role of erythropoietin in erythropoiesis
Glycosylated hemoglobin Sulfhemoglobin (Chapter 5).
Haptoglobin Tense (T) structure • Describe the site of erythropoiesis (Chapter 4).
Heme level ii
• Describe the metabolic pathways present in the mature eryth-
rocyte, and explain their role in maintaining viability of the
erythrocyte (Chapter 5).
• Summarize the development of hematopoiesis from the embry-
onic stage to the adult (Chapter 4).
that affect this function. The chapter also discusses structure, forma-
caSe StuDy tion, and laboratory detection of abnormal hemoglobins.
We will address this case study throughout the chapter.
Jerry, a 44-year-old male, arrived in the emergency room intrODuctiOn
by ambulance after a bicycle accident. Examination Hemoglobin is a highly specialized intracellular erythrocyte protein
revealed multiple fractures of the femur. He was oth- responsible for transporting oxygen from the lungs to tissue for oxi-
erwise healthy. The next day, he was taken to surgery dative metabolism and facilitating carbon dioxide transport from the
to repair the fractures. After surgery, his hemoglobin tissue to the lungs. Each gram of hemoglobin can carry 1.34 mL of
was 7 g/dL. He refused blood transfusions and was dis- oxygen. It is also a transporter of nitric oxide, which modulates vas-
charged 6 days later. Jerry called his doctor within days cular tone.
of being discharged and told him that he had difficulty Hemoglobin occupies approximately 33% of the volume of the
walking around the house on crutches because of short- erythrocyte and accounts for 90% of the cell’s dry weight. Each cell
ness of breath and lack of stamina. contains between 28 and 34 pg of hemoglobin, a concentration close
Consider why Jerry’s hemoglobin decreased after to the solubility limit of hemoglobin. This concentration is measured
surgery and how this could be related to his current by cell analyzers and reported as mean corpuscular hemoglobin
symptoms.
(MCH). In anemic states, the erythrocyte can contain less hemoglobin
(decreased MCH) and/or the individual can have fewer erythrocytes
present, both of which result in a decrease of the blood’s oxygen-car-
Overview rying capacity.
This chapter describes the synthesis and structure of hemoglobin and The erythrocyte’s membrane and its metabolic pathways are
factors that regulate its production. It compares the different types of responsible for protecting and maintaining the hemoglobin molecule
hemoglobin produced according to developmental stage, considers in its functional state. Abnormalities in the membrane that alter its
the function of hemoglobin in gaseous exchange, and analyzes factors permeability or alterations of the cell’s enzyme systems can lead to
M06_MCKE6011_03_SE_C06.indd 78 01/08/14 11:25 pm
ChapTEr 6 • hEmOgLObIN 79
changes in the structure and/or function of the hemoglobin molecule
and affect the capacity of this protein to deliver oxygen. a 2
Although a small amount of hemoglobin is synthesized as early a 1
as the pronormoblast stage, most hemoglobin synthesized in the Heme
developing normoblasts occurs at the polychromatophilic normo-
blast stage. In total, 75–80% of the cell’s hemoglobin is made before Globin
Chain
the extrusion of the nucleus. Because the reticulocyte does not have
a nucleus, it cannot make new RNA for protein synthesis. However,
residual RNA and mitochondria in the reticulocyte enable the cell
to make the remaining 20–25% of the cell’s hemoglobin. The mature
erythrocyte contains no nucleus, ribosomes, or mitochondria and is
unable to synthesize new protein.
Hemoglobin concentration in the body is the result of a fine bal-
ance between production and destruction of erythrocytes. The nor-
mal hemoglobin concentration in an adult male is about 15 g/dL with b
a total blood volume of about 5 L (50 dL). Therefore, the total body b 1 2
mass of hemoglobin is approximately 750 g:
■ Figure 6-1 Hemoglobin is a molecule composed of
15 g/dL * 50 dL = 750 g four polypeptide subunits. Each subunit has a globin chain
1 of with a heme nestled in a hydrophobic crevice that pro-
Because the normal erythrocyte life span is 120 days, 120 tects the iron from being oxidized. There are four different
the total amount of hemoglobin is lost each day through removal of types of globin chains:a, b, d, g. Two a@chains and two
senescent erythrocytes. Thus, an equivalent amount must be synthe- non@a-chains occur in identical pairs to form a tetramer.
sized each day to maintain a steady-state concentration. This amounts The types of globin chains present determine the type of
to approximately 6 g of new hemoglobin per day: hemoglobin. Depicted here is hemoglobin A, consisting
of two a- and two b-chains. The contacts between the
750 g (amount of hemoglobin lost
= 6.25 g/day a, b@chains in a dimer (i.e., a 1 b 1 ) are extensive and allow
120 days and synthesized each day) little movement. The contacts between the dimer pairs
(i.e., a 1 b 2 , a 2 b 1 ), however, are smaller and allow conforma-
If we divide the total amount of hemoglobin synthesized each day tional change of the molecule as it goes from oxyhemo-
(6.25 g) by the mean amount of hemoglobin in a red cell (MCH, 30 globin to deoxyhemoglobin.
pg), we can determine the daily production of new red blood cells:
12
6.25 g/day 10 pg
11
* = 2 * 10 cells/day
30 pg/cell g
★ taBle 6-1 The Structure of Hemoglobin
heMOglOBin Structure Structure conformational Description
Hemoglobin is a large tetrameric molecule, molecular weight 66,700 Primary Sequence of individual amino acids held
daltons, composed of four globular protein subunits (Figure 6-1 ■). together by peptide bonds in the globin
Each of the four subunits contains a heme group and a globin chain. chains; is critical to stability and function of
Heme, the prosthetic group of hemoglobin, is a tetrapyrrole ring with molecule; determines the overall structure
ferrous iron located in the center of the ring. Hemoglobin structure Secondary Arrangement of the amino acids resulting from
is described in Table 6-1 ★ (Figure 6-2 ■). Each heme subunit can hydrogen bonding between the peptide bonds
carry one molecule of oxygen bound to the central ferrous iron; thus, of the amino acids next to or near each other
(75% of the chain is in the form of an a@helix;
each hemoglobin molecule can carry four molecules of oxygen. each chain consists of 7 or 8 a@helix segments,
The composition of the globin chains is responsible for the dif- labeled A S H, separated by nonhelical
ferent functional and physical properties of hemoglobin. Two types [pleated] segments that do not participate in
of globin chains are produced: alpha-like (alpha [a], zeta [z]), and forming the a@helix but allow the polypeptide
non-alpha (epsilon [e], beta [b], delta [d], gamma [g]). The tetra- to fold on itself )
meric hemoglobin molecule consists of two pairs of unlike chains: Tertiary Folding superimposed on the helical and
pleated domains; forms the heme hydrophobic
two identical a@like and two identical non@a@chains. A pair of a@like pocket within globin chains and places polar
chains (a or z) combines with a pair of non@a@chains (P, b, d, or g) (hydrophilic) residues on the exterior of the
to form the various types of hemoglobin (Table 6-2 ★). The arrange- molecule; this tertiary structure changes upon
ment of each globin chain is similar. Each a@ and z@chain has 141 ligand binding
amino acids, and each P@, b@, d@, and @chain has 146 amino acids. Quaternary Relationship of the four protein subunits to
The b@ chain is composed of eight a@helical segments separated by one another; quaternary structural changes
that occur upon ligand binding result from the
seven short, nonhelical segments, and the a@chain has seven a@helical tertiary changes
segments. The helical segments are lettered A–H, starting at the amino
M06_MCKE6011_03_SE_C06.indd 79 01/08/14 11:26 pm
80 SECTION II • ThE hEmaTOpOIETIC SySTEm
Ala Ser Leu
Asp
Arg
Cys
Yal
Pro
Lys
Phe
Tyr Leu His Lys Asn
Thr
a Primary Leu Asp
structure Ala Ser Arg
Cys
Yal
Pro Tyr Thr
Phe Leu
Lys
His
Lys
Asn
b Secondary
Polypeptide structure
(globin)
Heme group
c Tertiary
structure
Fe ++
Heme
d Quaternary
structure
■ Figure 6-2 The structure of hemoglobin. (a) Primary structure is the
sequence of amino acids. (b) Secondary structure is the coiled a@helix and
b@pleated sheet formed by hydrogen bonding between the peptide bonds
in the chain. (c) Tertiary structure is the folding of the molecule into a three-
dimensional structure. (d) Quartenary structure is the combination of the
four polypeptide subunits, each of which contains a heme group, into a
larger protein.
end of the chain. The amino acids of the globin chains are identified ★ taBle 6-2 Normal Types of Hemoglobin According
by their helix location and amino acid number (e.g., F8 is the eighth to Developmental Stage
amino acid in the F helix). Amino acids between helices are identified
by amino acid number and the letters of the two helices (e.g., EF3). Developmental globin reference
The nonhelical segments allow the chains to fold upon themselves. Stage type chains interval
The four subunits of hemoglobin, each consisting of a heme Embryonic Gower 1 z 2 e 2 —
group surrounded by a globin chain, are held together by salt bonds, Gower 2 a 2 e 2 —
hydrophobic contacts, and hydrogen bonds in a tetrahedral forma- Portland z 2 g 2 —
tion giving the hemoglobin molecule a nearly spherical shape. When Fetal HbF a 2 g 2 90–95% before
ligands such as oxygen bind to hemoglobin, the number and strin- birth
gency of intersubunit contacts change and the shape of the molecule 50–85% at birth
changes. Mutations in the primary structure of globin chains can affect HbA a 2 b 2 10–40% at birth
subunit or dimer pair interactions and thus alter hemoglobin-oxygen HbA 2 a 2 d 2 61% at birth
affinity or the molecule’s stability. 71 year old HbF a 2 g 2 62%
HbA a 2 b 2 795%
CheCkpoint 6-1 HbA 2 a 2 d 2 63.5%
Adult HbA a 2 b 2 795%
Describe the quaternary structure of a molecule of hemoglo- HbA 2 a 2 d 2 1.5–3.7%
bin. How can a mutation in one of the globin chains at the HbF a 2 g 2 62%
subunit interaction site, a 1 b 2 , affect hemoglobin function? a = alpha; b = beta; g = gamma; d = delta; z = zeta
M06_MCKE6011_03_SE_C06.indd 80 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 81
heMOglOBin SyntheSiS final step, also occurring in the mitochondria, is the chelation of iron
Heme with protoporphyrin IX catalyzed by ferrochelatase to form heme
(Figure 6-4 ■). Heme then leaves the mitochondria to combine with
Heme is an iron-chelated porphyrin ring that functions as a pros- a globin chain in the cytoplasm. See Web Figure 6-1 for detailed
thetic group (nonamino acid component) of a protein. The porphyrin molecular structures of intermediates in heme synthesis.
ring, protoporphyrin IX, is composed of a flat tetrapyrrole ring with
++
ferrous iron (Fe ) inserted into the center. (Porphyrins are metaboli- Globin Chain Synthesis
cally active only when chelated.) Ferrous ions have six electron pairs Globin chain synthesis is directed by genes in two clusters on chromo-
per atom. In heme, four of these electron pairs are coordinately bound somes 11 and 16 (Figure 6-5 ■). These genes produce the seven differ-
to the N atoms of each of the four pyrrole rings. In hemoglobin, one of ent types of globin chains: zeta, alpha, epsilon, gamma-A, gamma-G,
G
A
the two remaining electron pairs (fifth) is coordinately bound with the delta, and beta (a, z, P, g , g , d, b). Two are found only in embryonic
N of the proximal histidine (F8) of the globin chain, and the other pair hemoglobins (z, e). The genes for the z@chain (the fetal equivalent of
(sixth) is the binding site for molecular oxygen. In the deoxygenated the a@chain) and a@chain are located on the short arm of chromo-
state, the sixth electron pair is occupied by a water molecule. Iron must some 16 (the a gene cluster). The z@chain is synthesized very early in
++
be in the ferrous (Fe ) state for oxygen binding to occur. Ferric iron embryonic development, but after 8–12 weeks, z@chain synthesis is
(Fe +++ ), which has lost an electron, cannot serve as an oxygen carrier replaced by a@chain synthesis. There are two a@loci (a@1, a@2), both
because the sixth potential binding site (electron pair) for oxygen is of which transcribe mRNA for a@chain synthesis. The protein product
no longer available. from each locus is structurally identical. The non@a@globin genes are
Heme synthesis begins in the mitochondria with the con- arranged in linear fashion in order of activation on chromosome 11
densation of glycine and succinyl coenzyme A (CoA) to form (the non@a@gene cluster).
5-aminolevulinic acid (ALA). This reaction occurs in the presence of The e@gene, the first non@a@gene to be activated, is located
the cofactor pyridoxal phosphate and the enzyme 5-aminolevulinate toward the 5 end of chromosome 11; during embryonic develop-
synthase (ALAS). This first reaction is the rate-limiting step in the ment, e@chain synthesis is switched off, and the two g@genes are acti-
synthesis of heme and occurs only when the cell has an adequate vated. One g@gene directs the production of a g@chain with glycine
1
G
supply of iron (Chapter 12). Synthesis continues through a series at amino acid position 136, g , and the other directs the production
A
G
of steps in the cytoplasm, eventually forming coproporphyrinogen. of a g@chain with alanine at position 136, g . The g @chain synthesis
A
G
Coproporphyrinogen then reenters the mitochondria and is further predominates before birth (3:1), but g @ and g @chain syntheses are
modified to form the protoporphyrin IX ring (Figure 6-3 ■). The equal (1:1) in adults. The next two genes on chromosome 11, d and b,
Mitochondria Cytoplasm
ALAS2 ALA dehydrase
Succinyl CoA ALA PBG
and Succinyl
glycine CoA synthase,
pyridoxal
phosphate PBG deaminase
Hydroxymethylbilane
Heme
Uroporphyrinogen III Spontaneous
cosynthase
Ferrochelatase
Protoporphyrin IX
Uroporphyringen III Uroporphyrinogen I
Protoporphyrinogen oxidase
Protoporphyrinogen III Coproporphyrinogen III Coproporphyrinogen I
Coproporphyrinogen
oxidase
■ Figure 6-3 Synthesis of heme. It begins in the mitochondria with the con-
densation of glycine and succinyl CoA catalyzed by 5-aminolevulinate synthase
2 (ALAS2) and succinyl CoA synthase and co-factor pyridoxal phosphate. The
product, 5-aminolevulinate (ALA), leaves the mitochondria to form the pyrrole
ring, porphobilinogen (PBG). The combination of four pyrroles to form a linear
tetrapyrrole (hydroxymethylbilane), the cyclizing of the linear form to uroporphy-
rinogen, and the decarboxylation of the side chains to form coproporphyrinogen
occur in the cytoplasm. The final reactions, the formation of protoporphyrin IX,
and the insertion of iron into the protoporphyrin ring occur in the mitochondria.
M06_MCKE6011_03_SE_C06.indd 81 01/08/14 11:26 pm
82 SECTION II • ThE hEmaTOpOIETIC SySTEm
CH 2
H CH 3
C
H 3 C CH 2
N N
HC Fe CH
N N
H CH 3
C
H
HOOC COOH
Hemoglobin Heme structure
■ Figure 6-4 The hemoglobin molecule on the left reveals the quartenary struc-
ture of hemoglobin with four protein chains, each folded around a heme molecule.
On the right is a heme molecule. Heme is composed of a flat tetrapyrrole ligand
(porphyrin) and iron. The iron has six coordinate sites. Nitrogen atoms of porphyrin
occupy four coordination sites in a square planar arrangement around the iron.
Iron in the ferrous state has two other coordinate sites, one of which is occupied by
the N of the proximal histidine (F8) of globin and one with molecular oxygen
or H 2 O.
are switched on to a small degree when the g@genes are activated, into this hydrophobic pocket where it is readily accessible to oxygen.
but they are not fully activated until g@chain synthesis diminishes A newly formed a@chain@heme subunit and a non@ @chain@heme
at about 35 weeks of gestation. The rate of synthesis of the d@chain subunit combine spontaneously, facilitated by electrostatic attrac-
1 that of the b@chain, due to differences in the promoter tion, to form a dimer (e.g., ab). Charge differences exist among the
is only 140
regions of the two genes. After birth, most cells produce predomi- non@a@globin chains. This promotes a hierarchy of affinity of these
nantly a@ and b@chains for the formation of HbA, the major adult chains for the a@globin chains. The b@globin chain has the greatest
hemoglobin (97%). affinity for a@globin chains followed by g@ and d@chains. Then two
The synthesis of globin peptide chains occurs on polyribo- dimers combine to form the tetrameric hemoglobin molecule (e.g.,
somes in the cytoplasm of developing erythroblasts (Figure 6-6 ■). a b ). The protein alpha hemoglobin-stabilizing protein (AHSP)
2 2
Globin chains are released from the polyribosomes and combine plays an important role in coordinating heme and globin assembly.
with heme molecules released from the mitochondria. The globin AHSP binds free a@chains and stabilizes their structure, increasing
chains are folded to create a hydrophobic pocket near the exterior their affinity for b@chains. This accelerates the formation of hemo-
surface of the chain between the E and F helices. Heme is inserted globin tetramers. 1
Chromosome 16 Chromosome 11
G A
Globin G A
chain
■ Figure 6-5 The genes for the globin chains are located on chromo-
somes 11 and 16. The z@chain appears to be the embryonic equivalent of the
a@chain, both of which are located on chromosome 16. Note that the a@gene
is duplicated. The other globin genes are located on chromosome 11.
M06_MCKE6011_03_SE_C06.indd 82 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 83
chains, protoporphyrin, and iron are not. Normally, the production of
Globin
mRNA a@globin subunits, non@a globin subunits, and heme are nearly equal.
This indicates that tight regulatory mechanisms exist, controlling the
production of hemoglobin. Hemoglobin synthesis is regulated by sev-
NH 2 NH 2 eral mechanisms including:
Fe
Fe
Fe
Fe Heme Fe
Fe
Fe
Fe
e
Fe
Fe
F
Fe
Fe
Fe
Fee
F
Fe
Fe
Mitochondria Mitochondria • Activity and concentration of the erythroid enzyme 5-aminolevulinate
synthase (ALAS2)
Fe Fe • Activity of porphobilinogen deaminase (PBGD)
• Concentration of iron
• Regulation of globin chain synthesis
Fe
The first step in the heme synthetic pathway, catalyzed by ALAS,
Encounter complex is the rate-limiting step in heme synthesis and takes place in the mito-
Fe chondria. ALAS is synthesized on ribosomes in the cytosol, and must
be imported into the mitochondria to catalyze the reaction. This
Fe mitochondrial import can be inhibited by high concentrations of free
2
Stable dimer heme. Heme also inhibits uptake of iron from transferrin into the cell.
Fe When iron is scarce, the synthesis of ALAS is decreased.
Iron entering the developing erythroblast can be in either the
pool available for metabolic processes (heme synthesis) or the storage
pool (ferritin and hemosiderin). The amount of iron in these pools
is regulated by proteins that control transcription and translation of
Fe
2 2 Tetramer proteins involved in heme synthesis and the formation of ferritin as
1–5
well as transferrin receptors. Iron metabolism is discussed in detail
Fe in Chapter 12.
The expression of globin genes occurs only in erythroid cells
during a narrow period of differentiation. Synthesis of globin chains
■ Figure 6-6 Assembly of hemoglobin. The a@ and begins in the pronormoblast and continues until the reticulocyte loses
b@globin polypeptides are translated from their respec-
tive mRNAs. Upon heme binding, the protein folds into its remnants of mRNA. The rate of globin synthesis is governed primar-
native three-dimensional structure. The binding of a@ and ily by the rate at which the DNA is transcribed to mRNA, but it is
b@hemoglobin subunits to each other is facilitated by elec- also modified by the processing of globin pre-mRNA to mRNA, the
trostatic attraction. An unstable intermediate encounter translation of mRNA to protein, and the stability of globin mRNA.
complex can rearrange to form the stable ab dimer. Two The individual globin genes have separate promoter regions available
dimers combine to form the functional a 2 b 2 tetramer. for activation at variable times during embryonic and fetal develop-
ment. In addition, the b@gene cluster has a locus control region (LCR),
located upstream (5 ) of the genes, which plays an important role in
The heme is positioned between two histidines of the globin regulating the entire gene cluster. The a@gene cluster has a similar
chain, the proximal (F8) and distal (E7) histidines. The proximal control region, the HS40, thought to have a similar function. Heme
histidine is bonded with the heme iron. The iron is protected in the plays an important role in controlling the synthesis of globin chains.
reduced ferrous state in this hydrophobic pocket. The exterior of the It stimulates globin synthesis by inactivating an inhibitor of transla-
chain is hydrophilic, which makes the molecule soluble. tion. A slight excess of a@chain mRNA is produced, but the mRNA
of b@chains is more efficiently translated, resulting in almost equal
synthesis of a@ and b@chains.
CheCkpoint 6-2
What globin chains are synthesized in the adult?
caSe StuDy (continued from page 78)
Jerry’s doctor gave him iron supplements to take
every day.
regulatiOn OF heMOglOBin 1. If Jerry is iron deficient, what is the effect of this defi-
SyntheSiS cient state on the synthesis of ALAS, transferrin recep-
Balanced synthesis of globin chains and heme is important to the tor, and ferritin?
survival of the erythrocyte because hemoglobin tetramers are soluble, 2. What was the rationale for giving Jerry the iron?
but individual components of hemoglobin such as unpaired globin
M06_MCKE6011_03_SE_C06.indd 83 01/08/14 11:26 pm
84 SECTION II • ThE hEmaTOpOIETIC SySTEm
OntOgeny OF heMOglOBin birth. After birth, the percentage of HbA continues to increase with
the infant’s age until normal adult levels (795%) are reached by the
The type of hemoglobin is determined by its composition of globin end of the first year of life.
chains (Table 6-2). Individual globin chains are expressed at different HbF production constitutes less than 2% of the total hemoglobin
levels in developing erythroblasts of the human embryo, fetus, and of adults. In normal adults, most if not all HbF is restricted to a few
adult. Some hemoglobins (Gower 1, Gower 2, Portland) occur only in erythrocytes, referred to as F cells. F cells constitute 2–5% of adult
the embryonic stage of development. HbF is the predominant hemo- RBCs, and from 13–25% of the hemoglobin within each F cell is HbF.
globin in the fetus and newborn, and hemoglobin A is the predomi- The switch from HbF to HbA after birth is incomplete and in part
nant hemoglobin after 1 year of age. reversible. For example, patients with hemoglobinopathies or severe
The synthesis of different globin chains occurs in sequence
depending on the developmental stage. This appears to be due to anemia can have increased levels of HbF, often proportionate to the
decrease in HbA. In bone marrow recovering from suppression and in
the sequential activation and then inactivation of transcription (i.e., some neoplastic hematologic diseases, HbF levels often rise.
“switching”) among the a@ and non@a@globin gene clusters. Globin HbA (a d ) appears late in fetal life, composes 6 1% of the total
2 2
2
gene expression is also affected by cellular, microenvironmental, hemoglobin at birth, and reaches normal adult values (1.5–3.7%) after
and humoral influences that affect the proliferation and differentia- one year. The d@gene locus is transcribed very inefficiently compared
tion of stem cells. In vitro cultures of burst forming unit—erythroid with the b@locus due to changes in the promoter region of the d@gene
(BFU-E; Chapter 4) from fetal liver, neonatal (umbilical cord) blood, that is recognized by erythroid-specific transcription factors (e.g.,
and adult blood show HbF production from these three sources to GATA-1). HbA has a slightly higher oxygen affinity than HbA; oth-
2
decrease in concentration from fetal to neonatal to adult. The most erwise, the two hemoglobins have similar or identical ligand binding
important determinant of the switch from fetal to adult hemoglobin curves, Bohr effect, and response to 2,3-BPG.
synthesis appears to be postconceptual age and is unaffected by the
time of birth: Premature infants do not switch over to adult hemoglo-
bin synthesis any earlier than they would if they had been carried full
term. The developmental control of the perinatal switch from HbF CheCkpoint 6-3
to HbA synthesis appears to be intrinsic to the erythroid cell and is What are the names and globin composition of the
6,7
probably time controlled by a developmental clock. The progenitor embryonic, fetal, and adult hemoglobins?
cells are gradually reprogrammed during the perinatal period, leading
to a switching from g@chain production to predominantly b@chain
production. This can involve not only preferential stimulation of the
b@globin gene but also active repression of the g@globin gene. Glycosylated Hemoglobin
Prolonged exposure of hemoglobin to chemically active compounds
Embryonic Hemoglobins in the blood can result in nonenzymatic modification of hemoglobin.
Embryonic erythropoiesis is associated with the production of the HbA is a minor component of normal adult hemoglobin (HbA) that
1
embryonic hemoglobins Gower 1, Gower 2, and Portland and appar- has been modified post-translationally in which a component has
ently synthesized in succession as globin synthesis switches from been added to (usually) the N terminus of the b@chain. Also known
z S a and from d S g in the first trimester of gestation. Embryonic as “fast hemoglobin” or glycated hemoglobins, HbA consists of three
1
hemoglobins are made from the combination of pairs of embryonic subgroups, HbA , HbA , and HbA . The clinically most important
1c
1a
1b
globin chains, z and P (z e ) or embryonic chains in combination subgroup of HbA is HbA , which is produced throughout the eryth-
2 2
1c
1
with a@ and g@chains (a P , z g ). These primitive hemoglobins are rocyte’s life and is proportional to the concentration of blood glucose.
2 2
2 2
detectable during early hematopoiesis in the yolk sac and liver and are Older erythrocytes typically contain more HbA than younger eryth-
1c
not usually detectable after the third month of gestation. rocytes, having been exposed to plasma glucose for a longer period of
time. However, if young cells are exposed to extremely high concentra-
Fetal Hemoglobin tions of glucose (7400 mg/dL) for several hours, the concentration of
1c
As embryonic erythropoiesis shifts to fetal erythropoiesis, hemoglo- HbA increases with both concentration and time of exposure.
Measurement of HbA is routinely used as an indicator of con-
1c
bin F (HbF; a g ) becomes the predominant hemoglobin formed dur- trol of blood glucose levels in diabetics because it is proportional
2 2
ing liver and bone marrow erythropoiesis in the fetus. HbF composes to the average blood glucose level over the previous 2–3 months.
90–95% of the total hemoglobin production in the fetus until 34936 Average levels of HbA are 7.5% in diabetics and 3.5% in normal
weeks of gestation. At birth, the infant has 50–85% HbF. 1c
individuals.
Adult Hemoglobins
The fetal to adult shift in erythropoiesis reflects transcription of the CheCkpoint 6-4
b@globin chain. In adults, hemoglobin A (HbA; a b ) is the major
2 2
hemoglobin. Although HbA is found as early as 9 weeks gestation, A patient has an anemia caused by a shortened RBC
b@chain synthesis occurs at a low level until the third trimester of life span (hemolysis); how would this affect the HBA 1c
pregnancy. b@chain synthesis steadily increases from gestational measurement?
week 30 onward but does not exceed g@chain synthesis until after
M06_MCKE6011_03_SE_C06.indd 84 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 85
heMOglOBin FunctiOn not readily give it up; decreased oxygen affinity means the hemoglobin
has a low affinity for oxygen and releases its oxygen more readily.
The function of hemoglobin is to transport and exchange respiratory Oxyhemoglobin and deoxyhemoglobin have different three-
gases. The air we breathe is a mixture of nitrogen, oxygen, water, and dimensional configurations. In the unliganded or deoxy state, the tetra-
carbon dioxide. Each of the gases contributes to the atmospheric pres- mer is stabilized by intersubunit salt bridges and is described as being in
sure (measured in mmHg) in proportion to its concentration. The par- the tense (T) structure or state. In oxyhemoglobin, the salt bridges are
tial pressure each gas exerts is referred to as P (e.g., PO ) and determines broken, and the molecule is described as being in the relaxed (R) struc-
2
the rate of diffusion of that gas across the alveolar-capillary membrane. ture or state. The change in conformation of hemoglobin (from T to R)
Arterialized blood leaves the lungs with a PO of 100 mmHg and a occurs as a result of a coordinated series of changes in the quaternary
2
PCO of 40 mmHg. In comparison, the PO of interstitial fluid in tis- structure of the tetramer as the subunits bind oxygen (see “The Allosteric
2
2
sues is about 40 mmHg, and the PCO is about 45 mmHg. Thus, when Property of Hemoglobin”). The T configuration is a low oxygen-affinity
2
blood reaches the tissues, oxygen diffuses out of the blood to the tissues conformation, and the R state is a high oxygen-affinity conformation.
and CO diffuses into the blood from tissue. The amount of dissolved Oxygen affinity of hemoglobin is usually expressed as the PO
2
2
O and CO that the plasma can carry is limited. Most O and CO at which 50% of the hemoglobin is saturated with oxygen (P ). The
2
2
2
2
diffuse into the erythrocyte to be transported to tissue or lungs. 50
P in humans is normally about 26 mmHg. If hemoglobin-oxygen
50
saturation is plotted versus the partial pressure of oxygen (PO ), a
2
Oxygen Transport sigmoid-shaped (S-shaped) curve results. This is referred to as the oxy-
Hemoglobin with bound oxygen is called oxyhemoglobin; hemoglo- gen dissociation curve (ODC) (Figure 6-7 ■). The shape of the curve
bin without oxygen is called deoxyhemoglobin. The amount of oxy- reflects subunit interactions between the four subunits of hemoglo-
gen bound to hemoglobin and released to tissues depends not only on bin (heme–heme interaction or cooperativity). Monomeric molecules
the PO and PCO but also on the affinity of Hb for O . The ease with such as myoglobin have a hyperbolic ODC indicating no cooperativity
2
2
2
which hemoglobin binds and releases oxygen is known as oxygen of oxygen binding. The sigmoid-shaped curve of hemoglobin disso-
affinity. Hemoglobin affinity for oxygen determines the proportion of ciation indicates that the deoxyhemoglobin tetramer is slow to take
oxygen released to the tissues or loaded onto the cell at a given oxygen up an O molecule, but binding one molecule of O to hemoglobin
2
2
pressure (PO ). Increased oxygen affinity means that the hemoglobin facilitates the binding of additional O . Thus, the “appetite” of hemo-
2
2
has a high affinity for oxygen, will bind oxygen more avidly, and does globin for oxygen grows with the addition of each oxygen molecule. 8
100 +
80
Percent saturation of Hb by O 2 60 Normal conditions 2
Increased H
Increased CO
Increased temperature
Increased 2.3-BPG
40
+
Decreased H
20
Decreased CO 2
Decreased temperature
Decreased 2.3-BPG
0 20 40 60 80 100 120
O partial pressure (mm Hg)
2
■ Figure 6-7 The oxygen affinity of hemoglobin is depicted by the
oxygen dissociation curve (ODC). The fractional saturation of hemoglobin
(y axis) is plotted against the concentration of oxygen measured as the PO 2
(x axis). At a pH of 7.4 and an oxygen tension (PO 2 ) of 26 mmHg, hemo-
globin is 50% saturated with oxygen (red line). The curve shifts in response
to temperature, CO 2 , O 2 , 2,3-BPG concentration, and pH. When the curve
shifts left (light blue line), there is increased affinity of Hb for O 2 . When the
curve shifts right (dark blue line), there is decreased affinity of Hb for O 2 .
“Figure 29.12” from Fundamentals of General, Organic and Biological Chemistry, 5E by
John McMurry, Mary E. Castellion, and David S. Ballantine. Copyright © 2007 by Pearson
Education. Reprinted and Electronically reproduced by permission of Pearson Education,
Inc., Upper Saddle River, New Jersey.
M06_MCKE6011_03_SE_C06.indd 85 01/08/14 11:26 pm
86 SECTION II • ThE hEmaTOpOIETIC SySTEm
The shape of the curve has certain physiologic advantages. The “flat- the allosteric Property of hemoglobin
tened” top of the S reflects the fact that 790% saturation of hemoglobin The sigmoid shape of the ODC is primarily due to heme–heme inter-
still occurs over a fairly broad range of PO . This enables us to survive actions described below. However, the relative position of the curve
2
and function in conditions of lower oxygen availability, such as living (or (shifted right or left) is due to other variables.
skiing) at high altitudes. Note that the steepest part of the curve occurs at Hemoglobin is an allosteric protein, meaning that its structure
oxygen tensions found in tissues. This allows the release of large amounts (conformation) and function are affected by other molecules. The
of oxygen from hemoglobin during the small physiologic changes in PO primary allosteric regulator of hemoglobin is 2,3-bisphosphoglycer-
2
encountered in the capillary beds of tissues. This is physiologically of great ate (2,3-BPG; also referred to as 2,3-diphosphoglycerate [2,3-DPG]).
importance, for it allows the overall transfer of oxygen from the lungs to A byproduct of the glycolytic pathway, 2,3-BPG, is present at almost
the tissues with relatively small changes in PO . The ODC shows that the equimolar amounts with hemoglobin in erythrocytes. In the presence
2
oxygen saturation of hemoglobin drops from 100% in the arteries to of physiologic concentrations of 2,3-BPG, the P of hemoglobin is
50
75% in the veins. This indicates that hemoglobin gives up about 25% of about 26 mmHg. In the absence of 2,3-BPG, the P of hemoglobin is
50
its oxygen to the tissues. When the curve is shifted to the right, the P is 10 mmHg, indicating a very high oxygen affinity. Thus, in the absence
50
increased, indicating that the oxygen affinity has decreased. This results of 2,3-BPG, little oxygen is released to the tissues.
in the release of more oxygen to the tissues. When the curve is shifted to Protons (H ), CO , and organic phosphates (2,3-BPG) are all
+
2
the left, the P is decreased, indicating that oxygen affinity has increased. allosteric effectors of hemoglobin that preferentially bind to deoxy-
50
In this case, less oxygen is released to the tissues. hemoglobin, forming salt bridges within and between the globin
chains and stabilizing the deoxyhemoglobin (T) structure. The ratio
in which 2,3-BPG binds to deoxyhemoglobin is 1:1. The binding site
caSe StuDy (continued from page 83) for 2,3-BPG is in a central cavity of the hemoglobin tetramer between
the b@globin chains. It binds to positive charges on both b@chains,
Jerry was lethargic and pale and was having problems thereby crosslinking the chains and stabilizing the quaternary struc-
with activities of daily living. ture of deoxyhemoglobin (Figure 6-8 ■).
3. Explain why Jerry could have these symptoms. Hemoglobin also binds oxygen allosterically. Oxygen binds to
hemoglobin in a 4:1 ratio because one molecule of O binds to each
2
2,3-Bisphosphoglycerate
■ Figure 6-8 2,3-BPG binds in the central cavity of deoxyhemoglobin.
This cavity is lined with positively charged groups on the beta chains
that interact electrostatically with the negative charges on 2,3-BPG.
The a@globin chains are in pink, the b@chains are in blue, and the heme
prosthetic groups in red.
Source: Based on Principles of Biochemistry, 4E by H. R. Horton, L. A. Moran, K. G. Scrim-
geour and M. D. Perry. Published by Pearson Education, Inc., © 2006.
M06_MCKE6011_03_SE_C06.indd 86 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 87
of the four heme groups of the tetramer. The binding of oxygen by a the high oxygen–affinity R conformation, the third and fourth O 2
hemoglobin molecule depends on the interaction of the four heme molecules are added easily. The structural changes within succes-
groups, referred to as heme–heme interaction. This interaction of sive heme subunits facilitate binding the oxygen by the remaining
the heme groups is the result of movements within the tetramer trig- heme subunits because fewer subunit crosslinks need to be broken
gered by the uptake of a molecule of oxygen by one of the heme to bind subsequent oxygen molecules. Thus, hemoglobin performs
groups. like a “mini-lung,” changing shape as it takes up and releases O 2 to
In the deoxygenated state, the heme iron is 0.4–0.6Å out of the the tissue.
plane of the porphyrin ring because the iron atom is too large to Oxygen interacts weakly with heme iron, and the two can dis-
align within the plane. The iron is displaced toward the proximal sociate easily. As O 2 is released by hemoglobin in the tissues, the heme
histidine of the globin chain to which it is linked by a coordinate pockets narrow and restrict entry of O , and the space between the
2
bond. Fully deoxygenated hemoglobin (T state) has a low oxygen b@chains widens and 2,3-BPG binds again in the central cavity. Thus,
affinity, and loading the first oxygen onto the tetramer does not as 2,3-BPG concentration increases, the T configuration of hemoglo-
occur easily. On binding of an oxygen molecule, the atomic diam- bin is favored and the oxygen affinity decreases.
eter of iron becomes smaller due to changes in the distribution of This cooperative binding of oxygen makes hemoglobin a
electrons, and the iron moves into the plane of the porphyrin ring, very efficient oxygen transporter. Cooperativity ensures that once
pulling the histidine of the globin chain with it (Figure 6-9 ■). a hemoglobin tetramer begins to accept oxygen, it promptly is
These small changes in the tertiary structure of the molecule near fully oxygenated. In the process of oxygen release to the tissues,
the heme group result in a large shift in the quaternary structure, the same general principle is followed. Individual hemoglobin
altering the bonds and contacts between chains and weakening the molecules are generally either fully deoxygenated or fully oxygen-
intersubunit salt bridges. Likewise, loading a second O 2 onto the ated. Only a small portion of the molecules exists in a partially
tetramer while it is still in the T conformation does not occur eas- oxygenated state.
ily. However, the iron atom of the second heme is likewise shifted,
further destabilizing the salt bridges. During the course of load-
ing the third O onto hemoglobin, the salt bridges are broken, and adjustments in hemoglobin–Oxygen affinity
2
the hemoglobin molecule shifts from the T to the R configuration, Variations in environmental conditions or physiological demand for
pulling the b@chains together. Consequently, the size of the central oxygen result in changes in erythrocyte and plasma parameters that
cavity between the b@chains decreases, and 2,3-BPG is expelled. In directly affect hemoglobin–oxygen affinity. In particular, PO 2 , pH
+
(H ), PCO 2 , 2,3-BPG, and temperature affect hemoglobin–oxygen
affinity (Table 6-3 ★).
Several physiologic mechanisms of oxygen delivery can be
explained by the hemoglobin–2,3-BPG interaction. When going
from sea level to high altitudes the body adapts to the decreased
PO 2 by releasing more oxygen to the tissues. This adaptation is
mediated by increases of 2,3-BPG in the erythrocyte, usually
noted within 36 hours of ascent. EPO and erythrocyte mass also
increase as a part of the body’s adaptive mechanism to decreased
PO but this adaptation can take several days to improve tissue
2
oxygenation. 9
Fetal hemoglobin (HbF) has a higher oxygen affinity compared
with adult hemoglobin, HbA. The g@globin chain has a serine residue
at the helical H21 position. In the b@globin chain, a histidine residue
occupies this position. This change results in weak binding of 2,3-BPG
Porphyrin Fe ++ Porphyrin
plane plane
★ taBle 6-3 Factors That Affect Hemoglobin–
O 2
Oxygen Affinity
■ Figure 6-9 Changes in the conformation of hemoglo- increase affinity Decrease affinity
bin occur when the molecule takes up O 2 . In the deoxy-
hemoglobin state, the heme iron of a hemoglobin subunit c O 2 c CO 2
is below the porphyrin plane (green). On uptake of an O 2 T CO 2 c H +
molecule, the iron decreases in diameter and moves into T H + c Temperature
the plane of the porphyrin ring, pulling the proximal histi- T Temperature c 2,3-BPG
dine with it (yellow). The helix containing the histidine also
shifts, disrupting ion pairs that link the subunits. 2,3-BPG is T 2,3-BPG
expelled, and the remaining subunits are able to combine Key: c = increased; T = decreased
with O 2 more readily.
M06_MCKE6011_03_SE_C06.indd 87 01/08/14 11:26 pm
88 SECTION II • ThE hEmaTOpOIETIC SySTEm
and increased oxygen affinity in HbF. The more efficient binding of Plasma transport
2,3-BPG to HbA facilitates the transfer of oxygen from the maternal A small amount of carbon dioxide is dissolved in the plasma and car-
(HbA) to the fetal (HbF) circulation. ried to the lungs. There it diffuses out of the plasma and is expired.
Rapidly metabolizing tissue as occurs during exercise produces
+
CO and acid (H ) as well as heat. These factors decrease the oxy- carbonic acid
2
gen affinity of hemoglobin and promote the release of oxygen to the Most of the carbon dioxide transported by the blood is in the form of
tissue. In the alveolar capillaries of the lungs, the high PO and low bicarbonate ions (HCO ), which are produced when carbon dioxide
-
2
3
+
PCO drive off the CO in the blood and reduce H concentration, diffuses from the plasma into the erythrocyte. In the presence of the
2
2
promoting the uptake of O by hemoglobin (increasing oxygen affin- erythrocyte enzyme carbonic anhydrase (CA), CO reacts with water
2
+
ity). Thus, PO , PCO , and H facilitate the transport and exchange to form carbonic acid (H CO ). 2
2
2
of respiratory gases. 2 3
The effect of pH on hemoglobin–oxygen affinity is known as the H 2 O + CO 2 d CA S H 2 CO 3
Bohr effect, an example of the acid–base equilibrium of hemoglobin
that is one of the most important buffer systems of the body. A molecule Subsequently, hydrogen ion and bicarbonate are liberated from
+
+
of hemoglobin can accept H when it releases a molecule of oxygen. carbonic acid and the H is accepted by deoxyhemoglobin:
+
Deoxyhemoglobin accepts and holds the H better than oxyhemo-
+
globin. In the tissues, the H concentration is higher because of the HHb
presence of lactic acid and CO . When blood reaches the tissues, hemo- c
2
+
+
globin’s affinity for oxygen is decreased by the high H concentration, H 2 CO 3 — CA ¡ H + HCO 3 -
thereby permitting the more efficient unloading of oxygen at these sites.
The bicarbonate ions do not remain in the RBC because the cell
# + # + can hold only a small amount of bicarbonate. Thus, the free bicarbon-
Hb 4O 2 + 2H N Hb 2H + 4O 2
ate diffuses out of the erythrocyte into the plasma. The cell cannot tol-
erate a loss in negative ions, so in exchange for the loss of bicarbonate,
Thus, proton binding facilitates O release and helps minimize -
2
changes in the hydrogen ion concentration of the blood when tissue Cl diffuses into the cell from the plasma, a phenomenon called the
metabolism is releasing CO and lactic acid. Up to 75% of the hemo- chloride shift. This occurs via the anion exchange channel (band 3).
2
+
globin oxygen can be released if needed (as in strenuous exercise) as The bicarbonate combines with Na (NaHCO ) in the plasma and
3
2
the erythrocytes pass through the capillaries. is carried to the lungs where the PCO is low. There the bicarbonate
diffuses back into the erythrocyte, is rapidly converted back into CO
2
and H O, and is expired.
2
CheCkpoint 6-5
hemoglobin Binding
What factors influence an increase in the amount of oxy-
gen delivered to tissue during an aerobic workout? Approximately 23% of the total CO exchanged by the erythrocyte
2
in respiration is through carbaminohemoglobin. Deoxyhemoglobin
directly binds 0.4 moles of CO per mole of hemoglobin. Carbon
2
dioxide reacts with uncharged N-terminal amino groups of the four
Carbon Dioxide Transport globin chains to form carbaminohemoglobin. At the lungs, the plasma
PCO decreases, and the CO bound to hemoglobin is released and
2
2
After diffusing into the blood from the tissues, carbon dioxide is diffuses out of the erythrocyte to the plasma. It then is expired as it
carried to the lungs by three separate mechanisms: dissolution in enters the alveolar air space.
-
the plasma, as HCO in solution, and binding to the N-terminal
3
amino acids of hemoglobin (carbaminohemoglobin) (Table 6-4 ★;
Figure 6-10 ■). caSe StuDy (continued from page 86)
★ taBle 6-4 Carbon Dioxide Transport in Blood After a week at home, Jerry called his doctor, who
sent him back to the hospital where he was given
Mechanism Percent of transportation 2 units of packed red cells. Within a day, he had more
energy.
Dissolved in plasma 7
Formation of carbonic acid, H 2 CO 3 70 4. Explain why Jerry would have had more energy after
Bound to Hb 23 the transfusions.
M06_MCKE6011_03_SE_C06.indd 88 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 89
Arterial blood
Lungs Venous blood
Red blood cell Cells Red blood cell
O 2
(in) O 2 HHb HbO 2 H CO 2 CO H O H 2 CO 3
2
2
(in)
HCO 3 HCO 3 HHb CO 2 HHbCO 2
H HCO 3 HCO 3
CO 2 CO H O H CO 3
2
2
2
(out) Cl O 2 O HHb HbO
2
2
CO HHb HHbCO 2 Cl plasma (out) Cl Cl
2
a From lungs to arterial blood b From blood to cell
■ Figure 6-10 Transport of oxygen and carbon dioxide in the erythrocyte is depicted. (a) In
-
+
-
the lungs, O 2 and HCO 3 enter the red blood cell. O 2 combines with Hb, releasing H . HCO 3
+
combines with H to form H 2 CO 3 , which dissociates into H 2 O and CO 2 , and CO 2 is expired.
-
To maintain electrolyte balance, at the same time that HCO 3 flows into the red blood cells,
-
Cl flows out (the reverse chloride shift). The cell membrane anion-exchange protein (band
3) controls this ion exchange. Carbaminohemoglobin (HHbCO 2 ) releases CO 2 in the lungs
+
(where the PCO 2 decreases) and is expired. The HHb releases the H as Hb takes up oxygen.
(b) CO 2 diffuses from the tissue into the venous blood and then into the erythrocyte. Within
the erythrocyte, CO 2 reacts with water to form bicarbonic acid, H 2 CO 3 . The bicarbonic acid
-
+
-
dissociates into a bicarbonate ion (HCO 3 ) and a proton (H ). The HCO 3 leaves the cell
-
and enters the plasma. In exchange, chloride (Cl ) from the plasma enters the erythrocyte
(chloride shift). The proton facilitates the dissociation of oxygen from oxyhemoglobin (HbO 2 )
+
through the Bohr effect. When O 2 enters the tissues, the H is taken up by deoxyhemoglobin.
Nitric Oxide and Hemoglobin Artificial Oxygen Carriers
Nitric oxide (NO) is a critical component for the maintenance of Efforts to reduce allogeneic blood transfusions and improve oxygen
blood vessel homeostasis. NO derived its name as the endothelium- delivery to tissues have resulted in development of artificial oxy-
derived relaxing factor (EDRF) because of its ability to relax smooth gen carriers (AOCs). Two groups of AOCs include hemoglobin-
10
muscle and dilate blood vessels. It is important in other aspects of based oxygen carriers (HBOCs) in solution and perfluorocarbons
normal vessel physiology as well as inhibition of platelet activation. (PFCs). The HBOCs consist of purified human or bovine hemoglo-
NO is produced in the endothelium from arginine by the action of bin or recombinant hemoglobin. The hemoglobin is altered chemi-
NO synthase. NO can diffuse from the plasma across the erythrocyte cally or genetically or is microencapsulated to decrease oxygen
membrane where it is picked up by oxyhemoglobin. Reaction with affinity and to prevent its breakdown into dimers that have signifi-
12
oxyhemoglobin destroys the NO and forms methemoglobin and cant nephrotic toxicity. The oxygen dissociation curve of HBOCs
nitrate, a process known as dioxygenation. is similar to that of native human blood. Adverse side effects of these
AOCs are Hb-induced vasoconstriction and resulting hypertension.
- These side effects are related to NO scavenging and inactivation by
HbO 2 + NO S MetHb + NO 3
the free hemoglobin as well as endothelin (a vasoconstrictor) release
13
Reaction of NO and hemoglobin is limited because of hemo- and sensitization of peripheral adrenergic receptors. Because
globin compartmentalization in the erythrocyte, slow diffusion of hemoglobin in solution imparts color to plasma, it might not be
NO across the RBC membrane, and the laminar blood flow that possible to perform laboratory tests based on colorimetric analysis
pushes the erythrocytes inward away from the vessel endothelium of patients receiving this product because measurements could give
11
where the NO is concentrated. The rate of reaction of NO with erroneous results.
cell-free hemoglobin is increased by at least 1000 fold. This extra- PFCs are fluorinated hydrocarbons with high gas-dissolving
cellular reaction is responsible for complications such as vasocon- capacity. They do not mix in aqueous solution and must be emulsified.
striction and increase of blood pressure that are encountered when In contrast to HBOCs, a linear relationship between PO and oxygen
2
using artificial hemoglobin-based oxygen carriers in solution. The content in PFCs exists. This means that relatively high O partial pres-
2
reaction also appears to be responsible for complications (e.g., high sure is required to maximize delivery of O by PFCs. The PFC droplets
2
blood pressure) that accompany some hemolytic anemias such as are taken up by the mononuclear phagocyte (MNP) system, broken
sickle cell disease. down, bound to blood lipids, transported to the lungs, and exhaled. 13
M06_MCKE6011_03_SE_C06.indd 89 01/08/14 11:26 pm
90 SECTION II • ThE hEmaTOpOIETIC SySTEm
AOCs are not approved for use in the United States although Heme iron can be stored as ferritin or hemosiderin within the mac-
13
HBOCs are approved for compassionate use. Phase III trials are rophage or released to the iron transport protein, transferrin, for
complete or in progress for HBOCs. No PFC has yet been approved delivery to developing normoblasts in the bone marrow. This endog-
for clinical use. 14 enous iron exchange is responsible for about 80% of the iron passing
through the transferrin pool. Thus, iron from the normal erythrocyte
heMOglOBin cataBOliSM aging process is conserved and reutilized. The globin portion of the
hemoglobin molecule is broken down and recycled into the amino
When the erythrocyte is removed from circulation by macrophages acid pool.
(extravascular hemolysis) or is lysed in the blood stream (intravascu- Heme, the porphyrin ring, is further catabolized by the macro-
lar hemolysis), hemoglobin is released and catabolized. phage and eventually excreted in the feces. The a@methane bridge
of the porphyrin ring is cleaved, producing a molecule of carbon
Extravascular Destruction monoxide and the linear tetrapyrrole biliverdin. Carbon monoxide
In extravascular hemolysis, erythrocyte removal by macrophages in is released to the blood stream, carried to the lungs, and expired. The
the spleen, bone marrow, and liver conserves and recycles essential biliverdin is rapidly reduced within the cell to bilirubin. Released
erythrocyte components such as amino acids and iron (Figure 6-11 ■). from the macrophage, bilirubin is bound by plasma albumin and
Most extravascular destruction of erythrocytes takes place in the mac- carried to the liver (this is called unconjugated or “indirect” bilirubin).
rophages of the spleen. Upon uptake by the liver, bilirubin is conjugated with two molecules
Within the macrophage, the hemoglobin molecule is broken of bilirubin glucuronide by the enzyme bilirubin UDP-glucuron-
down into heme, iron, and globin. Iron and globin (a polypeptide) are yltransferase present in the endoplasmic reticulum of the hepato-
conserved and reused for new hemoglobin or other protein synthesis. cyte. Once conjugated, bilirubin becomes polar and lipid insoluble.
Extravascular hemoglobin degradation
Hemoglobin Heme + Globin Plasma protein and
amino acid pool
Macrophage
Lungs
Biliverdin + CO + Fe Transferrin + Fe Bone marrow
Blood
Bilirubin
Plasma albumin
Bilirubin-Albumin (unconjugated)
Liver
Bilirubin diglucuronide (conjugated)
Bile duct to duodenum
Urobilinogen Blood
Kidney
Stool Urobilinogen (urine)
Urobilinogen + Stercobilinogen
■ Figure 6-11 Most hemoglobin degradation occurs within the macrophages of the
spleen. The globin and iron portions of the molecule are conserved and reutilized. Heme is
reduced to bilirubin, eventually degraded to urobilinogen, and excreted in the feces. Thus,
indirect indicators of erythrocyte destruction include the blood bilirubin level and urobilino-
gen concentration in the urine.
M06_MCKE6011_03_SE_C06.indd 90 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 91
Bilirubin diglucuronide (called conjugated or “direct” bilirubin) is Hepatocytes, which have haptoglobin receptors, take up the HpHb
excreted into the bile, eventually reaching the intestinal tract where and process it in a manner similar to that of hemoglobin released by
intestinal bacterial flora convert it into urobilinogen. Most urobilino- extravascular destruction.
gen is excreted in the feces where it is quickly oxidized to urobilin or The HpHb complex is cleared very rapidly from the bloodstream
stercobilin. However, 10–20% of the urobilinogen is reabsorbed from with a T disappearance rate of 10–30 minutes. The haptoglobin
1/2
the gut back to the plasma. The reabsorbed urobilinogen is either concentration can be depleted very rapidly in acute hemolytic states
excreted in urine or returned to the gut via an enterohepatic cycle. because the liver is unable to maintain plasma haptoglobin levels.
In liver disease, the enterohepatic cycle is impaired and an increased Haptoglobin, however, is an acute-phase reactant, and increased con-
amount of urobilinogen is excreted in the urine. centrations can be found in inflammatory, infectious, or neoplastic
conditions. (An acute phase reactant is a protein whose plasma con-
centration increases in response to inflammation and serves a func-
Intravascular Destruction tion in the immune response.) Therefore, patients with hemolytic
The small amount of hemoglobin released into the peripheral blood anemia (anemia caused by increased destruction of erythrocytes)
circulation through intravascular erythrocyte breakdown undergoes accompanied by an underlying infectious or inflammatory process
dissociation into ab dimers, which are quickly bound to the plasma can have normal haptoglobin levels.
glycoprotein haptoglobin (Hp) in a 1:1 ratio (Figure 6-12 ■). Hap- When haptoglobin is depleted, as in severe hemolysis, free ab
toglobin is an a2@globulin present in plasma at a concentration of dimers can be filtered by the kidney and reabsorbed by the proxi-
35–164 mg/dL (males) or 40–175 mg/dL (females). The haptoglobin– mal tubular cells. ab dimers passing through the kidney in excess
hemoglobin (HpHb) complex is too large to be filtered by the kidney, of the reabsorption capabilities of the tubular cells appear in the
so haptoglobin carries hemoglobin dimers in the blood to the liver. urine as free hemoglobin. Dimers reabsorbed by the tubular cells
Intravascular hemoglobin degradation
Free Hb in blood
Haptoglobin
Hb-haptoglobin Liver (catabolism same as extravascular)
Hb
in excess of haptoglobin
αβ dimers
Methemoglobin
Kidney Globin Amino acid pool
Tubular
reabsorption
Heme
(Fe***)
Urine Urine
hemoglobin hemosiderin Hemopexin Hemopexin-heme
Albumin
Methemalbumin
Albumin
Heme
RE cells in liver
■ Figure 6-12 When the erythrocyte is destroyed within the vascular system, hemoglobin is
released directly into the blood. Normally, the free hemoglobin quickly complexes with hapto-
globin, and the complex is degraded in the liver. In severe hemolytic states, haptoglobin can
become depleted, and free hemoglobin dimers are filtered by the kidney. In addition, with
haptoglobin depletion, some hemoglobin is quickly oxidized to methemoglobin and bound to
either hemopexin or albumin for eventual degradation in the liver.
M06_MCKE6011_03_SE_C06.indd 91 01/08/14 11:26 pm
92 SECTION II • ThE hEmaTOpOIETIC SySTEm
are catabolized to bilirubin and iron, both of which can reenter Methemoglobin
the plasma pool. However, some iron remains in the tubular cell Methemoglobin is hemoglobin with iron in the ferric (Fe +++ )
and is complexed to storage proteins forming ferritin and hemo- state and is incapable of combining with oxygen. Methemoglobin
siderin. Eventually, tubular cells loaded with iron are sloughed off not only decreases the oxygen-carrying capacity of blood but also
and excreted in the urine (hemosiderinuria). The iron inclusions results in an increase in oxygen affinity of the remaining normal
can be visualized with the Prussian blue stain. Thus, the pres- hemoglobin. This results in an even higher deficit of O delivery.
2
ence hemosiderinuria is a sign of recent increased intravascular Normally, methemoglobin composes 6 3% of the total hemoglobin
hemolysis. in adults. At this concentration, the abnormal pigment is not harm-
15
Hemoglobin not excreted by the kidney or bound to haptoglo- ful because the reduction in oxygen-carrying capacity of the blood
bin is either cleared directly by hepatic uptake or oxidized to methe- is insignificant.
moglobin. Heme dissociates from methemoglobin and avidly binds Clinically important methemoglobinemia can be due to the fol-
to a b@globulin glycoprotein, hemopexin. Hemopexin is synthe- lowing (Table 6-6 ★):
sized in the liver and combines with heme in a 1:1 ratio. The hemo-
pexin–heme complex is cleared from the plasma slowly with a T 1. Deficiencies of enzymes that reduce Fe +++ @hemoglobin to
1/2
++
disappearance of 7–8 hours. When hemopexin becomes depleted, Fe @hemoglobin; of these, the most important, accounting for
the dissociated oxidized heme combines with plasma albumin in a 760% of the reduction of methemoglobin, is NADH methemo-
1:1 ratio to form methemalbumin. Methemalbumin clearance by the globin reductase (Table 6-7 ★).
liver is also very slow. In fact, methemalbumin may be only a tempo- 2. Globin chain mutations that that stabilize heme iron in the
rary carrier for heme until more hemopexin or haptoglobin becomes Fe +++ state (hemoglobin M; Chapter 18). This structural variant
available. Heme is transferred from methemalbumin to hemopexin of hemoglobin is characterized by amino acid substitutions in
for clearance by the liver as it becomes available. When present in the globin chains near the heme pocket that stabilize the iron in
large quantity, methemalbumin and hemopexin–heme complexes the oxidized Fe +++ state.
impart a brownish color to the plasma. The Schumm ’s test is designed 3. Exposure to toxic substances that oxidize hemoglobin and over-
to detect these abnormal compounds spectrophotometrically. whelm the normal reducing capacity of the cell. Increased levels
of methemoglobin are formed when an individual is exposed
to certain oxidizing chemicals or drugs. Even small amounts of
CheCkpoint 6-6 these chemicals and drugs can cause oxidation of large amounts
of hemoglobin. If the offending agent is removed, methemoglo-
What lab tests would diagnose an increase in RBC
destruction (i.e., hemolysis), and what would be the binemia returns to normal levels within 24–48 hours.
expected results? Infants are more susceptible to methemoglobin production than
adults because HbF is more readily converted to methemoglobin and
because infants’ erythrocytes are deficient in reducing enzymes. Expo-
sure to certain drugs or chemicals that increase oxidation of hemoglo-
acquireD nOnFunctiOnal bin or water high in nitrates can cause methemoglobinemia in infants.
Color crayons containing aniline can cause methemoglobinemia if
heMOglOBinS ingested.
The acquired, nonfunctional hemoglobins are hemoglobins that have Cyanosis develops when methemoglobin levels exceed 10%
been altered post-translationally to produce molecules with compro- (71.5 g/dL) hypoxia is produced at levels exceeding 30–40%. Toxic
mised oxygen transport, thereby causing hypoxia and/or cyanosis levels of methemoglobin can be reduced by medical treatment with
(Table 6-5 ★). Hypoxia is a condition in which there is an inadequate methylene blue or ascorbic acid, which speeds up reduction by
amount of oxygen at the tissue level. (Hypoxemia is an inadequate NADPH-reducing enzymes. The NADPH reductase system requires
amount of oxygen in the blood; arterial PO 680 mmHg). Cyanosis G6PD and therefore this method of treatment is not effective in
2
refers to a bluish or slate-gray color of the skin due to the presence of patients with G6PD deficiency. In some cases of severe methemo-
more than 5 g/dL of deoxyhemoglobin in the blood. globinemia, exchange transfusions are helpful.
★ taBle 6-5 Abnormal Acquired Hemoglobins
hemoglobin acquired change abnormal Function lab Detection
methemoglobin Hb iron in ferric state Cannot combine with oxygen Demonstration of maximal absorption band
at wave length of 630 nm; chocolate color
blood
Sulfhemoglobin Sulfur combined with hemoglobin 1 100 oxygen affinity of HbA Absorption band at 620 nm
Carboxyhemoglobin Carbon monoxide combined with Affinity for carbon monoxide is 200 Absorption band at 541 nm
hemoglobin times higher than for oxygen
M06_MCKE6011_03_SE_C06.indd 92 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 93
★ taBle 6-6 Differentiation of Types of Methemoglobinemia
cause of Methemoglobinemia inherited/acquired enzyme activity hb electrophoresis
Exposure to oxidants Acquired Normal Normal
Decreased enzyme activity Inherited Decreased Normal
presence of hemoglobin m Inherited Normal Abnormal
In the congenital methemoglobinemias, cyanosis is observed of hemoglobin able to combine with oxygen (oxyhemoglobin plus
from birth, and methemoglobin levels reach 10–20%. Normal deoxyhemoglobin). FhbO and oxygen saturation are the same if no
2
hemoglobin’s oxygen affinity is increased in these children, result- abnormal hemoglobin is present. 16
ing in increased erythropoiesis and subsequently higher than nor-
mal hemoglobin levels and erythrocytosis. Even in the homozygous Sulfhemoglobin
state, individuals with HbM or defects in the reducing systems rarely Sulfhemoglobin is a stable compound formed when a sulfur atom
have methemoglobin levels of 725% and are usually asymptomatic combines with the heme group of hemoglobin. The sulfur atom binds
except for mild cyanosis. They do not usually require treatment. How- to a pyrrole carbon at the periphery of the porphyrin ring. Sulfura-
ever, cyanosis can be improved by treatment with methylene blue or tion of heme groups results in a drastically right-shifted oxygenation
ascorbic acid. Laboratory diagnosis of methemoglobinemia involves dissociation curve, which renders the heme groups ineffective for
demonstration of a maximum absorbance band at a wavelength of oxygen transport. This appears to be due to the fact that even half-
630 nm at pH 7.0–7.4. The blood sample can be chocolate brown in sulfurated, half-oxygen–liganded tetramers exist in the T configu-
color when compared with a normal blood specimen, and the color ration (the low oxygen-affinity form) of hemoglobin. Although the
15
does not change to red upon exposure to oxygen. Differentiation heme iron is in the ferrous state, sulfhemoglobin binds to oxygen
of acquired types from hereditary types of methemoglobin requires with an affinity only one-hundredth that of normal hemoglobin.
assay of NADH methemoglobin reductase and hemoglobin electro- Thus, oxygen delivery to the tissues can be compromised if there is
phoresis (Table 6-7). Enzyme activity is reduced only in hereditary an increase in this abnormal hemoglobin. The bright green sulfhe-
NADH-methemoglobin reductase deficiency, and hemoglobin elec- moglobin compound is so stable that the erythrocyte carries it until
trophoresis is abnormal only in HbM disease. Acquired methemoglo- the cell is removed from circulation. Ascorbic acid or methylene
binemia shows normal enzyme activity and a normal electrophoresis blue cannot reduce it; however, sulfhemoglobin can combine with
pattern. carbon monoxide to form carboxysulfhemoglobin. Normal levels of
In the presence of methemoglobinemia, oxygen saturation sulfhemoglobin do not exceed 2.2%. Cyanosis is produced at levels
obtained by a cutaneous pulse oximeter (fractional oxyhemoglobin, exceeding 3–4%.
FhbO ) can be lower than the oxygen saturation reported from a Sulfhemoglobin has been associated with occupational expo-
2
blood gas analysis. This is because FhbO is calculated as the amount sure to sulfur compounds, environmental exposure to polluted air,
2
of oxyhemoglobin compared with the total hemoglobin (oxyhe- and exposure to certain drugs. Sulfhemoglobinemia is formed dur-
moglobin, deoxyhemoglobin, methemoglobin, and other inactive ing the oxidative denaturation of hemoglobin and can accompany
hemoglobin forms) whereas oxygen saturation in a blood gas analy- methemoglobinemia, especially in certain drug- or chemical-induced
sis is the amount of oxyhemoglobin compared with the total amount hemoglobinopathies. Sulfhemoglobin is formed on exposure of blood
to trinitroluene, acetanilid, phenacetin, and sulfonamides. It also is
elevated in severe constipation and in bacteremia with Clostridium
welchii. Diagnosis of sulfhemoglobinemia is made spectrophotomet-
★ taBle 6-7 Erythrocyte Systems Responsible for rically by demonstrating an absorption band at 620 nm. Confirma-
Methemoglobin Reduction tion testing is done by isoelectric focusing. This is the only abnormal
rank in Order hemoglobin pigment not measured by the cyanmethemoglobin
of Decreasing method, which is used to measure hemoglobin concentration.
Methemoglobin
reduction System
First NADH methemoglobin reductase (also known Carboxyhemoglobin
as cytochrome b5 methemoglobin reductase, Carboxyhemoglobin is formed when hemoglobin is exposed to
diaphorase I, DPNH-diaphorase, DPNH dehy- carbon monoxide. Hemoglobin’s affinity for carbon monoxide is
drogenase I, NADH dehydrogenase, NADH
methemoglobin-ferrocyanide reductase) 7200 times higher than its affinity for oxygen. Carboxyhemoglobin
Second Ascorbic acid is incapable of transporting oxygen because CO occupies the same
Third Glutathione ligand-binding position as O . As is the case with methemoglobin-
2
Fourth NADPH methemoglobin reductase emia, carboxyhemoglobin has a significant impact on oxygen deliv-
ery because it destroys the molecule ’s cooperativity. CO also has a
M06_MCKE6011_03_SE_C06.indd 93 01/08/14 11:26 pm
94 SECTION II • ThE hEmaTOpOIETIC SySTEm
pronounced effect on the oxygen dissociation curve, shifting it to the Carboxyhemoglobin is commonly measured in whole blood by
left, resulting in increased affinity and a decreased release of O by a spectrophotometric method. Sodium hydrosulfite reduces hemo-
2
remaining normal hemoglobin molecules. High levels of carboxyhe- globin to deoxyhemoglobin, and the absorbances of the hemolysate
moglobin impart a cherry red color to the blood and skin. However, are measured at 555 and 541 nm. Carboxyhemoglobin has a greater
high levels of it together with high levels of deoxyhemoglobin can give absorbance at 541 nm.
blood a purple-pink color.
Blood normally carries small amounts of carboxyhemoglobin
formed from the carbon monoxide produced during heme catabo-
lism. The level of carboxyhemoglobin varies depending on individu-
als’ smoking habits and their environment. City dwellers have higher CheCkpoint 6-7
levels than country dwellers as a result of the carbon monoxide pro- A 2-year-old child was found to have 15% methemoglobin
duced from automobiles and industrial pollutants in cities. by spectral absorbance at 630 nm. What tests would you
Acute carboxyhemoglobinemia causes irreversible tissue damage suggest to help differentiate whether this is an inherited
and death from anoxia. Chronic carboxyhemoglobinemia is charac- or acquired methemoglobinemia, and what results would
terized by increased oxygen affinity and polycythemia. In severe cases you expect with each diagnosis?
of carbon monoxide poisoning, patients can be treated in hyperbaric
oxygen chambers.
Summary
Hemoglobin is the intracellular protein of erythrocytes respon- shifted to the right reflects decreased oxygen affinity; when it
sible for transport of oxygen from the lungs to the tissues. A fine has shifted to the left, oxygen affinity has increased. Increased
balance between production and destruction of erythrocytes CO 2 , heat, and acid decrease oxygen affinity; high O 2 concen-
serves to maintain a steady-state hemoglobin concentration. trations increase oxygen affinity.
Hemoglobin is a globular protein composed of four sub- Hemoglobin is an allosteric protein, which means that other
units. Each subunit contains a porphyrin ring with an iron mol- molecules affect hemoglobin structure and function. In particu-
ecule (heme) and a globin chain. The four globin chains are lar, the uptake of 2,3-BPG or oxygen can cause conformational
arranged in identical pairs, each composed of two different changes in the molecule. The structure of deoxyhemoglobin is
globin chains (e.g., a 2 b 2 ). Hemoglobin synthesis is controlled known as the T structure and that of oxyhemoglobin is known
by iron concentration within the cell, synthesis and activity of as the R structure.
the first enzyme in the heme synthetic pathway (ALAS), activity When hemoglobin is exposed to oxidants or other com-
of PBGD, and globin chain synthesis. pounds, the molecule can be altered, which can compromise
The oxygen affinity of hemoglobin depends on PO 2 , pH, its ability to carry oxygen. High concentrations of these abnor-
PCO 2 , 2,3-BPG, and temperature. Hemoglobin–oxygen affin- mal hemoglobins can cause hypoxia and cyanosis, which can be
ity can be graphically depicted by the ODC. A curve that has detected by spectrophotometric methods.
Review Questions
Level i 3. When iron is depleted from the developing erythrocyte,
the: (Objective 7)
1. Which of the following types of hemoglobin is the major
component of adult hemoglobin? (Objective 4) A. synthesis of heme is increased
A. HbA B. activity of ALAS is decreased
B. HbF C. formation of globin chains stops
C. HbA 2
D. heme synthesis is not affected
D. Hb Portland
+
4. When the H concentration in blood increases, the oxygen
2. One of the most important buffer systems of the body is affinity of hemoglobin: (Objective 3)
the: (Objective 5)
A. increases
A. chloride shift
B. Bohr effect B. is unaffected
C. heme–heme interaction C. decreases
D. ODC D. cannot be measured
M06_MCKE6011_03_SE_C06.indd 94 01/08/14 11:26 pm
ChapTEr 6 • hEmOgLObIN 95
5. Which of the following is the correct molecular structure 2. Which of the following is the major hemoglobin in the new-
of hemoglobin? (Objective 1) born? (Objective 2)
A. four heme groups, two iron, two globin chains A. a 2 b 2
B. two heme groups, two iron, four globin chains B. a 2 g 2
C. two heme groups, four iron, four globin chains
C. a 2 d 2
D. four heme groups, four iron, four globin chains
D. a 2 e 2
6. 2,3-BPG combines with which type of hemoglobin? 3. A 2-year-old patient who had been cyanotic since birth was
(Objectives 3, 5)
seen by a pediatrician. Blood was drawn for analysis of
A. oxyhemoglobin NADH methemoglobin reductase and results were normal.
What follow-up test would you suggest to the physician?
B. relaxed structure of hemoglobin
(Objective 6)
C. deoxyhemoglobin
A. hemoglobin electrophoresis
D. ab dimer
B. bone marrow aspiration and examination
7. During exercise, the oxygen affinity of hemoglobin is:
(Objective 3) C. haptoglobin and sulfhemoglobin determination
A. increased in males but not females D. glycosylated hemoglobin measurement by column
chromatography
B. decreased due to production of heat and lactic acid
4. A 25-year-old male was found unconscious in a car with
C. unaffected in those who are physically fit
the motor running. Blood was drawn and sent to the
D. affected only if the duration is more than 1 hour chemistry lab for spectral analysis. The blood was cherry
red in color. Which hemoglobin should be tested for?
8. Which of the following is considered a normal hemoglobin (Objective 6)
concentration in an adult male? (Objective 6)
A. sulfhemoglobin
A. 11.0 g/dL
B. methemoglobin
B. 21.0 g/dL
C. 15.0 g/dL C. carboxyhemoglobin
D. 9.0 g/dL D. oxyhemoglobin
9. Haptoglobin can become depleted in: (Objective 10) 5. The oxygen dissociation curve in a case of chronic carboxy-
hemoglobin poisoning would show: (Objective 7)
A. inflammatory conditions
A. a shift to the right
B. intravascular hemolysis
B. a shift to the left
C. infectious diseases
D. kidney disease C. a normal curve
10. A patient with an anemia due to increased extravascular D. decreased oxygen affinity
hemolysis would likely present with which of the following 6. A college student from Louisiana vacationed in Colorado
lab results? (Objective 9) for spring break. He arrived at Keystone Resort on the first
A. increased haptoglobin day. The second day, he was nauseated and had a head-
ache. He went to the medical clinic at the resort and was
B. hemoglobinuria told he had altitude sickness. The doctor told him to rest
C. normal hemoglobin and hematocrit for another 24 hours before participating in any activities.
What is the most likely reason he will overcome this condi-
D. increased serum bilirubin tion in the next 24 hours? (Objective 4)
Level ii A. His level of HbF will increase to help release more oxy-
gen to the tissues.
1. Which of the following hemoglobins is not found in the
normal adult? (Objective 2) B. The amount of carboxyhemoglobin will decrease to
normal levels.
A. a 2 b 2
C. The levels of ATP in his blood will reach maximal
B. a 2 g 2 levels.
C. a 2 d 2
D. The level of 2,3-BPG will increase and, in turn,
decrease oxygen affinity.
D. a 2 e 2
M06_MCKE6011_03_SE_C06.indd 95 01/08/14 11:26 pm
96 SECTION II • ThE hEmaTOpOIETIC SySTEm
7. When iron in the cell is replete, the translation of ferritin 9. In the lungs, a hemoglobin molecule takes up two oxygen
mRNA is: (Objective 3) molecules. What effect will this have on the hemoglobin
molecule? (Objectives 5, 8)
A. decreased
A. It will increase oxygen affinity.
B. increased
B. It will narrow the heme pockets blocking entry of addi-
C. unaffected tional oxygen.
D. variable C. The hemoglobin molecule will take on the tense structure.
8. An aerobics instructor just finished an hour of instruction. D. The center cavity will expand, and 2,3-BPG will enter.
Blood is drawn from her for a research study, and the oxy- 10. An anemic patient has hemosiderinuria, increased serum
gen dissociation is measured. What would you expect to bilirubin, and decreased haptoglobin. This is an indication
find? (Objective 4)
that there is: (Objective 10)
A. a shift to the left A. increased intravascular hemolysis
B. a shift to the right B. decreased extravascular hemolysis
C. no shift C. hemolysis accompanied by renal disease
D. an increased oxygen affinity D. a defect in the Rapoport-Leubering pathway
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. Find additional information to help
organize information and figures to help understand concepts.
References
1. Ferreira GC, Gong J. 5-aminolevulinate synthase and the first step of heme 9. boning D, maassen N, Jochum F et al. after effects of a high altitude expe-
biosynthesis. J Bioenerg Biomembr. 1995;27:151–59. dition on blood. Int J Sports Med. 1997;18:179–85.
2. Rouault TA. The role of iron regulatory proteins in mammalian iron homeo- 10. Schechter aN, gladwin mT. hemoglobin and the paracrine and endocrine
stasis and disease. Nature Chem Biol. 2006;2(8):406–14. functions of nitric oxide. N Engl J Med. 2003;348(15):1483–85.
3. Klausner RD, Rouault TA, Harford JB. Regulating the fate of mRNA: The 11. Kim-Shapiro Db, Schechter aN, gladwin mT. Unraveling the reactions of
control of cellular iron metabolism. Cell. 1993;72:19–28. nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. Arte-
4. Kawasaki N, morimoto K, Tanimoto T et al. Control of hemoglobin synthesis rioscler Thromb Vasc Biol. 2006;26:697–705.
in erythroid differentiating K562 cells. I. Role of iron in erythroid cell heme 12. henkel-hanke T, Oleck m. artificial oxygen carriers: a current view. AANA
synthesis. Arch Biochem Biophys. 1996;328:289–94. J. 2007;75(3):205–11.
5. Kawasaki N, morimoto K, hayakawa T. Control of hemoglobin synthesis in 13. Spahn DR. Blood substitutes artificial oxygen carriers: perfluorocarbon
erythroid differentiating K562 cells. II. Studies of iron mobilization in ery- emulsions. Crit Care. 1999;3:R93–97.
throid cells by high-performance liquid chromatography-electrochemical 14. Castro CI, Briceno JC. Perfluorocarbon-based oxygen carriers: Review of
detection. J Chromatogr B Biomed Sci Appl. 1998;705:193–201. products and trials. Artificial Organs. 2010;34(8):622–34.
6. peschle C, migliaccio ar, migliaccio g et al. regulation of hb synthesis in 15. Benz EJ Jr, Ebert BL. Hemoglobin variants associated with hemolytic
ontogenesis and erythropoietic differentiation: in vitro studies on fetal liver, anemia, altered oxygen affinity, and methemoglobinemias. In Hoffman
cord blood, normal adult blood or marrow, and blood from HPFH patients. R, Benz EJ Jr, Silberstein LE et al. Hematology: Basic Principles and
In: Stamatoyannopoulos G, Nienhuis AW, eds. Hemoglobins in Develop- Practice. Philadelphia: Elsevier Churchill Livingstone, 2013. (accessed
ment and Differentiation. New york: alan r. Liss; 1980:359–71. electronically)
7. Papayannopoulou T, Nakamoto B, Agostinelli F et al. Fetal to adult hemo- 16. Wentworth p, roy m, Wilson b et al. Clinical pathology rounds: toxic methe-
poietic cell transplantation in humans: Insights into hemoglobin switching. moglobinemia in a 2-year-old child. Lab Med. 1999;30:311–15.
Blood. 1986;67:99–104.
8. perutz mF. molecular anatomy, physiology, and pathology of hemoglobin.
In Stamatoyannopoulos g, Neinhuis aW, Leder p, majerus pW, eds. The
Molecular Basis of Blood Diseases. Philadelphia: WB Saunders; 1987.
M06_MCKE6011_03_SE_C06.indd 96 01/08/14 11:26 pm
7 Granulocytes
and Monocytes
Kristin Landis-Piwowar, Phd
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Identify terms associated with increases and decreases in granulocytes Objectives—Level I and Level II 97
and monocytes.
2. Differentiate morphological features of the granulocyte and monocyte Key Terms 98
precursors found in the proliferative compartment of the bone marrow. Background Basics 98
3. Describe the development, including distinguishing maturation and cell
features, of the granulocytic and monocytic-macrophage cell lineages. Case Study 98
4. Describe and differentiate the morphologic and other distinguishing Overview 98
cell features of each of the granulocytes and monocytes found in the
peripheral blood. Introduction 98
5. Explain the function of each type of granulocyte and monocyte found Leukocyte Concentration in the
in the peripheral blood. Peripheral Blood 99
6. Summarize the process of neutrophil migration and phagocytosis.
7. List the adult reference intervals for the granulocytes and monocytes Leukocyte Surface Markers 100
found in the peripheral blood. Leukocyte Function 100
8. Calculate absolute cell counts from data provided.
9. Differentiate and interpret absolute values and relative values of cell Neutrophils 100
count data. Eosinophils 110
10. List causes/conditions that increase or decrease absolute numbers of
individual granulocytes and monocytes found in the peripheral blood. Basophils 112
11. Compare and contrast pediatric and newborn reference intervals with Monocytes 113
adult reference intervals.
Summary 117
Objectives—Level II Review Questions 117
At the end of this unit of study, the student should be able to: Companion Resources 120
1. Summarize the kinetics of the granulocytic and monocytic-macrophage
cell lineages. References 120
2. Describe the processes that permit neutrophils to leave the peripheral
blood circulation and move to a site of infection and propose how
defects in these processes affect the body’s defense mechanism.
3. Compare and contrast the immunologic features and functions of each
of the granulocytes and monocytes found in the peripheral blood.
(continued)
M07_MCKE6011_03_SE_C07.indd 97 01/08/14 11:24 am
98 SECTION II • ThE hEmaTOpOIETIC SySTEm
5. Correlate the laboratory data that pertain to
Objectives—Level II (continued)
granulocytes and monocytes with the clinical informa-
4. Explain the physiological events that alter the number of tion for a patient.
circulating granulocytes and monocytes in the peripheral
blood.
Key Terms Background Basics
Agranulocytosis Marginating pool (MP) In addition to the basics from previous chapters, it is helpful to
Azurophilic granule Monocyte-macrophage have a general understanding of immunology (immune system
Charcot-Leyden crystal system and function); biochemistry (proteins, carbohydrates, lipids);
Chemokine Mononuclear phagocyte (MNP) algebra; and the use of percentages, ratios, proportions, and the
Chemotaxis system metric system.
Circulating Pool (CP) Neutropenia To maximize your learning experience, you should review these
Cluster of differentiation (CD) Neutrophilia concepts from previous chapters before starting this unit of study:
Degranulation Pathogen-associated molecular Level i
Diapedesis pattern (PAMP) • Identify components of the cell and describe their function.
Drumstick (Barr body) Pattern recognition receptor (Chapter 2)
Erythrophagocytosis (PRR) • Summarize the function of growth factors and the hierarchy
Granulocytosis Phagocytosis of hematopoiesis. (Chapter 4)
Leukocytosis Polymorphonuclear • Describe the function of the hematopoietic organs. (Chapter 3)
Leukopenia
Level ii
• List the growth factors and identify their function in leukocyte
differentiation and maturation. (Chapter 4)
• Describe the structure of the hematopoietic organs. (Chapter 3)
case study introduction
With the exception of T lymphocytes, leukocyte precursors proliferate,
We will refer to this case study throughout the chapter. differentiate, and mature in the bone marrow. Mature leukocytes are
released into the peripheral blood where they circulate briefly until they
harry, a 30-year-old male in good physical condition,
had a routine physical examination as a requirement for move into the tissues in response to stimulation. They perform their
purchasing a life insurance policy. A CBC was ordered function of host defense primarily in the tissues. The neutrophil, band
with the following results: hb 15.5 g/dL (155 g/L), hct neutrophil, eosinophil, basophil, monocyte, and lymphocyte are the leu-
12
47% (0.47 L/L), RBC count 5.2 * 10 /L, platelet count kocytes normally found in the peripheral blood of children and adults.
9
9
175 * 10 /L, and WBC count 12 * 10 /L. Leukocytes are nearly colorless in an unstained blood smear—
Consider how you could explain these results in a hence, the term leuko-, meaning “white.” The era of morphologic
healthy male. hematology began in 1877 with Paul Ehrlich’s discovery of a triac-
idic stain that allowed for the differentiation of leukocytes on fixed
1
blood smears. Today, Wright stain, a Romanowsky-type stain, utilizes
overview methylene blue and eosin to stain the cellular components of blood and
The terms leukocyte and white blood cell (WBC) are the synony- bone marrow that are smeared on glass slides. Basic cellular elements
mous names given to the nucleated blood cells that are involved in react with the acidic dye (eosin), and acidic cellular elements react with
the defense against foreign pathogens or antigens. Leukocytes develop the basic dye (methylene blue). The eosinophil contains large amounts
from the pluripotential hematopoietic stem cell in the bone marrow. of basic protein in its granules that react with the eosin dye—hence, the
In the presence of infection or inflammation, leukocytes can increase name eosinophil—whereas the basophil has granules that are acidic
in number and can display morphologic changes. Thus, an important and react with the basic dye, methylene blue—hence, the name baso-
screening test for a wide variety of conditions is the leukocyte count, phil. The neutrophil reacts with both acid and basic components of the
more commonly referred to as the WBC count. Leukocytes are classi- stain, giving the cell cytoplasm a clear or tan to pinkish appearance with
fied as granulocytes (neutrophils, eosinophils, basophils), monocytes, pink to violet stained granules. The nuclear DNA and cytoplasmic RNA
and lymphocytes. This chapter is a study of the normal differentiation of cells are acidic and pick up the basic stain, methylene blue. The eosin-
and maturation of granulocytes and the nongranulocytic monocyte. ophil, basophil, and neutrophil are polymorphonuclear (their nuclei
Each of these cells is discussed in terms of cell morphology, concen- have many lobes) and because their cytoplasm contains many granules,
tration in the peripheral blood, and function. Lymphocytes will be they are classified as granulocytes. Monocytes are mononuclear cells
discussed in Chapter 8. and contain small numbers of fine granules in a bluish-gray cytoplasm.
M07_MCKE6011_03_SE_C07.indd 98 01/08/14 11:24 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 99
William Hewson, the father of hematology, first observed leuko-
cytes in the eighteenth century. In the nineteenth century, the studies case study (continued from page 98)
of inflammation and bacterial infection intensified interest in leuko-
2
cytes. Many researchers studied the similarity of pus cells in areas harry’s CBC results were hb 15.5 g/dL (155 g/L), hct
12
of inflammation and the leukocytes of the blood. Ilya Metchnikov 47% (0.47 L/L), RBC count 5.2 * 10 /L, platelet count
9
9
observed the presence of nucleated blood cells surrounding a thorn 175 * 10 /L, and WBC count 12 * 10 /L.
introduced beneath the skin of a larval starfish. 1 1. Are any of these results outside the reference interval?
Many of Ehrlich’s observations and Metchnikov’s experiments If so, which one(s)?
provided the groundwork for understanding the leukocytes as defend- 2. If this were a newborn, would you change your evalu-
ers against infection. Ehrlich recognized that variations in numbers ation? If so, why?
of leukocytes accompanied specific pathologic conditions, such as
eosinophilia in allergies, parasitic infections, and dermatitis as well as
neutrophilia in bacterial infections.
Leukocytes function to fight infection by two separate but inter- An altered concentration of all leukocyte types or, more com-
related events: phagocytosis (innate immune response) and develop- monly, an alteration in one specific type of leukocyte can cause an
ment of the adaptive immune response. Granulocytes and monocytes increase or decrease in the total WBC count. For this reason, an abnor-
are the primary cells responsible for phagocytosis whereas monocytes mal total WBC count should be followed by a leukocyte differential
and lymphocytes interact to produce an effective adaptive immune count (commonly referred to as a WBC differential, or simply diff ). A
response (Chapter 8). Eosinophils and basophils interact in mediating manual WBC differential is performed by enumerating each leukocyte
allergic and hypersensitivity reactions. type within a total of 100 leukocytes on a stained blood smear using
a microscope. The differential results are reported as the percentage
of each cell type counted. To accurately interpret whether an increase
LeuKocyte concentration or decrease in cell types exists, however, it is necessary to calculate
the absolute concentration using the results of the WBC count and
in the PeriPheraL BLood the differential (relative concentration) in the following manner:
Leukocytes develop from pluripotential hematopoietic stem cells
(HSCs) in the bone marrow. Upon specific hematopoietic growth Differential count (in decimal form) * WBC count
9
9
factor stimulation, the stem cell proliferates and differentiates into * (10 /L) = Absolute cell count (* 10 /L)
the various types of leukocytes: granulocytes (neutrophils, eosino-
phils, basophils), monocytes, and lymphocytes. Once these cells have The application of this calculation is emphasized in the following
matured, they can be released into the peripheral blood or remain in example. Two different blood specimens from two different patients
the bone marrow storage pool until needed. were found to have a relative neutrophil concentration of 85%. The
9
An individual’s age and various physiologic and pathologic condi- total WBC count in one patient was 3 * 10 /L and in the other was
9
tions predominantly affect the WBC count. The total WBC count is high 9 * 10 /L. The relative neutrophil concentration on both specimens
9
at birth, ranging from 9–30 * 10 /L. A few immature granulocytic appears elevated (reference interval is 40–80%); however, calculation
9
cells (myelocytes, metamyelocytes) can be seen in the circulation during of the absolute concentration (reference interval 1.897.0 * 10 /L)
the first few days of life. However, immature leukocytes are not present shows that only one specimen has an absolute increase in neutrophils,
in the peripheral blood after this age except in certain diseases. Within whereas the other is within the reference interval:
9
the first week after birth, the leukocyte count drops to 5921 * 10 /L.
9
9
A gradual decline continues until the age of 8 years at which time the 0.85 * (3 * 10 /L) = 2.6 * 10 /L (within the reference interval)
9
9
9
leukocyte concentration averages 8 * 10 /L. Adult values average from 0.85 * (9 * 10 /L) = 7.7 * 10 /L (increased)
9
4.5–11.0 * 10 /L, and generally do not decline with aging. 3
In addition to age, physiologic and pathological events affect the Neutrophils comprise the largest portion of WBCs in peripheral
concentrations of leukocytes. Pregnancy, time of day, and an indi- blood followed by lymphocytes (Chapter 8), monocytes, eosinophils,
vidual’s activity level affect the WBC concentration. Infections and and basophils, respectively. In an adult, neutrophils make up 40–80%
immune-regulated responses cause significant changes in leukocytes. of total leukocytes. At birth, the neutrophil concentration is about
Many other pathologic disorders can also cause quantitative and/or 50–60%; this level drops to 30% by 4–6 months of age. After 4 years
qualitative changes in white cells. Considerable heterogeneity in leu- of age, the concentration of neutrophils gradually increases until adult
9
kocyte concentration has been found among racial, ethnic, and sex values are reached at 6 years of age (1.897.0 * 10 /L). Most periph-
subgroups, suggesting the need for unique reference intervals for spe- eral blood neutrophils are mature segmented forms. However, up to
4
cific populations. Thus, when WBC counts are evaluated, the patient’s 5% of the less mature, nonsegmented forms, called neutrophil bands,
age, and possibly race/ethnicity and sex, provide useful information. It can be seen in normal specimens. Most variations in the total WBC
also is helpful to assess the accuracy of cell counts by correlating them count are due to an increase or decrease in neutrophils.
9
with the patient’s previous cell counts and clinical history. Additional Monocytes usually compose 2–10% (0.190.8 * 10 /L)
testing, called reflex testing, can be indicated as a result of abnormali- of circulating leukocytes. Occasionally, reactive lymphocytes
ties in the WBC count. Changes associated with diseases and disorders (Chapter 8) resemble monocytes in morphology, posing clas-
will be discussed in subsequent chapters on leukocytes. sification difficulty even for the experienced hematologist.
M07_MCKE6011_03_SE_C07.indd 99 01/08/14 11:24 am
100 SECTION II • ThE hEmaTOpOIETIC SySTEm
Monocytes are functionally more similar to the granulocytes than to Other pathogens can be eliminated without the step of recognition
the nongranulocytic lymphocyte. just described. This is accomplished when a pathogen contains certain
Peripheral blood eosinophil concentrations are maintained at structures shared by many different pathogens or common alterations
9
0–5% (up to 0.4 * 10 /L) throughout life. It is possible that no eosino- that the pathogen makes to the body’s cells. The shared structures or
phils can be seen on a 100-cell differential. However, careful scanning common cellular alterations are called pathogen-associated molec-
of the entire smear should reveal an occasional eosinophil. ular patterns (PAMPs). Examples include bacterial lipopolysaccha-
Basophils are the least plentiful cells in the peripheral blood, 0–1% ride, viral RNA, and bacterial DNA. Leukocytes are able to remove
9
(up to 0.2 * 10 /L). It is common to find no basophils on a 100-cell these pathogens by interaction with the leukocyte’s surface receptors
5
9
differential. The finding of an absolute basophilia (70.2 * 10 /L), for PAMPs, referred to as pattern recognition receptors (PRRs).
however, is very important because it can indicate the presence of a Once a pathogen has been recognized, effector cells can attack, engulf,
hematologic malignancy. and kill it. Neutrophils, monocytes, and macrophages play a major role
in the innate immune system. The innate IR is rapid but limited.
LeuKocyte surface MarKers The adaptive immune response (adaptive IR) is initiated in lymphoid
tissue where pathogens encounter lymphocytes, the major cells involved
Leukocytes and other cells express a variety of molecules on their sur- in this response. This IR is slower to develop than the innate IR, but it pro-
faces that can be used as markers to help identify the lineage of a cell vides long-lasting immunity (memory) against the pathogen with which
as well as subsets within the lineage. These markers can be identified it interacts. The adaptive IR will be discussed in more detail in Chapter 8.
by reactions with specific monoclonal antibodies. A nomenclature In addition to its role in protection against infections, the cells
system was developed to identify antibodies with similar character- of the innate immune system possess mechanisms to recognize the
istics using the term cluster of differentiation (CD) followed by a products of damaged and dead host cells, eliminating those cells and
number. The CD designation is now used to identify the molecule rec- initiating tissue repair. These substances are called damage-associated
ognized by the monoclonal antibody. In addition to using CD mark- molecular patterns (DAMPs) and include stress-associated heat
ers to identify cell lineage, some surface markers are used to identify shock proteins (HSPs), crystals, and nuclear proteins. 6
stages of maturation as they are transiently expressed at a specific stage
of development. Other markers are expressed only after the cell has
been stimulated and thus can be used as a marker of cell activation. neutroPhiLs
CD markers are very helpful in differentiating neoplastic hematologic Neutrophils are the most numerous leukocyte in the peripheral blood.
disorders (Chapter 23) and can be identified by flow cytometry or They are easily identified on Romanowsky-stained peripheral blood
cytochemical stains (Chapters 37, 40). smears as cells with a segmented nucleus and fine pink to lavender
granules.
case study (continued from page 99) Differentiation, Maturation, and Morphology
The WBC differential performed on the specimen from Leukocytes develop from HSCs in the bone marrow. The common
harry had the following results: myeloid progenitor (CMP) cell gives rise to the committed precursor
cells for the neutrophilic, eosinophilic, basophilic, and monocytic
Neutrophils 58% lineages, whereas the common lymphoid progenitor (CLP) cell gives
Lymphocytes 32% rise to committed precursor cells for T, B, and natural killer (NK)
Monocytes 6% lymphocytes (Chapter 4). When lineage commitment has occurred,
7
Eosinophils 3% maturation begins. Myeloid and lymphoid cells go through unique
Basophils 1%
maturation processes. The myeloid cells include the granulocytes
3. Are any of the WBC concentrations outside the refer- and their precursor cells (granulocyte monocyte progenitor [GMP],
ence interval (relative or absolute)? colony-forming unit-granulocyte [CFU-G]), the eosinophilic and
basophilic cells and their precursors (CFU-Eo, CFU-Ba) and the
monocytic cells including monocytes and their precursors (GMP,
CFU-M). The lymphoid cells include the lymphocytes and their
LeuKocyte function precursors (CFU-T/NK, CFU-T, CFU-B).
The primary function of leukocytes is to protect the host from infec- Normally, the life span of the neutrophil is spent in three compart-
tious agents or pathogens by employing defense mechanisms called ments: the bone marrow (site of proliferation, differentiation, and mat-
the innate (natural) and/or the adaptive (acquired) immune uration), the peripheral blood (where they circulate for a few hours),
systems. The innate immune response (innate IR) is the body’s first and the tissues (where they perform their function of host defense).
response to common classes of invading pathogens. When a pathogen Neutrophilic production is primarily regulated by three cytokines,
enters the body, it must be recognized as foreign, or nonself, by soluble interleukin-3 (IL-3), granulocyte monocyte-colony-stimulating fac-
proteins (e.g., antibody or complement). The pathogen interacts with tor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF).
cell-surface receptors for IgG (FcgR) or complement (CR1, CR3) GM-CSF and G-CSF also regulate survival and functional activity of
on leukocytes before the pathogen can be eliminated. The leukocyte mature neutrophils. The neutrophil undergoes six morphologically
receptors that participate in the innate IR are always available and do identifiable stages in the process of maturation. The stages from the first
not require cell activation to be expressed. morphologically identifiable cell to the mature segmented neutrophil
M07_MCKE6011_03_SE_C07.indd 100 01/08/14 11:24 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 101
include (1) myeloblast, (2) promyelocyte, (3) myelocyte, (4) metamyelo-
cyte, (5) band or nonsegmented neutrophil, and (6) segmented neutro-
phil, also referred to as the polymorphonuclear neutrophil (PMN). a
During the maturation process, progressive morphological
changes occur in the nucleus. The nucleoli disappear, the chromatin
condenses, and the once round nuclear mass indents and eventually
segments. These nuclear changes are accompanied by distinct cyto-
plasmic changes. The scanty, agranular, basophilic cytoplasm of the
earliest stage is gradually replaced by pink-to-tan-staining granu-
lar cytoplasm in the mature differentiated stage (Figures 7-1 ■ and
7-2 ■, Table 7-1 ★). The four subsets of granules/organelles (primary, b
secondary, secretory, tertiary) are produced at specific times during
neutrophil development and contain specific molecules of physi- ■ figure 7-2 In the center are a myelocyte (a) and a
ologic importance. The biosynthesis of the granule content is primar- promyelocyte (b). Note the changes in the nucleus and
ily determined by activation or inhibition of transcription factors at cytoplasm. The myelocyte has a clear area next to the
certain time points during neutrophil development. Leukopoiesis is nucleus, which represents the Golgi apparatus. Note the
9
11
an amazing process that generates 195 * 10 cells per hour or 10 azurophilic granules in the promyelocyte. Also present are
7
cells per day. However, the marrow has the capacity to significantly two bands and in the top right corner a metamyelocyte.
increase the neutrophil production over this baseline level in response Orthochromatic normoblasts are present (bone marrow,
to infectious or inflammatory stimuli. The morphology of the stages Wright-Giemsa stain, 1000* magnification).
of maturation is discussed in the following sections.
the lineage of blasts (Chapter 37). Myeloblast CD markers include
Myeloblast CD33, CD13, CD38, and CD34. 8
The myeloblast (Table 7-1, Figures 7-1 and 7-3 ■) is the earliest Promyelocyte
morphologically recognizable precursor of the myeloid lineage. The
myeloblast size varies from 14–20 mcM (mm) in diameter, and it The promyelocyte/progranulocyte (Table 7-1, Figures 7-1 and 7-2)
has a high nuclear to cytoplasmic (N:C) ratio. The nucleus is usually varies in size from 15–21 mcM. The nucleus is still quite large, and
round or oval and contains a delicate, lacy, evenly stained chroma- the N:C ratio is high. The nuclear chromatin structure, although
tin. One to five nucleoli are visible. The small amount of cytoplasm coarser than that of the myeloblast, is still open and rather lacy, stain-
is agranular, staining from deep blue to a lighter blue. A distinct ing purple to dark blue. The color of the nucleus varies somewhat
unstained area adjacent to the nucleus representing the Golgi appa- depending on the stain used, and several nucleoli can still be vis-
ratus can be seen. Myeloblasts can stain faintly positive for peroxi- ible. The basophilic cytoplasm is similar to that of the myeloblast
dase and esterase enzymes and for lipids (Sudan black B) although but is differentiated by the presence of prominent, reddish-purple
granules are not evident by light microscopy. Staining reactions primary granules, also called nonspecific or azurophilic granules,
with peroxidase and esterase help differentiate myeloblasts from which are synthesized during this stage. The primary granules are
monoblasts and lymphoblasts. CD markers also aid in identifying
a
b
■ figure 7-3 (a) Indicates a pronormoblast and
(b) indicates a myeloblast. Note that the myeloblast
has more lacy, lighter-staining chromatin with distinct
■ figure 7-1 Stages of neutrophil development. nucleoli and bluish cytoplasm whereas the pronormoblast
Compare the chromatin pattern of the nucleus and the chromatin is more smudged with indistinct nucleoli and
cytoplasmic changes in the various stages. From left: a very deep blue-purple cytoplasm. Also pictured are bands,
very early band, myelocyte, promyelocyte, myeloblast, metamyelocyte, myelocytes, basophilic normoblast,
and very early band; above the myeloblast are two polychromatophilic normoblast, and orthochromatic
segmented neutrophils (bone marrow; Wright-Giemsa normoblast (bone marrow, Wright-Giemsa stain;
stain; 1000* magnification). 1000* magnification).
M07_MCKE6011_03_SE_C07.indd 101 01/08/14 11:24 am
Maturation transit time ~1 day 1–3 days 1–5 days 0.5–4 days 0.5–4 days 1–5 days
Markers CD13, CD33, CD34, CD33, CD15, CD16, CD11b/
cd CD38 CD38 CD18
Large, reddish-purple (azurophilic) primary or nonspecific granules Small pinkish-red to specific granules; azurophilic granules; secretory vesicles Predominance of small pinkish-lavender specific granules; some azurophilic granules present; secretory Abundant small, pinkish- lavender specific granules; some azurophilic granules present; secretory vesicles; tertiary granules
granules Absent vesicles As in band
n:c ratio; size (mcM) high; 14–20 high but less than myeloblast; 15–21 Decreased from promyelocyte; 12–18 Decreased 10–18 Decreased Decreased
Characteristics of Cells in the Maturation Stages of the Neutrophil
cytoplasm Light blue Deep blue Light blue, more mature shows tan to pink Pinkish-tan Pink to tan to clear Pink or tan to clear
Round or oval; delicate, lacy chromatin; nucleoli chromatin lacy but more condensed than blast; nucleoli present chromatin more condensed; nucleoli Chromatin condensed; stains dark purple; kidney bean shape condensed at ends of horseshoe-shaped nucleus; stains dark Nucleus segmented chromatin condensed; stains deep purple/black
nucleus Round or oval, Round to oval; usually absent to oval Chromatin purple into 2–4 lobes;
figure
taBLe 7-1 cell stage (% in bone marrow) Myeloblast (0.2–1.5) Promyelocyte (2–4) Myelocyte (8–16) Metamyelocyte (9–25) Band (nonsegmented) Segmented Neutrophil (polymorphonuclear)
★ (9–15)
102
M07_MCKE6011_03_SE_C07.indd 102 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 103
surrounded by a phospholipid membrane and contain peroxidase but nuclear shape is variable and is not the most reliable identifying
and a number of antimicrobial compounds. See Table 7-2 ★ for a feature. Care should be taken to review other cellular features such as
list of the contents of primary granules. the degree of the chromatin clumping, color of the cytoplasm, pre-
dominant granules present, and the cell size. The nuclear chromatin
Myelocyte is coarse and clumped and stains dark purple. Nucleoli are not visible.
The myelocyte (Table 7-1, Figures 7-1 through 7-3) varies in size from The cytoplasm has a predominance of secondary and secretory gran-
12–18 mcM. The nucleus is reduced in size (as is the N:C ratio) due ules. The ratio of secondary to primary granules is 2:1. The meta-
to nuclear chromatin condensation and appears more darkly stained myelocyte’s cytoplasm resembles the color of the cytoplasm of a fully
than the chromatin of the promyelocyte. Nucleoli can be seen in the mature neutrophil (pinkish-tan). Tertiary or gelatinase-containing
early myelocyte but are usually indistinct. The myelocyte nucleus can granules are synthesized mainly during the metamyelocyte and band
9
be round, oval, slightly flattened on one side, or slightly indented. The neutrophil stages. 9
clear light area next to the nucleus, representing the Golgi apparatus,
can still be seen. The myelocyte goes through two to three cell divi- Band neutrophil
sions; this is the last stage of the maturation process in which the cell The band neutrophil, also called nonsegmented neutrophil or stab,
is capable of mitosis. The early myelocyte has a rather basophilic cyto- is slightly smaller in diameter than the metamyelocyte. The metamy-
plasm, whereas the later, more mature myelocyte, has a more tan to elocyte becomes a band when the indentation of the nucleus is more
pink cytoplasm as the cell begins to lose cytoplasmic RNA. than half the diameter of the hypothetical round nucleus (Table 7-1
The hallmark for the myelocyte stage is the appearance of specific and Figure 7-1). The indentation gives the nucleus a horseshoe shape.
or secondary granules. Synthesis of peroxidase-positive primary gran- The chromatin displays increased condensation at either end of the
ules is halted, and the cell switches to synthesis of peroxidase-negative nucleus. The cytoplasm appears pink to tan, resembling both the pre-
secondary granules. Secondary granules are detected first near the vious stage and the fully mature segmented forms. The band neutro-
nucleus in the Golgi apparatus. This has sometimes been referred to phil is the first stage that normally is found in the peripheral blood. All
as the dawn of neutrophilia. These neutrophilic secondary granules four types of granules (primary, secondary, secretory, tertiary) can be
are small and sandlike with a pinkish-red to pinkish-lavender tint. found at this stage, but primary granules are not usually differentiated
Like the primary granules, a phospholipid membrane surrounds the with Wright stain in band neutrophils.
secondary granules. Large primary azurophilic granules can still be
apparent, but their concentration decreases with each successive cell segmented neutrophil
division because their synthesis has ceased. Their ability to pick up Although similar in size to the band form, the neutrophil, or PMN, is
stain also decreases with successive mitotic divisions. See Table 7-2 recognized, as its name implies, by a segmented nucleus with two or more
for a partial list of secondary granule contents. 9 lobes connected by a thin nuclear filament (Table 7-1, Figure 7-1). The
Secretory vesicles are scattered throughout the cytoplasm of chromatin is condensed and stains a deep purple black. Most neutrophils
myelocytes, metamyelocytes, band neutrophils, and segmented neu- have three or four nuclear lobes, but a range of two to five lobes is pos-
10
trophils (Table 7-2). Secretory vesicles are formed by endocytosis in sible. Fewer than three lobes are considered hyposegmented. A cell with
the later stages of neutrophil maturation and contain plasma proteins more than five lobes is considered abnormal and referred to as a hyper-
including albumin. When neutrophils are stimulated, the cytoplas- segmented neutrophil. Observing three or more five-lobed neutrophils
mic secretory vesicles fuse with the plasma membrane to increase in a 100-cell differential is usually considered pathologic (megaloblastic
the neutrophil surface membrane and expression of adhesion and anemia; Chapter 15). Nuclear lobes are often touching or superimposed
chemotactic receptors. on one another, sometimes making it difficult to differentiate the cell as
a band or PMN.
Metamyelocyte Individual laboratories and agencies such as the Clinical and Labo-
The metamyelocyte (Table 7-1, Figures 7-1 and 7-3) varies in size ratory Standards Institute (CLSI) have outlined criteria for differentiat-
11
from 10–18 mcM in diameter and is not capable of cell division. ing bands from PMNs in manual differentials. A band is defined as
Nuclear indentation that gives the nucleus a kidney bean shape can be having a nucleus with a connecting strip or isthmus with parallel sides
a characteristic that differentiates a metamyelocyte from a myelocyte, and having width enough to reveal two distinct margins with nuclear
★ taBLe 7-2 Neutrophil Granule Contents
Primary granules secondary granules secretory vesicles tertiary granules
Myeloperoxidase Lactoferrin Alkaline phosphatase Gelatinase
Lysozyme Lysozyme Complement receptor 1 Lysozyme
Cathepsin G, B, and D histaminase Cytochrome b 558
Defensins (group of cationic proteins) Collagenase
Bactericidal permeability increasing protein (BPI) Gelatinase
Esterase N heparinase
Elastase
M07_MCKE6011_03_SE_C07.indd 103 01/08/14 11:25 am
104 SECTION II • ThE hEmaTOpOIETIC SySTEm
chromatin material visible between the margins. If a margin of a given
lobe can be traced as a definite and continuing line from one side
across the isthmus to the other side, a filament is assumed to be present
although it is not visible. If a laboratory professional is not sure whether
a neutrophil is a band form or a segmented form, it is arbitrarily classified
as a segmented neutrophil. From a traditional clinical viewpoint, deter-
mining whether young forms of neutrophils (band forms and younger)
9
are increased has been useful. However, differentials performed by
automated hematology instruments do not differentiate between band
and segmented neutrophils. Band neutrophils are fully functional
phagocytes and often are included with the total neutrophil count. 11
The cytoplasm of the mature PMN stains a pink or tan to clear
color. It contains many secondary and tertiary granules and secre-
tory vesicles. Primary granules are present, but because of their loss of
staining quality, might not be readily evident. The ratio of secondary
to primary granules remains 2:1.
Neutrophilic granules contain protein, lipids, and carbohydrates. ■ figure 7-4 The segmented neutrophil on the right
Many of the proteins (enzymes) have already been discussed. About has an X chromatin body (arrow) (peripheral blood,
one-third of the lipids in neutrophils consist of phospholipids. Much Wright-Giemsa stain, 1000* magnification).
of the phospholipid is present in the plasma membrane or membranes
of the various granules. Cholesterol and triglycerides constitute most Bone Marrow
of the nonphospholipid neutrophil lipid. Although cytoplasmic non- Neutrophils in the bone marrow are derived from the stem cell pool
membrane lipid bodies can also be found in neutrophils, their role and can be divided into two pools: the mitotic pool and the postmi-
in cell function is unclear. Lipid material is likewise found in neutro- totic pool (Figure 7-5 ■). The mitotic pool, also called the proliferating
philic precursors and a cytochemical stain for lipids, Sudan black B, pool, includes cells capable of DNA synthesis: myeloblasts, promyelo-
is used to differentiate myeloid precursors from lymphoid precursors cytes, and myelocytes. Cells spend about 3–6 days in this proliferating
(Chapter 23). Carbohydrate in the form of glycogen is also found in pool and undergo four to five cell divisions. Although two to three of
neutrophils and some myeloid precursors. Neutrophils utilize gly- these divisions occur in the myelocyte stage, the number of cell divi-
cogen to obtain energy by glycolysis when required to function in sions at each stage is variable. The postmitotic pool, also known as the
hypoxic conditions (e.g., an abscess site). The periodic acid-Schiff maturation and storage pool, includes metamyelocytes, bands, and
(PAS) stain is used to detect glycogen in cells. CD markers on the segmented neutrophils. Cells spend about 5–7 days in this compart-
neutrophil include CD13, CD15, CD16, CD11b/CD18, and CD33. 8 ment before they are released to the peripheral blood. However, dur-
In normal females with two X chromosomes or males with XXY ing infections, the myelocyte-to-blood transit time can be as short as
chromosomes (Klinefelter syndrome), one X chromosome is randomly 2 days. The number of cells in the postmitotic storage pool is almost
inactivated in each somatic cell of the embryo and remains inactive in three times that of the mitotic pool. 10
all daughter cells produced from that cell. The inactive X chromosome The largest compartment of neutrophils is found within the
appears as an appendage of the neutrophil nucleus and is called a drum- bone marrow and is referred to as the mature neutrophil reserve. The
stick (Barr body) or an X chromatin body (Figure 7-4 ■). The number number of neutrophils circulating in the peripheral blood, the blood
of chromatin bodies detected in the neutrophil is one less than the num- compartment, is about one-third the size of the bone marrow compart-
ber of X chromosomes present; however, chromatin bodies are not visible ment. Once precursor cells have matured in the bone marrow, they are
in every neutrophil. The X chromatin bodies can be identified in 2–3% of released into the peripheral blood (Chapter 4). Normally, the input of
the circulating PMNs of 46, XX females, and Klinefelter males (47, XXY). 9 neutrophils from the bone marrow to the peripheral blood equals the
output of neutrophils from the blood to the tissues, maintaining a rela-
tive steady-state blood concentration. However, when the demand for
CheCkpoint 7-1 neutrophils increases, as in infectious states, the neutrophil concentra-
An adult patient’s peripheral blood smear revealed tion in the peripheral blood can increase quickly by their release from
many myelocytes, metamyelocytes, and band forms of the bone marrow storage (reserve) pool. Depending on the strength and
neutrophils. Is this a normal finding? duration of the stimulus, the marrow myeloid precursor cells (GMP,
CFU-G) also can be induced to proliferate and differentiate to form
additional neutrophils. The transit time between development in the
bone marrow and release to the peripheral blood can be decreased as a
Distribution, Concentration, and Kinetics result of several mechanisms: (1) acceleration of maturation, (2) skipped
The kinetics of a group of cells—their production, distribution, and cellular divisions, and (3) early release of cells from the marrow.
destruction—also is described as the cell turnover rate. For the The mechanisms regulating the production and release of neu-
neutrophil, kinetics follows the movement of the cell through a series trophils from the bone marrow to the peripheral blood are not com-
of interconnected compartments (the marrow, blood, tissues). pletely understood but likely include a feedback loop between the
M07_MCKE6011_03_SE_C07.indd 104 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 105
The release mechanism of the bone marrow storage pool is selec-
Bone marrow
tive in normal, steady-state kinetics, releasing only segmented neutro-
phils and a few band neutrophils. The mechanisms controlling this
Stem cell pool regulated release are not fully understood. The release is partially regu-
• Hematopoietic stem cells lated by the small pore size in the vascular endothelium lining the bone
• Progenitor cells marrow sinusoids and by the mature segmented neutrophil’s ability to
deform enough to squeeze through the narrow opening. Immature cells
are larger and less deformable and cannot penetrate the small pores;
however, when an increased demand for neutrophils exists, a higher
Mitotic pool proportion of less mature neutrophils is released into the peripheral
(3–6 days) blood. Glucocorticoids, endotoxin (bacterial lipopolysaccharide), and
• Myeloblasts G-CSF can increase neutrophil release from the marrow.
• Promyelocytes
• Myelocytes
Peripheral Blood
Neutrophils are released from the bone marrow to the peripheral blood,
but not all neutrophils are circulating freely at the same time. About
Postmitotic/storage pool half the total blood neutrophil pool is temporarily marginated along
(5–7 days) the vessel walls and is called the marginating pool (MP), whereas the
• Metamyelocytes
• Bands other half is freely circulating and is referred to as the circulating pool
10
• Segmented neutrophils (CP). Thus, if all marginated neutrophils were to circulate freely, the
total neutrophil count would double. Marginating neutrophils roll on the
endothelial surface at a slow rate caused by a loose binding interaction
between selectin adhesion proteins on neutrophils (L-selectin) and the
Peripheral blood
(<10 hours) L-selectin ligand on endothelial cells. The two pools are in equilibrium
CP and rapidly and freely exchange neutrophils. Stimulants such as strenu-
ous exercise, epinephrine, or stress can induce a shift from the MP to the
MP
CP, temporarily increasing the neutrophil count. The average neutro-
phil circulates 7.5 hours in the blood before diapedesing (transendo-
thelial migration) to the tissues, although a few die of apoptosis while in
the circulation. These are occasionally seen as “necrobiotic neutrophils”
Tissues with a pyknotic nucleus on the peripheral blood smear (Chapter 10).
(1–5 days)
• Segmented neutrophils tissues
Most neutrophils move into the tissues from the MP in response to
■ figure 7-5 Neutrophils are produced from stem cells chemotactic stimulation (see section on Neutrophil Function). In the
in the bone marrow and spend about 1–2 weeks in this tissues, the neutrophil is either destroyed by trauma (cell necrosis) or
maturation compartment. Most neutrophils are released lives until programmed cell death, apoptosis, occurs (Chapter 2). Neu-
to the peripheral blood as segmented forms. When the trophils that do not receive activation signals generally die within 1–2
demand for these cells is increased, more immature days. However, elevated levels of GM-CSF or G-CSF associated with an
forms can be released. One-half of the neutrophils in the infection or inflammatory process can prolong the neutrophil life span
peripheral blood are in the marginating pool (MP); the to 3–5 days by blocking apoptosis. Senescent or apoptotic neutrophils
other half are in the circulating pool (CP). Neutrophils are phagocytosed by macrophages. 9
spend 610 hours in the blood before marginating and
exiting randomly to tissue.
neutrophil Kinetics
Neutrophils constitute the majority of circulating leukocytes. The
9
absolute concentration varies between 1.8 and 7.0 * 10 /L. A num-
circulating neutrophils and the bone marrow. In normal conditions, ber of physiologic and pathologic variations affect the concentration
this mediator is likely G-CSF, produced by marrow stromal cells and of circulating leukocytes. Pathologic causes of changes in leukocyte
macrophages. Inflammatory cytokines such as IL-1 and tumor necro- numbers are discussed in subsequent chapters on white cell disorders
sis factor (TNF) are important in causing an increase in the neutrophil including Chapters 21–28.
concentration in pathologic conditions by inducing the macrophage Alteration in the concentration of peripheral blood leukocytes is
to increase the release of G-CSF and GM-CSF. The vascular endo- often the first sign of an underlying pathology. A normal leukocyte count
thelial cells (VECs) that form the inner lining of blood vessels also does not rule out the presence of disease, but leukocytosis (an increase
generate cytokines that govern activation and recruitment of leuko- in leukocytes) or leukopenia (a decrease in leukocytes) is an important
cytes. Endothelial cells can be important in recruiting neutrophils in clue to disease processes and deserves further investigation including a
the earliest phases of inflammation and injury. leukocyte differential count to identify the concentration of the different
M07_MCKE6011_03_SE_C07.indd 105 01/08/14 11:25 am
106 SECTION II • ThE hEmaTOpOIETIC SySTEm
9
types of leukocytes. Granulocytopenia (granulocytes 62.0 * 10 /L) function primarily in the tissues where microbial invasion typically
defines a decrease in all types of granulocytes (i.e., eosinophils, baso- occurs. Monocytes-macrophages help in this process but are slower to
phils, neutrophils). Neutropenia is a more specific term denoting a arrive at the site. The four steps in the innate immune response can be
decrease in only neutrophils. Neutropenia in adults exists if the absolute described as adherence, migration (chemotaxis), phagocytosis, and bac-
9
neutrophil count falls below 1.8 * 10 /L The condition of absence of terial killing.
granulocytes is called agranulocytosis, and the patient is at high risk
of developing an infection. Granulocytosis is a term used to denote an adherence
increase in all granulocytes. Neutrophilia is a more specific term indi- Neutrophils flow freely along the vascular endothelium when neither
cating an increase in neutrophils. Neutrophilia in adults occurs when the the neutrophil nor VEC is activated. Neutrophil adherence and migra-
9
absolute concentration of neutrophils exceeds 7.0 * 10 /L. This condi- tion to the site of infection begin with a series of interactions between
tion is most often the result of the body’s reactive response to bacterial the neutrophils and VEC when these cells are activated by a variety of
infection, metabolic intoxication, drug intoxication, or tissue necrosis. inflammatory mediators (cytokines).
Although the WBC count and absolute neutrophil count are used Several different families of cell adhesion molecules (CAM) and
to evaluate neutrophil production, they reflect a transient moment in their ligands play a major role in the adherence process. Adhesion
overall neutrophil kinetics and do not provide accurate, quantitative molecules include (Table 7-3 ★):
information on the rate of production or destruction, status of marrow • b2 (CD18) family of leukocyte integrins and their ligands
reserves, or abnormalities in cell distribution in the tissues. (immunoglobulin-like CAM)
• Selectins and their ligands
CheCkpoint 7-2
• Intercellular adhesion molecules (ICAMs)
9
An adult patient’s WBC count is 10 * 10 /L and there are Adhesion molecules and their ligands located on the leukocytes and
90% neutrophils. What is the absolute number of neutro- VEC act together to induce activation-dependent adhesion events.
phils? Is this within the reference interval for neutrophils? They are critical for every step of neutrophil recruitment to sites of
If not, what term would be used to describe it? tissue injury including margination along vessel walls, diapedesis
(passage of cells through intact vessel walls), and chemotaxis (migra-
tion in response to a chemical stimulation). Adhesion molecules are
Function transmembrane proteins with three domains: extracellular, transmem-
To be effective in its role in host defense, the neutrophil must move to brane, and intracellular. A ligand’s binding to the CAM extracellular
the site of the foreign agent, engulf it, and destroy it. Thus, neutrophils domain on a neutrophil sends a signal across the membrane to the
★ taBLe 7-3 Adhesion Molecules Important in Leukocyte–Endothelial Cell Interactions
Molecules cd designation expressed By counter-receptor/Ligand
1. b 2 @Integrins/neutrophils CD11a/CD18 Activated leukocytes ICAM-1 on VEC (CD54)
a 1 b 2 (LFA-1) ICAM-2 on VEC (CD102)
ICAM-3 (CD50)
a M b 2 (Mac-1) CD11b/CD18 Activated leukocytes ICAM-1 on EC
iC3b, fibrinogen, factor X
a x b 2 (p150, 95) CD11c/CD18 Activated leukocytes iC3b, fibrinogen
2. Selectins
L-selectins CD62L Leukocytes Sialylated carbohydrates; PSGL-1
x
E-selectin CD62E Activated VEC Sialylated carbohydrates (SLe ) and
L-selectin on activated leukocytes
P-selectin CD62P Activated VEC and platelets PSGL-1; sialylated carbohydrates
3. Immunoglobulin supergene family
ICAM-1 CD54 VEC LFA-1 (CD11a-CD18)
ICAM-2 CD102 VEC LFA-1 (CD11a-CD18)
ICAM-3 CD50 Neutrophils VEC
PECAM-1 CD31 VEC CD31, a v b 3
LFA-2 CD2 T lymphocytes LFA-3 (CD58) on EC
LFA-3 CD58 VEC CD2 on T lymphocytes
VCAM-1 CD106 VEC VLA-4 (CD49d) on monocytes,
lymphocytes, eosinophils,
basophils
LFA = leukocyte function-related antigen; CD = cluster of differentiation; VEC = vascular endothellial cell; ICAM = intercellular adhesion moleecules;
PSGL-1 = P selectin glcoprotein ligand-1
M07_MCKE6011_03_SE_C07.indd 106 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 107
cell’s interior, which activates secondary messengers within the cell. The rolling neutrophils are thus situated to respond to additional signals
These secondary messengers affect calcium flux, NADPH oxidase from chemoattractants (chemotactic substances—chemical messengers
activity, cytoskeleton assembly, and phagocytosis. that cause directional migration of cells along a concentration gradient)
Neutrophils and endothelial cells are transformed from a basal generated by infectious agents or an inflammatory response. 10
state to an activated state by inflammatory mediators (cytokines and Stage 2 is the activation of neutrophils. Chemokines (cytokines
chemokines). The initial result is activation of all three classes of with chemotactic activity) or other chemoattractants bind to the
CAMs. Springer has proposed a model of the neutrophil-endothelial endothelial cell surface where they interact with the loosely bound
12
cell adhesion and migration process that can be divided into four neutrophils and result in activation of neutrophil integrins. Chemoat-
stages: (1) activation of VEC, (2) activation of neutrophils, (3) binding tractants are released by tissue cells, microorganisms, and activated
of neutrophils to inner vessel linings, and (4) transendothelial migra- VEC and include specific and nonspecific proinflammatory media-
tion (Figure 7-6 ■). tors such as C5a (complement activation peptide), bacterial products,
Stage 1 involves the activation of VECs that allows for a loose lipid mediators (e.g., platelet-activating factor [PAF]), and chemokines.
association of VECs with neutrophils. Inflammatory cytokines induce Upon neutrophil activation, activation-dependent adhesion receptors
the expression of E- and P-selectins and L-selectin ligands on VEC. including the b2-integrin molecules are expressed. Leukocyte plasma
E- and P-selectin molecules on the VEC surface interact with their membranes have at least three b2@integrins: CD11a/CD18 (leukocyte
ligands on the neutrophil. Additionally, L-selectin, which is constitu- function associated antigen-1 [LFA-1]), CD11b/CD18 (Mac-1, also
tively present in the neutrophil membrane, interacts with its L-selectin known as the complement receptor 3 [CR3]), and CD11c/CD18. Each
ligands that are upregulated on the surface of the activated VEC. These has an a subunit (CD11a, CD11b, CD11c) noncovalently linked to a
10
interactions induce the neutrophil to transiently associate and dissoci- b subunit (CD18). The neutrophil molecule, L-selectin, is down-
ate with the VEC, causing the neutrophil to “roll” on the VEC surface. regulated at this time.
Leukocyte “rolling” Adhesion integrins
selectin mediated and lg-like-mediated Diapedesis
Stage 1 Cytokines Stage 2 Stage 3 Stage 4
tissue Chemoattractants
PMN
b2 integrin
RBC
Blood
vessel
VEC
receptor
(for L-
VEC E-, P-selectin selectin)
PMN-receptor ICAM Basement membrane
L-selectin (for E-, P-selectin) of vessel wall
■ figure 7-6 Adhesion of neutrophils to the vascular endothelium and eventual migra-
tion of neutrophils into the tissue occur as a result of activation of endothelial cells (EC) and
neutrophils by exposure to chemoattractants. When the cells are activated, they are induced
to express adhesion molecules. These transmembrane molecules send a signal across the
membrane to the interior of the cell when they attach to their receptor. The process occurs
in four stages: In Stage 1, E-, P-selectin and L-selectin (VEC) receptor are expressed on acti-
vated EC. The neutrophil’s L-selectin and receptors for E-, P-selectin cause the neutrophil to
attach loosely to the EC and roll along the endothelium. Neutrophils in Stage 2 are activated
by the presence of chemoattractants in the local environment and express the b2@integrins.
These chemoattractants also activate the ECs. The neutrophils in Stage 3 attach to the acti-
vated ECs via the attachment of their b2@integrins to ICAMs of the EC, resulting in a firmer
attachment than in Stage 1 and halting the rolling of the neutrophil. The neutrophil in Stage
4 migrates through the endothelium and basement membrane (diapedesis) to the area of
inflammation. They move in the direction of the chemoattractants (chemotaxis).
M07_MCKE6011_03_SE_C07.indd 107 01/08/14 11:25 am
108 SECTION II • ThE hEmaTOpOIETIC SySTEm
Stage 3 involves arrest of neutrophil rolling because the acti- Phagocytosis
vated neutrophils are more tightly bound to the VECs. Expression of After arriving at the site of infection, phagocytosis by neutrophils can
activation-dependent b2-integrin adhesion molecules on neutrophils begin. Monocytes and macrophages also arrive at the site of injury and
mediates firm adherence to ICAMs expressed by activated VECs near continue to accumulate and contribute to the inflammatory process.
the site of infection or inflammation. This induces a cytoskeletal and Phagocytes must recognize the pathogen as foreign before attachment
morphologic change in the leukocyte required for cellular migration. occurs and phagocytosis is initiated (Figure 7-7 ■). Once a pathogen
Stage 3 ends with a strong, sustained attachment of the leukocyte to is recognized, ingestion of the particle, fusion of the neutrophil gran-
the VEC. At this time, leukocyte NADPH oxidase membrane com- ules with the phagosome (degranulation), and finally the process
plexes are assembled and activated (discussed in “Bacterial Killing of bacterial killing and digestion occur. Phagocytosis is an active
and/or Digestion”). process that requires a large expenditure of energy by the cells. The
Stage 4 involves the transendothelial migration phase that occurs energy required for phagocytosis can result from anaerobic glycolysis
when neutrophils move through the vessel wall at the borders of VECs or aerobic processes.
by the process of diapedesis. As neutrophils pass out of the vessel and The principal factor in determining whether phagocytosis can
into the tissue, VECs modify their cell-to-cell adherent junctions. The occur is the physical nature of the surfaces of both the foreign par-
neutrophils use pseudopods to squeeze between endothelial cells, ticle and the phagocytic cell. Phagocytes recognize unique molecu-
leaving the vascular space and passing through the subendothelial lar characteristics of the pathogen’s surface (PAMP) and bind to the
basement membranes and periendothelial cells. Subendothelial base- invading organism via specific PRR (see the earlier section “Leu-
ment membranes are presumably eroded by the secretion of the neu- kocyte Function”). Some pathogens are recognized by the process
trophil enzymes gelatinase B and elastase from neutrophil granules. of opsonization (the coating of a particle with a soluble factor that
Migration is enhanced when IL-1 and/or TNF activate the VEC. 10 enhances the recognition process). Enhancement of phagocytosis
An autosomal recessive disorder, leukocyte adhesion deficiency through the process of opsonization speeds the ingestion of particles.
type-I (LAD-I), has partial or total absence of expression of the Two well-characterized opsonins are immunoglobulin G (IgG) and
b2-integrins on leukocyte membranes, often due to a mutation of the complement component C3b (see Chapter 8 for a description of
CD18 gene. This results in absence of leukocyte adhesion to the VEC IgG structure). The antibody IgG binds to the microorganism/par-
as well as lack of mobility and migration into the tissues and can result ticle by means of its Fab region, and the Fc region of the antibody
13
in life-threatening bacterial infections. An inability to synthesize the attaches to three classes of Fc receptors on the neutrophil membrane
E- and L-selectin ligand CD15s is seen in leukocyte adhesion deficiency (FcgRI[CD64], FcgRII[CD32], FcgRIII[CD16]). Thus, the antibody
type 2 (LAD-2) and also results in reoccurring infections (Chapter 21). forms a connecting link between the microorganism/particle and the
neutrophil. The neutrophil also has receptors for activated comple-
Migration ment component C3b (CR1/CD35, CR3/CD11b/CD18, CR4/CD11c/
Once in the tissues, neutrophil migration (chemotaxis) is guided CD18). Some bacteria with polysaccharide capsules avoid recognition,
by chemoattractant gradients. Neutrophils continue their migra- thus reducing the effectiveness of phagocytosis.
tion through the extravascular tissue, moving by directed ameboid Following recognition and attachment, the particle is surrounded
motion toward the infected site. Locomotion of neutrophils (and by neutrophil cytoplasmic extensions or pseudopods (Figure 7-7). As
other leukocytes) is a process of “crawling,” not “swimming.” During the pseudopods touch, they fuse, encompassing the particle within
locomotion along a chemotactic gradient, the neutrophils acquire a a phagosome that is bound by the cytoplasmic membrane turned
characteristic asymmetric shape, a process made possible by altera- “inside out.” A plasma membrane–bound oxidase is activated during
tions of the cytoskeleton triggered by neutrophil activation. There is ingestion, which plays an important role in microbicidal activities.
an extension of a broad pseudopodium (protopod) at the anterior of
the cell (containing the nucleus and organelles) and a narrow knob- Bacterial Killing and/or digestion
like tail (uropod) at the rear of the cell. Neutrophil migration through Bacterial killing and/or digestion follows ingestion of particles. After
the tissues requires b1 and b2 integrins with the continuous forma- formation of the phagosome, neutrophil granules migrate toward
tion of new adhesive contacts at the cell front and detachment from and fuse with the phagosome membrane, discharging their granule
the adhesive substrate at the rear of the cell. Chemotaxis is induced contents into the phagocytic vesicle (degranulation), forming a pha-
by a variety of chemoattractant molecules including bacterial formyl golysosome. The microbicidal and digestive proteins contained within
peptides (fMLP), C5a, IL-8, and PAF, many of the same molecules that both primary and secondary granules, which are normally sequestered
activated the neutrophil during Stage 2. 12 from the cytoplasm of the neutrophil, are thus selectively released and
activated within a membrane-enclosed system.
Microbicidal mechanisms that follow ingestion can be
CheCkpoint 7-3 divided into oxygen-dependent/oxidative or oxygen-independent/
nonoxidative activities (Table 7-4 ★). Oxygen-dependent micro-
A patient with life-threatening recurrent infections is bicidal activity is most important physiologically. Phagocytosis is
found to have a chromosomal mutation that results in a accompanied by an energy-dependent “respiratory burst” that gener-
loss of active integrin molecules on the neutrophil surface. ates oxidizing compounds produced from partial oxygen reduction.
Why would this result in life-threatening infections? The respiratory burst is described as including a significant increase
in glycolysis, a 2–3-fold increase in oxygen consumption, a 2–10-fold
M07_MCKE6011_03_SE_C07.indd 108 01/08/14 11:25 am
Chapter 7 • GranuloCytes and MonoCytes 109
Pseudopodia
2
Pathogen
1
3
Engulfment
Recognition
and binding
7 Formation of
phagocytic vacuole
Phagosome
Lysosome 4
Exocytosis
6
5
Killing and
digestion
Phagolysosome
fusion
■ Figure 7-7 Phagocytosis begins with (1) recognition and attachment of the
pathogen to the neutrophil (or macrophage). The pathogen is then internalized
(2), forming a phagocytic vacuole (phagosome [3]). Next, a primary granule (lysosome)
fuses with the vacuole (4), forming a phagolysosome (5). The granule releases its
contents (6) into the vacuole to help kill and digest the microbe (degranulation).
This is followed by extrusion of undigested vacuole contents from the neutrophil
(exocytosis [7]).
★ Table 7-4 Neutrophil Antimicrobial Systems
Oxygen Dependent Oxygen independent
• Myeloperoxidase independent • acid ph of phagosome: antibacterial; enhances some oxygen-dependent antimicrobial mechanisms and
hydrogen peroxide (H 2 o 2 ) other enzymes
-
superoxide anion (o 2 ) • lysozyme (primary and secondary granules): hydrolyzes cell wall of some bacteria; digests killed microbes
-
hydroxyl radicals (oH ) • lactoferrin (secondary granules): binds iron necessary for bacterial growth; also directly bactericidal
1
singlet oxygen ( o 2 )
• BpI (bactericidal permeability-inducing protein; primary granules); Coat microbes; alters cell permeability
• Myeloperoxidase dependent • defensins: small cationic peptides; broad spectrum of bactericidal activity
(forms oxidized halogens)
• Collagenase (secondary granules): degrades microbe macromolecules
• hydrolases (primary granules): digests microbe
M07_MCKE6011_03_SE_C07.indd 109 01/08/14 10:20 pm
110 SECTION II • ThE hEmaTOpOIETIC SySTEm
increase in hexose monophosphate (HMP) shunt activity and genera- are also subject to killing by reactive oxygen metabolites such as H O
2
2
-
tion of NADPH, and the production of a series of reactive oxygen spe- that form from the O secreted by active plasma membrane NADPH
2
14
cies (ROS). The oxidizing compounds—ROS—are important agents complexes into the tissue matrices. Neutrophils are not resistant to the
in killing ingested organisms. The enzyme activity needed to generate toxic effects of the oxidants they secrete and thus have high mortality
ROS is provided by NADPH oxidase, also known as respiratory burst during any sustained inflammatory response. 16
oxidase. In resting cells, NADPH oxidase is found as separate compo- In patients with chronic granulomatous disease, neutrophils are
1
nents of the plasma membrane (gp 91phox , p 22phox , Rap ) and intracellular missing one of the components of the NADPH oxidase complex and
). When phagocytosis takes place, the plasma
stores (p 47phox , p 67phox 15 therefore fail to produce the respiratory burst. They are still capable
membrane is internalized so that what was originally the outer plasma of eliminating infection caused by strains of bacteria susceptible to
membrane surface is now the lining of the phagocytic vesicle and faces killing by oxygen-independent mechanisms, but this antimicrobial
the interior of the phagosome. When the resting cell is exposed to any system is not very effective alone. Often these patients eventually die
of a wide variety of activating stimuli, activated NADPH oxidase is of multiple infections with bacteria resistant to the killing actions of
assembled from the cytoplasmic and membrane-associated subunits these granule proteins (Chapter 21).
at the phagosome membrane. From this location, the NADPH oxidase In addition to their primary functions of phagocytosis and kill-
generates and pours ROS into the phagosome. ing of microorganisms, neutrophils interact in other physiologic pro-
-
Once assembled, the oxidase produces superoxide anion (O ) cesses. Neutrophils stimulate coagulation by releasing a substance that
2
+
+
and NADP . The NADP activates the HMP shunt (Chapter 5), gen- activates prekallikrein to kallikrein, which in turn cleaves kinins from
-
erating more NADPH. O is further metabolized to produce addi- high-molecular-weight kininogen. Kinins are responsible for vascular
2
tional ROS with increasing microbicidal potency. dilation and increased vessel permeability. Kinins are also chemotactic
molecules that attract neutrophils to sites of inflammation. Neutro-
NADPH oxidase phils initially activate kinin production, but as the cells accumulate,
-
+
2 NADPH + 2 O 2 S 2 NADP + 2 O 2 + 2 H + they break down kinins. Neutrophils also secrete interleukin-1 (IL-1),
a pyrogen that acts on the hypothalamus to produce fever.
Superoxide oxidase
+ - -
2 H + O 2 + O 2 S H 2 O 2 + O 2
- - 1
H 2 O 2 + O 2 S 2 OH + O 2
CheCkpoint 7-4
The activated oxidase can be detected in the laboratory by a A patient has a compromised ability to utilize the oxygen-
nitroblue tetrazolium (NBT) test, cytochrome reduction, or chemi- dependent pathway in neutrophils. What two important
luminescence test. microbial killing mechanisms could be affected?
The second oxygen-dependent microbicidal system involves
the neutrophil’s primary granule enzyme myeloperoxidase (MPO).
Myeloperoxidase catalyzes the interaction of hydrogen peroxide pro-
-
duced during the respiratory burst with halide ions (e.g., chloride/Cl ) eosinoPhiLs
giving rise to oxidized halogens (e.g., hypochlorous acid/HOCl) that
+
increase bacterial killing. 14 The eosinophil originates from the IL-5–responsive CD34 myeloid
progenitor cells (CFU-Eo). Cytokines that influence proliferation
MPO and differentiation of the eosinophil lineage include GM-CSF, IL-3,
-
-
H 2 O 2 + Cl S OCl + H 2 O
17
and IL-5. However, it is now recognized that IL-5, released largely
The oxidants generated by the respiratory burst have potent micro- by activated T 2 lymphocytes and in small amounts by eosinophils,
H
bicidal activity against a wide variety of microorganisms such as bac- mast cells, NK cells, and natural killer T (NKT) cells, has relative lin-
teria, fungi, and multicellular and unicellular parasites. However, the eage specificity for eosinophils and is the major cytokine required for
18
phagocyte and surrounding tissues are also susceptible to damage. eosinophil production and terminal differentiation.
To detoxify the oxidant radicals, phagocytes use a variety of mecha-
nisms such as superoxide dismutase, catalase, and a variety of other Differentiation, Maturation, and Morphology
antioxidants. The eosinophil undergoes a morphologic maturation similar to the
Oxygen-independent granule proteins present in primary, sec- neutrophil with the same six stages of maturation identified. How-
ondary, and tertiary granules of neutrophils can successfully kill ever, it is not possible to morphologically differentiate eosinophilic
and degrade many strains of both gram-negative and gram-positive precursors from neutrophilic precursors with the light microscope
bacteria. See Table 7-4 for a list of the most important nonoxygen- until the myelocyte stage. At this stage, the typical acidophilic crys-
dependent antimicrobial proteins of neutrophils. Initially, the pH talloid granules of the eosinophil appear. Granule formation begins
within the phagolysosome decreases and inhibits bacterial growth, in the promyelocyte with small primary granules that lack the crys-
but this alone is insufficient to kill most microbes. Acidic conditions, talloid core of the specific granules. The first two stages (eosinophilic
however, can enhance the activity of some granule proteins such as myeloblast and promyelocyte) will not be described because they
hydrolases and lactoferrin, which perform optimally at low pH. In the are morphologically identical to the neutrophilic myeloblast and
extracellular environment, microorganisms that escape phagocytosis promyelocyte.
M07_MCKE6011_03_SE_C07.indd 110 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 111
eosinophilic Myelocyte to Mature eosinophil
The eosinophilic myelocyte contains large, eosin-staining, crystal-
loid granules. Maturation from the myelocyte to the metamyelocyte,
band, and segmented eosinophil stage is similar to that described for
the neutrophils with gradual nuclear indentation and segmentation.
No appreciable change occurs in the cytoplasm in these later stages
of development. The reddish orange spherical granules are larger
than neutrophilic granules, uniform in size, and evenly distributed
throughout the cell. Because of the low percentage of eosinophils in
the bone marrow, differentiating the eosinophil into its maturational
stages (e.g., eosinophilic myelocyte) serves no useful purpose when
the count is normal. Bone marrow maturation and storage time are
about 9 days.
The mature eosinophil (Figure 7-8 ■) is 12–15 mcM in diam-
eter. The nucleus usually has no more than two or three lobes, and the
cytoplasm is completely filled with granules. The mature eosinophil ■ figure 7-8 Eosinophil (peripheral blood, Wright-Giemsa
contains three types of granules: primary granules, small granules, and stain, 1000* magnification).
specific or secondary granules.
Primary granules contain Charcot–Leyden crystal proteins
(also called galectin-10) that possess lysophospholipase activity. The (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neu-
Charcot–Leyden crystal proteins are found in a variety of tissues, body rotoxin (EDN) (Table 7-5 ★). The MBP is located in the crystalloid
fluids, and secretions in association with eosinophilic inflammatory core; the other three proteins are found in the granule matrix. The
19
reactions. Small granules contain the enzymes acid phosphatase and crystalloid core also appears to store a number of proinflammatory
arylsulphatase but are not well characterized. cytokines such as IL-2, IL-4, and GM-CSF, and the matrix contains
Specific granules are the primary source of the cytotoxic and IL-5 and TNF@a. The eosinophil has the capacity to synthesize and
18
proinflammatory properties of the eosinophil. Specific granules are elaborate a number of other cytokines as well. The eosinophil’s capac-
large, bound by a phospholipid membrane, and have a central crystal- ity to produce cytokines has led to increased interest and research
loid core surrounded by a matrix. These granules contain four major into the eosinophil’s role as an effector cell in allergic inflammation.
proteins: major basic protein (MBP), eosinophil cationic protein In addition to granules, the eosinophil, like the neutrophil, contains a
★ taBLe 7-5 Major Constituents of Eosinophil Granules
Protein characteristics
Major basic protein (MBP) Is cytotoxic for protozoans and helminth parasites
Stimulates release of histamine from mast cells and basophils
Neutralizes mast cell and basophil heparin
Eosinophil cationic protein (ECP) Is capable of killing mammalian and nonmammalian cells
Stimulates release of histamine from mast cells and basophils
Inhibits T lymphocyte proliferation
Activates plasminogen
Enhances mucus production in the bronchi
Stimulates glycosaminoglycan production by fibroblasts
Eosinophil-derived neurotoxin (EDN) Can provoke cerebral and cerebellar dysfunction in animals
Inhibits T-cell responses
Eosinophil peroxidase (EPO) Combines with h 2 O 2 and halide ions to produce a potent bactericidal and helminthicidal action
Is cytotoxic for tumor and host cells
Stimulates histamine release and degranulation of mast cells
Diminishes roles of other inflammatory cells by inactivating leukotrienes
Lysophospholipase Forms Charcot-Leyden crystals
Miscellaneous enzymes Phospholipase D: inactivates mast cell PAF
Arylsulphatase: inactivates mast cell (leukotriene D4)
histaminase: neutralizes mast cell histamine
Acid phosphatase, catalase, nonspecific esterases
Lipid-derived mediators Promote smooth muscle contraction and mucus secretion and inhibit mast cell degranulation
PAF; thromboxane B2
M07_MCKE6011_03_SE_C07.indd 111 01/08/14 11:25 am
112 SECTION II • ThE hEmaTOpOIETIC SySTEm
number of lipid bodies that increase during eosinophil activation in surface of the parasite via exocytosis. A number of eosinophil proteins,
vitro. 17,18 Eosinophils express CD9, CD-11a, -11b, -11c, and CD13 including MBP, ECP, and EPO, are highly toxic for larval parasites.
molecules on their cytoplasmic membrane. These molecules function Eosinophils are also capable of phagocytizing bacteria (although less
in antigen presentation, VEC adhesion, and transmigration into the efficiently than neutrophils and macrophages) and have been shown
tissues, respectively. Additionally, the primary receptors that impart to function as antigen-presenting cells. 25
the unique functional features of eosinophils are interleukin-5 recep- Eosinophils respond weakly to IL-3, IL-5, and GM-CSF as
tor subunit-α ([IL-5Rα] responsible for proliferation, activation, and chemotaxins, but IL-5 synthesized by T lymphocytes has been
survival), CC-chemokine receptor 3 ([CCR3] promoting chemotaxis shown to strongly prime eosinophils for a chemotactic response
in response to eotaxins), and sialic acid-binding immunoglobulin-like to PAF, leukotriene B , or IL-8. Products released from basophils
4
lectin 8 ([SIGLEC8] whose signaling induces apoptosis). 18,20 and mast cells (eosinophil chemotactic factor [ECF]), lymphokines
from sensitized lymphocytes, and allergy-related antigen–antibody
Distribution, Concentration, and Kinetics complexes are strongly chemotactic for eosinophils. Eosinophils
express Fc receptors for IgE, the immunoglobulin that is prevalent
Eosinophils in adults have a concentration in the peripheral blood
9
…0.40 * 10 /L. The cell shows a diurnal variation with highest in the response to parasitic infections and mediates activation of
concentration in the morning and the lowest concentration in the eosinophil killing mechanisms. The cytokines IL-3, IL-5, and GM-
9
21
evening. Eosinophilia in adults is defined as 70.40 * 10 /L and CSF promote the adherence of eosinophils to VEC; transendothelial
is associated with allergic diseases, parasitic infections, toxic reac- migration is 10 times higher in the presence of these cytokines.
tions, gastrointestinal diseases, respiratory tract disorders, neoplastic Eosinophils have a b2 integrin-independent mechanism for
disorders, and other conditions. (See Chapter 21 for a complete list.) recruitment into the tissues that appears to be modulated by the
Eosinophilia is T-cell dependent because T cells are the predominant eosinophil adhesion receptor, VLA-4, and its ligand VCAM-1, found
source of IL-5. Persistent eosinophilia is seen in hypereosinophilic on VECs that have been activated by IL-1, TNF, or IL-4. Changes in
syndrome, a myeloproliferative disorder (Chapter 24). eosinophil adhesion molecule expression occur during eosinophil
Very little is known about the kinetics of eosinophils. Most of migration. This implies that dynamic changes in cell adhesion mol-
the body’s eosinophil population lies in connective tissue below the ecules are involved in cell recruitment to areas of inflammation.
epithelial layer in tissues that are exposed to the external environment The eosinophil liberates substances that can neutralize mast cell
22
such as the nasal passages, lung, skin, gastrointestinal tract, and uri- and basophil products, thereby down modulating the allergic response
nary tract. These cells spend 18 hours in the peripheral blood before (Table 7-5). Increasing evidence suggests a direct correlation between
migrating to the tissues where they can live for several weeks. Once in the degree of eosinophilia and severity of inflammatory diseases, such
the tissues, eosinophils do not re-enter the circulation. 18 as asthma, in which eosinophil activation and degranulation contribute
to the characteristic features of mucous production, bronchoconstric-
18
Function tion, and tissue remodeling. In inflammatory conditions, the cytotoxic
potential of eosinophils is turned against the host’s own tissue. 24
The cellular arm of the adaptive immune system (T lymphocytes;
Chapter 8) influences eosinophil production and function. 23,24
Eosinophils are pro-inflammatory cells associated with allergic dis- BasoPhiLs
+
eases, parasitic infections, and chronic inflammation. Their major role Basophils (Figure 7-9 ■) originate from the CD34 myeloid progeni-
is host defense against helminth parasites via a complex interaction of tors in the bone marrow. IL-3 is the main cytokine involved in human
eosinophils, the adaptive immune system, and parasite. The eosino- basophil growth and differentiation, but GM-CSF, stem cell factor
phil adheres to the organism and releases its granule contents onto the (SCF), IL-4, and IL-5 can also be involved. 26,27
a b
■ figure 7-9 (a) Basophil (peripheral blood, Wright-Giemsa stain, 1000* magnification).
(b) Basophil with washed out granules (Wright-Giemsa stain, staining artifact).
M07_MCKE6011_03_SE_C07.indd 112 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 113
Differentiation, Maturation, and Morphology Concentration, Distribution, and Destruction
Basophils undergo a maturation process similar to that described Basophils’ maturation in the bone marrow requires 2.5–7 days before
for the neutrophil. The first recognizable stage is the promyelocyte, they are released into circulation. In the peripheral blood, they num-
9
although this stage is very difficult to differentiate from the promyelo- ber 60.2 * 10 /L (61% of the total leukocytes). Basophilia in adults
9
cyte of the neutrophil or eosinophil. As with eosinophils and neutro- is defined as 70.2 * 10 /L in the peripheral blood. Basophils are
phils, the various stages of the maturing basophil are characterized by end-stage cells incapable of proliferation and spend only hours in the
a gradual indentation and segmentation of the nucleus. peripheral blood.
Basophilic Myelocyte to Mature Basophil Function
The basophilic myelocyte, metamyelocyte, band, and segmented form Both basophils and mast cells function as mediators of inflammatory
are easily differentiated from other granulocytes by the presence of responses, especially those of immediate hypersensitivity reactions
the large purple-black granules unevenly distributed throughout the such as asthma, urticaria, allergic rhinitis, and anaphylaxis. These cells
cytoplasm. The granules are described as metachromatic and contain have membrane receptors for IgE (FcεR). When IgE attaches to the
histamine, heparin, cathepsin G, major basic protein, and lysophos- receptor, the cell is activated and degranulation is initiated. Degranu-
26
pholipase. The mature basophil ranges in size from 10–15 mcM lation releases enzymes that are vasoactive, bronchoconstrictive, and
and has a segmented nucleus and many purple granules obscuring chemotactic (especially for eosinophils). This release of mediators ini-
both the background of the cytoplasm and the nucleus. Basophil gran- tiates the classic clinical signs of immediate hypersensitivity reactions.
ules contain peroxidase and are positive with the PAS cytochemical These cells can synthesize more granules after degranulation occurs.
reaction. Basophil granules are water-soluble and can dissolve on a Basophils and mast cells express CD40 ligand (CD40L) that interacts
well-rinsed Wright-stained smear, resulting in clear areas within the with CD40 molecules expressed on B lymphocytes. In conjunction
cytoplasm. Usually a few deep-purple–staining granules remain to aid with IL-4, the interaction of B lymphocyte CD40 and basophil CD40L
in the identification of the cell. Basophils express CD9, CD11a, and can induce IgE synthesis by B lymphocytes. Thus, basophils can play
CD13 molecules on their cytoplasmic membrane. an important role in inducing and maintaining allergic reactions. 27
Mast cell
The relationship between basophils and mast cells continues to be CheCkpoint 7-5
investigated. Research shows that basophils and mast cells represent dis- Indicate which of the granulocytes will be increased in the
+
tinct, terminally differentiated cells, separately derived from the CD34 following conditions: a bacterial infection, an immediate
common myeloid progenitor cell. Distinct committed progenitor cells hypersensitivity reaction, and an asthmatic reaction.
28
(CFU-Ba and CFU-MC) have been identified for each lineage. Mast
cells are found in the bone marrow and tissues but are not found in
peripheral blood. Mast cells have proliferative potential and live for
several weeks to months. At times, differentiating the mast cell and the Monocytes
basophil precursors in the bone marrow is difficult although some dif- The monocyte is produced in the bone marrow from a bipoten-
ferences exist (Table 7-6 ★). The mast cell nucleus is round and sur- tial progenitor cell, the GMP, which is capable of producing either
rounded by a dense population of granules. The mast cell granules mature monocytes or neutrophils. The differentiation and prolifera-
contain acid phosphatase, protease, and alkaline phosphatase. Mast tion of GMP into monocytes depend on the action of GM-CSF, IL-3,
cells have a membrane antigen profile similar to that of macrophages. and M-CSF. The primary role of monocytes is host defense and this
★ taBLe 7-6 Comparison of the Characteristics of Basophils and Mast Cells
characteristics Basophils Mast cells
Origin hematopoietic stem cell hematopoietic stem cell
Site of maturation Bone marrow Connective or mucosal tissue
Proliferative potential No yes
Life span Days Weeks to months
Size Small Large
Nucleus Segmented Round
Granules Few, small (peroxidase positive) Many, large (acid phosphatase, alkaline phosphatase positive)
Key cytokine regulating development IL-3 SCF
Surface receptors:
IL-3-R Present Absent
c-kit (SCF-R) Absent Present
IgE receptor (FcPR) Present Present
M07_MCKE6011_03_SE_C07.indd 113 01/08/14 11:25 am
114 SECTION II • ThE hEmaTOpOIETIC SySTEm
role is fulfilled in the tissues. Monocytes continue to differentiate in Monoblast
the tissues, transforming into macrophages. Monocytes and macro- The monoblast (Figure 7-10a ■) nucleus is most often ovoid or round
phages can be stimulated by T lymphocytes and endotoxin to liberate but can be folded or indented. Monoblasts are large (12–20 mcM in
endogenous M-CSF, which can be one mechanism for the mono- diameter). The pale blue-purple nuclear chromatin is finely dispersed
cytosis associated with some infections. M-CSF also activates the (lacy), and several nucleoli are easily identified. The monoblast has
29
secretory and phagocytic activity of monocytes and macrophages. abundant agranular blue-gray cytoplasm.
Monocytes and macrophages make up the monocyte-macrophage
system, also called the mononuclear phagocyte (MNP) system. Promonocyte
The promonocyte (Figure 7-10b ■) is an intermediate form between
Differentiation, Maturation, and Morphology the monoblast and the monocyte. The promonocyte is usually the first
stage to develop morphologic characteristics that allow it to be clearly
The morphologically recognizable monocyte precursors in the bone differentiated as a monocyte precursor by light microscopy. The cell is
marrow are the monoblast and the promonocyte. These cells are large, 12–20 mcM in diameter. The nucleus is most often irregular and
present in a very low concentration in normal bone marrow and are indented with a fine chromatin network. Nuclear chromatin is coarser
found in abundance only in leukemic processes involving the MNP than the monoblast, and nucleoli can be present. The promonocyte’s
system. The monoblast of the marrow cannot be morphologically cytoplasm is abundant with a blue-gray color; azurophilic granules
distinguished from the myeloblast by light microscopy unless pro- can be present. Cytochemical stains for nonspecific esterase, peroxi-
liferation of the monocytic series is marked as occurs in monocytic dase, acid phosphatase, and arylsulfatase are positive.
leukemia. Because myeloblasts and monoblasts are indistinguishable
by light microscopy, cytochemical stains (Chapters 23, 26, 37), and Monocyte
immunophenotyping (Chapters 23, 26, 40) frequently are used to Mature monocytes (Figure 7-10c ■) range in size from 12–20 mcM
help differentiate myeloblasts and monoblasts in suspected cases of with an average size of 18 mcM, making them the largest mature
leukemia. cells in peripheral blood. The nucleus is frequently horseshoe- or
a b
c
■ figure 7-10 Stages of monocyte maturation: (a) monoblast: Note lacy chromatin,
nucleoli, and high N:C ratio; (b) promonocyte: The chromatin is somewhat more coarse and
the amount of cytoplasm is increased; (c) monocyte: The nucleus is more lacy than that of a
neutrophil or lymphocyte and is irregular in shape ([a, b]: bone marrow, Wright-Giemsa stain,
1000* magnification. [c]: peripheral blood, Wright-Giemsa stain, 1000* magnification).
M07_MCKE6011_03_SE_C07.indd 114 01/08/14 11:25 am
ChapTEr 7 • GraNuLOCyTES aND mONOCyTES 115
bean-shaped and possesses numerous folds, giving it the appear- metabolism. These cells can live for months in the tissues. Macro-
ance of brainlike convolutions or chewed gum. The chromatin phages do not normally re-enter the blood, but in areas of inflam-
is loose and linear, forming a lacy pattern in comparison to the mation, some can gain access to the lymph, eventually entering the
clumped dense chromatin of mature lymphocytes or granulocytes. circulation.
Monocytes, however, are sometimes difficult to distinguish from Tissue macrophages, also known as histiocytes, develop dif-
large lymphocytes, especially in reactive states when there are ferent cytochemical and morphologic characteristics that depend
many reactive lymphocytes. The monocyte cytoplasm has vari- on the site of maturation and habitation in tissue. These cells are
able morphologic characteristics depending on its activity. The cell widely distributed in the body and have been given specific names
adheres to glass and “spreads” or sends out numerous pseudopods, depending on their anatomic location. For example, macrophages in
resulting in a wide variation of size and shape on blood smears. the liver are known as Kupffer cells, those in the lung as alveolar
The blue-gray cytoplasm is evenly dispersed with fine, dustlike macrophages, those in the skin as Langerhans cells, and those in
membrane-bound granules, which give the cell cytoplasm the the brain as microglial cells. The osteoclasts in the bone are also of
appearance of ground glass. Vacuoles are frequently observed in MNP derivation. 31
the cytoplasm. Macrophages can proliferate in the tissues, especially in areas
Electron-microscopic cytochemistry reveals two types of gran- of inflammation, thereby increasing the number of cells at these
ules present in monocytes. One type contains peroxidase, acid phos- sites. Occasionally, two or more macrophages fuse to produce giant
phatase, and arylsulfatase, suggesting that these granules are similar multinucleated cells. This occurs in chronic inflammatory states
to the lysosomes (primary azurophilic granules) of neutrophils. and granulomatous lesions where many macrophages are tightly
Less is known about the content of the other type of granule except packed together. Fusion also occurs when particulate matter is too
that they do not contain alkaline phosphatase and are therefore dis- large for one cell to ingest or when two cells simultaneously ingest
30
similar to specific granules of neutrophils. The lipid membrane a particle.
of the granules stains faintly with Sudan black B. Many CD markers
including CD11b/CD18, CD13, CD14, and CD15 are expressed by Distribution, Concentration, and Kinetics
monocytes.
Before maturing into monocytes, the promonocyte undergoes
two or three divisions. Bone marrow transit time is 54 hours. In
Macrophage
The monocyte leaves the blood and enters the tissues where it matures contrast to the large neutrophil storage pool, there is no significant
reserve pool of monocytes in the bone marrow. Most monocytes
into a macrophage (Figure 7-11 ■). The transition from monocyte are released within a day after their maturation from promonocytes.
to macrophage is characterized by progressive cellular enlargement, Monocytes diapedese into the tissue from the peripheral blood in
reaching a size of 15–80 mcM. The nucleus becomes round with a a random manner after circulating for an average transit time of
reticular (netlike) appearance, nucleoli appear, and the cytoplasm 8 hours. 29
appears blue-gray with irregular edges and many vacuoles present. As Similar to neutrophils, the total vascular monocyte pool consists
it matures, the macrophage loses peroxidase, but the amount of endo- of a marginated pool and a circulating pool. However, unlike neutro-
plasmic reticulum (ER), lysosomes, and mitochondria all increase. phils, the marginating pool is about three times the size of the circulat-
In addition, distinct granules are noted in the maturing macrophage ing pool. Monocytes in the circulating peripheral blood number about
and are found to contain lysosomal hydrolases. Macrophages acquire 0.190.8 * 10 /L in the normal adult, or 2910% of the total leu-
9
the expression of CD68, a glycoprotein that can be important in lipid
kocytes. Children have a slightly higher concentration. Monocytosis
(increase in monocytes) in adults occurs when the absolute monocyte
9
count is 70.8 * 10 /L.
Function
Monocytes and macrophages are active in both the innate and adap-
tive IR. In addition to their phagocytic function, they secrete a variety
of substances that affect the function of other cells, especially lympho-
cytes. Lymphocytes in turn secrete soluble products (lymphokines)
that modulate monocytic functions.
Monocytes and macrophages ingest and kill microorganisms.
They are particularly effective in inhibiting the growth of intracel-
lular microorganisms, a process that first requires monocyte acti-
vation. Activation results in the production of many large granules,
enhanced phagocytosis, and an increase in the activity of the HMP
shunt. Monocyte activation occurs in the presence of lymphokines
■ figure 7-11 Arrow indicates a macrophage. Note produced by T lymphocytes. Killing by activated monocytes is non-
the numerous vacuoles and cellular debris (bone marrow, specific (i.e., the secretions from Listeria-sensitized T cells activate a
Wright-Giemsa stain, 1000* magnification). killing mechanism in monocytes not only to Listeria but also to other
M07_MCKE6011_03_SE_C07.indd 115 01/08/14 11:25 am
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