Buttermore ED, Thaxton CL, Bhat MA 2013 Organization and maintenance Kempermann G 2011 Adult Neurogenesis 2. New York: Oxford University
of molecular domains in myelinated axons. J Neurosci Res 91:603–22. Press.
Cairns NJ, Lee VM-Y, Trojanowski JQ 2004 The cytoskeleton in neurodegen- A summary of the knowledge on adult neurogenesis.
erative disease. J Pathol 204:438–49. Kettenmann H, Hanisch UK, Noda M et al 2011 Physiology of microglia.
Cardoso FL, Brites D, Brito MA 2010 Looking at the blood–brain barrier: Phys Rev 91:461–553.
molecular anatomy and possible investigation approaches. Brain Res Kettenmann H, Ransom BR 2012 Neuroglia. New York: Oxford University
Rev 64:328–63. Press.
Catterall WA 2010 Ion channel voltage sensors: structure, function, and A comprehensive textbook on glial cells.
pathophysiology. Neuron 67:915–28.
Kidd GJ, Ohno N, Trapp BD 2013 Biology of Schwann cells. Handb Clin
Contractor A, Mulle C, Swanson GT 2011 Kainate receptors coming of age: Neurol 115:55–79.
milestones of two decades of research. Trends Neurosci 34:154–63.
Kim H-J, DiBernardo AB, Sloane JA et al 2006 TK WAVE1 is required for
Day R, Salzet M 2002 The neuroendocrine phenotype, cellular plasticity, and oligodendrocyte morphogenesis and normal CNS myelination. J Neuro-
the search for genetic switches: redefining the diffuse neuroendocrine sci 26:5849–59.
system. Neuroendocrinol Lett 23:379–84.
Kole MH, Stuart GJ 2012 Signal processing in the axon initial segment.
Deller T, Korte M, Chabanis S et al 2003 Synaptopodin-deficient mice lack Neuron 73:235–47.
a spine apparatus and show deficits in synaptic plasticity. Proc Natl Acad
Kulkarni VA, Firestein BL 2012 The dendritic tree and brain disorders. Mol


Sci U S A 100:10494–9.
Cell Neurosci 50:10–20.
Dieni S, Matsumoto T, Dekkers M et al 2012 BDNF and its pro-peptide are
Leung CT, Coulombe PA, Reed RR 2007 Contribution of olfactory neural
stored in presynaptic dense core vesicles in brain neurons. J Cell Biol
stem cells to tissue maintenance and regeneration. Nat Neurosci 10:
196:775–88.
720–6.
Eggen BJ, Raj D, Hanisch UK 2013 Microglial phenotype and adaptation.
Levitan IB, Kaczmarek LK 2001 The Neuron: Cell and Molecular Biology.
J Neuroimmune Pharmacol 8:807–23.
New York: Oxford University Press.
Eggermann E, Bucurenciu I, Goswami SP et al 2012 Nanodomain coupling A basic neuroscience textbook with the focus on neurones.
between Ca2+ channels and sensors of exocytosis at fast mammalian
synapses. Nature Rev Neurosci 13:7–21. Lucarz A, Brand G 2007 Current considerations about Merkel cells. Eur J
Cell Biol 86:243–51.
Finger S 2001 Origins of Neuroscience: A History of Explorations into Brain
Function. New York: Oxford University Press. Matsuzaki M, Honkura N, Ellis-Davies GC et al 2004 Structural basis of
A historic introduction to the neuroscience field. long-term potentiation in single dendritic spines. Nature 429:761–6.
Matyash V, Kettenmann H 2010 Heterogeneity in astrocyte morphology and
Forni PE, Wray A 2012 Neural crest and olfactory system: new prospective.
physiology. Brain Res Rev 63:2–10.
Mol Neurobiol 46:349–60.
Murakoshi H, Yasuda R 2012 Postsynaptic signaling during plasticity of
Furness JB 2012 The enteric nervous system and neurogastroenterology. Nat
dendritic spines. Trends Neurosci 35:135–43.
Rev Gastroenterol Hepatol 9:286–94.
Nave KA 2010 Myelination and support of axonal integrity by glia. Nature


Gassmann M, Bettler B 2012 Regulation of neuronal GABA(B) receptor func-
468:244–52.
tions by subunit composition. Nature Rev Neurosci 13:380–94.
Neher E, Sakaba T 2008 Multiple roles of calcium ions in the regulation of
Gershon MD 1998 The Second Brain. New York: HarperCollins.
neurotransmitter release. Neuron 59:861–72.
Gray EG 1959 Axo-somatic and axo-dendritic synapses of the cerebral
Neunlist M, Van Landeghem L, Mahé MM et al 2013 The digestive neuronal–
cortex: an electron microscopic study. J Anat 83:420–33.
glial–epithelial unit: a new actor in gut health and disease. Nat Rev
Gulbransen BD, Sharkey KA 2012 Novel functional roles for enteric glia in Gastroenterol Hepatol 10:90–100.
the gastrointestinal tract. Nat Rev Gastroenterol Hepatol 9:625–32.


NERvOuS SySTEm
67.e2
1
NOITCES
Nicholls JG, Martin AR, Fuchs PA et al 2011 From Neuron to Brain, 5th ed. Schmieg N, Memendez G, Schiavo G et al 2014 Signalling endosomes in
Sunderland, MA: Sinauer Associates. axonal transport: travel updates on the molecular highway. Semin Cell
A basic and comprehensive neuroscience textbook. Dev Biol 27:32–43.
Nishiyama A, Komitova M, Suzuki R et al 2009 Polydendrocytes (NG2 cells): Segal V, Vlachos A, Korkotian E 2010 The spine apparatus, synaptopodin,
multifunctional cells with lineage plasticity. Nature Rev Neurosci 10: and dendritic spine plasticity. Neuroscientist 16:125–31.
9–22. Seifert G, Schilling K, Steinhäuser C 2006 Astrocyte dysfunction in neuro-
Nolte J 2008 The Human Brain: An Introduction to Functional Anatomy, logical disorders: a molecular perspective. Nature Rev Neurosci 7:
6th ed. Edinburgh: Elsevier, Mosby; Ch. 9, pp. 197–222. 194–206.
Norrmén C, Suter U 2013 Akt/mTOR signalling in myelination. Biochem Shah MM, Hammond RS, Hoffman DA 2010 Dendritic ion channel traffick-
Soc Trans 41:944–50. ing and plasticity. Trends Neurosci 33:307–16.
Pakkenberg B, Gundersen HJG 1988 Total number of neurons and glial cells Shepherd GM 2003 The Synaptic Organization of the Brain. New York:
in human brain nuclei estimated by the disector and the fractionator. Oxford University Press.
J Microsc 150:1–20. A description of the circuitry of the brain.
Parpura V, Zorec R 2010 Gliotransmission: exocytotic release from astrocytes. Shi L, Fu AKY, Ip NY 2012 Molecular mechanisms underlying maturation
Brain Res Rev 63:83–92. and maintenance of the vertebrate neuromuscular junction. Trends


Peles E, Salzer JL 2000 Molecular domains of myelinated axons. Curr Op Neurosci 35:441–53.
Neurobiol 10:558–65. Sine SM 2012 End-plate acetylcholine receptor: structure, mechanism, phar-
Perea G, Navarrete M, Araque A 2009 Tripartite synapses: astrocytes process macology, and disease. Phys Rev 92:1189–234.
and control synaptic information. Trends Neurosci 32:421–31. Spruston N 2008 Pyramidal neurons: dendritic structure and synaptic inte-
Pereira JA, Lebrun-Julien F, Suter U 2012 Molecular mechanisms regulating gration. Nature Rev Neurosci 9:206–21.
myelination in the peripheral nervous system. Trends Neurosci 35: Squire L, Kandel E 2008 Memory: From Mind to Molecules. Greenwood
123–34. Village, CO: Roberts.
Poliak S, Peles E 2003 The local differentiation of myelinated axons at nodes A description of the mechanism of memory formation.
of Ranvier. Nat Rev Neurosci 4:968–80. Squire L, Berg D, Bloom FE et al 2012 Fundamental Neuroscience, 4th ed.
Proske U, Gandevia SC 2012 The proprioceptive senses: their roles in signal- Oxford: Elsevier, Academic Press.
ing body shape, body position and movement, and muscle force. A basic but comprehensive neuroscience textbook.
Physiol Rev 92:1651–97.
Suedhof TC 2012 The presynaptic active zone. Neuron 75:11–25.
Robel S, Berninger B, Goetz, M 2011 The stem cell potential of glia: lessons
Tait MJ, Saadoun S, Bell BA et al 2008 Water movements in the brain: role
from reactive gliosis. Nature Rev Neurosci 12:88–104.
of aquaporins. Trends Neurosci 31:37–43.
Ryan TJ, Grant SGN 2009 The origin and evolution of synapses. Nature Rev
Willard SS, Koochekpour S 2013 Glutamate, glutamate receptors, and down-
Neurosci 10:701–26.


stream signaling pathways. Int J Biol Sci 9:948–59.
Sakmann B, Neher E 2009 Single-channel Recording. New York: Springer.
Wong RO, Ghosh A 2002 Activity-dependent regulation of dendritic growth
An introduction to the patch-clamp technique and electrophysiology.
and patterning. Nature Rev Neurosci 10:803–12.
Sanai N, Nguyen T, Ihrie RA et al 2011 Corridors of migrating neurons in Zujovic V, Thibaud J, Bachelin C et al 2011 Boundary cap cells are peripheral
the human brain and their decline during infancy. Nature 478:382–6. nervous system stem cells that can be redirected into central nervous
Sanes DH, Reh TA, Harris WA 2011 Development of the Nervous System, system lineages. Proc Natl Acad Sci USA 108:10714–19.
3rd ed. Oxford: Elsevier, Academic Press.
A textbook on developmental neurobiology.


68
1
NOITCES
CHAPTER Blood, lymphoid tissues
4
and haemopoiesis
Blood is an opaque fluid with a viscosity greater than that of water defensive cells (white blood cells, or leukocytes). The latter include
(mean relative viscosity 4.75 at 18°C), and a specific gravity of 1.06 at neutrophil, eosinophil and basophil granulocytes, B lymphocytes and
15°C. It is bright red when oxygenated, in the systemic arteries, and dark monocytes. T lymphocytes develop in the thymus from bone marrow-
red to purple when deoxygenated, in systemic veins. Blood is a mixture derived progenitors. These cells all contribute to the immune system of
of a clear liquid, plasma and cellular elements, and consequently the the human (for an overview of the immune system, see Murphy (2011)).
hydrodynamic flow of blood in vessels behaves in a complex manner Platelets are produced in the bone marrow as cellular fragments of
that is not entirely predictable by simple Newtonian equations. megakaryocytes. Only erythrocytes and platelets are generally confined
Plasma is a clear, yellowish fluid that contains many substances in to the blood vascular system, whereas all leukocytes can leave the cir-
solution or suspension: low-molecular-weight solutes give a mean culation and enter extravascular tissues. The numbers of cells doing so
freezing-point depression of 0.54°C. Plasma contains high concentra- increases greatly during inflammation caused by local infections or
tions of sodium and chloride ions, potassium, calcium, magnesium, tissue damage.
phosphate, bicarbonate, traces of many other ions, glucose, amino acids The lymphoid tissues are the thymus, lymph nodes, spleen and the
and vitamins. It also includes high-molecular-weight plasma proteins, lymphoid follicles associated mainly with the alimentary and respira-
e.g. clotting factors, particularly prothrombin; immunoglobulins and tory tracts. Lymphocytes populate lymphoid tissues and are concerned
complement proteins involved in immunological defence; glycopro- with immune defence. Lymphoid tissue also contains supportive stro-
teins, lipoproteins, polypeptide and steroid hormones, and globulins mal cells, which are non-haemopoietic in origin (e.g. thymic epithe-


for the transport of hormones and iron. The plasma is involved in the lium); non-haemopoietic follicular dendritic cells of lymph nodes and
transport of most molecules that are released or secreted by cells in splenic follicles; haemopoietically derived dendritic cells; and macro-
response to pathological or physiological stimuli and so the routine phages of the mononuclear phagocyte system. Dendritic cells and blood
chemical analysis of plasma is of great diagnostic importance. There is monocyte-derived macrophages are found additionally in most tissues
increasing interest in using metabolomics approaches for the high- and organs, where they function as antigen-presenting cells (APCs).
throughput analysis of small molecules or metabolites in the serum, as
a potential aid to diagnosis and understanding of disease (Psychogios
CELLS OF PERIPHERAL BLOOD
et al 2011).
The precipitation of the protein fibrin from plasma to form a clot
(Fig. 4.1) is initiated by the release of specific materials from damaged ERYTHROCYTES
cells and blood platelets in the presence of calcium ions. If blood or
plasma samples are allowed to stand, they will separate into a clot and Erythrocytes (red blood cells, RBCs) account for the largest proportion
a clear yellowish fluid, the serum. Clot formation is prevented by of blood cells (99% of the total number), with normal values of
removal of calcium ions, e.g. by addition of citrate, oxalate or various 4.1–6.0 × 106/µl in adult males and 3.9–5.5 × 106/µl in adult females.
organic calcium chelators (EDTA, EGTA) to the sample. Heparin is also Polycythaemia (increased red cell mass) can occur in individuals living
widely used as an anticlotting agent because it interferes with fibrin clot at high altitude, or pathologically in conditions resulting in arterial
formation. hypoxia. Reduction in red cell mass (anaemia) has many underlying
In postnatal life, blood cells are formed in the bone marrow. Hae- causes but in rare instances can be due to structural defects in erythro-
mopoiesis produces red cells (erythrocytes) and a wide variety of cytes (see below).


Each erythrocyte is a biconcave disc (see Fig. 4.1; Fig. 4.2) with a
mean diameter in dried smear preparations of 7.1 µm; in fresh prepara-
tions the mean diameter is 7.8 µm, decreasing slightly with age. Mature
erythrocytes lack nuclei. They are pale red by transmitted light, with
Fig. 4.2 A human heart muscle biopsy specimen, showing an erythrocyte
within a capillary. The erythrocyte biconcave disc is typically electron-
Fig. 4.1 Erythrocytes enmeshed in filaments of fibrin in a clot. (Courtesy dense and almost fills the capillary lumen. (Courtesy of Dr Bart Wagner,
of Michael Crowder MD.) Histopathology Department, Sheffield Teaching Hospitals, UK.)


Cells of peripheral blood
69
4
RETPAHC
paler centres because of their biconcave shape. The properties of their exposed on the aged erythrocyte. These lead to the cells being recog-
cell coat cause them to adhere to one another by their rims to form nised and taken up by macrophages. Red cells are destroyed at the rate
loose piles of cells (rouleaux). In normal blood, a few cells assume a of 5 × 1011 cells a day (or nearly 6 million a second) and are normally
shrunken, star-like, crenated form; this shape can be reproduced by replaced from the bone marrow (see Fig. 4.12) at the same rate.
placing normal biconcave erythrocytes in a hypertonic solution, which
causes osmotic shrinkage. In hypotonic solutions erythrocytes take up Blood groups
water and become spherical; they may eventually lyse to release their
haemoglobin (haemolysis), leaving red-cell ghosts.
Over 300 red cell antigens are recognizable with specific antisera. They
Erythrocytes have a plasma membrane that encloses mainly a single
can interact with naturally occurring or induced antibodies in the
protein, haemoglobin, as a 33% solution. The plasma membrane of
plasma of recipients of an unmatched transfusion, causing agglutina-
erythrocytes is 60% lipid and glycolipid, and 40% protein and
tion and lysis of the erythrocytes. Erythrocytes of a single individual
glycoprotein.
carry several different types of antigen, each type belonging to an anti-
More than 15 classes of protein are present, including two major
genic system in which a number of alternative antigens are possible in
types. Glycophorins A and B (each with a molecular mass of approxi-
different persons. So far, 19 major groups have been identified. They
mately 50 kDa) span the membrane, and their negatively charged car-
vary in their distribution frequencies between different populations,
bohydrate chains project from the outer surface of the cell. Their sialic
and include the ABO, Rhesus, MNS, Lutheran, Kell, Lewis, Duffy, Kidd,
acid groups confer most of the fixed charge on the cell surface. A second


Diego, Cartwright, Colton, Sid, Scianna, Yt, Auberger, Ii, Xg, Indian and
transmembrane macromolecule, band 3 protein, forms an important
Dombrock systems. Clinically, the ABO and Rhesus groups are of most
anion channel, exchanging bicarbonate for chloride ions across the
importance.
membrane and allowing the release of CO in the lungs.
2 Leukocytes also bear highly polymorphic antigens encoded by allelic
The filamentous protein, spectrin, is responsible for maintaining the
gene variants. These belong to the group of major histocompatibility
shape of the erythrocyte. A dimer is formed of α1 and β1 spectrin
complex (MHC) antigens, also termed human leukocyte antigens
monomers, and two dimers then come together to form a tetramer
(HLA) in humans. HLA class I antigens are expressed by all nucleated
(Machnicka et al 2013). These are joined by junctional complexes that
cells. Class II antigens are expressed on antigen-presenting cells (APCs)
contain (among other proteins) ankyrin, short actin filaments, tropo-
of the immune system, but can also be induced on many parenchymal
myosin and protein 4.1, forming a hexagonal lattice that supports the
cell types, e.g. after exposure to interferon. HLA class I and II antigens
plasma membrane (Mankelow et al 2012). The junctional complex also
play important roles in cell–cell interactions in the immune system,
interacts with transmembrane proteins. This structure gives the mem-
particularly in the presentation of antigens to T lymphocytes by APCs.
brane great flexibility; red cells are deformable but regain their bicon-
cave shape and dimensions after passing through the smallest capillaries,
which are 4 µm in diameter (Mohandas and Gallagher 2008). Erythro- LEUKOCYTES
cyte membrane flexibility also contributes to the normally low viscosity
of blood. Molecular defects in the cytoskeleton result in abnormalities Leukocytes (white blood cells) belong to at least five different categories
of red cell shape, membrane fragility, premature destruction of eryth- (see Fig. 4.12) and are distinguishable by their size, nuclear shape and


rocytes in the spleen and haemolytic anaemia (Iolascon et al 1998). cytoplasmic inclusions. In practice, leukocytes are often divided into
Fetal erythrocytes up to the fourth month of gestation differ mark- two main groups: namely, those with prominent stainable cytoplasmic
edly from those of adults, in that they are larger, are nucleated and granules, the granulocytes, and those without.
contain a different type of haemoglobin (HbF). After this time they are
progressively replaced by the adult type of cell. Granulocytes
Haemoglobin
This group consists of eosinophil granulocytes, with granules that bind
acidic dyes such as eosin; basophil granulocytes, with granules that bind
Haemoglobin (Hb) is a globular protein with a molecular mass of basic dyes strongly; and neutrophil granulocytes, with granules that
67 kDa. It consists of globulin molecules bound to haem, an iron- stain only weakly with either type of dye. Granulocytes (Fig. 4.3) all
containing porphyrin group. The oxygen-binding power of haemo- possess irregular or multilobed nuclei and belong to the myeloid series
globin is provided by the iron atoms of the haem groups, and these are of blood cells (see Fig. 4.12).
maintained in the ferrous (Fe++) state by the presence of glutathione
Neutrophil granulocytes
within the erythrocyte. The haemoglobin molecule is a tetramer, made
up of four subunits, each a coiled polypeptide chain holding a single Neutrophil granulocytes (neutrophils) are also referred to as polymor-
haem group. phonuclear leukocytes (polymorphs) because of their irregularly
Mutations in the haemoglobin chains can result in a range of pathol-
ogies (Forget and Bunn 2013).
Lifespan
Erythrocytes last between 100 and 120 days before being destroyed. As
erythrocytes age, they become increasingly fragile, and their surface
charges decrease as their content of negatively charged membrane glyco-
proteins diminishes. The lipid content of their membranes also reduces.
Aged erythrocytes are taken up by the macrophages of the spleen N


(Mebius and Kraal 2005) and liver sinusoids, usually without prior
lysis, and are hydrolysed in phagocytic vacuoles where the haemoglobin
is split into its globulin and porphyrin moieties. Globulin is further
degraded to amino acids, which pass into the general amino-acid pool.
Iron is removed from the porphyrin ring and either transported in the
circulation bound to transferrin and used in the synthesis of new hae- B
moglobin in the bone marrow, or stored in the liver as ferritin or
haemosiderin. The remainder of the haem group is converted in the
liver to bilirubin and excreted in the bile. Haemoglobin that is released
by destruction of erythrocytes in the body binds to haptoglobin, and is
taken up via CD163 receptors expressed on the surface of macrophages
(Kristiansen et al 2001).
The recognition of effete erythrocytes by macrophages appears to
take place by a number of mechanisms (Bratosin et al 1998). These Fig. 4.3 Neutrophil (N) and basophil (B) granulocytes within a renal
include the exposure of phospholipids (such as phosphatidyl serine) glomerular capillary in a human kidney biopsy. The neutrophil nucleus is
that are normally found on the inner leaflet of the membrane bilayer, more segmented (four lobes are visible) and the granules are smaller and
alterations in the carbohydrates expressed by the cells (most notably more electron-dense than in the basophil. (Courtesy of Dr Bart Wagner,
the loss of sialic acid) and the binding of autoantibodies to antigens Histopathology Department, Sheffield Teaching Hospitals, UK.)


Blood, lymphoid tissues and haemopoiesis
69.e1
4
RETPAHC
In normal blood, five types of polypeptide chain can occur: namely, antibodies in the donated blood are diluted to insignificant levels.
α, β and two β-like polypeptides, γ and δ. A third, β-like η chain is Normally, however, blood is only transfused between persons with cor-
restricted to early fetal development. Each haemoglobin molecule con- responding groups because anomalous antibodies of the ABO system
tains two α-chains and two others, so that several combinations, and are occasionally found in blood and may cause agglutination or lysis.
hence a number of different types of haemoglobin molecule, are pos- The anti-ABO agglutinins, unlike those of the Rhesus system, belong to
sible. For example, haemoglobin A (HbA), which is the major adult the immunoglobulin M (IgM) class and do not cross the placenta
class, contains 2 α- and 2 β-chains; a variant, HbA with 2 α- and 2 during pregnancy.
2
δ-chains, accounts for only 2% of adult haemoglobin. Haemoglobin F The Rhesus antigen system is determined by three sets of alleles:
(HbF), found in fetal and early postnatal life, consists of 2 α- and 2 namely, Cc, Dd and Ee. The most important clinically is Dd. Inheritance
γ-chains. Adult red cells normally contain less than 1% of HbF. of the Rh factor also obeys simple Mendelian laws; it is therefore pos-
In the genetic condition thalassaemia, only one type of chain is sible for a Rhesus-negative mother to bear a Rhesus-positive child.
expressed normally, the mutant chain being absent or present at much Under these circumstances, fetal Rh antigens can stimulate the produc-
reduced levels. Thus, a molecule may contain 4 α-chains (β-thalassaemia) tion of anti-Rh antibodies by the mother; as these belong to the IgG
or 4 β-chains (α-thalassaemia). In haemoglobin S (HbS) of sickle-cell class of antibody they are able to cross the placenta. For most of the


disease, a point mutation in the β-chain gene (valine substituted for pregnancy the stroma stops the blood group antibodies from crossing
glutamine) causes the haemoglobin to polymerize under conditions of into the fetal circulation. However, immediately prior to birth, the
low oxygen concentration, thus deforming the red blood cell. antibodies can cross this barrier and cause destruction of fetal erythro-
In the ABO system, two allelic genes are inherited in simple Mende- cytes. In the first such pregnancy little damage usually occurs because
lian fashion. Thus the genome may be homozygous and carry the AA anti-Rh antibodies have not been induced, but in subsequent Rh-positive
complement, the blood group being A, or the BB complement, which pregnancies massive destruction of fetal red cells may result, causing
gives blood group B, or it may carry neither (OO), producing blood fetal or neonatal death (haemolytic disease of the newborn). Sensitiza-
group O. In the heterozygous condition the following combinations tion of the maternal immune system can also result from abortion or
can occur: AB (blood group AB), AO (blood group A) and BO (blood miscarriage, or occasionally even from amniocentesis, which may intro-
group B). The ABO blood group antigens are all membrane glycolipids. duce fetal antigens into the maternal circulation. Treatment is by
Individuals with group AB blood lack antibodies to both A and B exchange transfusion of the neonate or, prophylactically, by giving
antigens, and so can be transfused with blood of any group; they are Rh-immune (anti-D) serum to the mother after the first Rh-positive
termed universal recipients. Conversely, those with group O, universal pregnancy, which destroys the fetal Rh antigen in her circulation before
donors, can give blood to any recipient, since anti-A and anti-B sensitization can occur.


BlOOd, lymPHOId TISSuES ANd HAEmOPOIESIS
70
1
NOITCES
segmented (multilobed) nuclei. They form the largest proportion of the capacity to neutrophils, but are present only in small numbers in
white blood cells (40–75% in adults, with a normal count of 2500– normal blood (100–400/µl). The nucleus has two prominent lobes
7500/µl) and have a diameter of 12–14 µm. The cells may be spherical connected by a thin strand of chromatin. Their cytoplasmic specific
in the circulation, but they can flatten and become actively motile granules are uniformly large (0.5 µm) and give the living cell a slightly
within the extracellular matrix of connective tissues. yellowish colour. The cytoplasm is packed with granules, which are
The numerous cytoplasmic granules are heterogeneous in size, spherical or ellipsoid and membrane-bound. The core of each granule
shape and content, but all are membrane-bound and contain hydro- is composed of a lattice of major basic protein, which is responsible
lytic and other enzymes. Two major types can be distinguished accord- for its strong eosinophilic staining properties. The surrounding
ing to their developmental origin and contents. Non-specific or matrix contains several lysosomal enzymes including acid phosphatase,
primary (azurophilic) granules are formed early in neutrophil matura- ribonuclease, phospholipase and a myeloperoxidase unique to
tion. They are relatively large (0.5 µm) spheroidal lysosomes contain- eosinophils.
ing myeloperoxidase, acid phosphatase, elastase and several other Like other leukocytes, eosinophils are motile. When suitably stimu-
enzymes. Specific or secondary granules are formed later, and occur in lated, they are able to pass into the extravascular tissues from the circu-
a wide range of shapes including spheres, ellipsoids and rods. These lation. They are typical minor constituents of the dermis, and of the
contain strong bacteriocidal components including alkaline phos- connective tissue components of the bronchial tree, alimentary tract,


phatase, lactoferrin and collagenase, none of which is found in primary uterus and vagina. The total lifespan of these cells is a few days, of which
granules. Conversely, secondary granules lack peroxidase and acid some 10 hours is spent in the circulation, and the remainder in the
phosphatase. Some enzymes, e.g. lysozyme, are present in both types extravascular tissues.
of granule. Eosinophil numbers rise (eosinophilia) in worm infestations and
In mature neutrophils the nucleus is characteristically multilobed also in certain allergic disorders, and it is thought that they evolved as
with up to six (usually three or four) segments joined by narrow nuclear a primary defence against parasitic attack. They have surface receptors
strands; this is known as the segmented stage. Less mature cells have for IgE that bind to IgE-antigen complexes, triggering phagocytosis and
fewer lobes. The earliest to be released under normal conditions are release of granule contents. However, they are only weakly phagocytic
juveniles (band or stab cells), in which the nucleus is an unsegmented and their most important function is the destruction of parasites too
crescent or band. In certain clinical conditions, even earlier stages in large to phagocytose. This antiparasitic effect is mediated via toxic mol-
neutrophil formation, when cells display indented or rounded nuclei ecules released from their granules (e.g. eosinophil cationic protein and
(metamyelocytes or myelocytes) may be released from the bone marrow. major basic protein). They also release histaminase, which limits the
In mature cells the edges of the nuclear lobes are often irregular. inflammatory consequences of mast cell degranulation. High local con-
In females 3% of the nuclei of neutrophils show a conspicuous ‘drum- centrations of eosinophils, e.g. in bronchial asthma and in cutaneous
stick’ formation, which represents the sex chromatin of the inactive X contact sensitivity and allergic eczema, can cause tissue destruction as
chromosome (Barr body). Neutrophil cytoplasm contains few mito- a consequence of the release of molecules such as collagenase from their
chondria but abundant cytoskeletal elements, including actin filaments, granules.


microtubules and their associated proteins, all characteristic of highly
motile cells. Basophil granulocytes
Neutrophils are important in the defence of the body against micro- Slightly smaller than other granulocytes, basophil granulocytes are
organisms. They can phagocytose microbes and small particles in the 10–14 µm in diameter, and form only 0.5–1% of the total leukocyte
circulation and, after extravasation, they carry out similar activities in population of normal blood, with a count of 25–200/µl. Their distin-
other tissues. They function effectively in relatively anaerobic condi- guishing feature is the presence of large, conspicuous basophilic gran-
tions, relying largely on glycolytic metabolism, and they fulfil an impor- ules. The nucleus is somewhat irregular or bilobed, and is usually
tant role in the acute inflammatory phase of tissue injury, responding obscured in stained blood smears by the similar colour of the basophilic
to chemotaxins released by damaged tissue. Phagocytosis of cellular granules. The granules are membrane-bound vesicles, which display a
debris or invading microorganisms is followed by fusion of the phago- variety of crystalline, lamellar and granular inclusions: they contain
cytic vacuole with granules, which results in bacterial killing and diges- heparin, histamine and several other inflammatory agents, and closely
tion. Actively phagocytic neutrophils are able to reduce oxygen resemble those of tissue mast cells. Both basophils and mast cells have
enzymatically to form reactive oxygen species including superoxide high-affinity membrane receptors for IgE and are therefore coated with
radicals and hydrogen peroxide, which enhance bacterial destruction IgE antibody. If this binds to its antigen it triggers degranulation of the
probably by activation of some of the granule contents (Segal 2005, cells, producing vasodilation, increased vascular permeability, chemo-
Nathan 2006). Neutrophils can also produce neutrophil extracellular tactic stimuli for other granulocytes, and the symptoms of immediate
traps (NETs), which are web-like structures composed of DNA and hypersensitivity, e.g. in allergic rhinitis (hay fever). Despite these simi-


proteolytic enzymes that can trap bacteria and kill them (Kaplan and larities, basophils and mast cells develop as separate lineages in the
Radic 2012). myeloid series, from haemopoietic stem cells in the bone marrow.
Phagocytosis is greatly facilitated by circulating antibodies to mol- Evidence from experimental animal models suggests that they are
ecules such as bacterial antigens, which the body has previously closely related (see Fig. 4.12) but studies on mast cell disorders in
encountered. Antibodies coat the antigenic target and bind the plasma humans indicate that their lineages diverge from a more distant ances-
complement protein, C1, to their non-variable Fc regions. This activates tral progenitor (Kocabas et al 2005). The role of mast cells in the regula-
the complement cascade, which involves some 20 plasma proteins tion of responses to pain is of interest clinically as a therapeutic target
synthesized mainly in the liver, and completes the process of opsoniza- (Chatterjea and Martinov 2015).
tion. The complement cascade involves the sequential cleavage of the
complement proteins into a large fragment, which generally binds to
Mononuclear leukocytes
the antigenic surface, and a small bioactive fragment, which is released.
The final step is the recognition of complement by receptors on the
Monocytes
surfaces of neutrophils (and macrophages), which promotes phagocy-
tosis of the organism. Monocytes are the largest of the leukocytes (15–20 µm in diameter) but
Neutrophils are short-lived; they spend some 6–7 hours circulating they form only a small proportion of the total population (2–8% with
in the blood and a few days in connective tissues. The number of cir- a count of 100–700/µl of blood). The nucleus, which is euchromatic,
culating neutrophils varies, and often rises during episodes of acute is relatively large and irregular, often with a characteristic indentation
bacterial infection. They die after carrying out their phagocytic role; on one side. The cytoplasm is pale-staining, particulate and typically
dead neutrophils, bacteria, tissue debris (including tissue damaged by vacuolated. Near the nuclear indentation it contains a prominent Golgi


neutrophil enzymes and toxins) and interstitial fluid form the charac- complex and vesicles. Monocytes are actively phagocytic cells and
teristic, greenish-yellow pus of infected tissue. The colour is derived contain numerous lysosomes. Phagocytosis is triggered by recognition
from the natural colour of neutrophil myeloperoxidase. of opsonized material, as described for neutrophils. Monocytes are
Granules may also be released inappropriately from neutrophils. highly motile and possess a well-developed cytoskeleton.
Their enzymes are implicated in various pathological conditions, e.g. Monocytes express class II MHC antigens and share other similarities
rheumatoid arthritis, where tissue destruction and chronic inflamma- to tissue macrophages and dendritic cells. Most monocytes are thought
tion occur. to be in transit via the blood stream from the bone marrow to the
peripheral tissues, where they give rise to macrophages and dendritic
Eosinophil granulocytes cells; different monocyte subsets may target inflamed tissues. Like other
Eosinophil granulocytes (eosinophils; for a review, see Rothenberg and leukocytes, they pass into extravascular sites through the walls of capil-
Hogan (2006)) are similar in size (12–15 µm), shape and motile laries and venules.


Cells of peripheral blood
71
4
RETPAHC
Fig. 4.5 Tubular glands
in the appendix,
showing intraepithelial
lymphocytes (short
arrows). A lymphocyte
in anaphase is
indicated (long arrow).
Fig. 4.4 A small, resting lymphocyte in human peripheral blood. The
nuclear:cytoplasmic ratio is high and the cytoplasm contains few
organelles, indicative of its quiescent state. (Courtesy of Dr Bart Wagner,
Histopathology Department, Sheffield Teaching Hospitals, UK.)
Lymphocytes
Lymphocytes (Fig. 4.4; see Figs 4.6, 4.12) are the second most numer-
ous type of leukocyte in adulthood, forming 20–30% of the total popu-
lation (1500–2700/µl of blood). In young children they are the most
numerous blood leukocyte. Most circulating lymphocytes are small,
6–8 µm in diameter; a few are medium-sized and have an increased
cytoplasmic volume, often in response to antigenic stimulation. Occa-
sionally, cells up to 16 µm are seen in peripheral blood. Lymphocytes,
like other leukocytes, are found in extravascular tissues (including lym-
phoid tissue); however, they are the only white blood cells that return
to the circulation. The lifespan of lymphocytes ranges from a few days
(short-lived) to many years (long-lived). Long-lived lymphocytes are
necessary for the maintenance of immunological memory.
Blood lymphocytes are a heterogeneous collection mainly of B and
T cells, and consist of different subsets and different stages of activity
and maturity. About 85% of all circulating lymphocytes in normal
blood are T cells. Primary immunodeficiency diseases can result from
molecular defects in T and B lymphocytes (reviewed in Cunningham-
Rundles and Ponda (2005)). Included with the lymphocytes, but prob-
ably constituting a separate lineage subset, are the natural killer (NK)
cells. NK cells most closely resemble large T cells morphologically.
Small lymphocytes (both B and T cells) contain a rounded, densely Fig. 4.6 A mature B cell (plasma cell) in human connective tissue. The


staining nucleus that is surrounded by a very narrow rim of cytoplasm, abundant rough endoplasmic reticulum is typical of a cell actively
barely visible in the light microscope. In the electron microscope (see synthesizing secretory protein, in this case immunoglobulin. The cell to
the left is a fibroblast. (Courtesy of Dr Bart Wagner, Histopathology
Fig. 4.4), few cytoplasmic organelles can be seen apart from a small
Department, Sheffield Teaching Hospitals, UK.)
number of mitochondria, single ribosomes, sparse profiles of endoplas-
mic reticulum and occasional lysosomes; these features indicate a low
metabolic rate and a quiescent phenotype. However, these cells become germinal centres in the lymphoid tissues. Following this, some B cells
motile when they contact solid surfaces, and can pass between endothe- differentiate into large basophilic (RNA-rich) plasma cells, either within
lial cells to exit from, or re-enter, the vascular system. They migrate or outside the lymphoid tissues. Plasma cells produce antibodies in
extensively within various tissues, including epithelia (Fig. 4.5). their extensive rough endoplasmic reticulum (Fig. 4.6) and secrete
Larger lymphocytes include T and B cells that are functionally acti- them into the adjacent tissues. They have a prominent pale-staining
vated or proliferating after stimulation by antigen, and NK cells. They Golgi complex adjacent to an eccentrically placed nucleus, typically
contain a nucleus, which is, at least in part, euchromatic; a basophilic with peripheral blocks of condensed heterochromatin resembling the
cytoplasm, which may appear granular; and numerous polyribosome numerals of a clock (clock-faced nucleus) (see Fig. 4.12). Other germi-
clusters, consistent with active protein synthesis. The ultrastructural nal centre B cells develop into long-lived memory cells capable of
appearance of these cells varies according to their class and is described responding to their specific antigens not only with a more rapid and
below. higher antibody output, but also with an increased antibody affinity
compared with the primary response.
B cells Antibodies are immunoglobulins, grouped into five classes accord-


B cells and the plasma cells that develop from them synthesize and ing to their heavy polypeptide chain. Immunoglobulin G (IgG) forms
secrete antibodies that can specifically recognize and neutralize foreign the bulk of circulating antibodies. Immunoglobulin M (IgM) is nor-
(non-self) macromolecules (antigens), and can direct various non- mally synthesized early in immune responses. Immunoglobulin A (IgA)
lymphocytic cells (e.g. neutrophils, macrophages and dendritic cells) to is present in breast milk, tears, saliva and other secretions of the ali-
phagocytose pathogens. B cells differentiate from haemopoietic stem mentary tract, coupled to a secretory piece (a 70 kDa protein) that is
cells in the bone marrow. After deletion of autoreactive cells, the selected synthesized by the epithelial cells. This protects the immunoglobulin
B lymphocytes then leave the bone marrow and migrate to peripheral from proteolytic degradation and is part of the process by which the
lymphoid sites (e.g. lymph nodes). Here, following stimulation by molecule is transported across the epithelium; IgA thus contributes to
antigen, they undergo further proliferation and selection, forming mucosal immunity. IgA deficiency is relatively common, particularly in


BlOOd, lymPHOId TISSuES ANd HAEmOPOIESIS
72
1
NOITCES
some ethnic groups (reviewed in Woof and Kerr (2006)). Immunoglob- and enhancing the immune responses mediated by those cells. In addi-
ulin E (IgE) is an antibody which binds to receptors on the surfaces of tion to Th1 and Th2 cells, other subsets of helper T cells have been
mast cells, eosinophils and blood basophils; it is found only at low described. These include Th17 cells (which secrete the cytokine IL-17),
concentrations in the circulation. Immunoglobulin D (IgD) is found implicated in autoimmune diseases (Stockinger and Veldhoen 2007).
together with IgM as a major membrane-bound immunoglobulin on Other subsets (Th9, Th22) have been described and it is likely that many
mature, immunocompetent but naïve (prior to antigen exposure) B more subsets will be characterized in the future (Jiang and Dong 2013).
cells, acting as the cellular receptor for antigen. Helper T cells are also important in directing the destruction of
When circulating antibodies bind to antigens they form immune pathogens by recruiting accessory cells (e.g. macrophages, neutrophils,
complexes. If present in abnormal quantities, these may cause patho- eosinophils) to the site of infection and by activating their effector
logical damage to the vascular system and other tissues, either by inter- functions. This process is tightly coordinated. For example, Th1 helper
fering mechanically with the permeability of the basal lamina (e.g. T cells secrete cytokines that not only attract and activate macrophages
some types of glomerulonephritis), or by causing local activation of the but also provide help for B cells and guide their immunoglobulin pro-
complement system that generates inflammatory mediators (e.g. C5a), duction to the subclasses that fix complement. Thus these antibodies
attacks cell membranes and causes vascular disease. In pregnancy, opsonize the pathogen target, which can then be recognized, ingested
maternal IgG crosses the placenta and confers passive immunity on the and destroyed by the macrophage accessory cells that bear receptors for


fetus. Maternal milk contains secretory immunoglobulins (IgA) that complement and the Fc region of IgG. These Th1 cells are sometimes
help to combat bacterial and viral organisms in the alimentary tract of referred to as delayed-type hypersensitivity T cells. In contrast, Th2 cells
the baby during the first few weeks of postnatal life. secrete cytokines that induce the development and activation of eosi-
nophils, and also induce B cells to switch their immunoglobulins to
T cells non-complement-fixing classes (e.g. IgE). Pathogens such as parasitic
There are a number of subsets of T (thymus-derived) lymphocytes, all worms can then be coated with IgE antibody and hence recognized and
progeny of haemopoietic stem cells in the bone marrow. They develop destroyed by the effector functions of the eosinophil accessory cells,
and mature in the thymus, and subsequently populate peripheral sec- which bear receptors for the Fc region of IgE.
ondary lymphoid organs, which they constantly leave and re-enter via If helper T-cell activities are non-functional, a state of immunodefi-
the circulation. As recirculating cells, their major function is immune ciency results. This means that potentially pathogenic organisms, which
surveillance. Their activation and subsequent proliferation and func- are normally kept in check by the immune system, may proliferate and
tional maturation are under the control of antigen-presenting cells. T cause overt pathology, e.g. in acquired immune deficiency syndrome
cells undertake a wide variety of cell-mediated defensive functions that (AIDS), where a virus (human immunodeficiency virus, HIV) specifi-
are not directly dependent on antibody activity, and which constitute cally infects and kills (predominantly) helper T cells, though some
the basis of cellular immunity. T-cell responses focus on the destruction antigen-presenting cells are also killed.
of cellular targets such as virus-infected cells, certain bacterial infections,
fungi, some protozoal infections, neoplastic cells and the cells of grafts Regulatory T cells
from other individuals (allografts, when the tissue antigens of the A third population of T cells, ‘regulatory’ T or ‘Treg’ cells, are important
donor and recipient are not sufficiently similar). Targets may be killed in controlling the immune response. These CD4+, CD25+ cells have an


directly by cytotoxic T cells, or indirectly by accessory cells (e.g. macro- immunomodulatory function and can dampen the effector functions
phages) that have been recruited and activated by cytokine-secreting of both cytotoxic and helper T cells. Regulatory T cells (natural Tregs)
helper T cells. A third group, regulatory T cells, acts to regulate or limit are produced in the thymus and are an important additional mecha-
immune responses. nism for maintaining self-tolerance (Safinia et al 2013). Treg function
Functional groups of T cells are classified according to the molecules is antigen-specific and depends on direct cell–cell contact. Molecules
they express on their surfaces. The majority of cytokine-secreting helper secreted or induced by Treg cells, such as IL-10 or transforming growth
T cells express CD4, while cytotoxic T cells are characterized by CD8. factor beta (TGF-β), also play an important role in mediating Treg sup-
Regulatory T cells co-express CD4 and CD25. The CD (cluster of dif- pressive effects on the immune system. Similar induced regulatory T
ferentiation) prefix provides a standard nomenclature for all cell-surface cells can be induced in the periphery and may be important in the
molecules. At present, more than 330 different CD antigens have been induction of oral tolerance to ingested antigens, as well as tolerance to
designated; each one represents a cell surface molecule that can be tissue-specific molecules that are not expressed in the thymus (Schmitt
identified by specific antibodies. Further details of the classification are and Williams 2013).
beyond the scope of this publication and are given in Male et al (2012).
Structurally, T lymphocytes present different appearances depending Natural killer (NK) cells
on their type and state of activity. When resting, they are typically Natural killer (NK) cells have functional similarities to cytotoxic T cells
small lymphocytes and are morphologically indistinguishable from but they lack other typical lymphocyte features and do not express
B lymphocytes. When stimulated, they become large (up to 15 µm), antigen-specific receptors. They normally form only a small percentage
moderately basophilic cells, with a partially euchromatic nucleus and of all lymphocyte-like cells and are usually included in the large


numerous free ribosomes, rough and smooth endoplasmic reticulum, granular lymphocyte category. When mature, NK cells have a mildly
a Golgi complex and a few mitochondria, in their cytoplasm. Cytotoxic basophilic cytoplasm. Ultrastructurally, their cytoplasm contains ribo-
T cells contain dense lysosome-like vacuoles that function in cytotoxic somes, rough endoplasmic reticulum and dense, membrane-bound
killing. vesicles 200–500 nm in diameter with crystalline cores. These contain
the protein perforin, which is capable of inserting holes in the plasma
Cytotoxic T cells membranes of target cells, and granzymes, which trigger subsequent
Cytotoxic T lymphocytes (which express CD8) are responsible for the target cell death by apoptosis. NK cells are activated to kill target cells
direct cytotoxic killing of target cells (e.g. virus-infected cells); the by a number of factors. They can recognize and kill antibody-coated
requirement for direct cell–cell contact ensures the specificity of the target cells via a mechanism termed antibody-dependent cell-mediated
response. Recognition of antigen, presented as a peptide fragment on cytotoxicity (ADCC). They also have receptors that inhibit NK destruc-
MHC class I molecules, triggers the calcium-dependent release of lytic tive activity when they recognize MHC class I on normal cells. When
granules by the T cell. These lysosome-like granules contain perforin NK cells detect the loss or downregulation of MHC class I antigens
(cytolysin), which forms a pore in the target cell membrane. They also on certain virus-infected cells and some tumour cells, they activate
contain several different serine protease enzymes (granzymes), which apoptosis-inducing mechanisms that enable them to attack these
enter the target cell via the perforin pore and induce the programmed abnormal cells, albeit relatively non-specifically. For further reading, see
cell death (apoptosis; p. 26) of the target. Vivier et al (2008) and Chan et al (2014).
Helper T cells
Helper T cells (which express CD4) are characterized by the secretion PLATELETS
of cytokines. Two major populations have been identified according to
the range of cytokines produced. Th1 helper T cells typically secrete Blood platelets, also known as thrombocytes, are relatively small


interleukin (IL)-2, tumour necrosis factor alpha (TNF-α) and interferon (2–4 µm across) irregular or oval discs present in large numbers
gamma (IFN-γ), while Th2 cells produce cytokines such as IL-4, IL-5 (200,000–400,000/µl) in blood. In freshly harvested blood samples
and IL-13. These two CD4-expressing populations are termed ‘helper’ they readily adhere to each other and to all available surfaces, unless
T cells because one aspect of their function is to stimulate the prolifera- the blood is treated with citrate or other substances that reduce the
tion and maturation of B lymphocytes and cytotoxic T lymphocytes availability of calcium ions. Platelets are anucleate cell fragments,
(mediated via cytokines such as IL-4, IL-2 and IFN-γ), thus enabling derived from megakaryocytes in the bone marrow. They are surrounded


lymphoid tissues
73
4
RETPAHC
by a plasma membrane with a thick glycoprotein coat, which is respon- together to initiate immune responses to foreign antigens (Malhotra
sible for their adhesive properties. A band of 10 microtubules lies et al 2013). These secondary tissues include lymph nodes, spleen, and
around the perimeter of the platelet beneath the plasma membrane; lymphoid tissue associated with epithelial surfaces (mucosa-associated
the microtubules are associated with actin filaments, myosin and other lymphoid tissue, MALT), e.g. the palatine and nasopharyngeal tonsils,
proteins related to cell contraction. The cytoplasm also contains mito- Peyer’s patches in the small intestine, lymphoid nodules in the respira-
chondria, glycogen, scant smooth endoplasmic reticulum, tubular tory and urogenital systems, the skin. The microstructure of lymph
invaginations of the plasma membrane, and three major types of nodes and of MALT in general is described below. Details of all other
membrane-bound vesicle, designated α, δ and λ granules. lymphoid tissues and organs are included in the descriptions of the
Alpha granules are the largest, and have diameters of up to 500 nm. appropriate regional anatomy.
They contain platelet-derived growth factor (PDGF), fibrinogen and Lymphocytes enter secondary lymphoid tissues from the blood,
other substances. Delta granules are smaller (up to 300 nm) and usually by migration through the walls of capillaries or venules (high
contain 5-hydroxytryptamine (5-HT, serotonin) that has been endocy- endothelial venules, HEVs (see Ch. 6)) and leave by the lymphatic
tosed from the blood plasma. Lambda granules are the smallest (up to system. In the spleen, lymphocyte entry and exit take place via the
250 nm) and contain lysosomal enzymes. marginal zone and venous drainage respectively. Antigen-presenting
Platelets play an important role in haemostasis. When a blood vessel cells (dendritic cells) enter via the lymphatics, bringing with them
is damaged, platelets become activated, evert their membrane invagina- antigen from peripheral infected sites. In all the secondary tissues there


tions to form lamellipodia and filopodia, and aggregate at the site of are specific areas where either B or T cells are concentrated. After activa-
injury, plugging the wound. They adhere to each other (agglutination) tion, functionally competent lymphocytes migrate to other sites in the
and to other tissues. Adhesion is a function of the thick platelet coat body, where they combat the original infection. Organized lymphoid
and is promoted by the release of adenosine diphosphate (ADP) and structures, termed tertiary lymphoid organs, can also develop at sites of
calcium ions from the platelets in response to vessel injury. The contents chronic inflammation (Stranford and Ruddle 2012).
of released α granules, together with factors released from the damaged
tissues, initiate a complex sequence of chemical reactions in the blood
plasma, which leads to the precipitation of insoluble fibrin filaments LYMPH NODES
in a three-dimensional meshwork, the fibrin clot (see Fig. 4.1). More
platelets attach to the clot, inserting extensions of their surfaces, filopo- Lymph nodes are encapsulated centres of antigen presentation and
dia, deep into the spaces between the fibrin filaments, to which they lymphocyte activation, differentiation and proliferation, which are
adhere strongly. The platelets then contract (clot retraction) by actin– facilitated by complex trafficking of cells and lymphatic flow through
myosin interactions within their cytoplasm, and this concentrates the the structure (Girard et al 2012). They generate mature, antigen-primed
fibrin clot and pulls the walls of the blood vessel together, which limits B and T cells, and filter particles, including microbes, from the lymph
any further leakage of blood. After repair of the vessel wall, which may by the action of numerous phagocytic macrophages. A normal young
be promoted by the mitogenic activity of PDGF, the clot is dissolved by adult body contains up to 450 lymph nodes, of which 60–70 are found
enzymes such as plasmin. Plasmin is formed by plasminogen activators in the head and neck, 100 in the thorax and as many as 250 in the
in the plasma, probably assisted by lysosomal enzymes derived from abdomen and pelvis. Lymph nodes are particularly numerous in the


the λ granules of platelets. Platelets typically circulate for 10 days before neck, mediastinum, posterior abdominal wall, abdominal mesenteries,
they are removed, mainly by splenic macrophages. pelvis and proximal regions of the limbs (axillary and inguinal lymph
nodes). By far the greatest number lie close to the viscera, especially in
the mesenteries.
LYMPHOID TISSUES
Microstructure
Lymphocytes are located in many sites in the body, most obviously at
strategic sites that are liable to infection, e.g. the oropharynx. The main
areas of lymphocyte concentration are classified as primary or second- Lymph nodes (Fig. 4.7) are small, oval or kidney-shaped bodies,
ary lymphoid organs, according to whether they are involved in de novo 0.1–2.5 cm long, lying along the course of the lymphatic vessels. Each
lymphocyte generation (primary lymphoid organs, e.g. bone marrow, usually has a slight indentation on one side: the hilum, through which
thymus) or are the site of mature lymphocyte activation and initiation blood vessels enter and leave, and the efferent lymphatic vessel leaves.
of an immune response (secondary lymphoid organs, e.g. lymph nodes, Several afferent lymphatic vessels enter the capsule around the periph-
spleen). ery. Lymph nodes have a highly cellular cortex and a medulla (Fig. 4.8),
The secondary or peripheral lymphoid organs are the specialized which contains a network of minute lymphatic channels (sinuses)
sites where B and T lymphocytes and antigen-presenting cells come through which lymph from the afferent lymphatics is filtered, to be
Fig. 4.7 The structure of a lymph node.
Trabeculae Capsule Germinal centre
of secondary
Afferent lymphatics lymphoid follicle
Subcapsular sinus
Cortex
Primary lymphoid follicle
Paracortex
Medullary sinus
Medulla
Hilum
Artery
Vein Efferent lymphatic vessel
High endothelial
venule (HEV)


Blood, lymphoid tissues and haemopoiesis
73.e1
4
RETPAHC
All lymphocytes arise from pluripotent haemopoietic stem cells in
the bone marrow. The B lymphocyte lineage develops through a series
of differentiation stages within the bone marrow. The newly formed B
cells then leave through the circulation and migrate to peripheral sites.
In contrast, T-lymphocyte development requires the thymus; the bone
marrow-derived stem cells must therefore migrate via the blood circula-
tion to the thymus. After their differentiation and maturation into
immunocompetent T cells that have survived thymic selection processes
(1–3%), they re-enter the circulation and are transported to peripheral
sites where they join the pool of naïve lymphocytes that recirculate
through the secondary lymphoid organs via blood and lymphatic cir-
culation systems.


BlOOd, lymPHOId TISSuES ANd HAEmOPOIESIS
74
1
NOITCES
F
C
F
M
G
F
G V
Fig. 4.9 A germinal centre in a lymphoid follicle of the human palatine
tonsil, immunolabelled to show CD38-positive B cells in the germinal
Fig. 4.8 A lymph node (human) showing cortex (C) and medulla (M), and centre (red), IgD-positive naïve B cells (green) in the mantle zone and
lymphoid follicles (F), some with germinal centres (G). Also shown are the activated, transferrin receptor (CD71)-positive cells of various lineages
subcapsular sinus (arrow) and medullary blood vessels (V). The dark line (blue). (Courtesy of Dr Cécile Chalouni, Ludwig Institute for Cancer
(top, centre) is a small crease in the tissue section and is an artefact. Research, Yale University School of Medicine, USA.)
(Courtesy of Mr Peter Helliwell and the late Dr Joseph Mathew,
Department of Histopathology, Royal Cornwall Hospitals Trust, UK.)
packed and in the outer cortical area they form lymphoid follicles or
collected at the hilum by the efferent lymphatic. The cortex is absent at nodules (see Fig. 4.8), which are populated mainly by B cells and spe-
the hilum, where the medulla reaches the surface. cialized follicular dendritic cells (FDCs) (see Fig. 4.15). The number,
The capsule is composed mainly of collagen fibres, elastin fibres degree of isolation and staining characteristics of follicles vary according
(especially in the deeper layers) and a few fibroblasts. Trabeculae of to their state of antigenic stimulation. A primary follicle is uniformly
dense connective tissue extend radially into the interior of the node populated by small, quiescent lymphocytes, whereas a secondary fol-
from the capsule. They are continuous with a network of fine type III licle has a germinal centre (Fig. 4.9), composed mainly of antigen-
collagen (reticulin) fibrils, which branch and interconnect to form a stimulated B cells, which are larger, less deeply staining and more


very dense network in the cortex, providing attachment for various cells, rapidly dividing than those at its periphery.
mostly dendritic cells, macrophages and lymphocytes. There are fewer The role of the germinal centre is to provide a microenvironment
fibres in the germinal centres of follicles (see below). Reticulin and the that allows the affinity maturation of the B-cell response, so that as the
associated proteoglycan matrix are produced by fibroblasts associated immune response progresses, the affinity or strength with which anti-
with the fibrous network. bodies bind their antigen increases (Shlomchik and Weisel 2012,
Victora and Nussenzweig 2012). There are several zones in the germinal
Lymphatic and vascular supply
centre where this is allowed to happen. In the ‘dark zone’, the B cells
(centroblasts) undergo rapid proliferation, which is associated with
Lymph nodes are permeated by channels through which lymph perco- hypermutation of their antibody molecules. They then move into the
lates after its entry from the afferent vessels. The conduit system consists ‘light zone’ (as centrocytes), where they can interact with the FDCs,
of collagen fibres and associated fibrils surrounded by fibroblast reticu- which carry intact unprocessed antigen on their surface in the form of
lar cells, forming a sponge-like reticulum that provides not only spaces immune complexes (Rezk et al 2013). The centrocytes compete for
for the lymphocytes but also a system for the transport of antigen and binding to the antigen; those whose antibody has the highest affinity
signalling molecules (such as chemokines) that control the highly survive and the rest die. T cells are also present, helping the survival of
dynamic movement and interaction of the immune cells. Dendritic cells the B cells and inducing class switching. Macrophages in the germinal
can reach inside the conduits to sample antigen, and then present it to centre phagocytose apoptotic lymphocytes (e.g. those B cells that die as
immune cells (Roozendaal et al 2008). part of the process of affinity maturation), and consequently macro-
Afferent lymphatic vessels enter at many points on the periphery, phage cytoplasm becomes filled with engulfed lipid and nuclear debris


branch to form a dense intracapsular plexus, and then open into the forming sparkling intracellular inclusions (leading to the term tingible
subcapsular sinus, a cavity that is peripheral to the whole cortex except body macrophage).
at the hilum (see Fig. 4.7). Numerous radial cortical sinuses lead from The mantle zone (see Fig. 4.9) is produced as surrounding cells are
the subcapsular sinus to the medulla, where they coalesce as larger marginalized by the rapidly growing germinal centre. It is populated by
medullary sinuses. The latter become confluent at the hilum with the cells similar to those found in primary follicles: mainly quiescent B cells
efferent vessel that drains the node. All of these spaces are lined by a with condensed heterochromatic nuclei and little cytoplasm (hence the
continuous endothelium and traversed by fine reticular fibres. deeply basophilic staining of this region in routine preparations; Fig.
Arteries and veins serving lymph nodes pass through the hilum, 4.10), a few helper T cells, FDCs and macrophages. After numerous
giving off straight branches that traverse the medulla and send out mitotic divisions the selected B cells give rise to small lymphocytes,
minor branches as they do so. In the cortex, arteries form dense arcades some of which become memory B cells and leave the lymph node to
of arterioles and capillaries in numerous anastomosing loops, eventu- join the recirculating pool, while others leave to mature as antibody-
ally returning to highly branched venules and veins. Capillaries are secreting plasma cells either in the lymph node medulla or in peripheral
especially profuse around the follicles, which contain fewer vessels. tissues.
Postcapillary HEVs are abundant in the paracortical zones. They form The deep cortex or paracortex lies between the cortical follicles and
an important site of blood-borne lymphocyte extravasation into lym- the medulla, and is populated mainly by T cells, which are not organ-
phoid tissue, apparently by migration through labile endothelial tight ized into follicles. Both CD4 and CD8 T-cell subsets are present. The
junctions (occluding junctions, zonulae adherentes). The density of the paracortex also contains interdigitating dendritic cells. These dendritic


capillary beds increases greatly when lymphocytes multiply in response cells include Langerhans cells from the skin and other squamous epi-
to antigenic stimulation. Veins leave a node through its principal trabec- thelia, which have migrated as veiled cells via the afferent lymphatics
ulae and capsule, and drain them and the surrounding connective into the draining lymph nodes (see Fig. 4.14). Their role is to present
tissue. processed antigen to T cells. The region expands greatly in T cell-
dominated immune responses, when its cells are stimulated to prolifer-
Cells and cellular zones of lymph nodes
ate and disperse to peripheral sites.
In the medulla, lymphocytes are much less densely packed in irregu-
Although most of the cells in a lymph node are B and T lymphocytes, lar, branching medullary cords between which the reticulin network
their distribution is not homogeneous. In the cortex, cells are densely is easily seen. Other cells include macrophages, which are more numer-


Haemopoiesis
75
4
RETPAHC
Fig. 4.10 A germinal
centre in a follicle of
mucosa-associated
lymphoid tissue (MALT)
in the mucosa and
submucosa of the
appendix. The bases of
tubular glands of the
mucosal epithelium are
seen in the upper field.
H
S
S
Fig. 4.11 Haemopoietic tissue (H) in the marrow cavity of a fetal long
bone undergoing endochondral ossification (top). Islands of densely
ous in the medulla than in the cortex, plasma cells and a few packed nucleated haemopoietic cells of different lineages are separated
granulocytes. by large vascular sinusoids (S), which are filled with mature red blood
cells in the general circulation.
MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)
tonsils they include modified stratified squamous reticulated epithelial
Large amounts of unencapsulated lymphoid tissue exist in the walls of cells (see p. 576). The main function of B lymphocytes in MALT is to
the gastrointestinal, respiratory, reproductive and urinary tracts, and in produce IgA for secretion into the lumen of the tracts that they line
the skin; they are collectively termed mucosa-associated lymphoid (Cerutti et al 2011).
tissue (MALT), most of which is found in the gut as gut-associated Many of the lymphocytes migrating between cells in the basal
lymphoid tissue (GALT) (Koboziev et al 2010). regions of epithelia (see Fig. 4.5) are effector cytotoxic and helper T cells
Throughout the body, MALT includes an extremely large population that have already been selected in lymphoid nodules and are engaged
of lymphocytes, reflecting the size of the gastrointestinal tract. Lym- in immune responses. Similar cells, and activated IgA-producing B cells
phoid cells are located in the lamina propria and in the submucosa as and plasma cells, are also scattered throughout the entire mucosal
discrete follicles or nodules. More scattered cells, derived from these lamina propria.
follicles, are found throughout the lamina propria and in the base of


the epithelium (see Figs 4.5, 4.10). MALT includes macroscopically
HAEMOPOIESIS
visible lymphoid masses, notably the peripharyngeal lymphoid ring of
tonsillar tissue (palatine, nasopharyngeal, tubal and lingual), and the
Peyer’s patches of the small intestine, all of which are described else- Postnatally, blood cells are formed primarily in the bone marrow. Other
where. Most MALT consists of microscopic aggregates of lymphoid tissues, particularly the spleen and liver, may develop haemopoietic
tissue, which lack a fibrous capsule. Lymphocyte populations are sup- activity once more, if production from the marrow is inadequate.
ported mechanically by a network of fine type III collagen (reticulin)
fibres and associated fibroblasts, as they are in lymph nodes.
BONE MARROW
In common with lymph nodes, MALT provides centres for the activa-
tion and proliferation of B and T lymphocytes in its follicles and para-
follicular zones, respectively. The function of cells in these zones, Bone marrow is a soft pulpy tissue that is found in the marrow cavities
including antigen-presenting cells (follicular dendritic cells and inter- of all bones (Fig. 4.11) and even in the larger Haversian canals of lamel-
digitating dendritic cells) and macrophages, as well as T and B cells, is lar bone. It differs in composition in different bones and at different
similar to that found in lymph nodes. The close proximity of lym- ages, and occurs in two forms: yellow and red marrow. In old age the
phocytes within MALT to an epithelial surface facilitates their access to marrow of the cranial bones undergoes degeneration and is then termed
pathogens. MALT lacks afferent lymphatic vessels. Lymphocytes migrate gelatinous marrow.
into MALT through its HEVs and leave mainly via its efferent lymphat-
Yellow marrow
ics, which drain interstitial fluid as lymph; the lymphocyte population
in MALT is not fixed. Migration from MALT follows a different route
from the major peripheral route of recirculation. After antigen activa- Yellow marrow consists of a framework of connective tissue that sup-


tion, lymphocytes travel via the regional lymph nodes to disperse ports numerous blood vessels and cells, most of which are adipocytes.
widely along mucosal surfaces to provide protective T- and B-cell A small population of typical red marrow cells persists and may be
immunity. reactivated when the demand for blood cells becomes sufficiently great.
Follicle-associated epithelium Red marrow
The epithelium covering MALT varies in type according to its location. Red marrow is found throughout the skeleton in the fetus and during
It is unusual in possessing cells that are involved in sampling antigens the first years of life. After about the fifth year the red marrow, which
and transferring them to antigen-presenting cells in the underlying represents actively haemopoietic tissue, is gradually replaced in the long
tissues; appropriate clones of T and B cells in local lymphoid tissues are bones by yellow marrow. The replacement starts earlier, and is generally
then activated and amplified prior to their exit via the lymphatics. more advanced, in the more distal bones. By 20–25 years of age, red
Specialized epithelial cells in the small and large intestine have charac- marrow persists only in the vertebrae, sternum, ribs, clavicles, scapulae,
teristic short microvilli on their luminal surfaces and are known as pelvis and cranial bones, and in the proximal ends of the femur and
microfold (M) cells (Kraehenbuhl and Neutra 2000); in the palatine humerus.


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Red bone marrow consists of a network of loose connective tissue, To generate a complete set of blood cells from a single pluripotent
the stroma, which supports clusters of haemopoietic cells (haemopoi- cell may take some months. The later progenitor cells form mature cells
etic cords or islands) and a rich vascular supply in which large, thin- of their particular lineages more quickly. However, because they are not
walled sinusoids are the main feature (see Fig. 4.11). The vascular self-renewing, grafts of these later cells eventually fail because the cells
supply is derived from the nutrient artery to the bone, which ramifies they produce all ultimately die. This is of considerable importance in
in the bone marrow, and terminates in thin-walled arterioles from bone marrow replacement therapy. The presence of pluripotent stem
which the sinusoids arise. These, in turn, drain into disproportionately cells in the donor marrow is essential for success; only 5% of the normal
large veins. Lymphatic vessels are absent from bone marrow. The stroma number are needed to repopulate the marrow. Following replacement
contains a variable amount of fat, depending on age, site and the hae- therapy, T lymphocytes reconstitute more slowly than the other haemo-
matological status of the body, and small patches of lymphoid tissue poietic lineages, reflecting the progressive reduction in size of the
are also present. Marrow thus consists of vascular and extravascular thymus with age (chronic involution).
compartments, both enclosed within a bony framework from which Within the bone marrow there is also a population of mesenchy-
they are separated by a thin layer of endosteal cells. mal stem cells that can differentiate into a wide variety of non-
haemopoietic cells. These pluripotent stem cells can also be found in
Stroma
the circulation, and are being investigated for their use in repairing
Stroma is composed of a delicate network of fine type III collagen damaged organs (see Commentary 1.2).
(reticulin) fibres secreted by highly branched, specialized fibroblast-like
cells (reticular cells) derived from embryonic mesenchyme. When hae- Lymphocytes


mopoiesis stops, as occurs in most limb bones in adult life, these cells
(or closely related cells) become distended with lipid droplets and fill
Lymphocytes are a heterogeneous group of cells that may share a
the marrow with yellow fatty tissue (yellow marrow). If there is a later
common ancestral lymphoid progenitor cell, distinct from the myeloid
demand for haemopoiesis, the stellate stromal cells reappear. The
progenitor cell (see Fig. 4.12). The first identifiable progenitor cell is
stroma also contains numerous macrophages attached to extracellular
the lymphoblast, which divides several times to form prolymphocytes;
matrix fibres. These cells actively phagocytose cellular debris created by
both cells are characterized by a high nuclear:cytoplasmic ratio. B cells
haemopoietic development, especially the extruded nuclei of erythro-
undergo differentiation to their specific lineage subset entirely within
blasts, remnants of megakaryocytes and cells that have failed the
the bone marrow and migrate to peripheral or secondary lymphoid
B-lymphocyte selection process. Stromal cells play a major role in
tissues as naïve B cells, ready to respond to antigen. However, T cells
the control of haemopoietic cell differentiation, proliferation and
require the specialized thymic microenvironment for their develop-
maturation.
ment. During fetal and early postnatal life, and subsequently at lower
Marrow sinusoids are lined by a single layer of endothelial cells,
levels throughout life, progenitor cells migrate to the thymus where they
supported by reticulin on their basal surfaces. Although the endothelial
undergo a process of differentiation and selection as T cells, before
cells are interconnected by tight junctions, their cytoplasm is extremely
leaving to populate secondary lymphoid tissues.
thin in places, and the underlying basal lamina is discontinuous. The
passage of newly formed blood cells from the haemopoietic compart- B-cell development
ment into the blood stream appears to occur through an interactive
B cells start their development in the subosteal region of the bone
process with the endothelium, producing temporary apertures (large
marrow and move centripetally as differentiation progresses. Their
fenestrae) in their attenuated cytoplasm.
development entails the rearrangement of immunoglobulin genes to


Haemopoietic tissue create a unique receptor for antigen on each B cell, and the progressive
expression of cell-surface and intracellular molecules required for
Cords and islands of haematogenous cells consist of clusters of
mature B-lymphocyte function. Autoreactive cells that meet their self-
immature blood cells in various stages of development; several differ-
antigen within the bone marrow are eliminated. Overall, some 25% of
ent cell lineages are typically represented in each focal group. One or
B cells successfully complete these developmental and selection proc-
more macrophages lie at the core of each such group of cells. These
esses; those that fail die by apoptosis and are removed by macrophages.
macrophages engage in phagocytic functions, are important in trans-
Bone marrow stromal cells (fibroblasts, fat cells and macrophages)
ferring iron to developing erythroblasts for haemoglobin synthesis,
express cell-surface molecules and secreted cytokines that control
and may play a role, with other stromal cells, in regulating the rate of
B-lymphocyte development. The mature naïve B lymphocytes leave via
cell proliferation and maturation of the neighbouring haemopoietic
the central sinuses. They express antigen receptors (immunoglobulin)
cells.
of IgM and IgD classes. Class switching to IgG, IgA and IgE occurs in
the periphery following antigen activation in response to signals from
T helper cells.
CELL LINEAGES
T-cell (thymocyte) development
Haemopoietic stem cells
T cells develop within the thymus from blood-borne, bone marrow-
derived progenitors that enter the thymus via HEVs at the corticomedul-
Within the adult marrow there is a very small number (0.05% of hae- lary junction. They first migrate to the outer (subcapsular) region of the
mopoietic cells) of self-renewing, pluripotent stem cells that are capable thymic cortex and then, as in the bone marrow, move progressively
of giving rise to all blood cell types, including lymphocytes (Fig. 4.12). inwards towards the medulla as development continues. T-cell develop-
Although they cannot be identified morphologically in the marrow, ment involves gene rearrangements in the T-cell receptor (TcR) loci to


they can be recognized in aspirates by the expression of specific cell- create unique receptors for antigen on each cell, together with the pro-
surface marker proteins (e.g. CD34). It is thought that haemopoietic gressive expression of molecules required for mature T-cell function.
stem cells occupy specific environmental niches in the marrow associ- Selection of the receptor repertoire is more stringent for T cells than for
ated with the endosteum of trabecular bone or with sinusoidal endothe- B cells because of the way in which mature T cells recognize peptides
lium, and that their microenvironment is important in homeostasis, derived from protein antigens presented in conjunction with specific
the balance between self-renewal and differentiation. Stem cells can molecules of the major histocompatibility complex (MHC) expressed
also be found (at lower concentrations) in the peripheral blood, par- on the surfaces of cells. Thus mature CD8 (cytotoxic) T cells recognize
ticularly after treatment with appropriate cytokines. antigen in the form of short peptides complexed with the polymorphic
Progressively more lineage-restricted committed progenitor cells MHC class I molecules, while CD4 (helper/regulatory) T cells recognize
develop from these ancestors (see Laiosa et al (2006) for a review) to the peptides in the context of MHC class II molecules. As the TcR
produce the various cell types found in peripheral blood. The commit- recognizes both the peptide and the MHC molecule, the T cell will only
ted progenitor cells are often termed colony-forming units (CFU) of the recognize peptides bound to their own (self) type of MHC; it will not
lineage(s), e.g. CFU-GM cells give rise, after proliferation, to neutrophil ‘see’ peptides in combination with allelically different MHC molecules
granulocytes, monocytes and certain dendritic cells, whereas CFU-E (i.e. those from other individuals). This is termed MHC restriction of
produce only erythrocytes. Each cell type undergoes a period of matura- T-cell recognition. Selection of T cells in the thymus must ensure the
tion in the marrow, often accompanied by several structural changes, survival of those T cells that can respond only to foreign antigens,


before release into the general circulation. In some lineages, e.g. the bound to their own (self) class of MHC molecule. Cells that are incap-
erythroid series, the final stages of maturation take place in the circula- able of binding to self MHC molecules, or which bind to self-antigens,
tion, whereas in the monocytic lineage, they occur after the cells have are eliminated by apoptotic cell death (see p. 26); it is estimated that
left the circulation and entered peripheral tissues where they differenti- up to 95% of T-cell progenitors undergo apoptosis in this way. Cells
ate into macrophages and some dendritic cells. that express an appropriate TcR and have effective MHC-restricted


Haemopoiesis
77
4
RETPAHC
Neutrophil Megakaryocyte Platelet
CFU-GM CFU-Mk BFU-E
CFU-E
Eosinophil
CFU-GEMM
Myeloblast CFU-G
Common myeloid
progenitor
Basophil
Erythroblast
Monoblast CFU-M Haemopoietic
stem cell
Mast cell
Common lymphoid
progenitor
Monocyte Erythrocyte
NK cell T cell B cell
Osteoclast Macrophage Dendritic cell Lymphocyte Plasma cell
Fig. 4.12 The origins and lineage relationships of haemopoietically derived cells of the immune system. Mature cells and selected progenitors (all human)
are illustrated (magnifications vary). The dendritic cell was cultured from peripheral blood, immunolabelled to show HLA-DR and photographed using
Nomarski optics. The megakaryocyte and erythroblast are from a bone marrow smear, stained with May–Grünwald–Giemsa (MGG); the remaining cells
illustrated are from peripheral blood smears (Wright’s stain), sections of connective tissue (plasma cell, mast cell), bone (osteoclast) and lung alveolus
(macrophage). Platelets (one is arrowed) are subcellular fragments of bone marrow megakaryocytes. Note that circulating small lymphocytes cannot be
classified further with routine staining methods. For further explanation of cellular structure and staining properties, along with abbreviations, see the
text. (Dendritic cell image courtesy of Dr Cécile Chalouni, Ludwig Institute for Cancer Research, Yale University School of Medicine, USA. All other
images courtesy of Mr Peter Helliwell and the late Dr Joseph Mathew, Department of Histopathology, Royal Cornwall Hospitals Trust, UK.)


BlOOd, lymPHOId TISSuES ANd HAEmOPOIESIS
78
1
NOITCES
binding properties survive to become mature, naïve T cells that leave colony-forming unit for granulocytes and macrophages (CFU-GM). Dif-
the thymus and populate the periphery. ferent colony-stimulating factors (CSF) act on the common progenitor
Thymic stromal cells play a crucial role in T-cell development and to direct its subsequent differentiation pathway. Monocyte progenitors
selection. Thymic epithelial cells in the cortex express both MHC class pass through a proliferative monoblast stage (14 µm) and then form
I and II molecules and are unique in their ability to select T cells that differentiating promonocytes, which are slightly smaller cells in which
recognize self MHC with a moderate affinity (positive selection). Dele- production of small lysosomes begins. After further divisions, mono-
tion of self-antigen reactive cells (negative selection) is mainly control- cytes (up to 20 µm) are released into the general circulation. Most
led by thymic dendritic cells located at the corticomedullary junction migrate into perivascular and extravascular sites, which they then popu-
and in the medulla, although the epithelium can also perform this late as macrophages, while others may give rise to certain dendritic cells,
function. Apoptotic thymocytes are removed by thymic macrophages. including Langerhans cells.
The role of the thymic epithelium in thymocyte differentiation is
complex and involves cell–cell contact as well as the secretion of soluble Platelets
mediators such as cytokines, chemokines, neuroactive peptides (e.g.
somatostatin) and thymic hormones (e.g. thymulin). Thymic fibro- Platelets arise in a unique manner by the shedding of thousands of
blasts and the extracellular matrix also play a role. cytoplasmic fragments from the tips of processes of megakaryocytes in
the bone marrow. The first detectable cell of this line is the highly
Erythrocytes basophilic megakaryoblast (15–50 µm), followed by a promegakaryo-
cyte stage (20–80 µm), in which synthesis of granules begins. Finally,


Erythrocytes and granulocytes belong to the myeloid lineage. The earli- the fully differentiated megakaryocyte, a giant cell (35–160 µm) with a
est identifiable erythroid progenitor cells are capable of rapid bursts of large, dense, polyploid, multilobed nucleus, appears. Once differentia-
cell division to form numerous daughter cells; they have thus been tion is initiated from the CFU-Meg, DNA replicates without cytoplasmic
named burst-forming units of the erythroid line (BFU-E; see Fig. 4.12). division (endoreduplication), and the chromosomes are retained
They give rise to the CFU-E, which, with their immediate progeny, are within a single polyploid nucleus that may contain up to 256n chro-
sensitive to the hormone erythropoietin. This hormone, produced in mosomes (where n is the haploid complement present in gametes).
the kidney, induces further differentiation along the erythroid line. Megakaryocyte lineage characteristics and disorders are reviewed in Sun
The first readily identifiable cell of the erythroid series is the pro- et al (2006).
erythroblast, which is a large (about 20 µm) cell with a large euchro- The cytoplasm contains fine basophilic granules and becomes par-
matic nucleus and a moderately basophilic cytoplasm. It also responds titioned into proplatelets by invaginations of the plasma membrane.
to erythropoietin. The proerythroblast contains small amounts of fer- These are seen ultrastructurally as a network of tubular profiles, which
ritin and bears some of the protein spectrin on its plasma membrane. coalesce to form cytoplasmic islands 3–4 µm in diameter. Individual
Proerythroblasts proliferate to produce smaller (12–16 µm) basophilic platelets are shed into the circulation from a long, narrow process of
erythroblasts, rich in ribosomes, in which haemoglobin-RNA synthesis megakaryocyte cytoplasm that is protruded through an aperture in the
begins. The cytoplasm becomes partially, and then uniformly, eosi- sinusoidal endothelium.
nophilic (the polychromatic erythroblast and orthochromatic erythro-


blast respectively). These cells are only 8–10 µm in diameter and
PHAGOCYTES AND ANTIGEN-PRESENTING CELLS
contain very little cytoplasmic RNA. The nucleus becomes pyknotic
(dense, deep-staining, shrunken) and is finally extruded from the cell,
leaving an anucleate reticulocyte, which enters a sinusoid. Its reticular Macrophages and neutrophils (see above) are specialized phagocytes.
staining pattern, visible using special stains, results from residual cyto- Certain dendritic cells (see Fig. 4.12), e.g. Langerhans cells of the skin
plasmic RNA, which is usually lost within 24 hours of entering the and other stratified squamous epithelia, are ‘professional’ antigen-
peripheral blood circulation. Reticulocyte numbers in peripheral blood presenting cells (APCs); they take up foreign material by endocytosis
are therefore a good indicator of the rate of red-cell production. The and macropinocytosis, and are uniquely capable of efficiently activating
whole process of erythropoiesis takes 5–9 days. naïve as well as mature T lymphocytes. Macrophages are also able to
process and present antigen to lymphocytes, but are less effective than
Granulocytes dendritic cells. In addition, they play an important role in the effector
arm of the immune response, clearing the infectious agent by phagocy-
tosis. The third major cell type involved in antigen presentation and
Granulocyte formation involves major changes in nuclear morphology
T-cell activation is the B lymphocyte, which is particularly efficient at
and cytoplasmic contents, which are best known for the neutrophil.
taking up antigen that binds to its surface immunoglobulin (see above).
Initially, myeloid progenitor cells transform into large (10–20 µm)
Follicular dendritic cells of lymph nodes, MALT and the spleen are
myeloblasts that are similar in general size and appearance to proeryth-
capable of presenting non-processed antigen to B lymphocytes, but are
roblasts. These proliferative cells have large euchromatic nuclei and lack
not classic APCs because they cannot present antigen to helper T cells.
cytoplasmic granules. They differentiate into slightly larger promyelo-


APCs endocytose antigen, digest it intracellularly, mostly to peptide
cytes, in which the first group of specific proteins is synthesized in the
fragments, and present the fragments on their surfaces, generally in
rough endoplasmic reticulum and Golgi apparatus. The proteins are
conjunction with MHC class II molecules. (Class II molecules are nor-
stored in large (0.3 µm) primary (non-specific) granules, which are
mally found only on APCs, although many other cells can express class
large lysosomes containing acid phosphatase. Smaller secondary (spe-
II molecules in inflammatory situations.) Recognition of foreign antigen
cific) granules are formed in the smaller myelocyte, which is the last
is controlled by a variety of APC cell-surface receptors: Fc and comple-
proliferative stage. The nucleus is typically flattened or slightly indented
ment receptors mediate uptake of opsonized material, while pattern
on one side in myelocytes.
recognition receptors of the innate immune system, e.g. Toll-like recep-
In the next, metamyelocyte, stage, the cell size (10–15 µm) decreases,
tors and scavenger receptors, directly recognize pathogen-derived
the nucleus becomes heterochromatic and horseshoe-shaped, and
molecules.
protein synthesis almost stops. As the neutrophil is released, the nucleus
becomes first heavily indented (the juvenile stab or band form), and
subsequently segmented into up to six lobes, characteristic of the MACROPHAGES
mature neutrophil. The whole process usually takes 7 days to complete
(3 days proliferating and 4 days maturing). Neutrophils may then be
The mononuclear phagocyte system consists of the blood monocytes,
stored in the marrow for a further 4 days, depending on demand, before
from which the other types are derived, and various tissue macrophages,
their final release into the circulation.
some of which have tissue-specific names. Certain dendritic cells are
Eosinophils and basophils pass through a similar sequence but their
sometimes included in the mononuclear phagocyte system; although
nuclei do not become as irregular as that of the neutrophil. It is thought
they share a common lineage ancestor, they appear to form a discrete
that these cells each arise from distinct colony-forming units, which are


branch of the family tree. Most monocytes and macrophages express
separate from the CFU-GM.
class II MHC molecules.
Macrophages are very variable in size (generally 15–25 µm) and are
Monocytes
found in many tissues of the body, where they constitute a heterogene-
ous family of cells (reviewed in Gordon and Taylor (2005)). They
Monocytes are formed in the bone marrow. Monocytes and neutrophils are migrant cells in all general connective tissues, in bone marrow and
appear to be closely related cells; together with some of the antigen- all lymphoid tissues, and include alveolar macrophages in the lung
presenting dendritic cells, they arise from a shared progenitor, the and Kupffer cells in liver sinusoids. Macrophages often aggregate in


Phagocytes and antigen-presenting cells
79
4
RETPAHC
subserous connective tissue of the pleura and peritoneum, where they activity is greatly increased when the target has been coated in antibody
are visible as milky spots near small lymphatic trunks. They cluster (opsonized) or complement, or both. Once phagocytosis has occurred,
around the terminations of small (penicillar) arterioles in the spleen the vacuole bearing the ingested particle fuses with endosomal vesicles,
and are distributed, more diffusely, throughout the splenic cords. which contain a wide range of lysosomal enzymes, including many
Osteoclasts in bone are closely related to macrophages; they are hydrolases, and oxidative systems capable of rapid bacteriocidal action.
syncytial cells derived from the fusion of up to 30 progenitor monocytes These activities are much enhanced when macrophages are stimulated
in bone tissue, where they differentiate further. Microglia of the central (activated macrophages) by cytokines, e.g. IFN-γ, which are secreted by
nervous system originate from an embryonic monocyte precursor and other cells of the immune system, especially T lymphocytes.
migrate into the central nervous system during its development. They Close antibody-mediated binding may initiate the release of lyso-
differ from macrophages in that normally they are quiescent cells in somal enzymes on to the surfaces of the cellular targets to which the
which MHC class II expression is downregulated, and they display little macrophages bind. This process of cytotoxicity is also used by other
phagocytic activity. cells, including neutrophils and eosinophils, particularly if the targets
Macrophages vary in structure depending on their location in the are too large to be phagocytosed (e.g. nematode worm parasites).
body. All have a moderately basophilic cytoplasm containing some
rough and smooth endoplasmic reticulum, an active Golgi complex and Secretory activities
a large, euchromatic and somewhat irregular nucleus. These features are
consistent with an active metabolism: synthesis of lysosomal enzymes
Activated macrophages can synthesize and secrete various bioactive
continues in mature cells. All macrophages have irregular surfaces with
substances, e.g. IL-1, which stimulate the proliferation and maturation


protruding filopodia and they contain varying numbers of endocytic
of other lymphocytes, greatly amplifying the reaction of the immune
vesicles, larger vacuoles and lysosomes. Some macrophages are highly
system to foreign antigens. They also synthesize TNF-α, which is able
motile, whereas others tend to remain attached and sedentary, e.g. in
to kill small numbers of neoplastic cells. TNF-α depresses the anabolic
hepatic and lymphoid sinuses. Within connective tissues, macrophages
activities of many cells in the body, and may be a major factor mediat-
may fuse to form large syncytia (giant cells) around particles that are
ing cachexia (wasting), which typically accompanies more advanced
too large to be phagocytosed, or when stimulated by the presence of
cancers. Other macrophage products include plasminogen activator,
infectious organisms, e.g. Mycobacterium tuberculosis.
which promotes clot removal; various lysosomal enzymes; several com-
When blood-borne monocytes enter the tissues through the endothe-
plement and clotting factors; and lysozyme (an antibacterial protein).
lial walls of capillaries and venules, they can undergo a limited number
In pathogenesis, these substances may be released inappropriately and
of rounds of mitosis as tissue macrophages before they die and are
damage healthy tissues, e.g. in rheumatoid arthritis and various other
replaced from the bone marrow, typically after several weeks. There is
inflammatory conditions.
some evidence that alveolar macrophages of the lung are able to
undergo many more mitoses than other macrophages.
DENDRITIC CELLS
Phagocytosis
There are two distinct groups of dendritic cell: myeloid dendritic cells
The uptake of particulate material and microorganisms is carried out (also known as classic dendritic cells) and plasmacytoid dendritic cells
by macrophages in many tissues and organs. When present in general (Liu 2001, Merad et al 2013, Gerlach et al 2013). These cells can arise
connective tissue, they ingest and kill invading microorganisms and from both common lymphoid progenitors and common myeloid pro-
remove debris that has been produced as a consequence of tissue genitors. Both groups of cells are involved in antigen presentation,


damage. They recognize, engulf and rapidly ingest apoptotic cells in all though have somewhat different functional roles in controlling both
situations; the mechanism of apoptotic cell uptake does not activate the the adaptive and innate immune systems. The myeloid dendritic cells
phagocyte for antigen presentation, and so the process is immunologi- are professional APCs, which are able to process and present antigen to
cally silent. In the lung, alveolar macrophages constantly patrol the T lymphocytes, including naïve T cells. They are present as immature
respiratory surfaces, to which they migrate from pulmonary connective dendritic cells in the epidermis of the skin (Fig. 4.14) and other
tissue (Fig. 4.13). They engulf inhaled particles, including bacteria,
surfactant and debris, and many enter the sputum (hence their alterna- Langerhans Precursor Blood
tive names, dust cells or, in cardiac disease, heart failure cells, which are cell cell vessel
full of extravasated erythrocytes). They perform similar scavenger func-
tions in the pleural and peritoneal cavities. In lymph nodes, macro- Cornified layer
phages line the walls of sinuses and remove particulate matter from
lymph as it percolates through them. In the spleen and liver, macro-
phages are involved in particle removal and in the detection and
Prickle cell layer Epidermis
destruction of aged or damaged erythrocytes. They begin the degrada-
tion of haemoglobin for recycling iron and amino acids.
Macrophages bear surface receptors for the Fc portions of antibodies
and for the fragments of the C3 component of complement. Phagocytic Basal layer
Migrating veiled cell
Afferent lymphatic Dermis
Subcapsular sinus
Capsule
Lymphoid follicle
in cortex
Interdigitating dendritic Lymph node
cell in paracortex
Fig. 4.14 Dendritic cells in the skin and lymphoid tissues. Their migratory
routes are shown: from blood-borne, marrow-derived precursors to
Fig. 4.13 Alveolar macrophages (dust cells, arrows) containing ingested immature dendritic cells (Langerhans cells) in skin, and then to migrating


carbon particles, in alveoli and interalveolar septa of the human lung. veiled cells in afferent lymphatic vessels and interdigitating dendritic cells
(Courtesy of Mr Peter Helliwell and the late Dr Joseph Mathew, in lymph nodes. An example of each cell in the sequence (arrowed) is
Department of Histopathology, Royal Cornwall Hospitals Trust, UK.) shown in red.


BlOOd, lymPHOId TISSuES ANd HAEmOPOIESIS
80
1
NOITCES
stratified squamous epithelia, e.g. the oral mucosa (Langerhans cells),
and in the dermis and most other tissues (interstitial dendritic cells),
where they are concerned with immune surveillance. Immature den-
dritic cells have an antigen-capturing function. They respond to chemo-
tactic signals, e.g. defensins released by epithelial cells in the small
intestine, and they express pattern recognition receptors (e.g. Toll-like
receptors) on their surface. Binding of pathogen-associated molecular
pattern molecules (PAMPs) derived from bacteria (e.g. carbohydrate,
lipopolysaccharide or DNA) to these receptors stimulates the dendritic
cells to become activated and migrate via the lymphatics to nearby
secondary lymphoid tissues, where they can present antigen to T cells.
They can also be activated by recognition of damage-associated pattern
molecules (DAMPs), such as ATP, DNA, heat-shock proteins and high-
mobility group box 1 (HMGB1) released from injured or necrotic cells.
Mature dendritic cells are known as veiled cells when in the afferent
lymphatics and the subcapsular sinuses of lymph nodes, and as inter-
digitating dendritic cells once they are within the lymphoid tissue
proper. Their function within the secondary lymphoid tissue is to
present their processed antigen to T lymphocytes, and thus to initiate
Fig. 4.15 Follicular dendritic cells (brown) in a germinal centre of the
and stimulate the immune response. For a review of research on den-
human palatine tonsil (immunoperoxidase-labelled). (Courtesy of Marta
dritic cell function, see Colonna et al (2006). Perry MD, UMDS, London.)
Langerhans cells
respond to antigen presented by dendritic cells. The T cells are stimu-
Langerhans cells (see Fig. 4.14) are one of the best-studied types of lated not only by recognition of the antigen–MHC complex by the TcR,
immature dendritic cell (reviewed in Berger et al (2006), Chopin and but also by interaction with co-stimulatory molecules expressed by the