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CELL LINEAGES

Haemopoietic stem cells

Within the adult marrow there is a very small number (0.05% of haemopoietic cells) of self-renewing, pluripotent stem cells which are capable of giving rise to all blood cell types, including lymphocytes (Fig. 4.12). Although they cannot be identified morphologically in the marrow, they can be recognized in aspirates by the expression of specific cell surface marker proteins (e.g. CD34). It is thought that haemopoietic stem cells occupy specific environmental niches in the marrow associated with the endosteum of trabecular bone or with sinusoidal endothelium and that their microenvironment is important in homeostasis, the balance between self-renewal and differentiation. Stem cells can also be found (at lower concentrations) in the peripheral blood, particularly after treatment with appropriate cytokines.

Progressively more lineage-restricted committed progenitor cells develop from these ancestors (see Laiosa et al 2006 for a recent review) to produce the various cell types found in peripheral blood. The committed progenitor cells are often termed colony-forming units (CFU) of the lineage(s), e.g. CFU-GM cells give rise, after proliferation, to neutrophil granulocytes, monocytes and certain dendritic cells, whereas CFU-E produce only erythrocytes. Each cell type undergoes a period of maturation in the marrow, often accompanied by several structural changes, before release into the general circulation. In some lineages, e.g. the erythroid series, the final stages of maturation take place in the circulation, whereas in the monocytic lineage, they occur after the cells have left the circulation and entered peripheral tissues where they differentiate into macrophages and some dendritic cells.

To generate a complete set of blood cells from a single pluripotent cell may take some months. The later progenitor cells form mature cells of their particular lineages more quickly. However, because they are not self-renewing, grafts of these later cells eventually fail because the cells they produce all ultimately die. This is of considerable importance in bone marrow replacement therapy. The presence of pluripotent stem cells in the donor marrow is essential for success: only 5% of the normal number are needed to repopulate the marrow. Following replacement therapy, T lymphocytes reconstitute more slowly than the other haemopoietic lineages, reflecting the progressive reduction in size of the thymus with age (chronic involution).

Lymphocytes

Lymphocytes are a heterogeneous group of cells which may share a common ancestral lymphoid progenitor cell, distinct from the myeloid progenitor cell which gives rise to all of the cell types described above. The first identifiable progenitor cell is the lymphoblast, which divides several times to form prolymphocytes: both cells are characterized by a high nuclear to cytoplasmic ratio. B cells undergo differentiation to their specific lineage subset entirely within the bone marrow and migrate to peripheral or secondary lymphoid tissues as naïve B cells, ready to respond to antigen. However, T cells require the specialized thymic microenvironment for their development. During fetal and early postnatal life, and subsequently at lower levels throughout life, progenitor cells migrate to the thymus where they undergo a process of differentiation and selection as T cells, before leaving to populate secondary lymphoid tissues.

B cell development

B cells start their development in the subosteal region of the bone marrow, and move centripetally as differentiation progresses. Their development entails the rearrangement of immunoglobulin genes to create a unique receptor for antigen on each B cell, and the progressive expression of cell surface and intracellular molecules required for mature B lymphocyte function. Autoreactive cells which meet their self-antigen within the bone marrow are eliminated. Overall some 25% of B cells successfully complete these developmental and selection processes: those that fail die by apoptosis and are removed by macrophages. Bone marrow stromal cells (fibroblasts, fat cells and macrophages) express cell surface molecules and secreted cytokines which control B lymphocyte development. The mature naïve B lymphocytes leave via the central sinuses. They express antigen receptors (immunoglobulin) of IgM and IgD classes. Class switching to IgG, A and E occurs in the periphery following antigen activation in response to signals from T helper cells.

T-cell (thymocyte) development

T cells develop within the thymus from blood-borne bone marrow-derived progenitors which enter the thymus via HEVs at the corticomedullary junction. They first migrate to the outer (subcapsular) region of the thymic cortex and then, as in the bone marrow, move progressively inwards towards the medulla as development continues. T-cell development involves gene rearrangements in the T-cell receptor (TcR) loci to create unique receptors for antigen on each cell, together with the progressive expression of molecules required for mature T-cell function. Selection of the receptor repertoire is more stringent for T cells than for B cells because of the way in which mature T cells recognize cell-bound antigens presented in conjunction with specific proteins of the major histocompatibility complex (MHC) expressed on the surfaces of cells. Thus mature CD8 (cytotoxic) T cells recognize antigen in the form of short peptides complexed with the polymorphic MHC class I molecules, while CD4 (helper/regulatory) T cells recognize the peptides in the context of MHC class II molecules. Because the TcR recognizes both the peptide and the MHC molecule, the T cell will only recognize peptides bound to their own (self) type of MHC: they will not ‘see’ peptides in combination with allelically different MHC molecules (i.e. those from other individuals). This is termed MHC restriction of T-cell recognition. Selection of T cells in the thymus must ensure the survival of those T cells which can respond only to foreign antigens, bound to their own (self) class of MHC molecule. Cells which are incapable of binding to self MHC molecules, or which bind to self-antigens, are eliminated by apoptotic cell death: it is estimated that up to 95% of T cell progenitors undergo apoptosis in this way. Cells which express an appropriate TcR and have effective MHC-restricted binding properties survive to become mature, naïve T cells which leave the thymus and populate the periphery.

Thymic stromal cells play a crucial role in T-cell development and selection. Thymic epithelial cells in the cortex express both MHC class I and II molecules and are unique in their ability to select T cells which recognize self MHC (positive selection). Deletion of self-antigen reactive cells (negative selection) is mainly controlled by thymic dendritic cells located at the corticomedullary junction and in the medulla, although the epithelium can also perform this function. Apoptotic thymocytes are removed by thymic macrophages. The role of the thymic epithelium in thymocyte differentiation is complex and involves cell–cell contact as well as the secretion of soluble mediators such as cytokines, chemokines, neuroactive peptides (e.g. somatostatin) and thymic hormones (e.g. thymulin). Thymic fibroblasts and the extracellular matrix also play a role.

Erythrocytes

Erythrocytes and granulocytes belong to the myeloid lineage. The earliest identifiable erythroid progenitor cells are capable of rapid bursts of cell division to form numerous daughter cells; they have thus been named burst-forming units of the erythroid line (BFU-E). They give rise to the CFU-E, which, with their immediate progeny, are sensitive to the hormone erythropoietin. This hormone, produced in the kidney, induces further differentiation along the erythroid line.

The first readily identifiable cell of the erythroid series is the proerythroblast, which is a large (about 20 μm) cell with a large euchromatic nucleus and a moderately basophilic cytoplasm. It also responds to erythropoietin. The proerythroblast contains small amounts of ferritin and bears some of the protein spectrin on its plasma membrane. Proerythroblasts proliferate to produce smaller (12–16 μm) basophilic erythroblasts, rich in ribosomes, in which haemoglobin-RNA synthesis begins. The cytoplasm becomes partially, and then uniformly, eosinophilic (the polychromatic erythroblast and orthochromatic erythroblast respectively). These cells are only 8–10 μm in diameter and 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 staining pattern, visible using special stains, results from residual cytoplasmic RNA which is usually lost within 24 hours of entering the peripheral blood circulation. Reticulocyte numbers in peripheral blood are therefore a good indicator of the rate of red cell production. The whole process of erythropoiesis takes 5–9 days.

Granulocytes

Granulocyte formation involves major changes in nuclear morphology and cytoplasmic contents which are best known for the neutrophil. Initially, myeloid progenitor cells transform into large (10–20 μm) myeloblasts which are similar in general size and appearance to proerythroblasts. These proliferative cells have large euchromatic nuclei and lack cytoplasmic granules. They differentiate into slightly larger promyelocytes, in which the first group of specific proteins is synthesized in the rough endoplasmic reticulum and Golgi apparatus. The proteins are stored in large (0.3 μm) primary (non-specific) granules, which are large lysosomes containing acid phosphatase. Smaller secondary (specific) granules are formed in the smaller myelocyte, which is the last proliferative stage. The nucleus is typically flattened or slightly indented on one side in myelocytes.

In the next, metamyelocyte, stage, the cell size (10–15 μm) decreases, the nucleus becomes heterochromatic and horse-shoe shaped, and 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 mature neutrophil. The whole process usually takes 7 days to complete, of which 3 days are spent proliferating, and 4 days maturing. Neutrophils may then be stored in the marrow for a further 4 days, depending on demand, before their final release into the circulation.

Eosinophils and basophils pass through a similar sequence but their nuclei do not become as irregular as that of the neutrophil. It is thought that these cells each arise from distinct colony-forming units, which are separate from the CFU-GM.

Monocytes

Monocytes are formed in the bone marrow. Monocytes and neutrophils appear to be closely related cells: together with some of the antigen-presenting dendritic cells, they arise from a shared progenitor, the colony-forming unit for granulocytes and macrophages (CFU-GM). Different colony-stimulating factors (CSF) act on the common progenitor to direct its subsequent differentiation pathway. Monocyte progenitors pass through a proliferative monoblast stage (14 μm) and then form differentiating promonocytes, which are slightly smaller cells in which production of small lysosomes begins. After further divisions, monocytes (up to 20 μm) are released into the general circulation. Most migrate into perivascular and extravascular sites, which they then populate as macrophages, while others may give rise to certain dendritic cells, including Langerhans cells.

Platelets

Platelets arise in a unique manner by the shedding of thousands of cytoplasmic fragments from the tips of processes of megakaryocytes in the bone marrow. The first detectable cell of this line is the highly basophilic megakaryoblast (15–50 μm), followed by a promegakaryocyte stage (20–80 μm), in which synthesis of granules begins. Finally, the fully differentiated megakaryocyte, a giant cell (35–160 μm) with a large, dense, polyploid, multilobed nucleus, appears. Once differentiation is initiated from the CFU-Meg, DNA replicates without cytoplasmic division (endoreduplication), and the chromosomes are retained within a single polyploid nucleus which may contain up to 256n chromosomes (where n is the haploid complement present in gametes). Megakaryocyte lineage characteristics and disorders are reviewed in Sun et al (2006).

The cytoplasm contains fine basophilic granules and becomes partitioned into proplatelets by invaginations of the plasma membrane. These are seen ultrastructurally as a network of tubular profiles which coalesce to form cytoplasmic islands 3–4 μm in diameter. Individual platelets are shed into the circulation from a long, narrow process of megakaryocyte cytoplasm which is protruded through an aperture in the sinusoidal endothelium.

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