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Leukocytes, their range and function

Leukocytes (white blood cells) encompass all blood cells that contain a nucleus, and these cells basically constitute the cells of the immune system. They thus function to protect the body by inactivating and destroying foreign agents. Certain leukocytes are also capable of recognizing and destroying altered body cells, such as cancer cells. Most are not confined exclusively to blood, but can circulate/exchange between blood, lymph and body tissues. This renders them more functionally effective by facilitating migration and congregation at a site of infection.

Leukocytes have been subclassified into three families: mononuclear phagocytes, lymphocytes and granulocytes. These can be differentiated from each other on the basis of their interaction with a dye known as Romanowsky stain (Figure 8.B1).

Mononuclear phagocytes consist of monocytes and macrophages, and execute their defence function primarily by phagocytosis. Like all leukocytes, they are ultimately derived from bone marrow stem cells. Some such stem cells differentiate into monocytes, which enter the bloodstream from the bone marrow. From there, they migrate into most tissues in the body, where they settle and differentiate (mature) to become macrophages (sometimes called histocytes). Macrophages are found in all organs and connective tissue. They are given different names, depending upon in which organ they are located (hepatic macrophages are called Kupffer cells, central nervous system macrophages are called microglia, and lung macrophages are termed alveolar macrophages). All macrophages are effective scavenger cells, engulfing and destroying (by phagocytosis) any foreign substances they encounter. They also play an important role in other aspects of immunity by producing cytokines, and acting as antigen-presenting cells.

Lymphocytes are responsible for the specificity of the immune response. They are the only immune cells that recognize and respond to specific antigens, due to the presence on

Figure 8.B1 The range of white blood cell types

their surface of high-affinity receptors. In addition to blood, lymphocytes are present in high numbers in the spleen and thymus. They may be subcategorized into antibody-producing B-lymphocytes, T-lymphocytes (which are involved in cell-mediated immunity) and null cells.

T-lymphocytes may be subcategorized on a functional basis into T-helper, T-cytoxic and T-suppressor cells. T-helper cells can produce various cytokines which can stimulate and regulate the immune response. T-cytotoxic cells can induce the lysis of cells exhibiting foreign antigen on their surface. As such, their major target cells are body cells infected by viruses or other intracellular pathogens (e.g. some protozoa). T-suppressor cells function to dampen or suppress an activated immune response, thus functioning as an important 'off' switch.

Most T-helper cells express a membrane protein termed CD4 on their surface. Most T-cyto-toxic and T-suppressor cells produce a different cell surface protein, termed CD8. Monoclonal antibodies specifically recognizing CD4 or CD8 proteins can thus be used to differentiate between some T cell types.

Null cells are also known as 'large granular lymphocytes', but are best known as 'natural killer' (NK) cells. These represent a third lymphocyte subgroup. They are capable of directly lysing cancer cells and virally infected cells.

The third leukocyte cell type is termed granulocytes, due to the presence of large granules in their cytoplasm. Granulocytes, many of which can be activated by cytokines, play a direct role in immunity, and also in inflammation. Granulocytes can be subdivided into three cell types of which neutrophils (also known as polymorphonuclear leukocytes; PMN leukocytes) are the most abundant. Attracted to the site of infection, they mediate acute inflammation and phagocytose opsonized antigen efficiently due to the presence of an IgG Fc receptor on their surface. Eosinophils display a cell surface IgE receptor and, thus, seem to specialize in destroying foreign substances that specifically elicit an IgE response (e.g. parasitic worms). These cells also play a direct role in allergic reactions. Basophils also express IgE receptors. Binding of antigen-IgE complex prompts these cells to secrete their granule contents, which mediate hypersensitivity reactions.

CH8 THE CYTOKINES: THE INTERFERON FAMILY Table 8.2 Cytokines, as grouped on a structural basis

Cytokine family

Members

'P-Trefoil' cytokines

FGFs IL-1

Chemokines

IL-8

TNF family

Haematopoietins

EGF family

'Cysteine knot' cytokines

MlPs

TGFs

PDGF

TGF-a

G-CSF

GM-CSF

CNTF

As a consequence of the various approaches adopted in naming and classifying cytokines, it is hardly surprising to note that many are known by more that one name. IL-1, for example, is also known as lymphocyte activating factor (LAF), endogenous pyrogen, leukocyte endogenous mediator, catabolin and mononuclear cell factor. This has led to even further confusion in this field.

During the 1980s, rapid developments in the areas of recombinant DNA technology and monoclonal antibody technology contributed to a greater depth of understanding of cytokine biology:

• Genetic engineering allowed production of large quantities of most cytokines. These could be used for structural and functional studies of the cytokine itself, and its receptor.

• Analysis of cytokine genes established the exact evolutionary relationships between these molecules.

• Detection of cytokine mRNA and cytokine receptor mRNA allowed identification of the full range of sources and target cells of individual cytokines.

• Hybridoma technology (Chapter 13) facilitated development of immunoassays capable of detecting and quantifying cytokines.

• Inhibition of cytokine activity in vivo by administration of monoclonal antibodies (and, more recently, by gene knockout studies) continues to elucidate the physiological and pathophysi-ological effect of various cytokines.

The cytokine family continues to grow, and often a decision to include a regulatory protein in this category is not a straightforward one. The following generalizations may be made with regard to most cytokines:

• They are very potent regulatory molecules, inducing their characteristic effects at nanomolar to picomolar concentrations.

• Most cytokines are produced by a variety of cell types, which may be leukocytes or non-leukocytes, e.g. IL-1 is produced by a wide range of cells, including leukocytes (such as monocytes, macrophages, NK cells, B- and T-lymphocytes) and non-leukocytes (such as smooth muscle cells, vascular endothelial cells (a single layer of cells lining blood vessels), fibroblasts (cells found in connective tissue that produce ground substance and collagen fibre precursors), astrocytes (non-neural cells found in the central nervous system) and chondrocytes (cells embedded in the matrix of cartilage)).

• Many cell types can produce more than one cytokine. Lymphocytes, for example, produce a wide range of interleukins, CSFs, TNF, IFN-as and IFN-y Fibroblasts can produce IL-1, -6, -8, and -11, CSFs, IFN-P and TNF.

• Many cytokines play a regulatory role in processes other that immunity and inflammation. Neurotrophic factors, such as NGF and BDNF, regulate growth, development and maintenance of various neural populations in the central and peripheral nervous system. EPO stimulates the production of red blood cells from erythroid precursors in the bone marrow.

• Most cytokines are pleiotropic, i.e. can affect a variety of cell types. Moreover, the effect that a cytokine has on one cell type may be the same or different to its effect on a different cell type. IL-1, for example, can induce fever, hypotension and an acute phase response. G-CSF is a growth factor for neutrophils, but it is also involved in stimulating migration of endothelial cells and growth of haematopoietic cells. IFN-y stimulates activation and growth of T- and B-lymphocytes, macrophages, NK cells, fibroblasts and endothelial cells. It also displays weak anti-proliferative activity with some cell types.

• Most cytokines are inducible, and are secreted by their producer cell, e.g. induction of IL-2 synthesis and release by T-lymphocytes is promoted by binding of IL-1 to its receptor on the surface of T cells. IFN-as are induced by viral intrusion into the body. In general, potent cytokine inducers include infectious agents, tissue injury and toxic stimuli. The bodies main defence against such agents, of course, lies with the immune system and inflammation. Upon binding to target cells, cytokines can often induce the target cell to synthesize and release a variety of additional cytokines.

• In contrast, some cytokines (e.g. some CSFs and EPO) appear to be expressed constitutively. In yet other instances cytokines such as PDGF and TGF-P are stored in cytoplasmic granules and can be rapidly released in response to appropriate stimuli. Other cytokines (mainly ones with growth factor activity, e.g. TGF-P, FGF and IL-1) are found bound to the extracellular matrix in connective tissue, bone and skin. These are released, bringing about a biological response upon tissue injury.

• Many cytokines exhibit redundancy, i.e. two or more cytokines can induce a similar biological effect. Examples include TNF-a and -P, both of which bind to the same receptor and induce very similar, if not identical, biological responses. This is also true of the IFN-a family proteins and IFN-P, all of which bind the same receptor.

Although all cytokines are polypeptide regulatory factors, not all polypeptide regulatory factors are classified as cytokines. Classical polypeptide hormones, such as insulin, FSH and GH are not considered members of the cytokine family. The distinguishing features between these two groups is ill defined, and in many ways artificial. Originally, one obvious distinguishing feature was that hormones

Table 8.3 The cytokine receptor superfamilies. Refer to text for further details and to Table 8.1 for explanation of cytokine abbreviations

Receptor superfamily name

Alternative name

Main members

The haematopoietic receptor superfamily

The cytokine receptor

Receptors for:

superfamily

IL-2-IL-7, IL-9, IL-12, G-CSF, GM-

CSF, EPO, LIF, CNTF, GH

The interferon receptor superfamily

Cytokine receptor type

Receptors for:

II family

IFN-a, -P, -y, IL-10

The immunoglobulin superfamily

-

Receptors for:

IL-1, IL-6, FGF, PDGF, M-CSF

PTK receptor superfamily

-

Receptors for:

EGF, insulin, IGF-1

The nerve growth factor superfamily

-

Receptors for:

NGF, TGF

The seven transmembrane spanning receptor

-

Receptors for various chemokines,

superfamily

including IL-8 and MIP

The complement control protein superfamily

-

IL-2 receptor (a-chain)

were produced by a multicellular, anatomically distinguishable gland (e.g. the pancreas, the pituitary, etc.) and functioned in a true endocrine fashion, affecting cells far distant from the site of their production. Many initially described cytokines are produced by white blood cells (which do not constitute a gland in the traditional sense of the word), and often function in an autocrine/paracrine manner.

However, even such distinguishing characteristics have become blurred. EPO, for example, is produced in the kidney and liver and acts in an endocrine manner, promoting production of red blood cells in the bone marrow. EPO could thus also be considered to be a true hormone.

8.1.1 Cytokine receptors

Recombinant DNA technology has also facilitated detailed study of cytokine receptors. Based upon amino acid sequence homology, receptors are usually classified as belonging to one of six known superfamilies (Table 8.3). Individual members of any one superfamily characteristically display 20-50 per cent homology. Conserved amino acids normally occur in discrete bands or clusters, which usually correspond to a discrete domain in the receptor. Most receptors exhibit multiple domains. In some cases a single receptor may contain domains characteristic of two or more superfamilies. For example, the IL-6 receptor contains domains characteristic of both the haematopoietic and immunoglobulin superfamilies, making it a member of both.

Some cytokine receptors are composed of a single transmembrane polypeptide (e.g. receptors for IL-8, -9 and -10). Many contain two polypeptide components (including the IL-3, -4, and -5 receptors), and a few contain three or more polypeptide components (e.g. the IL-2 receptor contains three polypeptide chains). In some instances a single cytokine may be capable of initiating signal transduction by binding two or more distinct receptors (e.g. IL-1 has two distinct receptors (types I and II), both of which are transmembrane glycoproteins).

In many cases where a receptor consists of multiple polypeptides, one of those polypeptides (which will be unique to that receptor) will interact directly with the ligand. The additional polypeptide(s), responsible for initiation of signal transduction, may be shared by a number of receptors (Figure 8.1).

Figure 8.1 Cytokine receptors usually display a unique cytokine ('Ligand')-binding domain, but they share additional receptor components that are normally responsible for signal transduction. This explains the molecular basis of pleiotropy. IL-6, IL-11 and LIF receptors, for example, are all composed of a distinct ligand-specific binding domain and a separate subunit (gp 130). gp 130 is responsible for initiating signal transduction and is identical in all three receptors. This is depicted schematically above

Figure 8.1 Cytokine receptors usually display a unique cytokine ('Ligand')-binding domain, but they share additional receptor components that are normally responsible for signal transduction. This explains the molecular basis of pleiotropy. IL-6, IL-11 and LIF receptors, for example, are all composed of a distinct ligand-specific binding domain and a separate subunit (gp 130). gp 130 is responsible for initiating signal transduction and is identical in all three receptors. This is depicted schematically above

Some cytokine receptors can directly initiate signal transduction upon binding of ligand. In other cases additional elements are involved. For many receptors, the exact intracellular events triggered upon ligand binding remain to be elucidated. However, the molecular details of signal transduction pathways for others (e.g. the interferons) are now understood

8.1.2 Cytokines as biopharmaceuticals

Cytokines, in many ways, constitute the single most important group of biopharmaceutical substances. As coordinators of the immune and inflammatory response, manipulation of cytokine activity can have a major influence on the body's response to a variety of medical conditions. Administration of certain cytokines can enhance the immune response against a wide range of infectious agents and cancer cells. EPO has proven effective in stimulating red blood cell production in anaemic persons. Growth factors have obvious potential in promoting wound healing. And neurotrophic factors display some clinical promise in the abatement of certain neurodegenerative diseases.

A better understanding of the molecular principles underlining cytokine biology may also provide new knowledge-based strategies aimed at defeating certain viral pathogens. These pathogens appear to establish an infection successfully, at least in part, by producing specific proteins that thwart the normal cytokine-based immunological response. The cowpox virus, for example, produces an IL-1-binding protein, and the shope fibroma virus produces a TNF-binding protein. The Epstein-Barr virus, on the other hand, produces a protein homologous to IL-10.

A variety of medical conditions are now believed to be caused or exasperated by overproduction of certain cytokines in the body. A variety of pro-inflammatory cytokines, including IL-6, -8 and TNF, have been implicated in the pathogenesis of both septic shock and rheumatoid arthritis. Inhibiting the biological activity of such cytokines may provide effective therapies for such conditions. This may be achieved by administration of monoclonal antibodies raised against the target cytokine, or administration of soluble forms of its receptor that will compete with cell surface receptors for cytokine binding.

Some cytokines have already gained approval for medical use. Many more are currently undergoing clinical or preclinical trials. Over the next few chapters the biology and potential medical applications of these cytokines will be discussed in detail. The remainder of this chapter concerns itself with the prototypic cytokine family, namely the interferons.

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