Eosinophil Granulocytes

2.3.2.1.1. Structure and Differentiation

Mature eosinophils found in blood and tissues are characterised by their segmented nucleus and by large intracytoplasmic granules, which stain a yellow-pink colour with acidic dyes such as eosin (WARDLAW and MOQBEL 1992).

The bone marrow is the main production site for eosinophils as well as basophils and neutrophils. They all share a common precursor cell, the myeloblast, which divides and further develops into promyelocytes. In the following myelocytic stage, specific granules appear and the distinction into neutrophil, eosinophil and basophil myelocytes can be made. In the subsequent metamyelocytic stage, division ceases and after further maturation the cells obtain their characteristic granulocyte features (LIEBICH 1990). In the rat, this cell cycle is completed in 22-30 hours and the mature cells are released from the bone marrow after 36-40 h. In parasitised animals, the first takes only about 9 h and the latter 18 h (MCEWEN 1992). Several cytokines have been implicated in eosinopoiesis, with IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulating the development of early precursor cells, whereas IL-5, produced by activated T cells and mast cells, is the principal cytokine that specifically stimulates proliferation and differentiation of eosinophil progenitors (JONES 1993).

In sheep, eosinophil granulocytes represent about 3-5% of peripheral blood leukocytes (LIEBICH 1990), but the circulating cells are only in transit between the sites of production and the tissue sites of consumption, mainly the skin and mucosal sites (CAPRON and DESREUMAUX 1996). It is estimated that for every circulating eosinophil there are 200 mature cells in the bone marrow and 500 in

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loose submucosal connective tissues (KAY 1985). The life span of eosinophils is not known exactly; it has been estimated to be several days in healthy tissues (WELLER 1991) but can be prolonged during helminth infection or allergic reactions (BEHM and OVINGTON 2000). Apoptotic cells are phagocytosed by other cells, such as macrophages, or excreted after transepithelial migration in the gut or lungs (KAY 1985).

Two populations of eosinophils of different densities can be separated from humans with eosinophilia (VADAS et al. 1979; BASS et al. 1980) or from parasitised sheep (CHAMBERS 1990). The eosinophils with lighter density, called hypodense eosinophils, are metabolically more active (increased production of mediators, increased chemotaxis) than normal (normodense) eosinophils (WINQVIST et al. 1982). They are also more cytotoxic for helminth larvae in vitro (CAPRON et al. 1984) and show morphological alterations and increased surface cell receptor expression (WELLER 1991).

2.3.2.1.2. Function

Although eosinophils are able to phagocytose particulate matter (e.g. mast cell granules and immune complexes) and release bactericidal substances (GLEICH and ADOLPHSON 1986), they cannot effectively defend against bacterial infections as neutrophils can. The main role of eosinophils is seen in the defence against larger pathogens that cannot be phagocytosed, such as helminth parasites (JONES 1993). At the site of helminth infection, eosinophils become activated and secrete immunomodulatory and proinflammatory mediators and cytokines. They degranulate and release cytotoxic products. Eosinophils are also thought to take part in wound healing, tissue repair and fibrosis and may act as antigen-presenting cells (WELLER and LIM 1997).

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2.3.2.1.3. Granule Proteins

Eosinophil granules contain several basic proteins, as described by JONES (1993): a crystalloid core is composed of major basic protein (MBP), surrounded by a matrix containing mainly eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil-derived neurotoxin (EDN). Of the granule proteins, MBP, ECP and EPO were shown to be cytotoxic for helminth parasites as well as mammalian cells in vitro (GLEICH 1990). BUTTERWORTH et al.

(1979) demonstrated the cytotoxity of purified MBP for larvae of Schistosoma mansoni. MBP and EPO have also been shown to be strong agonists for platelet activation (ROHRBACH et al. 1990) and ECP inhibits lymphocyte proliferation in vitro (PETERSON et al. 1986). The bactericidal and helmithicidal activity of EPO is intensified when it is combined with hydrogen peroxide (H2O2) and halide ions, e.g. bromide (GLEICH and ADOLPHSON 1986). EDN has a marked ribonuclease activity and was shown to be neurotoxic in rabbits and guinea pigs, causing the so-called “Gordon phenomenon” (GLEICH and ADOLPHSON 1986).

Eosinophils also contain various enzymes, such as histaminase, collagenase, arylsulphatase, acid phosphatase and lysophospholipase. The latter makes up the Charcot-Leyden crystals, which are found at sites of eosinophil degranulation (JONES 1993). The role of lysophospholipase is unknown, but it may be a protection against the toxic effects of endogenous or parasite-derived lysophospholipids (JONES 1993). The granule contents are released through degranulation. This process is initiated by cross-linking of surface receptors after binding of molecules such as immunoglobulins and complement (BALIC et al.

2000a).

2.3.2.1.4. Eosinophil-derived Mediators

Eosinophils are also able to newly synthesise and release a range of cytokines and membrane-derived lipid mediators. The secreted lipid mediators leukotriene (LT) C4, LTD4, prostaglandin (PG) E2, thromboxane (TX) B2 and platelet

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activating factor (PAF) stimulate vasoactivity, smooth muscle contraction and secretion of mucus (WARDLAW and MOQBEL 1992; BEHM and OVINGTON 2000). PAF also has chemotactic activity for eosinophils and stimulates the effector functions of eosinophils, neutrophils, macrophages and platelets (BEHM and OVINGTON 2000). Other mediators secreted by eosinophils are various cytokines and growth factors (GM-CSF, IL-3, IL-5, eotaxin), regulators of the immune response [IL-2, IL-4, IL-10, IL-12, IL-16, Interferon (IFN)-γ] and other substances involved in inflammation, fibrosis, wound healing and tissue repair, such as transforming growth factor (TGF)-α, TGF-β, TNF-α, IL-1α, IL-1β, IL-6 and IL-8 (WARDLAW and MOQBEL 1992; CAPRON and DESREUMAUX 1996;

BEHM and OVINGTON 2000). The cytotoxity of eosinophils is also partly due to their ability to generate substances like superoxide and hydrogen peroxide in the oxidative burst (WARDLAW and MOQBEL 1992).

2.3.2.1.5. Migration and Activation

Eosinophils react towards a variety of signals, transmitted through chemical ligands that bind to receptors expressed on the cell surface. This leads to activation of the cells and their migration to the source of the chemotactic stimulus, where they synthesise and secrete their stored or newly formed biologically active molecules.

The first step in eosinophil migration from blood into tissues involves adhesion to endothelial cells through interaction between their surface receptors and eosinophil adhesion molecules. Receptor expression increases when the endothelial cells are stimulated with inflammatory mediators (e.g. IL-1, TNF-α) and eosinophil adhesion is up-regulated by mediators such as PAF, 3 and IL-5; the latter two selectively enhance eosinophil but not neutrophil adhesion (WALSH et al. 1990; BEHM and OVINGTON 2000).

Following adhesion to the endothelium, migration of eosinophils into the tissues is controlled by cytokines and chemokines. Apart from its role in eosinophil

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differentiation in the bone marrow, IL-5 also is an important cytokine in eosinophil recruitment and activation (SANDERSON 1992; MOULD et al. 1997). During eosinophilia in allergic conditions and helminthosis, IL-5 mobilises eosinophils from the bone marrow, while the chemokine eotaxin induces sequestration of eosinophils from blood into the tissues. Both substances selectively regulate eosinophil trafficking and are also generated in inflamed tissue, where IL-5 is critical for eosinophil homing and migration into tissues in response to eotaxin (COLLINS et al. 1995). Other substances possibly involved in the complex process of eosinophil recruitment are the cytokines IL-1, IL-3, IL-4, GM-CSF, TNF-α and other chemotactic substances such as leukotrienes, PAF, macrophage inflammatory protein (MIP) 1α, monocyte chemoattractant protein (MCP) 3 and the anaphylatoxins histamine, C3a and C5a (MCEWEN 1992;

WARDLAW and MOQBEL 1992). Some parasites also secrete factors with direct chemotactic potential for eosinophils, as described below in Section 2.5.

Eosinophils possess Fc-receptors for antibody, and receptor binding of the immunoglobulins IgE, IgG and IgA was shown to trigger degranulation in vitro (CAPRON and CAPRON 1990; ABU-GHAZALEH et al. 1989). Binding of the secretory component of IgA provides a potent stimulus for eosinophil degranulation (LAMKHIOUED et al. 1995).