TÉCNICAS DE PASTEURIZACIÓN DE MEZCLA PARA HELADO

1.6.1 Eosinophils

Eosinophils are bone marrow-derived granulocytes with a bilobed nucleus and contain characteristic specific granules, as well as primary granules and small granules

(Dvorak, 1994). IL-3, IL-5 and GM-CSF are important in eosinophilopoesis and the prolongation of mature eosinophil survival (see above).

1.6.2 Granule contents (preformed mediators)

Specific granules contain the basic eosinophil granule proteins (Dvorak, 1994). Major basic protein (MBP) constitutes the core of the specific granule and represents 50% of the total granule protein (Gleich and Adolphson, 1986). It is a 117 amino acid single polypeptide chain with a molecular weight of 14,000 and its arginine-richness accounts for its basicity (Gleich and Adolphson, 1986). MBP has no enzymatic activity but is toxic to helminths, protozoa, bacteria, tumour cells and host cells. It is also able to activate neutrophils and platelets and causes non-cytotoxic release of histamine from mast cells, and other actions include non-specific complement activation and induction of bronchospasm in animal lungs (Gleich and Adolphson, 1986; Weller, 1991;

Wardlaw and Kay, 1987; Jones, 1993). Its toxic actions may stem from disruption of cell membranes (Weller, 1994).

The other granule proteins are found in the specific granule matrix (Gleich and

Adolphson, 1986). Eosinophil cationic protein (ECP) is a 133 amino acid protein with a molecular weight between 18,000 and 21,000 (Gleich and Adolphson, 1986). It has ribonuclease activity and shows homology with eosinophil-derived neurotoxin (EDN) and pancreatic ribonuclease. ECP is toxic to bacteria, helminths, protozoa, host cells, is a potent neurotoxin and it exerts its toxicity by forming membrane pores (Gleich and

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Adolphson, 1986; Weller, 1991; Wardlaw and Kay, 1987; Jones, 1993; Young et al., 1986). ECP also can stimulate histamine release from mast cells, stimulate fibroblasts to produce glycosaminoglycans and inhibits T cell and B cell proliferation via non- cytotoxic actions (Gleich and Adolphson, 1986; Wardlaw and Kay, 1987; Jones,

1993). EDN is a 104 amino acid polypeptide with a molecular weight of 18,000 and is a potent ribonuclease and neurotoxin but is only weakly toxic to parasites and host cells (Gleich and Adolphson, 1986; Jones, 1993; Weller, 1991). Eosinophil peroxidase (EPO) is a cationic toxin but in the presence of peroxide and halide is more effective in killing bacteria, parasites, viruses and tumour cells (Gleich and Adolphson, 1986; Jones, 1993). It also has other effects including the non-cytotoxic release of histamine from mast cells.

Charcot-Leyden crystal (CLC) protein, which has a molecular weight of 17,000, comprises up to 1 0% of eosinophil protein and is found in the cell membrane and in

primary granules (Gleich and Adolphson, 1986; Jones, 1993; Weller, 1991). It is a lysophospholipase and therefore acts as a surfactant (Gleich et al., 1993). Other granule contents and preformed mediators in eosinophils include collagenases, arylsulphatase, histaminase, P-glucuronidase, cathepsin, acid phosphatase, catalase, phospholipases and non-specific esterases (Spry, 1988; Kroeger et al., 1992).

1.6.3 Newly-synthesised eosinophil mediators

Upon degranulation, eosinophils synthesise a range of lipid mediators, including PAF (which has a wide range of actions including stimulation of platelet aggregation and activation, eosinophil chemotaxis, activation and degranulation and activation of other leukocytes such as neutrophils and mast cells), LTs (most prominently LTC4, also

LTB4), PGs (PGE2, PGD2, PGF2) and TxB2, all with many proinflammatory actions

(Weller, 1993). Eosinophils also produce oxygen metabolites such as superoxide anion, hydroxyl radicals, peroxide and singlet oxygen, which are important in killing micro-organisms and tumour cells but are also toxic to host cells (Petreccia et al., 1987; Kanofsky et al., 1988).

Eosinophils also synthesise cytokines (see below).

1.6.4 Eosinophil cell surface antigens and receptors

Eosinophils possess a number of immunoglobulin receptors: FCyRII (CD32, the low affinity receptor for IgG), FCeRII (CD23, the low affinity receptor for IgE), FCaR and FCÔR; FCyRII (CD64, high affinity IgG receptor) and FCyRIII (CD 16, low affinity) are inducible by IFN-y (Weller, 1991; Wardlaw, 1994; Kay, 1991; Hartnell et al..

1992). Binding of immunoglobulin to Ig receptors (especially IgA) stimulates eosinophil degranulation (Weller, 1991).

Eosinophils also bear receptors for complement (e.g. for C5a, C3b), for lipid mediators (PAF, LTs and PGs), cytokines (including IL-2 [inducible], IL-3, IL-4, IL-5, IL-8,

GM-CSF, IFN-y, -p, -a, TNF-a, RANTES, MCP-3, M IP-la, eotaxin, LCF) and others (e.g. glucocorticoid, p-adrenoceptor and oestrogen receptors) (Weller, 1991; Gleich et al., 1993; Kroegel et al., 1992). Due to the presence of such receptors, eosinophil chemotaxis, activation, degranulation, mediator production and adhesion are influenced by many cytokines and mediators (Table 1.2). Many of these mediators are not specific for eosinophils, and also act upon other leukocytes, such as neutrophils. Because of the selective recruitment of eosinophils in allergic disease, there has been interest in mediators that show more specificity for eosinophils. Such mediators include RANTES, M IP-la, IL-5 and LCF as chemoattractants, IL-3 and IL-4 (indirectly, by inducing VCAM-1 on endothelium) in eosinophil adherence, and IL-3 and IL-5 in eosinophil activation (Wardlaw, 1994; Nourshargh, 1993; Resnick and Weller, 1993).

A number of adhesion molecules may be expressed by eosinophils, including GDI la (LFA-la), CD 11b (MAC-la), C D llc (P150/95a), CD 18, VLA-4a (CD49d), L- selectin, ligands for E- and P-selectins, PECAM (platelet endothelial cell adhesion molecule) and SiLew X (Sialyl-Lewis X), which mediate different aspects of eosinophil adherence and emigration (Table 1.3) (Nourshargh, 1993; Resnick and Weller, 1993; Wardlaw, 1994). The VLA-4 / VCAM-1 interaction is thought to be particularly important in selective eosinophil recruitment (Resnick and Weller, 1993). Other surface antigens that may be seen on eosinophils are CD4, HLA-DR, CD9, CD69 and CD45 (Weller, 1993; Wardlaw, 1994; Gleich et al., 1993). Certain antigens, including CD4, ICAM-1, IL-2R and HLA-DR, are induced under certain conditions, such as eosinophil activation by cytokine (Weller, 1991; Wardlaw, 1994; Gleich et al., 1993; Weller, 1992).

1.6.5 Eosinophil activation

In normal individuals, eosinophils are in a resting state. Exposure of eosinophils to eosinophil-active mediators causes a priming of eosinophils, with enhancement of certain effector functions, such as chemotaxis, adhesion and cytotoxicity, and greater responsiveness to inflammatory mediators (Gleich et al., 1993; Jones, 1993; Wardlaw,

1994; Weller, 1991). Such eosinophils show greater survival, are more metabolically active, show greater expression of surface receptors (e.g. Ig receptors, complement

receptors and adhesion molecules) and expression of other surface antigens is induced (e.g. CD4, ICAM-1, IL-2R, HLA-DR, CD69, FCyRI and FCyRIII). Activated eosinophils convert granule proteins from storage to secretory forms, show enhanced degranulation upon stimulation and can produce more leukotrienes and superoxide anions; they demonstrate increased cytotoxicity, and chemotaxis to mediators such as PAF or LTB4 is increased. In eosinophilic diseases such as asthma, allergic rhinitis and

hypereosinophilic syndrome, there is a shift towards a less dense (hypodense)

eosinophil phenotype, and such eosinophils show certain morphologic changes such as vacuolation and smaller-sized specific granules with loss of the granule core (Bass et al., 1980; Peters et al., 1988). Many authors believe that hypodense eosinophils represent the morphological change of activation (Weller, 1991; Wardlaw, 1994), but the published relationship of hypodensity to functional activation status has been variable and apparently partially dependent on experimental design (Prin et al., 1986; Hartnell et al., 1990).

1.6.6 Eosinophils, allergy and cytokine production 1.6.6.1 Eosinophils are central to allergy

It is well established that tissue (and sometimes blood) eosinophilia is a hallmark of allergic diseases such as asthma, allergic rhinitis and AD. Disease is associated with the presence of eosinophils and eosinophil-derived granule proteins in tissue, the blood, mucosal secretions and lavage fluids (Frigas et al., 1981; Dor et al., 1984; Kroegel et al., 1991; Frigas and Gleich, 1986; Uehara et al., 1990; Wassom et al., 1981;

Leiferman et al., 1985; B ascom et al., 1989; Bentley et al., 1992). Blood eosinophils show an activated phenotype (Fukuda et al., 1985; Frick et al., 1989; Bass et al., 1980). The levels of eosinophils and their products can be correlated with direct and indirect measures of disease severity (e.g. clinical severity, bronchial

hyperresponsiveness and lung function values in asthma) (Taylor and Luksza, 1987; Durham and Kay, 1985; Horn et al., 1975; Walker et al., 1993; Kagi et al., 1992). The eosinophils are believed to mediate epithelial damage in asthma, via their granule proteins (Gleich, 1990; Gleich et al., 1988; Kroegel et al., 1992). Deposition of eosinophil granule proteins is observed at areas of epithelial damage and MBP, ECP and EPO can all cause impaired function, damage and destruction of airway epithelial cells (Filley et al., 1982; Azzawi et al., 1990; Frigas et al., 1980; Motojima et al.,

1989).

1.6.6.2 Eosinophils can produce cytokines

Eosinophils are capable of storage and release of cytokines, which contradicts the conventional view of the eosinophil as a simple effector leukocyte (Venge and

Hakansson, 1991). Cytokine mRNA and product may be expressed constitutively in peripheral blood eosinophils from normal humans (IL-6, IL-8, RANTES, TGF-a and

TNF-a) (Hamid et al., 1992b; Melani et al., 1993; Yousefi et al., 1995; Costa et al., 1993; Walz et al., 1994; Lim et al., 1995) or from patients with hypereosinophilia (IL- 5, EL-6, TNF-a, TGF-a, TGF-g and M lP-la) (Melani et al., 1993; Costa et al., 1993;

Desremaux et al., 1992; Dubucquoi et al., 1994; Wong et al., 1990; Wong et al., 1991; Weller et al., 1993). In atopic disease, blood eosinophils express IL-5, increased levels of IL- 8 and can be stimulated to synthesise IL-3 (Yousefi et al., 1995; Tanaka et al.,

1994; Fujisawa et al., 1994).

L6.6.3 Eosinophil cytokine production in allergy and disease

Tissue eosinophils have been shown to produce cytokines in disease, including allergic disorders. In nasal allergy and polyposis, IL-4, IL-5, TNF-a, TGF-a, TGF-p, and M IP-la mRNA or protein have been co-localised to eosinophils (Costa et al., 1993; Ying et al., 1993; Saito et al., 1994; Kay et al., 1995; Howarth et al., 1995; Finotto et al., 1994; Elovic et al., 1994; Ohno et al., 1992). In asthma, BAL eosinophils contain IL-5 mRNA (Broide et al., 1992), mucosal eosinophils contain IL-5 protein (Bradding et al., 1994) and IL-4 mRNA (Kay et al., 1995) and in AD eosinophils contain IL-5 mRNA (Tanaka et al., 1994). Other disorders in which eosinophil cytokines have been identified include coeliac disease (IL-5) (Desremeaux et al., 1992), eosinophilic cystitis (EL-5) (Dubucquoi et al., 1994), dermatitis herpetiformis (EL-5) (Desremeaux et al.,

1995), necrotising enterocolitis (TNF-a) (Tan et al., 1993), colonic adenocarcinoma and oral squamous cell carcinoma (TGF-a) (Wong et al., 1990), Hodgkin's disease (TGF-P) (Kadin et al., 1993) and pemphigoid (IL-10, IFN-y) (Lamkhioued et al.,

1995).

Eosinophils may therefore be involved in amplifying and orchestrating the allergic response by secretion of pro-allergic cytokines such as IL-3, IL-4, IL-5 and may also be involved in tissue repair functions through the release of TGF-a and -P (Moqbel et al., 1994) in addition to the effects mediated through eicosanoids and other mediators. The relationship between eosinophils and other cytokine-producing cells in allergy (T cells, mast cells) appears complex, although eosinophils themselves are likely to be regulated by cytokines such as IL-3, EL-5 and GM-CSF released from T cells (Moqbel et al., 1994).