Clinical and Experimental Allergy. 1990. Voiume 20, Supplement 4, pages 31 -34

Modulation of eosinophil function in vitro A. B. KAY Department of Allergy and Clinical Imtnunology. National Heart & Lung Institute. Dovehou.se Street. London SW3 6LY, U.K. Introduction

It is well established that a blood and tissue eosinophilia is associated with helminthic infections and that eosinophils are also a feature of allergic inflarnmation [1]. Eosinophilmediated damage might be an important factor in denudation of the bronchial epithelium in asthma [2], Following adhesion to the vascular endothelium and subsequent diapedesis, eosinophils, like neutrophils, tnay become activated as a result of changes in the microenvironment. In vitro, stimulation of eosinophils with various agonists results in the generation of lipid mediators [3,4] and partial reductive products of oxygen [5] together with exocytosis of basic granule proteins [6], The effector function of eosinophiis in adaptive itnmunity, i.e. the extracellular killing of non-phagocytosable targets such as opsonized helminthic larvae, is triggered via complement and/or immunoglobulin receptors present on the eell membrane [7].

Reeeptors for immunogiobulins and adhesion glycoproteins

There are a number of specific receptors which may play a role in the recognition of opsonized particles and the activation of cellular responses [8]. Among those weil characterized for the neutrophil and the monocyte are the three types of Fey receptor (FcyRT/CD64, FcyRII/ CDw32, FcyRIII/CDI6), complement receptors (CRl/ CD35 and CR3/CD1 tb), and other membrane proteins important for intercellular adhesion (LFA-1 -alpha/ CDI la, pl50,95-alpha/CDl Ic and ICAM-1/CD54), FcyRl is the only receptor that will bind monomerie IgG, while FcyRTI and FcyRIII only exhibit detectable binding with multivalent ligand, such as immune complexes [9], Complement receptor type one (CRl) is the receptor for the third component of complement, C3b. The leucocyte integrins are a family of three receptors, LFA-1, CR3 and pi 50,95, which are heterodimers composed of two non-covalently linked polypeptide chains. Each receptor has a distinct alpha chain (molecular weights 190 kD, 185 kD and 150 kD, respectively) which is associated with a common beta-chain (MW 95 kD) [10]. Complement receptor type three (CR3) is the receptor for

C3bi and has binding sites for other tnolecLiles [II]; pl50,95 binds C3bi and may also bind other Hgands [12]. Lyinphocytefunction-associated antigen-1 (LFA-1) interacts with intercellular adhesion molecule-1 (ICAM-1) on other cells to facilitate intercellular adhesion [13]. Eosinophils exhibit considerable heterogeneity and ean be separated into distinct populations by density gradient centrifugation. There is evidence that the lower density ceils are activated in terms of metabolic activity and IgG Fc and complement receptor expression [14-16], Much of the work characterizing IgG Fc and complement receptors on eosinophils has used the rosette technique [17], which is a relatively imprecise method for determining receptor expression. There are few data on the expression of the leucocyte integrins on eosinophits [18 20]. We have used specific monoclonal antibodies and flow cytometry to investigate the expression of these receptors on normal density and low density eosinophils. The findings were compared with the neutrophil and the monocyte in order to identify differences which may be relevant to the specialized function of the eosinophil. Several novel properties of the human eosinophil were established. These were: (1) that the eosinophil expressed FcyRTT (CDw32) only (unlike monocytes which bear FcyRII and FcyRIII); (2) that the absence of FcyRIII (CD 16) on eosinophils served as a basis for distinguishing eosinophil and neutrophil populations by itntnunofluoreseence; (3) that the leucocyte adhesion glycoproteiiLS, LFA-1-alpha (CDI la), CR3-alpha (CDllb), pl50,95alpha(CDllc)and the common beta-chain (CD 18), were expressed on the eosinophil as well as the neutrophil; and (4) that CDI 8 expression was signilicantiy reduced on low density eosinophils from the hypereosinophiiic syndrome [21]. Thus, our findings emphasize the unique phenotype of the human eosinophil in terms of Fey receptor expression, the similarity of the eosinophil and neutrophil with regard to the leucocyte integrins and thateo.sinophils of low density do not differ greatly from those of normal density in terms of receptor expression.

Mechanisms of local eosinophitia

Eosinophil leucocytes are known to accumulate and persist preferentially at the site of allergic tissue reactions 31

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[1,22]. For example, Frew and Kay [23], employing skin biopsies from late-phase cutaneous reactions (LPCR) in human atopic subjects, demonstrated activated (BG2+) eosinophils 48 hr after challenge with specific allergen. In contrast, neutrophils appeared early and their numbers decreased with time. The mechanism of local tissue eosinophilia is largely unexplained. Although platelet activating factor (PAF) is a potent eosinophil chemoattractant, it was shown to have even greater effects on neutrophil locomotion [24]. Inter!eukin-5 (IL-5) has been shown to be a selective chemotactic agent for the eosinophil, but the activity was very weak compared with that of PAF [25]. Furthermore, the general concept that the elaboration of selective chemotactic factors explains the local accumulation of various cell types in inflammatory reactions remains largely unproven. For this reason other mechanisms need to be considered to explain the local eosinophilia observed in allergy, asthma and helminthic infections. The role of interleukin-5 Using in-.siui hybridization, we have recently identified niRNA for IL-5 in both allergen-induced LPCR and bronchial mucosal biopsies from allergic asthma patients [26]. In addition, lL-5 [27], IL-3 [28] and granulocyte/ macrophage-colony stimulating factor (GM-CSF) [29] prolong the survival of human eosinophils />/ vitro and this may provide a partial explanation for Ihe eosinophilic response in vivo. TL-5 has a number of selective effects on eosinophils. These include terminal differentiation ofthe committed eosinophil precursor [30,31]. enhanced cytotoxicity and increased respiratory burst activity [32]. We recently examined the role ofIL-5 in the eosinophil adhesion reaction. We proposed that IL-5 promotes preferential hyperadhesion of eosinophils, as compared with the neutrophi] [33]. Lamas et al. [34] had previously reported that eosinophil adherence to cultured human vascular endothelial cells (HVEC) or gelatinized plates could be enhanced by prior incubation with FMLP or TNF. Furthermore, a number of cytokines acted directly on cultured HVEC to give enhanced adhesion of untreated eosinophils. PAF was also shown to stimulate increased eosinophil adherence lo HVEC or plastic surfaces. Enhanced adhesion was inhibited by prior incubation with a monoclonal antibody directed against the common beta-chain ofthe CD! 1/18 leucocyte integrin family [20]. Thus, we have compared IL-5 with IL-3, GM-CSF and PAF to determine whether this cytokine preferentially influences eosinophil as opposed to neutrophil, hyperadhesiveness to plasma-coated glass (PCG) or human microvascular endothelial cells (HMVEC). Furthermore, the contribution ofthe CD 11/18 complex to this process

was assessed using both flow cytometry and inhibition assays. We found that IL-5, IL-3 and GM-CSF also induced a time-dependent increase in adherence of normal density eosinophils to PCG, optimal at 60 min, whereas the effect of PAF was greater at 15 min. Similar results were obtained with neutrophils with the exception that IL-5 had minimal and non-significant effects on this cell type. Unstimulated eosinophils and neutrophils also adhered to PCG or HMVEC, but in low numbers. Pre-incubation of eosinophils with IL-5, GM-CSF or PAF resulted in dose-dependent increases in the numbers of cells adherent to PCG. IL-3 had a smaller but significant effect on enhanced eosinophil adhesion to PCG, while IL-2 and lyso-PAF were.ineffective. Neutrophils gave similar levels of baseline and stimulated adhesion to PCG as eosinophils, IL-5 again had no significant stimulatory effect. IL-5 also increased eosinophil, but not neulrophil, adherence to HMVEC in a concentration-depend ent manner. Pre-incubation with the protein synthesis inhibitor cycloheximide had no effect on TL-5-, GM-CSF- or PAFstimulated eosinophil adhesion. The contribution ofthe CDll/18 leucocyte integrins to IL-5 and PAF-induced eosinophilic hyperadherence was investigated by inhibition experiments utilizing monoclonal antibodies was also assessed. Enhanced adhesion to PCG (by PAF) or HMVEC (by IL-5) was inhibited by (ranked in order of potency) anti-CR3-aIpha = common beta-chain > LFA1-alpha. Anti-pl50,95-alpha had no measurable effect. Baseline adhesion by unstimulated eosinophils was not significantly influenced by prior incubation with these monoclonal antibodies. Using flow cytometry, IL-5 and IL-3 were found to upregulate eosinophil but not neutrophil CR3 expression. These findings demonstrate that TL5 enhances eosinophil, but not neutrophil adherence reactions, by a mechanism dependent, at least in part, on the CDl 1/18 family of adhesion glycoproteins. Conclusions The density of expression of a number of functionally relevant receptors appears to be very similar on eosinophils and neutrophils; there may be differences in intracellular signalling mechanisms of these two cell types which give rise to a difference in the quality of the response to a given stimulus. In addition, while the use of specific monoclonal antibodies and flow cytometry gives an accurate measure of the number of receptors on the surface ofthe cell, it gives no indication ofthe affinity of the receptor. Therefore, there are a number of ways in which an apparently similar level of receptor expression could give rise to a very different level of response to a stimulus in different cell types.

Modulation of eo.sinophit furiclion

Investigations of these well characterized cell surface receptors showed only one major difference between eosinophils and neutrophils, i.e. lack of CD 16 expression on eosinophils. The functional consequences of this difference are unclear. Eosinophils have very distinctive cytotoxic proteins within their granules which are crucial to their effeetor function and which clearly distinguish them from other inflammatory cells. It is possible that they also possess specialized cell surface receptors which require further characterization. Our data also suggest that in vivo., eosinophil hyperadherence is similar to that ofthe neutrophil in terms of the dependence on CD 11/18 and involves the utilization of CR3 and to a lesser extent LFA-I. Although we observed enhanced expression of CR3-alpha by stimulated eosinophils, the possibility that enhanced eosinophil adhesion is dependent upon an affinity or conformational change in the receptor and not increased expression cannot be excluded. The observation that IL-5 selectively enhanced eosinophil adherence to PCG and HMVEC may provide some insight into the preferential eosinophil accumulation observed in atopic allergic inflammation and infection with helminthic parasites.

References 1 Gieich GJ, Adolphson CR. The eosinophilic leukoeyte: structure and function. Adv Immunol 1986; 39:177-253. 2 Laitinen LA, Heino M, Laitinen A, Kava T. Haahtela T. Damage to the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599606. 3 Weller PF, Lee CW, Foster DW, Corey EJ, Austen KF, Lewis RA. Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of LTC4. Proc Natl Acad Sci U.S.A. 1983; 80:7626-30. 4 Shaw RJ, Walsh GM, Cromwell O, Moqbei R, Spry CJF, Kay AB. Activated human eosinophils generate SRS-A leukotrienes following IgG-dependent stimulation. Nature 1985; 316:150-2. 5 Pincus SH. Hydrogen peroxide release from eosinophiis: quantitative, comparative studies of human and guinea pig eosinophils. J Invest Dermatol 1983; 80:278-8L 6 Spry CJF. Synthesis and secretion of eosinophil granule substances, immunoi Today 1985; 6:332-5. 7 Anwar ARE, Smithers SR, Kay AB. Killing of schistosomula of Sctiisto.soma mamoni coated with antibody and/or complement by human leucocytes in vitro: requirement for complement in preferential killing by eosinophils. J Immunol 1979; 112:628-37. 8 Hogg N. Human mononuclear phagocyte molecules and the use of monoclonal antibodies in their detection. Clin Exp Immunol 1987; 69:687-94.

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9 Anderson AL, Looney RJ. Human leukocyte IgG Fc receptors. Immunol Today 1986; 7:264-6. 10 Springer TA, Anderson DA. The importance of the Mac~l, LFA-1 giycoprotein family in monocyte and granulocyte adherence, chemotaxis and migration into inflammatory sites: insights from an experiment of nature. In: Evened D, Nugent J, O'Connor M, eds. Biochemistry of Macrophages. London: Pitman, 1980:102-26. 11 Ross GD, Cain JA, Lachmann PJ. Membrane complement receptor type three (CR3) has lectin-likc properties analogous to bovine conglutinin and functions as a receptor for zymosan and rabbit erythrocytes as wel! as a receptor for iC3b. J Immunol 1985; 134:3307-15. 12 Myones BL, Diazell JG. Hogg N, Ross GD. Neulrophil and monocyte cell surface pi 50,95 has iC3b-rcccptor (CR4) activity resembling CR3. J Clin Invest 1988; 82:640-51. 13 Dustin ML, Rothlein R, Bhan AK, Dinarello CA. Springer TS. Induction by IL-1 and interferon-gamma: tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-1). J Immunol 1986; 137:245-54. 14 Bass DA, Grover WH. Lewis JC, Szejda P, DeChatelet LR. McCall CE. Comparison of eosinophils from normals and patients with eosinophilia. j Clin Invest 1980; 66:1558-64. 15 PincusSH,SchoolcyWR. DiNapoli M, Brodcr S. Metabolic heterogeneity of eosinophils from normal and hypcreosinophilic patients. Blood 1981; 58:1175-81. 16 Winqvist L Olofsson T, Majpersson A, Hallbcrg T. Altered density, metabohsm and surface receptors of eosinophils in eosinophiha. Immunology 1982; 47:531-9. 17 Anwar ARE, Kay AB. Membrane receptors for IgG and complement (C4, C3b and C3d) on human eosinophils and neutrophils and their relation to eosinophilia. J Immunol 1977; 119:976-82. 18 Fischer E, Capron M, Prin L, Kusnierz, J-P, Kazatchkine M D. Human eosinophils express CR I and CR3 complement receptors for the cleavage fragments of C3. Cell Immunol 1986; 97:297-306. 19 Capron M, Kazatchkine MD, Fischer E. ei al. Functional role of the alpha-chain of complement receptor type 3 in human eosinophil-dependent antibody-mediated cytotoxicity against schistosomes. J Immunol 1987; 139:2059-65. 20 Kimani G, Tonnescn MG, Henson P. Stimulation of eosinophil adherence to human vascular endothelial cells In vitro by platelet activating factor. J Immunol 1988: 140:3161-6. 21 Hartnell A, Moqbel R, Walsh GM, Bradley B, Kay AB. Fcgamma and CD1I/CD18 receptor expression on normal density and low density human cosinophits. Immunology 1990; 69:264-70. 22 Spry CJF. Eosinophils. Oxford: Oxford Medical Publications, 1988. 23 Frew AJ, Kay AB. The relationship between Infiltrating CD4 lymphocytes, activated eosinophiis and the magnitude of the allergen-induced iate phase cutaneous reaction in man. J Immunol 1988; 141:4158-64. 24 Wardlaw AJ, Moqbel R, Cromwell O, Kay AB. Platcict activating factor. A potent chemotactic and chemokinetic factor for human eosinophiis. J Ciin Invest 1986; 78:1701-6.

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25 Wang JM, Rambaldi A, Biondi A, Chen ZG, Sanderson CJ, Mantovani A. Recombinanl hLiman interleukin-5 is a selective eosinophil chcmoattractant. Eur J Immunol 1989; 19:701 5. 26 Hamid Q. Azzawi M, Moqbel R, Ying S. JetTery PK, Kay .'XB. Expression of mRNA for lnterleukin-5 in mucosal bronchial biopsies from asthma. In: Proceedings of 1990 Meeting of the Collegium Internationale Aliergologicuum. Basel: Kurger, 1990; in press. 27 Yamaguchi Y, Hayashi Y, Sugama Y, ci aL Highly purified murine intcrleukin-5 (IL-5) stimulates eosinophil function ;tnd prolongs/nr/O'o survival. J Exp Med 1988; 176:1737-42. 28 Rothenberg ME, Owen WF, Silberstein DS, el al. Human eosinophils have prolonged survival, enhanced funcliona! properties and become hypodense when exposed to human interleukin'3. J Ciin Invest 1988; 81:1986-92. 29 Lojiez AF, Williamson J, Gamble JR, ei ai Recombinanl human granulocyte/macrophage eolony-stimiilating factor stimulates in vitro mature neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986; 78:1220 8. 30 Clutferbuck EJ, Hirst EMA, Sanderson CJ. Human intcr-

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teukin-5 (IL-5) regulates the produclion of eosinophiis in human bone marrow cultures. Comparison and interaction with IL-1, TL-3, IL-6 and GM-CSF. Blood 1988; 73:150413. Saito H.Flatake K, Dvorak AM, eM/. Selective dilfcrentiation and proliferation of haematopoietic cells induced by recombinant human interleukins. Proc Nail Acad Sci U.S.A. 1988; 85:2288-92. Lopez AF, Sanderson CJ, Gamble JR, Campbell HR. Young IG, Vadas MA. Recombinant human interleukin-5 is a selective activator of human eosinophil function. J Exp Med 1988; 167:219-24, Walsh GM, Hartnell A, Wardlaw AJ, Kurihara K, Sanderson CJ, Kay AB. IL-5 enhances the iu vitro adhesion of human eosinophiis but not neutrophils, in a leucocyte integrin (CDI l/l8)-dependent manticr. Itmnunology 1990; 71:258-65. Lamas AM, Mulroney CM, Sehleimer RP, Studies ofthe adhesive interaction between puriiied human eosinophils and cultured vaseular endothetial eells. J Immunol 1988; 140:1500-5,

Modulation of eosinophil function in vitro.

Clinical and Experimental Allergy. 1990. Voiume 20, Supplement 4, pages 31 -34 Modulation of eosinophil function in vitro A. B. KAY Department of All...
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