Chemokines and IL‐5: major players of eosinophil recruitment in asthma (1999)

It has long been recognized that eosinophils play a major role in the pathogenesis of bronchial asthma. These cells are considered to cause tissue damage leading to disruption of the bronchial epithelium, enhanced bronchial responsiveness, and airway obstruction. Recruitment of eosinophils at the allergic inflammatory site involves a series of events including rolling, adhesion to endothelial cells, transendothelial migration and subsequent chemotactic movement. The first three steps of eosinophil recruitment are mediated by a different set of adhesion molecules involved in various specific time-points while the last step is predominantly regulated by the local production of chemoattractants. In this regard a number chemotactic factors including phospholipids (e.g. PAF) and leucotrienes (e.g. LTB4) have been associated with allergic disease [ 1]. However, these chemoattractants are rapidly metabolized or taken up by the surrounding cells and their involvement in asthma is not well understood. There is now plenty of evidence to suggest that chemokines and IL-5 play a prominent role in eosinophil recruitment. Chemokines are a small group of chemotactic cytokines with molecular weights in the range of 8–10 kDa. These cytokines have been divided into four subfamilies on the basis of the position of either one or two cysteine residues located near the amino terminus of the protein (CXC, CC, C and CXXXC subfamilies) [ 2]. Among the chemokines, RANTES, MCP-3, MCP-4, eotaxin and eotaxin-2, members of the CC subfamily, have attracted most interest in allergic inflammation [ 3] ( Fig. 1). RANTES, MCP-3 and MCP-4 are chemotactic for monocytes, lymphocytes and eosinophils, while eotaxin and eotaxin-2 are predominantly chemotactic for eosinophils, basophils and lymphocytes of the TH2 type. . Eosinophil recruitment into asthmatic airways is mediated by the local release of both chemokines and interleukin-5 (IL-5). Analyses of bronchoalveolar (BAL) fluid of asthmatic subjects exposed to endobronchial allergen challenge using high performance liquid chromatography has allowed RANTES to be identified as a major eosinophil attractant [ 4]. Interestingly, concentrations of BAL RANTES as low as 0.5 ng/mL were able to induce eosinophil chemotaxis and concentrations of this chemokine correlated with eosinophil numbers in BAL fluid suggesting this cytokine may play a major role in the recruitment of eosinophils into asthmatic airways. Moreover, RANTES inmunoreactivity has been localized to endothelial vessel cells in nasal polyp biopsies derived from atopic and nonatopic subjects [ 5]. MCP-3 and MCP-4 are also potent eosinophil attractants and they have been implicated in the allergic asthmatic reaction. These cytokines have the ability to induce histamine release from IL-3-primed basophils, however, compared with MCP-1, both MCP-3 and MCP-4 are slightly less potent basophil activators. Increased mRNA expression for MCP-3 and MCP-4 has been reported on bronchial biopsies derived from asthmatics [ 6] and on nasal biopsies from patients suffering allergic and nonallergic sinusitis [ 7]. MCP-3 mRNA expression has been predominantly localized to the submucosa while MCP-4 mRNA expression was mostly confined to the epithelium, and to a less extent the submucosa. MCP-4 mRNA expression has also been found in BAL cells from asthmatic and normal subjects. However, there was no significant difference between these two groups [ 8]. Eosinophil chemotaxis assays have failed to detect biologically active MCP-3 and MCP-4 in BAL fluid suggesting that these chemokines may be released in low concentrations into the airway epithelial lining fluid of asthmatic subjects [ 4]. However, as chemokines bind to heparan sulphate proteoglycans present on the extracellular matrix, the possibility cannot be excluded that these chemokines could not be washed efficiently from the bronchial tissue during the BAL procedure. Eotaxin and eotaxin-2 are potent eosinophil attractants which are functionally similar but structurally different. They are only 39% identical at amino acid level. Eotaxin was identified in the BAL fluid of ovalbumin-sensitized guinea pigs [ 9]. Subsequently, the gene encoding human eotaxin was described and found to exhibit 58% identity with guinea pig eotaxin [ 10]. Compared with MCP-3 and MCP-4, eotaxin shows 58% and 57.7% amino acid homology, respectively. Eotaxin has also been reported to be a chemoattractant for basophils and lymphocytes of the TH2 subset. Both increased eotaxin mRNA expression and elevated levels of eotaxin protein have been reported in the airways of asthmatic patients [ 11, 12]. Endobronchial allergen challenge in asthmatics further induces eotaxin release in BAL fluid. However, this cytokine is released in lower concentrations than RANTES. Eotaxin-2 is a novel eosinophil attractant also known as CKβ6 and MPIF-2 [ 13, 14]. Like eotaxin, eotaxin-2 is also chemotactic for basophils and induces both histamine and LTC4 release from IL-3-primed basophils. Intradermal injection of eotaxin-2 in the rhesus monkey induces eosinophil recruitment at the injection site [ 14]. While it is suspected that eotaxin-2 may be involved in the recruitment of eosinophils that characterizes bronchial asthma, this remains to be shown. Another chemokine which has been implicated in the allergic asthmatic reaction is interleukin-8. This cytokine is predominantly chemotactic for neutrophils and it has also been reported to chemoattract eosinophils from atopic, but not from normal subjects [ 15]. Similarly, long-term IL-5-primed eosinophils migrate in response to IL-8 [ 16]. While these in vitro studies are informative, in vivo studies have failed to support these findings [ 4, 17]. For example, bronchoscopy studies in asthmatics have shown that bronchoscopy with BAL induce IL-8 release and neutrophil but not eosinophil recruitment [ 17]. In contrast, allergen challenge induces eosinophil recruitment which is associated with RANTES release but not with IL-8 [ 4]. Moreover, analysis of BAL fluid by HPLC showed that the BAL fraction containing IL-8 was chemotactic for neutrophils, but not for eosinophil derived from atopic subjects [ 4]. These in vivo findings questions the role of IL-8 as a eosinophil attractant in asthma. Interleukin-5 is a well-recognized mediator of eosinophil recruitment. The in vitro effects of IL-5 on eosinophils include chemoattraction, transendothelial migration, induction of superoxide production, degranulation, and prolongation of eosinophil survival [ 18]. Evidence for an in vivo role of IL-5 as a eosinophil-specific cytokine derives from a study showing that transgenic mice expressing the IL-5 gene exhibit significant eosinophilia which can be inhibited by the administration of an antibody to the IL-5 receptor [ 19]. Similarly, administration of neutralizing antibodies to IL-5 to presensitized guinea pigs, suppresses eosinophil accumulation and hyperreactivity when these animals are exposed to the appropriate allergen. Interleukin-5 mRNA expression has been found to be upregulated on bronchial biopsies of asthmatic subjects, and elevated levels of IL-5 protein has been detected in the BAL fluid asthmatic patients. Recently, this group has investigated the kinetics of release of IL-5 and demonstrated that levels of this cytokine increase significantly 4 h after allergen challenge, reaching maximal concentrations at 24 h [ 20]. In contrast, chemokines such as RANTES, MIP-1α and MCP-1 peak at 4 h in BAL fluid and concentrations of these cytokines return towards baseline values at 24 h [ 21]. These findings suggest that both, chemokines and IL-5 may recruit eosinophils in concert into the asthmatic airways [ 20]. In this issue of Clinical and Experimental Allergy, Lampinen, Rak and Venge have examined the role of IL-5, IL-8 and RANTES as eosinophil attractants in the BAL fluid derived from patients with birch pollen allergy [ 22]. This study shows increased eosinophil chemotactic activity (ECA) in the BAL fluid obtained during season when compared with that collected out of season. The use of anti-IL-5, anti-IL-8 and anti-RANTES MoAbs substantially inhibited the BAL-induced ECA. To evaluate the contribution of each of the cytokines, authors combined IL-5, IL-8 and RANTES and performed chemotaxis assays. While either anti-IL-5 or anti-RANTES blocked the eosinophil migration of eosinophils derived from both allergic and normal donors the anti-IL-8 MoAb significantly inhibited the eosinophil migration of eosinophils derived from allergic but not normal donors. It must be pointed out, however, that the difference in the migratory response of eosinophils derived from both groups was minor (≈ 82 vs ≈ 76 [buffer ≈ 98]) suggesting the IL-8 is only a weak attractant for eosinophils from allergic patients. Consistent with previous reports, authors showed that recombinant IL-8 was chemotactic for eosinophils derived from allergic subjects but not from normal subjects. In this study the priming effects of IL-5 was also studied (eosinophils were incubated with IL-5 over 30 min). However, IL-5 did not prime eosinophils to respond to either IL-8 or RANTES. Heath et al. have previously shown that short-term IL-5-primed eosinophils do not migrate in response to IL-8 [ 16], while long-term primed eosinophils show a good chemotactic response. Two independent groups have previously shown that priming eosinophils with IL-5 predominantly induces nonspecific eosinophil migration (chemokinesis) but does not enhance the eosinophil chemotactic response to either RANTES or eotaxin [ 16, 23]. Interleukin-5 is a weak eosinophil attractant. However, in this study IL-5 was found to be the most potent attractant followed by RANTES. This finding could be explain by the fact that platelets release RANTES (major cellular source of this cytokine [ 24]) during the eosinophil isolation procedure which in turn may desensitize these cells to migrate to RANTES in the chemotaxis assays. To evaluate desensitization of eosinophils to this cytokine, Schroeder et al. use other attractants as positive controls (i.e. PAF) [ 4, 24]. In this way it can be easily observed that while eosinophils migrate poorly in response to RANTES these cells usually show a good chemotactic response to PAF. The demonstration that both eosinophil-activating chemokines and IL-5 are implicated in the eosinophil recruitment that characterizes the asthmatic reaction make these cytokines an attractive target for the generation of new anti-asthma drugs (Fig. 1). Indeed, the endobronchial allergen challenge studies described in this editorial clearly show that chemokines predominantly recruit eosinophils during the early phase of the late asthmatic response (4–8 h) while IL-5 undertakes this task at later stage (> 24 h). Chemokines activate leucocytes by binding to selective, G-protein-coupled receptors present on different cell types while IL-5 bind the human IL-5 receptor present on eosinophil which consists of a unique alpha subunit (hIL-5R alpha) and a common beta subunit (betac). The chemokine receptor CCR3 is the most highly expressed in eosinophils (19 000–30 000 receptors) and to lower extent CCR1 [ 25] (CCR3 is also expressed in both basophils and only small fraction of T cell of the TH2 phenotype). Both, eotaxin and eotaxin-2 specifically activate eosinophils via the CCR3 receptor, while the other eosinophil-activating chemokines in addition to CCR3 use other chemokine receptors: RANTES acts on CCR1 and CCR3, MCP-3 acts on CCR1, CCR2 and CCR3 and MCP-4 acts on CCR1, CCR2 and CCR3. Interestingly, pretreatment of eosinophils with a specific anti-CCR3 MoAb completely blocked the eosinophil chemotactic response to a number of the eosinophil-activating chemokines including RANTES, MCP-2, MCP-3 MCP-4 and eotaxin [ 16]. Taken together these observations suggest that the development of drugs to neutralize the effects of either chemokines or IL-5 on eosinophils might represent a novel therapeutic approach in asthma. The author is grateful to Dr Gustavo Reyes-Terán for designing the figure presented in this editorial; to both The National Asthma Campaign and The Alexander von Humbolt Foundation for financial support (grants).