Eotaxin Timothy J. Williams* and Ian Sabore Leukocyte Biology Section, Sir Alexander Fleming Building Imperial College School of Medicine, Sir Alexander Fleming Building, London, South Kensington, SW7 2AZ, UK * corresponding author tel: 0207-594-3159, fax: 0207-594-3119, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.11006.
SUMMARY Eosinophil leukocytes accumulate in high numbers in the lungs of asthmatic patients and are believed to be important contributors to the tissue damage and lung dysfunction seen in this disease. Eotaxin was originally discovered as a potent eosinophil chemoattractant present in bronchoalveolar lavage fluid of allergen-challenged/sensitized guinea pigs. Purification and sequencing of the protein revealed it to be a 73 amino acid CC chemokine. Mouse, rat, and human eotaxins have since been discovered, all signaling through a single receptor, CCR3, which is highly expressed on eosinophils, and also on basophils and a subpopulation of TH2-type T lymphocytes. Two other CC chemokine eotaxins, `eotaxin 2' and `eotaxin 3', have recently been discovered which have similar activity, signaling via CCR3. The eotaxins are believed to be important in regulating eosinophil recruitment in the asthmatic lung and in allergic reactions in general. Thus, the eotaxin receptor CCR3 is a prime target for the development of anti-asthma/anti-allergy drugs.
BACKGROUND
Discovery The allergic inflammation of human asthma is characterized by a significant influx of eosinophils into the lung tissue that are thought to be involved in the pathogenesis of the disease (Spry and Tai, 1989; Bousquet et al., 1990; Cieslewicz et al., 1999). Selective eosinophil recruitment is also a feature of other diseases characterized by allergic inflammation, including allergic rhinitis (Durham et al., 1992), atopic
dermatitis (Spergel et al., 1999; Yawalkar et al., 1999), and helminthic infections (Butterworth, 1984; Mochizuki et al., 1998; del Pozo et al., 1999). Indeed, it has been postulated that the inflammatory response of allergic asthma on exposure to inhaled antigens is a misdirection of the same mechanism that is induced as part of the defense against parasitic infection. The phases of selective eosinophil recruitment that occur in allergic reactions in vivo imply the existence of selective endogenous eosinophil chemoattractants. In keeping with this, Schlecht and Schwenker observed in 1912 that sensitized guinea pigs showed allergen-induced bronchoconstriction associated with eosinophil recruitment upon inhaled allergen challenge (Schlecht and Schwenker, 1912). Many subsequent studies using similar models identified roles for lipid-derived mediators such as LTB4 and PAF in eosinophil recruitment; however, none of the mediators described are truly eosinophil selective. New impetus for the search for eosinophil-selective mediators of leukocyte recruitment was provided by the identification of the first chemoattractant cytokines (chemokines), including MIP-1 (Wolpe et al., 1988), RANTES (Schall et al., 1988), and MCP-1 (Yoshimura et al., 1989). These molecules are small proteins (molecular weight 8±10 kDa), with the ability to mediate relatively selective recruitment of inflammatory leukocytes including monocytes and neutrophils. By 1992 the chemokine family had expanded rapidly. Chemokines such as RANTES had activity on eosinophils in addition to other cell types, but no chemokines had been identified that were exclusively active on eosinophils. In the early 1990s, work was undertaken to identify candidate selective eosinophil-recruiting proteins, using a pharmacological approach. Guinea pigs sensitized to ovalbumin were challenged by aerosolized
1216 Timothy J. Williams and Ian Sabore allergen, resulting in immediate bronchoconstriction and an inflammatory infiltrate that developed over the subsequent 6±12 hours. Bronchoalveolar lavage fluid (BALF) was taken from these guinea pigs and purified by high-pressure liquid chromatography (HPLC). Each of the resulting HPLC fractions was injected intradermally into naõÈ ve bioassay animals, which had received intravenous injections of radiolabeled eosinophils. The resulting local intradermal eosinophil recruitment was measured by determining the radioactivity of the skin sites. These studies determined that BALF from allergen-challenged, sensitized guinea pigs contained a single eosinophil-stimulating entity. This activity was purified and identified as a small protein; full-length sequencing revealed a novel chemokine of the CC class. This was named eotaxin, as it induced the selective recruitment of eosinophils in vivo (Griffiths-Johnson et al., 1993; Jose et al., 1994b). The guinea pig cDNA was subsequently cloned using primers based on the protein sequence (Jose et al., 1994a). Based on these studies, eotaxin homologs have been identified using molecular techniques in several species including mouse (Rothenberg et al., 1995a), rat (Williams et al., 1998), and humans (Ponath et al., 1996b). Ongoing work in animal models and in studies of human disease continues to underscore the potential importance of this cytokine in the allergic inflammatory response.
the recruitment of eosinophils in allergic inflammation. In this regard, eotaxin also regulates the release of bone marrow eosinophils, in conjunction with IL-5 (Palframan et al., 1998b). CCR3 is also expressed at high levels on basophils (Uguccioni et al., 1997), and therefore may also regulate their recruitment. Recently, CCR3 expression has been shown on T cells differentiated to a TH2 type phenotype in vitro (Gerber et al., 1997; Sallusto et al., 1997; Bonecchi et al., 1998), and also on mast cells (Romagnani et al., 1999), reinforcing the role of eotaxin as a key regulatory molecule in eosinophilic and allergic inflammation. Eosinophils are predominantly tissue-resident cells, with a potential role in immune surveillance, for example in the defense against parasites. There is increasing evidence that the constitutive expression of this chemokine in mucosal tissues, particularly in the gut, may play a major role in the regulation of basal trafficking of eosinophils (Rothenberg, 1999). CCR3 is also expressed on macrophages and microglia, where it can function as a coreceptor for HIV (Alkhatib et al., 1997). The expression of CCR3 can also be induced on human neutrophils by treatment with IFN , but the functional significance of this is not clear (Bonecchi et al., 1999).
Alternative names
Accession numbers
There are no alternative names for eotaxin.
Human eotaxin gene: U46572, Z92709 Human eotaxin cDNA: U34780, NM002986, U46572, D49372, Z75668, Z75669, Z69291, U46573 Mouse eotaxin cDNA: U40672, U77462, U26426 Brown Norway rat eotaxin cDNA: Y08358, U96637 Guinea pig eotaxin cDNA: U18941, X77603
Structure Human eotaxin is a chemokine of the CC class, comprising 74 amino acids and with a molecular weight of 8.3 kDa.
Main activities and pathophysiological roles Eotaxin was originally identified as a selective stimulator of eosinophil recruitment in vivo. Unlike most chemokines, it signals through one receptor alone (CCR3), although it may also function as an antagonist for CXCR3 (Weng et al., 1998). Thus, the principle actions of eotaxin are to date defined by the distribution of CCR3. This receptor is highly expressed on eosinophils (Ponath et al., 1996a), and eotaxin is therefore thought to play a major role in
GENE AND GENE REGULATION
Chromosome location The eotaxin gene has been localized to chromosome 17 q21.1-21.2 (Garcia-Zepeda et al., 1997).
Relevant linkages The CC chemokine gene cluster on chromosome 17 q11 has been linked with atopy (Nickel et al., 1999). Considerable variations in the 30 UTR of the eotaxin gene have been identified (Nickel et al., 1999); however, direct linkages of eotaxin gene polymorphisms to asthma have yet to be identified.
Eotaxin 1217
Regulatory sites and corresponding transcription factors Potential binding sites for the following transcription factors have been identified in the eotaxin gene: SP-1, CF1, E2A, NFB, gIRE, GRE, GM-CSF, AP-2, CK-2, NF-IL6/CEBP, IRF-1, AP-1, and PEA3 (Garcia-Zepeda et al., 1997). Hein et al. identified a very similar pattern of transcription factors with potential binding sites in the gene: SP-1, CF1, E2A, NFB-(like), gIRE, GRE, GM-CSF, AP-2, TCF1, PEA3, AP-1, NF-IL6/CEBP, GATA, and AP-3 (Hein et al., 1997).
Cells and tissues that express the gene Numerous cell types have been shown to express eotaxin. The most important sources of this chemokine in vivo have yet to be unequivocally determined, but the respiratory epithelium and endothelium are probably major sources of eotaxin in asthma. Eotaxin expression has been identified in many tissues including the heart, intestines, lungs, testes, thymus, and kidney. Expression has been shown in lymphoid tissues in Hodgkin's lymphoma, in nasal polyposis, and in experimental autoimmune thyroiditis. The cells and lines expressing eotaxin described to date are: epithelial cells, endothelial cells, smooth muscle, cardiac muscle, eosinophils, dermal fibroblasts, macrophages, Reed±Sternberg cells (in Hodgkin's lymphoma), A549 cells, and BEAS 2B cells.
JC4912, 2208449A, CAA99997, CAA99998, CAA93258, AAA98957 Mouse eotaxin: P48298, AAA99776 Brown Norway rat eotaxin: P97545, CAA69645, AAB65775, JC2478 Guinea pig eotaxin: AAC52180, P80325, I48099, CAA54698
Sequence See Figure 1.
Description of protein CC chemokines such as eotaxin contain four highly conserved cysteine residues (see alignment in Figure 1). The two N-terminal cysteine residues are adjacent to each other, and bonded by disulfide bridges to the third and fourth cysteines in the distal end of the molecule. In common with many other chemokines, the molecule readily forms dimers, but it is unclear whether the monomer or dimer is the active form in vivo.
Discussion of crystal structure
PROTEIN
The NMR structure of eotaxin has been resolved recently (Crump et al., 1998). In solution, eotaxin exists in equilibrium between monomeric and dimeric forms. In common with other CC chemokines, the structure is that of a three-stranded -pleated sheet with an overlying helix. However, in contrast to RANTES and MCP-1 whose N-termini are very structured, that of eotaxin is considerably less structured.
Accession numbers
Important homologies
Human eotaxin: AAC50369, NP002977, P51671, 2EOT, 1EOT, AAC51297, BAA08370, CAB07027,
Two other CC chemokines that show the same receptor selectivity as eotaxin have been identified in
Figure 1 Protein sequences of human, mouse, guinea pig, and rat eotaxin. Note that the mature protein sequences and signal peptides are shown. Amino acids conserved within all four species are indicated in bold.
1218 Timothy J. Williams and Ian Sabore humans, and named eotaxin 2 (Forssmann et al., 1997; Patel et al., 1997; White et al., 1997) and eotaxin 3 (Kitaura et al., 1999; Shinkai et al., 1999). Despite the close functional similarities of these proteins, their levels of similarity at the protein level are very low. Based on the mature protein sequences, the similarity between eotaxin and eotaxin 2 is 37.5% and between eotaxin and eotaxin 3 is 42%.
Posttranslational modifications Eotaxin proteins are formed with a signal peptide that is removed by proteolytic cleavage. There is also evidence that eotaxin is glycosylated, and that this glycosylation may modify function. Eotaxin was originally purified from the BALF of allergen-challenged, sensitized guinea pigs. On reverse-phase HPLC, biologically active protein was present in three closely related fractions, each giving a single band of different molecular mass on SDS-PAGE. These eotaxin mass variants were in keeping with Oglycosylation variants (Jose et al., 1994b). Ongoing unpublished research has shown that natural glycosylated and synthetic guinea pig eotaxin show similar activities in vitro, but different activities in vivo (L. Bodman, unpublished data). Further studies will characterize the importance of these sugar groups in modifying the biological activity of eotaxin.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce The following cell types produce eotaxin: epithelial cells, endothelial cells, smooth muscle, cardiac muscle, eosinophils, dermal fibroblasts, mast cells, macrophages, Reed±Sternberg cells (in Hodgkin's lymphoma), A549 cells, and BEAS 2B cells.
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Eotaxin expression in vivo in animal models and human asthma is induced by allergic stimuli including allergen challenge (Griffiths-Johnson et al., 1993; Jose et al., 1994a, 1994b; Rothenberg et al., 1995b; Gonzalo et al., 1996a; Humbles et al., 1997; Lamkhioued et al., 1997; Li et al., 1997; Ying et al.,
1997; Brown et al., 1998; Taha et al., 1999). Following allergen challenge, airway epithelial cells and macrophages are significant sources of eotaxin (Humbles et al., 1997; Li et al., 1997; Cook et al., 1998; Taha et al., 1999), as are the infiltrating eosinophils themselves (Gauvreau et al., 1999). Similarly, eotaxin expression in allergic rhinitis is predominantly evident in epithelial cells and macrophages, and is upregulated by local allergen challenge (Minshall et al., 1997). In the guinea pig, respiratory viral infection also induces eotaxin expression, suggesting a link with viral-induced exacerbations of asthma (Scheerens et al., 1999). Studies of allergic lung disease defined a regulatory role for T lymphocytes in eotaxin generation (MacLean et al., 1996). This may be explained by the recent findings that IL-4 and IL-13 have significant roles in the induction of bronchial hyperresponsiveness in allergic lung inflammation, in part through their ability to induce eotaxin generation from cells including fibroblasts (Mochizuki et al., 1998; Li et al., 1999; Zhu et al., 1999). Generation of eotaxin has also been observed as part of the phenomenon of IL-4induced tumor suppression (Rothenberg et al., 1995a). In a mouse model of atopic dermatitis, IL-4 and IL-5 also regulated local eotaxin generation (Spergel et al., 1999), and eotaxin expression is also upregulated in human atopic dermatitis (Yawalkar et al., 1999). Ozone inhalation induces lung expression of eotaxin in rats (Ishi et al., 1998). Eotaxin generation is also observed in intestinal parasitic infection (del Pozo et al., 1999), and in response to onchocercal larvae killed in vivo by diethylcarbamazine treatment (Pearlman et al., 1999). Various infective stimuli are associated with eotaxin generation in lymph nodes (Tedla et al., 1999), and in the fibroblasts, smooth muscle cells, and Reed±Sternberg cells of lymph nodes involved by Hodgkin's disease (TeruyaFeldstein et al., 1999). The expression of eotaxin by pulmonary epithelial cells is enhanced in vitro by TNF and IL-1 <, particularly in combination with IFN stimulation, and suppressed by dexamethasone (Lilly et al., 1997). In nasal polyposis, local generation of eotaxin is inhibited by dexamethasone (Jahnsen et al., 1999). A similar pattern of cytokine regulation of eotaxin generation is seen in airway smooth muscle, although in these cells IFN does not appear to enhance cytokineinduced eotaxin generation (Chung et al., 1999; Ghaffar et al., 1999). Indeed, in some studies eotaxin generation by fibroblasts is stimulated by TNF, but potently inhibited by IFN (Miyamasu et al., 1999). TNF, C5a, and ionomycin also induce eotaxin generation by eosinophils (Nakajima et al., 1998; Han et al., 1999), and TNF-induced eosinophil eotaxin
Eotaxin 1219 expression is downregulated by IL-5 (Han et al., 1999). Human mast cells can also be induced to secrete eotaxin by stimulation with stem cell factor (SCF) coating cell culture plates in vitro, and also by SCF presented on the surface of primary lung fibroblasts (Hogaboam et al., 1998).
RECEPTOR UTILIZATION Eotaxin is unusual among CC chemokines in that it exhibits stimulatory effects through one receptor alone, CCR3 (Daugherty et al., 1996; Ponath et al., 1996a; Heath et al., 1997; Sabroe et al., 1999). Eotaxin has also been reported to bind to, but not stimulate, CXCR3, and may act as a natural antagonist of this receptor (Weng et al., 1998).
IN VITRO ACTIVITIES
In vitro findings The cellular targets of eotaxin are highly restricted by the exclusive expression of its sole receptor, CCR3. Table 1 illustrates the ranges of activities described on CCR3-expressing cells. A recent report has also shown that eotaxin may stimulate chemotaxis, but not degranulation, in human mast cells (Romagnani et al., 1999).
Regulatory molecules: Inhibitors and enhancers Those molecules regulating the expression of eotaxin are listed in Table 1. To date, relatively little is known about the regulation of eotaxin actions on its target cells. There is evidence that the expression of the eotaxin receptor, CCR3, is regulated on eosinophiliclike HL-60 cells by IL-5, a cytokine that is known to prime eosinophil responses to chemokines including IL-8 (Sehmi et al., 1992). IL-5 also enhances the ability of eotaxin to induce chemotaxis of eosinophils through basement membrane in the form of matrigel (Okada et al., 1997). We have shown that IL-5 can potentiate eosinophil shape change induced by eotaxin (I. Sabroe, unpublished data). We have also shown that MIP-1 can potentiate the ability of eotaxin to upregulate CD11b expression on eosinophils isolated from normal human subjects (I. Sabroe, unpublished data).
Bioassays used Actin polymerization, shape change, chemotaxis, Ca2 mobilization, adhesion molecule upregulation (flow cytometry), adhesion assays, CCR3 internalization, superoxide generation, leukotriene generation, histamine release, enzyme release (degranulation/ exocytosis), apoptosis. Chemokines are predominantly associated with the regulation of spatial positioning of leukocytes. As such, chemokines may mediate leukocyte release from the bone marrow, recruitment into tissues, and positioning of these leukocytes within the tissue. Eotaxin stimulates many signaling pathways involved in the regulation of eosinophil trafficking, including actin polymerization (Tenscher et al., 1996), shape change (Sabroe et al., 1999), chemotaxis (Ponath et al., 1996b; Heath et al., 1997), and the stimulation of adhesion to activated cell monolayers (Burke-Gaffney and Hellewell, 1996; Hohki et al., 1997). Adhesion to bronchial epithelial cells may also be relevant in the eosinophil-induced epithelial damage seen in asthma, and this too can be stimulated by eotaxin (BurkeGaffney and Hellewell, 1998). Eotaxin has also been shown to stimulate eosinophil respiratory burst (Elsner et al., 1996) and superoxide generation (Tenscher et al., 1996). Similarly, in basophils eotaxin is a potent stimulator of basophil chemotaxis, and is also able to induce basophil histamine release and leukotriene generation (Uguccioni et al., 1997).
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles There is increasing evidence that eotaxin plays a major role in both the acute allergic inflammatory response and also in the regulation of the basal trafficking of eosinophils into mucosal sites such as the lung and intestine. Constitutive expression of eotaxin has been described in the guinea pig lung and human airway smooth muscle cells (Jose et al., 1994a; Rothenberg et al., 1995b; Humbles et al., 1997; Li et al., 1997; Ghaffar et al., 1999) and the lamina propria of the mouse jejunum (Matthews et al., 1998; Mishra et al., 1999). In keeping with this, eosinophils are present in the normal guinea pig lung (Humbles et al., 1997), and eotaxin-deficient mice showed reduced levels of eosinophils in the jejunum and thymus in comparison with normal wild-type controls
Ranges of activities described on CCR3-expressing cells
Action of eotaxin
Target cell type Eosinophil
Basophil
TH2-type T cell
Macrophage or microglial cell
Neutrophil
Chemotaxis
Yes (Ponath et al., 1996b)
Yes (Uguccioni et al., 1997)
Yes (Jinquan et al., 1999)
Unknown
Yes (after IFN treatment) (Bonecchi et al. 1999)
Shape change
Yes (Sabroe et al., 1999)
Yes (Heinemann and Sabroe, unpublished data)
Unknown
Not tested
No (in neutrophils not treated with IFN ) (Sabroe et al., 1999)
Actin polymerization
Yes (Tenscher et al., 1996)
Unknown
Unknown
Unknown
Unknown for IFN -treated cells
Ca2 mobilization
Yes (Tenscher et al., 1996; Sabroe et al., 1999)
Unknown
Unknown
Unknown
Unknown for IFN -treated cells
Oxygen free radical generation
Yes (Tenscher et al., 1996); but possibly more effective as a chemoattractant than cell activator
Unknown
Unlikely
Unknown
Unknown for IFN -treated cells
Respiratory burst
Yes (Elsner et al., 1996)
Probably (Uguccioni et al., 1997)
Not applicable
Unknown
Unknown for IFN -treated cells
Degranulation
Unknown
Yes (Uguccioni et al., 1997)
Not applicable
Not applicable
Unknown for IFN -treated cells
Leukotriene generation
Unknown
Yes (Uguccioni et al., 1997)
Not applicable
Not applicable
Unknown for IFN -treated cells
Cytokine generation
No to MCP-1 (Izumi et al., 1997)
Differing reports (Devouassoux et al., 1999; Ochensberger et al., 1999)
Unknown
Unknown
Unknown for IFN -treated cells
Adhesion molecule upregulation
Yes (Tenscher et al., 1996)
Unknown
Unknown
Unknown
Unknown for IFN -treated cells
CCR3 internalization
Yes (Zimmermann et al., 1999)
Unknown
Unknown
Unknown
Unknown for IFN -treated cells
Antagonism of HIV entry
Probably (Choe et al., 1996)
Unknown
Probably
Yes (Alkhatib et al., 1997; He et al., 1997)
Unknown
1220 Timothy J. Williams and Ian Sabore
Table 1
Eotaxin 1221 (Matthews et al., 1998). These studies are consistent with a significant role for eotaxin in the basal trafficking of eosinophils to mucosal sites that interface with the external environment (Rothenberg, 1999). However, many body tissues including the heart constitutively express eotaxin mRNA (Rothenberg et al., 1995a; Kitaura et al., 1996) despite having no detectable constitutive eosinophil content. Therefore, although eotaxin plays a role in the basal trafficking of eosinophils from blood to tissues, other factors such as local adhesion molecule expression presumably also play an important role. There is now overwhelming evidence that eotaxin plays a role in the recruitment of eosinophils at sites of allergic inflammation. The first description of eotaxin was as a protein that stimulated eosinophil recruitment in a guinea pig model of allergic airways inflammation (Jose et al., 1994b). Subsequent studies in the same model demonstrated that all the eosinophil-stimulating chemokine activity in BALF from allergen-challenged guinea pigs could be neutralized by an anti-eotaxin antibody (Humbles et al., 1997). In the same model, eosinophil recruitment into the lung paralleled the generation of eotaxin, and eosinophil dispersal from the lung tissues to the airways paralleled a decrease in tissue eotaxin with preservation of eotaxin levels in the BALF (Humbles et al., 1997). Several mouse models of allergic inflammation have also shown a role for eotaxin in eosinophil recruitment into the lung and skin (Gonzalo et al., 1996a, 1996b; Teixeira et al., 1997; Campbell et al., 1998). The guinea pig eotaxin receptor has also been cloned and blocking antibodies generated for use in vivo (Sabroe et al., 1998). It is to be hoped that these reagents will further define the role of the eotaxin/CCR3 pathway in vivo. Eotaxin also has a further and probably important role in the regulation of tissue eosinophilia. Collins et al. demonstrated a dual role for eotaxin and IL-5 in eosinophil recruitment (Collins et al., 1995), whereby levels of local eosinophil recruitment in response to eotaxin were dependent upon the blood eosinophilia, which was regulated by IL-5. Using a novel assay system, Palframan et al. showed that perfusion of the guinea pig hindlimb with eotaxin resulted in the release of eosinophils from the femoral bone marrow (Palframan et al., 1998a, 1998b). In this respect, eotaxin acted in synergy with IL-5 (Palframan et al., 1998a, 1998b). In allergic inflammation, it is therefore likely that eotaxin has both local effects on eosinophil recruitment and distant effects on eosinophil release from the bone marrow. Unlike IL-5, eotaxin does not increase eosinophil survival (Wedi et al., 1998).
Species differences The data available to date support a central role for eotaxin in eosinophil recruitment in guinea pig, mouse, rat, and human. There are variations in chemokine receptor expression on eosinophils between species: although all species express a CCR3 homolog, there are marked variations in the level of expression of CCR1. This receptor is present on mouse eosinophils (Post et al., 1995), and is variably present on human eosinophils (Daugherty et al., 1996; Ponath et al., 1996a; Sabroe et al., 1999). There are variations in the ability of the eotaxins to activate CCR3 on other species: guinea pig eotaxin stimulates human eosinophils (Jose et al., 1994b), but not vice versa. Intradermal injection of human and murine eotaxin in the rat induces eosinophil accumulation (Kudlacz et al., 1999), and this eosinophil accumulation can be suppressed by antibodies to 1 integrins, 2 integrins, ICAM-1, and VCAM-1 (Sanz et al., 1998). Human RANTES also acts as an antagonist on the guinea pig CCR3 receptor in vitro and in vivo (Marleau et al., 1996).
Knockout mouse phenotypes Mouse eotaxin knockouts have been generated, and have revealed differing results. In one eotaxin knockout strain, eosinophil recruitment at sites of pulmonary allergen challenge was reduced (Rothenberg et al., 1997), whereas in another knockout strain, eosinophil recruitment was unaffected (Yang et al., 1998). However, mouse eosinophils express two receptors that are capable of mediating eosinophil recruitment (CCR1 and CCR3), which may in part explain these differences (Post et al., 1995). Preliminary data from CCR3 knockout mice have been presented (A. A. Humbles and C. J. Gerard, `Chemokines and Chemokine Receptors', Keystone Symposia 1999), and further data are eagerly awaited.
Interactions with cytokine network The principle interactions of eotaxin with the cytokine network are detailed above. IL-4 and IL-13, principal TH2 cytokines generated in allergic inflammation, are probably important in regulating eotaxin generation. IL-5 and eotaxin are both crucial factors for eosinophil recruitment in allergic inflammation, through their synergistic actions on eosinophil release from the bone marrow and the effects of IL-5 on the priming of eosinophil function and prolongation of
1222 Timothy J. Williams and Ian Sabore eosinophil lifespan in tissues. The actions of eotaxin on eosinophils may be modulated by other chemokines, such as RANTES which induces a prolonged internalization of the CCR3 receptor (Zimmermann et al., 1999), and MCP-4 that has, as yet poorly characterized, potentially inhibitory effects on eosinophil function (Sabroe et al., 1999). Furthermore, a role has been postulated for chemokines such as MIP1 to act in concert with eotaxin in the regulation of eosinophil function (I. Sabroe, unpublished data).
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects Normal levels of eotaxin in tissues or circulation remain to be defined. It is possible that, as in animal models, eotaxin may play a role in the regulation of basal eosinophil trafficking.
Role in experiments of nature and disease states In man, eotaxin expression is upregulated in the asthmatic lung, both at baseline and after allergen challenge (Lamkhioued et al., 1997; Ying et al., 1997; Brown et al., 1998; Gauvreau et al., 1999; Zeibecoglou et al., 1999). Recent data have also shown that exacerbations of mild asthma may be correlated with increased levels of eotaxin in the plasma (Lilly et al., 1999). Eotaxin expression has also been observed at other sites of disease where eosinophil recruitment is observed, such as ulcerative colitis (Garcia-Zepeda et al., 1996). In a well-described and characterized model of allergic inflammation in response to intradermal allergen, eotaxin generation by cells including macrophages and endothelial cells was associated with the early (6 hour) accumulation of eosinophils (Ying et al., 1999). These data tie in well with the time course of eotaxin-induced eosinophil recruitment in the guinea pig lung (Humbles et al., 1997). However, the later accumulation of eosinophils and the accumulation of basophils in the human intradermal allergen model was not associated with eotaxin expression, but was (at least for eosinophils) correlated with the local production of eotaxin 2 and MCP-4 (Ying et al., 1999).
Eotaxin expression has also been shown in blister fluid following local allergen challenge (Fernvik et al., 1999), and atopic dermatitis (Yawalkar et al., 1999), and in eosinophilic myeloproliferation and lymphoproliferative disorders, such as Hodgkin's disease (Jundt et al., 1999; Teruya-Feldstein et al., 1999). A role for eotaxin in the recruitment of eosinophils to sites of parasitic infection has also been described (del Pozo et al., 1999; Pearlman et al., 1999). As previously noted, eotaxin is active on other cell types important in allergic inflammation, most notably basophils (Uguccioni et al., 1997) and TH2type T cells (Sallusto et al., 1997; Bonecchi et al., 1998). T cells infiltrating human tissue in the company of eosinophils express CCR3 (Gerber et al., 1997). Thus the expression of eotaxin may control many aspects of the inflammatory process through the recruitment of regulatory T cells secreting cytokines including IL-4, IL-5, and IL-13, as well as the recruitment of effector inflammatory leukocytes such as eosinophils and basophils. At present, eotaxin measurements are not sufficiently standardized to be utilized as a diagnostic aid or measurement of the severity of allergic inflammation. There are numerous diseases other than asthma characterized by eosinophilic inflammation, including the vasculitic disease Churg Strauss syndrome and the hypereosinophilic syndrome. It will be interesting to determine if eotaxin levels correlate with eosinophil recruitment or severity of inflammation in these conditions. Rejection of transplanted organs may also be associated with an eosinophilic infiltrate, and this is a further area that is worthy of exploration (Le Moine et al., 1999).
IN THERAPY
Preclinical ± How does it affect disease models in animals? Antagonism of the eosinophil eotaxin receptor, CCR3, has been described using chemokines and chemokine analogs. Human RANTES is an effective antagonist of guinea pig CCR3 and inhibits guinea pig eosinophil recruitment in response to eotaxin administered intradermally (Marleau et al., 1996). In the mouse, met-RANTES also acts as an antagonist of CCR3 (although in this respect it is not selective) (Elsner et al., 1997), and inhibits eosinophil recruitment in response to intradermal allergen (Teixeira et al., 1997). Antibodies to eotaxin have been shown to partially neutralize eosinophil recruitment and inhibit bronchial hyperreactivity (BHR) in a mouse
Eotaxin 1223 model of allergic airways inflammation (Campbell et al., 1998; Gonzalo et al., 1998). However, the role of eosinophils in the generation of BHR remains controversial. Many groups have demonstrated a significant dependence upon eosinophils for the generation of BHR following allergen challenge or viral infections (Flavahan et al., 1988; Cieslewicz et al., 1999; Hisada et al., 1999; Schwarze et al., 1999), but this is not a universal finding (Corry et al., 1996).
Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. Antagonists of eotaxin actions have yet to be described in man, although at least one small molecule antagonist of CCR1 and CCR3 has been described in the patent literature, compound J113863 published by BANYU Pharmaceutical Company, patent W098/ 04554.
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LICENSED PRODUCTS See Table 2. This list is not intended to be a definitive statement of all reagents available.
Table 2 Licensed products Reagent
Use
Supplier
Human eotaxin
In vitro studies
Peprotech EC Ltd (www.peprotech.com) Gryphon Labs (www.gryphonsci.com) Research Diagnostics Inc. (www.researchd.com) Leinco Technologies (www.leinco.com)
Mouse eotaxin
In vitro and in vivo studies
Peprotech EC Ltd (www.peprotech.com) R&D Systems (www.rndsystems.com) Research Diagnostics Inc. (www.researchd.com)
Guinea pig eotaxin
In vitro and in vivo studies
Gryphon Labs (www.gryphonsci.com)
Anti-human eotaxin antibodies
In vitro studies
Peprotech EC Ltd (www.peprotech.com) R&D Systems (www.rndsystems.com) Leinco Technologies (www.leinco.com) Research Diagnostics Inc. (www.researchd.com)
Anti-mouse eotaxin antibodies
In vitro and in vivo studies
R&D Systems (www.rndsystems.com) Leinco Technologies (www.leinco.com)
Mouse eotaxin ELISA
Quantitation of eotaxin generation
R&D Systems (www.rndsystems.com)
Anti-human CCR3 antibody
Blockade of eotaxin actions in vitro
R&D Systems (www.rndsystems.com)
Radiolabeled human eotaxin
Receptor expression and affinities
Amersham (www.apbiotech.com)