Eotaxin

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