PERSPECTIVE
Eotaxin
An Essential Mediator of Eosinophil Trafficking into Mucosal Tissues

Marc E. Rothenberg

Department of Pediatrics, Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Children's Hospital Medical Center, Cincinnati, Ohio

Eosinophil accumulation in the peripheral blood and tissues is a hallmark feature of several important medical diseases, including atopic disorders (allergic rhinitis, asthma, and eczema), parasitic infections, and numerous systemic diseases (e.g., Churg Strauss syndrome, eosinophilic pneumonia, eosinophilic gastroenteritis, and the idiopathic hypereosinophilic syndrome) (1). The findings that eosinophils normally account for only a small percent of circulating or tissue-dwelling cells and that their numbers markedly and selectively increase under specific disease states indicate the existence of molecular mechanisms that regulate the selective generation and accumulation of these leukocytes. The pathologic role of eosinophils primarily occurs in tissues; therefore, a major focus of scientific investigation on eosinophils has been to elucidate the processes involved in eosinophil tissue recruitment. Numerous mediators have been identified as eosinophil chemoattractants, including diverse molecules, such as lipid mediators (platelet-activating factor, leukotrienes), bacterial products (formylmethionyl leucylphenylalanine [FMLP]), and recently, chemokines such as RANTES (regulated on activation, normal T cells expressed and secreted) and macrophage inflammatory protein (MIP)-1 (2). However, none of these mediators selectively promote eosinophil recruitment; they are therefore not considered to be the primary mediators of the tissue eosinophilia observed in numerous hypereosinophilic disorders.

In contrast to these molecules, eotaxin is an eosinophil-selective chemoattractant and therefore has been the subject of recent intensive research. Eotaxin was initially discovered using a biologic assay in guinea pigs designed to identify the molecules responsible for allergen-induced eosinophil accumulation in the lungs. Using an in vivo chemotaxis assay in guinea-pig skin, the partial amino acid sequence for the protein responsible for eosinophil chemoattraction in the bronchoalveolar fluid in allergen-challenged guinea pigs was determined (3). This facilitated the genetic cloning of the genes and complemenatry DNA (cDNA) for guinea pig, murine, and human eotaxin, and the identification of eotaxin as a member of the CC chemokine family most homologous to the macrophage chemoattractant protein (MCP) subfamily (4). This subfamily of eotaxin and MCP chemokines is clustered on human chromosome 17q11, a region clustered with other CC chemokines (such as MIP-1, I-309, RANTES, and HCC-1, -2) (8). Interestingly, this region has been recently linked to asthma susceptibility (9).

Recently, another chemokine, which is active primarily on eosinophils, has been identified, and this chemokine has been termed eotaxin-2 (10). Eotaxin-2 is only distantly related to eotaxin because it is only 39% homologous and is located in a different chromosomal position (7q11). The cloning of these genes and the production of recombinant protein enabled the biologic activity of eotaxin to be examined. Eotaxin was distinguished from all other chemokines because it was found to be a potent eosinophil-selective chemoattractant having minimal activity on other leukocytes. Eotaxin was also identified as a potent activator of eosinophils capable of inducing superoxide generation and the release of granule proteins. Eotaxin was subsequently shown to also be active on human peripheral blood basophils, stimulating chemoattraction in vitro and weak histamine release from IL-3-primed basophils (11). Administration of eotaxin in vivo has been performed in guinea pigs, rodents, and primates, and all of these studies have consistently demonstrated a strong preference of eotaxin for eosinophils in vivo because the eosinophil is the only cell that is recruited. Interestingly, the activity of eotaxin in vivo is enhanced by the copresence of interleukin (IL)-5 (12). IL-5, an eosinophil-selective hematopoietin that regulates eosinophil growth, differentiation, and survival, is a potent stimulus for the release of eosinophils and eosinophil precursors from the bone marrow into the peripheral circulation (13). The increased level of circulating eosinophils and the ability of IL-5 to prime eosinophils to have enhanced responsiveness to eotaxin probably accounts for the synergy between these two cytokines (14). The specific activity of eotaxin is mediated by the selective expression of the eotaxin receptor, CC chemokine receptor-3 (CCR-3), a seven-transmembrane-spanning G-protein-linked genetically polymorphic receptor primarily expressed on eosinophils and basophils (15). The binding of eotaxin to this receptor induces a series of biochemical changes, including activation of Gi proteins, transient increases in intracellular calcium concentration, cytoskeletal rearrangements, activation of the mitogen-activated protein (MAP)-kinase pathway, and rapid and prolonged receptor internalization into an endocytic compartment shared with the transferrin receptor (19, 20). CCR-3 is a promiscuous receptor; it interacts with multiple ligands, including MCP-2, -3, -4, RANTES, and HCC-2; however, the only ligand that signals exclusively through this receptor is eotaxin, accounting for eotaxin's cellular selectivity. Recently, the eotaxin receptor has been found on a subpopulation of T-helper (Th)2 lymphocytes (21). However, the biologic role of eotaxin in the regulation of Th2-cell function and differentiation has not been determined. It is interesting to speculate, however, that eotaxin orchestrates allergic inflammation by coregulating the accumulation of eosinophils, basophils, and Th2 cellsthe cells that are characteristic of allergic inflammation.

A variety of approaches have been used to determine the biologic role of eotaxin in vivo, including gene targeting and antibody neutralization. Eotaxin gene-deficient mice have a selective deficiency of tissue-dwelling eosinophils in the thymus and gastrointestinal tract, the only two nonhematopoietic organs with constitutive levels of eosinophils (22, 23). In contrast to the critical role of constitutive eotaxin, eotaxin-gene-deficient animals have only a partial reduction in allergen-induced eosinophil accumulation in the lung. In one mouse strain, this was limited to the early part of the late-phase response; whereas in another strain, no deficiency was seen (24, 25). Furthermore, eotaxin-deficient animals have normal allergen-induced airway hyperreactivity, indicating that eotaxin is not required for the end organ damage seen in these animal models of asthma.

Using a less specific approach, antibody neutralization studies have been conducted to determine the role of eotaxin in allergen-induced airway inflammation. In one report, a role for eotaxin was identified through repeated allergen challenges; whereas eosinophil recruitment was dependent on MIP-1 after a single allergen challenge (26). In another study, a role for eotaxin in both eosinophil recruitment and the development of airway hyperreactivity was demonstrated (27). It is interesting that in this study, neutralizing antibodies against a series of different chemokines reduced differential leukocyte recruitment and airway hyperreactivity. Taken together, these experiments indicate that allergic airway inflammation in the lung is orchestrated by a complex network of chemokines and cytokines (Figure 1). In the gastrointestinal tract, where eotaxin is constitutively expressed, this chemokine has a nonredundant, critical role in regulating eosinophil levels, indicating a probable role for eotaxin in innate immunity (Figure 2).

View larger version (31K): [in this window] [in a new window]   Figure 1.   Schematic representation of eosinophil recruitment into the lung. Eosinophils develop in the bone marrow where they differentiate from the hematopoietic progenitor cell into mature eosinophils. Factors that control this process have not been fully defined; however, IL-3, IL-5, and GM-CSF are important in eosinophil expansion during conditions of hypereosinophilia. Eosinophil migration from the bone marrow into the circulation is primarily regulated by IL-5. Circulating eosinophils subsequently interact with the endothelium by processes involving rolling, adhesion, and diapedesis. These steps are regulated by eosinophil chemotactic factors and by the upregulation of adhesion molecules on endothelium by proinflammatory cytokines and IL-4. Eosinophils are mobilized into the tissue in response to a chemotactic gradient established primarily by chemokines (e.g., eotaxin, MCPs, RANTES) liberated from respiratory epithelial cells. The chemotactic response is enhanced by IL-5, generated from CD4+ Th2 cells and mast cells. IL-5 is also an important eosinophil cytokine for priming and survival. Additionally, eosinophils have the capacity to generate their own autocrine chemoattractants (eotaxin) and survival factors (e.g., GM-CSF). Depending upon the chemokine concentration gradient, pulmonary eosinophils also transmigrate through the respiratory epithelium and have the potential to degranulate, resulting in epithelial damage.

View larger version (61K): [in this window] [in a new window]   Figure 2.   Schematic representation of eosinophil trafficking in the gastrointestinal tract. Circulating eosinophils (E) adhere to the endothelium through a series of ordered steps involving rolling, adhesion, and diapedesis. This process is stimulated by chemotactic signals generated in the tissue, and which also regulate eosinophil homing. The chemokine eotaxin is predominantly produced by mononuclear cells (M), located in the crypt region. Eotaxin regulates local eosinophil levels. Gastrointestinal eosinophils reside throughout the lamina propria predominantly at the base of the crypt in proximity to crypt cells and Paneth cells (C). Eosinophils also reside in the lamina propria of the villus in proximity to lymphocytes (L).

The signals that modulate the expression of eotaxin have been an active area of research as reported by Han and colleagues in this issue of the Journal (28). Eotaxin has been shown to be an early gene product induced by proinflammatory cytokines in a variety of cell types in vitro. The airway epithelial cell, which is a major source of eotaxin in the respiratory tract, has been shown to express eotaxin messenger RNA (mRNA) and protein within 60 min after exposure to tumor necrosis factor (TNF)-, IL-1, or interferon (IFN)- (6). Furthermore, eotaxin is produced by fibroblasts, and IL-4 appears to be particularly important for eotaxin induction in cutaneous tissue (29). Analysis of the 5' flanking region of eotaxin reveals several regulatory elements that may explain the induction of eotaxin by cytokines and the inhibition of eotoxin by glucocorticoids (30). Of note, the eotaxin promoter in mice and humans has a nuclear factor (NF)-B-binding site, STAT-6-binding elements, IFN- response elements, and a glucocorticoid response element. This may explain the observed regulation of eotaxin by TNF-, IL-4, IFN-, and glucocorticoids, respectively. The expression of eotaxin is not restricted to a Th2 environment because IFN- is a strong inducer of eotaxin, and eotaxin expression is not altered in IL-4-deficient mice (31). The mechanism by which eotaxin mediates an eosinophil-selective response remains to be elucidated since the mere production of eotaxin is not associated with eosinophil accumulation. This dichotomy is best exemplified in the upper gastrointestinal tract (esophagus and tongue) and the heart, which express constitutive eotaxin but have no resident eosinophils. It is interesting to speculate that eosinophil tissue recruitment only occurs under conditions in which Th2 cytokines and eotaxin are coproduced. The recent observation that eotaxin-specific expression and pulmonary eosinophilia is seen only after adoptive transfer of Th2 clones (not Th1 clones) is consistent with this model (32). In addition to being expressed by structural cells (e.g., epithelial cells, fibroblasts, endothelial cells, smooth-muscle cells, and chondrocytes), eotaxin is also produced by infiltrative inflammatory cells. For example, in the healthy respiratory tract, eotaxin is predominantly produced by epithelial cells (33). However, after allergen challenge, the infiltrating macrophages and eosinophils, to a lesser extent, are major sources of eotaxin.

Signals that regulate the expression of eotaxin by leukocytes vary among different cell types. In macrophage cell lines in vitro, proinflammatory cytokines are potent inducers of eotaxin (34); in mast cells, the stem cell factor is a critical inducer (35). In eosinophils themselves, several different cytokines have been shown to influence eotaxin expression. In the present study by Han and associates, eosinophils are demonstrated to constitutively express eotaxin protein (~ 100 pg/10⁷ cells) and mRNA; and IL-5 and TNF- are shown to further induce eotaxin expression (28). IL-5 is paradoxically demonstrated to inhibit TNF-- induced eotaxin. Han and coworkers also demonstrated that dexamethasone inhibits the production of eotaxin mRNA in eosinophils. Taken together with previous studies demonstrating that IL-3 selectively induces the expression of eotaxin in eosinophils (6) and that eosinophils can secrete eotaxin in response to C5a (36), it is apparent there is a complex network of regulatory signals controlling eotaxin expression by eosinophils. It is also interesting to speculate that expression of eotaxin by eosinophils may be a marker for eosinophil activation and may have pathophysiologic consequences because this would allow for auto-amplification of eosinophil recruitment into sites of inflammation. (A summary of eotaxin expression and regulation in vitro and in vivo is presented in Table 1.)

Thus, the identification of two cytokines that synergistically regulate eosinophil accumulation in the peripheral blood and tissues can now offer us a molecular mechanism to help in our understanding of the occurrence of selective eosinophilia in multiple human diseases. Although much progress has been made in the last five years since the initial description of eotaxin, there is still a great deal of knowledge that needs to be further elucidated. It is imperative to determine the relative importance of eotaxin and its related homologues (eotaxin-2 and MCP-2, -3, and -4) in the pathogenesis of human inflammatory diseases. It is also critical to contrast the mechanisms regulating eosinophil accumulation in the human lung with those of the gastrointestinal tract in order to determine if eotaxin has a nonredundant role in either of these mucosal sites. This is particularly important, since an initial trial of a humanized antibody against human IL-5 failed to show the suspected improvement in asthma, indicating that the human lung may use accessory pathways independent of IL-5 for the induction of allergic airway inflammation (37). Multiple pharmaceutical companies have therefore undertaken research programs designed to identify therapeutic agents that will target the eotaxin and CCR-3 pathway. When these agents become available, and when we examine the consequences of genetic polymorphisms in eotaxin and CCR-3, we will be able to determine the relevance of these exciting new findings in regard to human diseases.

	Footnotes

Abbreviations: CC chemokine receptor-3, CCR-3; complementary DNA, cDNA; interleukin, IL; interferon-, IFN-; mitogen-activated protein kinase, MAPK; messenger RNA, mRNA; regulated on activation, normal T cells expressed and secreted, RANTES; T helper 2 cells, Th2; tumor necrosis factor-, TNF-.

(Received in original form July 1, 1999).

Acknowledgments: The author is grateful to Dr. S. P. Hogan for his critical review and graphic assistance and to the numerous colleagues and trainees who have contributed to this perspective.

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