PERSPECTIVE
Eotaxins
Contributing to the Diversity of Eosinophil Recruitment and Activation

Christianne Bandeira-Melo, Anne Herbst, and Peter F. Weller

Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Eosinophils are cellular hallmarks of allergic inflammation, and investigations to delineate the mechanisms responsible for eosinophil recruitment and activation have been important for understanding the pathogenesis of allergic diseases. Since the early finding that anaphylactically-challenged guinea pig lungs released an eosinophil chemoattractant activity (then termed, eosinophil chemotactic factor of anaphylaxis) (1), the search has been on to identify the primary chemoattractant agent with specificity for eosinophils. Over the years many agents have been identified that exhibit eosinophil chemoattractant activity (2), but more recently interest has focused on the chemokine, eotaxin. Eotaxin was first identified as the dominant eosinophil-chemoattractant in a guinea pig model of allergic airway inflammation (3), promoting both local eosinophil recruitment to the lung and, in cooperation with interleukin (IL)-5, rapid mobilization of a pool of bone marrow-retained, matured eosinophils (4). Subsequently, human eotaxin has been cloned, and its synthesis (mRNA and protein) demonstrated in allergic diseases, including asthma (5).

Chemokines are members of a very large superfamily of homologous small (8 to 10 kDa) peptides that typically regulate the chemoattraction of leukocytes. Chemokines are subdivided into families based on the relative positions of their first two cysteine residues. The CC (or ) chemokines have their cysteines located adjacent to each other and, in general, are chemoattractants not for neutrophils but for eosinophils, basophils, monocytes, and lymphocytes. Human eotaxin, an 8.4 kDa CC chemokine, is not the only CC chemokine with preferential activities on eosinophils. More recently, two additional new human eosinophil selective CC chemokines have been described and named eotaxin-2 (8) and eotaxin-3 (9, 10). Strikingly, in contrast to their similar functional chemoattractant activities, the overall sequence identities of these three members of eotaxin subfamily are only ~ 34-38%. On the other hand, eotaxin-1 (the initially recognized eotaxin) has higher sequence identity (> 60% amino acid sequence identity) with the CC chemokine subfamily of monocyte chemoattractant proteins (MCPs), that have no selectivity toward eosinophils, preferentially acting on monocytes and lymphocytes. For instance, eotaxin-1 has 65% identity with MCP-1, which binds and activates the receptors CCR2 and CCR4, but not CCR3, the CC chemokine receptor that mediates the relative specificity of eotaxin subfamily members for eosinophils (6).

The identification of three eotaxins, each acting via CCR3 receptors, raises the issue of what are the differential roles of these functionally analogous CC chemokines. One could posit that they might be differentially expressed perhaps in different tissues, such as the lung or the gut, to mediate organ preferential attraction of eosinophils (for review, see [11]). The study by Berkman and coworkers (53), however, provides clear evidence that, at least at the mRNA level, both eotaxin-1 and eotaxin-2 may be coexpressed in human lungs of nonchallenged asthmatic patients, and eotaxin-3 may further be expressed following allergen challenge.

In assessing the potential roles of the three eotaxins, their capacities not only to recruit eosinophils but also to activate their cellular responses within tissue sites of chemokine formation should be considered. High affinity binding of the eotaxins to pertussis-inhibitable Gi protein-coupled CCR3 receptors triggers a transient influx of Ca⁺⁺ followed rapidly by actin polymerization and shape change (12). Subsequent CCR3-elicited signaling for chemotaxis involves phosphorylation and activation of tyrosine kinases (16) and the MAP kinases, ERK1/2 and p38 (15). In addition to chemoattraction, eotaxins are potent activators of eosinophil effector functions, and all currently recognized effector responses (excepting ligand-induced internalization of CCR3) elicited by the eotaxins are mediated via CCR3, Gi coupled-responses including Ca⁺⁺ influx (Figure 1). Recognized effector responses and known signaling pathways elicited in eosinophils are summarized below:

View larger version (74K): [in this window] [in a new window]   Figure 1.   Schematic representation of eotaxin-induced, CCR3-mediated eosinophil activation. Binding of eotaxins to CCR3 receptors expressed on the eosinophil's surface causes: chemotaxis preceded by actin polymerization and cell shape change; production of reactive oxygen molecules; degranulation of ECP and EDN; LTC4 formation at lipid bodies; and vesicular transport-mediated release of preformed IL-4. These eosinophil effector responses could be blocked by CCR3 receptor antagonists (e.g., Met-RANTES) and monoclonal antibodies to CCR3 and were initially triggered by common signaling steps, including activation of Gi protein and a rapid influx of Ca++. Subsequent activation of distinct kinases has been identified to each phenomenon. In addition, eotaxin binding to CCR3 can inhibit cell infection by HIV-1 and induces ligand-dependent receptor internalization, whose signaling pathways are completely unknown but independent on Gi activation or Ca++ influx. Definition of abbreviations: 5-lypoxigenase, 5-LO; eosinophil cationic protein, ECP; eosinophil-derived neurotoxin, EDN; extracellular signal-regulated kinase, ERK; phosphatidylinositol 3-kinase, PI3; protein kinase C, PKC; pertussis toxin, PTX.

Respiratory burst. Both eotaxin-1 and eotaxin-2 elicit the production of reactive oxygen species in human eosinophils with potencies similar to optimal concentrations of C5a and greater than MCP-3, MCP-4, and RANTES (12, 14). Eotaxin-induced production of reactive oxygen species was blocked by staurosporin, genistein, and wortmannin, indicating the involvement of protein kinase C (PKC), tyrosine kinase(s), and phosphoinositide-3 kinase (PI3K) activation (13), respectively, likely in accord with their roles in the activation of NADPH oxidase.

LTC4 production and lipid body formation. Recently, we established that the CC chemokines, eotaxin-1, and RANTES, activate eosinophils and basophils for enhanced leukotriene C₄ (LTC₄) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles (17). Eotaxin-1-induced lipid body formation and enhanced LTC₄ release were both mediated by CCR3 receptor Gi protein-linked downstream signaling and by activation of PI3K and ERK1/2 and p38 MAP kinases. Eotaxin-1-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC₄ production. Likewise, as we now show, both eotaxin-2 and eotaxin-3, also correlatively induce eosinophil lipid body formation and enhanced production of LTC₄ (Figure 2). With specific immunolocalization of intracellular LTC₄, lipid bodies were the predominant sites of LTC₄ synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils (17). Therefore, CCR3 ligand-initiated signaling via PI3K and MAP kinases both elicits the formation of lipid body organelles and promotes LTC₄ formation at these specific extranuclear sites.

View larger version (33K): [in this window] [in a new window]   Figure 2.   Lipid body formation (A) and priming for LTC4 production (B) by eotaxin-stimulated human eosinophils. Eosinophils were incubated with indicated eotaxins for 1 h for induction of lipid bodies. Thereafter, eosinophils were incubated with 0.1 µM A23187 for 15 min for LTC4 production. To normalize for differences in baseline LTC4 release between different donors, results are presented as percent increase in the LTC4 released by A23187-stimulated cells. Results are mean ± SD from five independent assays. *P < 0.05 and **P < 0.01 compared with negative controls.

Release of eosinophil granule cationic proteins. Studies of the effects of eotaxin-1 on eosinophil degranulation, based on analyses of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) release detectable in eosinophil supernatant fluids, have yielded varying results. In some reports, physiological concentrations of eotaxin-1 were unable to elicit detectable release of these granule proteins (18, 19). On the other hand, high concentrations (> 20 nM) of eotaxin-1 have induced release of eosinophil granule-derived proteins, that depended on (a) CCR3-mediated signaling and (b) activation of ERK2 and p38 MAP kinases (20). Further studies are needed to clarify whether more sensitive techniques are able to detect release induced by physiological concentrations of eotaxin and to analyze the mechanisms of release, discriminating between exocytosis and piecemeal degranulation.

Vesicular-transport mediated release of preformed, granule-derived IL-4. Eotaxin-1 has been identified as a physiological stimulus to elicit release of preformed IL-4 stored within human eosinophil granules (23). In contrast to the cytolytic release of IL-4 from calcium ionophore-activated eosinophils, eotaxin-1, and RANTES, but not IL-8 or interferon-, elicited IL-4 release by noncytotoxic mechanisms. With a dual antibody capture and detection immunofluorescent microscopic assay, IL-4 was released at discrete spots close to cell surface (23). IL-5 enhanced eotaxin-induced IL-4 release, which was mediated by Gi-protein-coupled CCR3 receptors, detectable as early as 5 min and maximum within 1 h. IL-4 release was not diminished by transcription or protein synthesis inhibitors, but was suppressed by brefeldin A, an inhibitor of vesicle formation. Thus, CCR3-mediated signaling can rapidly mobilize IL-4 stored preformed in human eosinophils for release by vesicular transport to contribute to immune responses (23). Moreover, two specific inhibitors of the 5-lipoxygenase pathway, AA861 and MK886, inhibited eotaxin-induced IL-4 release indicating that eosinophil-derived 5-lipoxygenase metabolites, potentially cysteinyl leukotrienes, whose formation is also induced by eotaxin-1, might act as signaling molecules in mediating eotaxin-induced piecemeal degranulation.

Inhibition of infection by HIV-1. Members of the chemokine receptor family, mainly CCR5 but also CCR3, play critical roles in early events in HIV-1 infection, acting as coreceptors with CD4 (24). Eosinophils, besides their high expression of CCR3, also express CD4 (25) and are infectable by HIV-1 (26, 27). Like RANTES, eotaxin-1 inhibits CCR3-mediated HIV-1 infection (28), implicating CCR3 ligands as endogenous regulators of HIV transmission.

CCR3 internalization. Ligand-induced internalization of chemokine receptors (e.g., CCR1, CCR5, CXCR2, and CXCR4) is well documented. Likewise, CCR3 ligands, including eotaxin-1 and RANTES, induce CCR3 receptor internalization from eosinophil plasma membranes to an intracellular endocytic compartment. Ligand-induced CCR3 internalization involves molecular events that are dissociated from signaling involved in triggering eosinophil activation, since it is not dependent on Gi-protein coupling, intracellular Ca⁺⁺ transients or PKC activity (29). Down-regulation of CCR3 activation by eotaxins might be germane to the control not only of eosinophil infiltration into target tissues, but also to the regulation of CCR3-mediated activation of eosinophil effector responses.

Other potential regulators of CCR3-mediated activation elicitable by the eotaxins include: (a) Impairment of eotaxin expression and release. Systemic glucocorticoids have prominent antiinflammatory effects and are effective drugs when used in the treatment of eosinophil-related inflammatory diseases. Moreover, consensus sequences known to interact with glucocorticoid response elements have been identified at the human eotaxin gene (30), indicating that steroids may function as regulators of eotaxin gene expression. In agreement, many studies have been showing potent inhibitory effects of steroids on the production of eotaxins both by in vitro human cultured cells (e.g., lung epithelial and airway smooth muscle cells) (31, 32) and in different human tissues (e.g., nasal polyps and nasal mucosa of allergic rhinitis) (33, 34). (b) Degradation of eotaxin. Proteolytic processing by endogenous enzymes has been identified as a key regulator of chemokine activity. The membrane-associated serine protease dipeptidyl peptidase IV (DPP IV), also named CD26, can cleave dipeptides from proteins containing NH₂-terminus with a proline or alanine residue in the penultimate position. A recent report showed that eotaxin-1 belongs to a increasing group of CC chemokines that is terminally truncated by DPP IV/CD26 (35). This structural alteration resulted in impairment of eotaxin stimulatory activity. (c) Antagonism of CCR3. Because of the increasing number of CCR3 agonists and the consequent compensatory processes involved, direct inhibition of CCR3 molecules, rather than inhibition of the ligands, has emerged as an attractive therapeutical strategy for eosinophil-related diseases. Several kinds of CCR3 inhibitors/antagonists have been developed. In addition to neutralizing antibodies raised to CCR3, NH₂-terminus truncated RANTES analogues (e.g., Met-RANTES) have also been reported as potent antagonists of CCR3. Although Met-RANTES is more effective in preventing eosinophil recruitment in vivo in inflammatory models of allergy (36), it has been shown that Met-RANTES can inhibit eotaxin-elicited human eosinophil effector functions, such as Ca⁺⁺ influx, actin polymerization, chemotaxis, and release of reactive oxygen species (37). More recently, synthetic nonpeptides molecules have been developed as very selective CCR3 antagosinsts, being able to inhibit Ca⁺⁺ influx, shape change, and chemotaxis induced by eotaxin-1 and eotaxin-2 (38, 39). (d) Endogenous antagonism of CCR3. A pair of recent reports has revealed a new group of potential regulators of CCR3 activation. Nibbs and coworkers (40) showed that the CC chemokine MIP-4, known as a T cell chemoattractant with unknown receptor, exhibits CCR3 antagonistic activity by blocking eotaxin-induced eosinophil activation. Similarly, Loetscher and coworkers (41) showed that a group of CXC chemokine agonists of the CXCR3 receptor, including ITAC, Mig, and IP10, functions as antagonists for CCR3. These CXC chemokines compete for the binding of eotaxin to CCR3 and inhibit eotaxin- or eotaxin-2-induced Ca²⁺ influx and chemotaxis. Interestingly, CXCR3 is expressed preferentially on Th1 cells and therefore ITAC, Mig, and IP10 that chemoattracts commonly Th1 cells via CXCR3 can, on the other hand, impair the activation of CCR3-positive Th2 response-related cells in response to eotaxins, reinforcing the Th1 polarization. Even more interesting is the new evidence that questions eotaxin selectivity for CCR3. Analogous to the binding abilities of CXCR3 ligands on CCR3, low affinity binding of eotaxin has been shown on CXCR3 receptors (42, 43). This interaction between eotaxin and CXCR3 does not trigger, but rather blocks, IP10-mediated receptor activation in CXCR3 transfected cells. Surprisingly, opposite results were observed in eosinophils. Jinquan and coworkers (44) showed that human eosinophils could express surface CXCR3 that could be activated by IP10 and Mig stimulation, promoting Ca⁺⁺ influx, chemotaxis, and ECP release. But these breaking developments were not restricted to CXCR3 receptors. A recent study demonstrated that eotaxin can bind to CCR2 and CCR5 (45), causing respectively opposite effectsantagonistic on CCR2 and agonistic in CCR5. Therefore, the former selective CCR3 ligands named eotaxins may be natural CXCR3 and CCR2 antagonists and CCR5 agonists at least.

Although we have focused on the three eotaxins' activities on eosinophils mediated by their binding to CCR3, it must be recalled that other chemokines, including MCP-3, MCP-4, and RANTES, also signal via CCR3 but are not CCR3 selective and can signal via additional receptors. Moreover, CCR3 expression is not restricted to eosinophils. CCR3 initially found on eosinophils, basophils (46), and Th2 cells (47) is now being found on various and diverse other cell types including mast cells (48), astrocytes (49), airway epithelial cells (50), B cells in Hodgkin's disease lymph nodes (51), and others. Adding to this complexity is a still rudimentary understanding of the hierarchical cross talk and regulation that occurs amongst signaling pathways engaged by chemokine and related G-protein linked receptors. Finally, the "Holy Grail" of finding the single eosinophil chemoattractant agent pertinent to allergic inflammation becomes less likely. The mast cell-derived prostanoid, prostaglandin D2, has recently been identified as another potent chemoattractant for Th2 cells, basophils, and eosinophils (52). Thus, the recruitment of eosinophils to sites of allergic inflammation will utilize a combinatorial series of interactions of multitude potential chemoattractants and cell adherence mechanisms. Analogously, regulation of the activation of tissue eosinophils will also be dependent on multiple effects and interactions amongst agonists and antagonists.

	Footnotes

Address correspondence to: Peter F. Weller, M.D., Beth Israel Deaconess Medical Center, DA-617, 330 Brookline Avenue, Boston, MA 02215. E-mail: pweller{at}caregroup.harvard.edu

(Received in original form April 20, 2001).

Acknowledgments: Anne Herbst was funded by the "Studienstiftung des deutschen Volkes" (Germany). The authors thank Mojabeng Phoofolo for excellent technical assistance. Supported by NIH AI20241, AI22571, HL56386, and AI 41995.

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