Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 micrograms/ml and 1 microgram/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 micrograms/ml and 3 micrograms/ml. Moreover, eotaxin-induced [Ca2+]i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca2+]i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 micrograms/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 micrograms/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.

Chemokines play an important role in attracting granulocytes into sites of inflammation. Two chemokine subfamilies differ in their biologic activity for different granulocyte subsets. Whereas CXC chemokines such as interleukin-8 (IL-8) activate predominantly neutrophils, CC chemokines such as RANTES and eotaxin activate predominantly eosinophils. However, controversial results have been published in the past regarding the biologic role of IL-8 in eosinophil activation, particularly in allergic diseases. In this study, we investigated the functional evidence and expression of both IL-8 receptors, CXCR1 and CXCR2, on highly purified human eosinophils. In the first set of experiments, a chemotaxis assay was performed showing that IL-8 did not induce chemotaxis of eosinophils. In addition, and in contrast to neutrophils and lymphocytes, IL-8 did not induce a rapid and transient release of cytosolic free Ca2+([Ca2+]i) in eosinophils, even after preincubation with TH1- and TH2-like cytokines. To investigate whether neutrophil contamination might be responsible for the reported IL-8 effects on eosinophils, neutrophils were added to highly purified eosinophils from the same donor in different concentrations. Interestingly, as little as 5% of neutrophil contamination was sufficient to induce an increase of [Ca2+]iafter stimulation with IL-8. Flow cytometry experiments with monoclonal antibodies against both IL-8 receptors demonstrated no expression of CXCR1 and CXCR2 on eosinophils before or after cytokine activation. Reverse transcriptase-polymerase chain reaction experiments showed that eosinophils, in contrast to neutrophils and lymphocytes, did not express mRNA for CXCR1 and CXCR2. In summary, this study clearly demonstrates that CXCR1 and CXCR2 are not expressed on human eosinophils, even after priming with different bioactive cytokines. Because the CXC chemokine IL-8 did not induce in vitro effects on human eosinophils, IL-8 may also not contribute in vivo to the influx of eosinophil granulocytes into sites of allergic inflammation. Our results suggest that CC chemokines such as eotaxin, eotaxin-2, and MCP-4 are predominant for the activation of eosinophils.

Eosinophils play an important role in allergic and autoimmune diseases. They are activated by distinct chemokines, leading to the immigration into the inflamed tissue, and mediate tissue damage by releasing reactive oxygen species. Recently, eotaxin was found to have the broadest spectrum of activities of all eosinophil-activating CC chemokines. In this study we investigated the effect of the novel CC chemokine, eotaxin-2, on eosinophil effector functions and compared its activity with eotaxin. Using nitrobenzoxadiazole-phallacidin staining and flow cytometry, we show that eotaxin-2 induced rapid and transient actin polymeriza-tion, a prerequisite for cell migration and modulation of the respiratory burst, in eosinophils in the same range of efficacy as observed for eotaxin. Eotaxin-2 induced the release of reac-tive oxygen species in a dose-dependent manner; half maximal and maximal release were found at 50 ng/ml and 500 ng/ml, respectively. Surprisingly, the efficacy of eotaxin-2 was comparable to that of eotaxin and C5a. Release of reactive oxygen species was inhibited by pertussis toxin, indicating the involvement of G i proteins in the signaling of eotaxin-2. Moreover , the anti-CC chemokine receptor 3 (CCR3) monoclonal antibody, 7B11, was able to inhibit transient rise in the cytosolic Ca 2+ concentration and the release of reactive oxygen species following stimulation with eotaxin-2. Therefore, eotaxin-2 represents a potent CC chemokine for human eosinophils activating chemotaxis-related events, such as actin poly-merization, and the respiratory burst via the CCR3. Moreover, the efficacy of eotaxin-2 seems to be in the same range as that of eotaxin which might re-evaluate the recent profile of activity of CC chemokines in the activation of human eosinophils.

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The C3a anaphylatoxin is a potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like Ca2+ transients and the activation of the respiratory burst. These findings support an important role for C3a in diseases typically associated with a peripheral blood or tissue eosinophilia. Synthetic human C3a analogue peptides with variations at the C-terminal effector domain have been evaluated with respect to their binding affinity and signaling potency on human eosinophils. Flow cytometrical analysis and RT-PCR revealed that the C3a receptor is constitutively expressed on human eosinophils. Peptides bearing an N-terminal 9-fluorenylmethoxycarbonyl and the 6-aminohexanoyl motif were the most powerful peptides tested. Amino acid replacements in the conserved C-terminal pentapeptide decreased binding affinity and functional potency substantially. In addition, synthetic C3a analogue peptides induced C3aR internalization, led to transient changes of intracellular Ca2+ concentration, and did release reactive oxygen species in human eosinophils indicating the in vivo relevance of C3a-related sequences. The tripeptide LAR was found to be essential for C3a receptor binding on human eosinophils. Moreover, the putative binding motif of C3a anaphylatoxin is also crucial for the induction of biologic effects in the human system such as changes of intracellular Ca2+ concentration and the release of reactive oxygen species. This study demonstrates that the carboxyl terminus is important for the interaction with the C3aR and the biologic potency of C3a anaphylatoxin in the human system and plays a key role in the activation process of human eosinophils.

Eosinophils play an important role in allergic and autoimmune diseases. They are activated by distinct chemokines, leading to the immigration into the inflamed tissue, and mediate tissue damage by releasing reactive oxygen species. Recently, eotaxin was found to have the broadest spectrum of activities of all eosinophil-activating CC chemokines. In this study we investigated the effect of the novel CC chemokine, eotaxin-2, on eosinophil effector functions and compared its activity with eotaxin. Using nitrobenzoxadiazole-phallacidin staining and flow cytometry, we show that eotaxin-2 induced rapid and transient actin polymerization, a prerequisite for cell migration and modulation of the respiratory burst, in eosinophils in the same range of efficacy as observed for eotaxin. Eotaxin-2 induced the release of reactive oxygen species in a dose-dependent manner; half maximal and maximal release were found at 50 ng/ml and 500 ng/ml, respectively. Surprisingly, the efficacy of eotaxin-2 was comparable to that of eotaxin and C5a. Release of reactive oxygen species was inhibited by pertussis toxin, indicating the involvement of G i proteins in the signaling of eotaxin-2. Moreover, the anti-CC chemokine receptor 3 (CCR3) monoclonal antibody, 7B11, was able to inhibit transient rise in the cytosolic Ca 2+ concentration and the release of reactive oxygen species following stimulation with eotaxin-2. Therefore, eotaxin-2 represents a potent CC chemokine for human eosinophils activating chemotaxis-related events, such as actin polymerization, and the respiratory burst via the CCR3. Moreover, the efficacy of eotaxin-2 seems to be in the same range as that of eotaxin which might re-evaluate the recent profile of activity of CC chemokines in the activation of human eosinophils.

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine–induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met–RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP–3 and eotaxin was investigated. Met–RANTES had no intrinsic activity on [Ca²⁺]_i transients in eosinophils and was able to dose–dependently inhibit [Ca²⁺]_i transients in eosinophils following stimulation with RANTES, MCP–3 and eotaxin. Besides its effect on [Ca²⁺]_i transients, Met–RANTES dose–dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP–3 and eotaxin. The results of this study lead to the conclusion that Met–RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP–3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.

CC chemokine receptors are expressed on hematopoietic cells, and these may impart selective homing of monocyte, leukocyte, and lymphocyte subsets to sites of inflammation. CC chemokine receptor 3 is the major receptor on eosinophils and is also expressed on other inflammatory cells suggesting its important role for allergic diseases such as atopic dermatitis and bronchial asthma. Eotaxin, eotaxin-2 and eotaxin-3 have been identified as ligands that only activate CC chemokine receptor 3. CC chemokine receptor 3 is also activated by other promiscuous ligands, however, such as RANTES and monocyte chemotactic protein 4. To date, CC chemokine receptor 3 has not been reported to be expressed on nonhematopoietic cells. In this study, we investigated whether keratinocytes possess autocrine and paracrine mechanisms for CC chemokine secretion and receptor expression as reported for the expression of interleukin 8 and its receptors. Reverse transcriptase polymerase chain reaction analysis demonstrated that CC chemokine receptor 3 mRNA is expressed constitutively in cultured keratinocytes. The signal quantities of the CC chemokine receptor 3 amplicons showed lower intensities for keratinocytes than for eosinophils. In situ hybridization techniques exhibited that basal cell layers of the epidermis were stained homogeneously for CC chemokine receptor 3 mRNA with a decreasing signal to the upper epidermis showing that differentiating and proliferating keratinocytes did express mRNA specific for CC chemokine receptor 3. Immunohistochemical studies confirmed low expression of CC chemokine receptor 3 protein on epidermal keratinocytes compared to the high level observed on infiltrating eosinophils. Furthermore, stimulation of cultured keratinocytes with eotaxin resulted in an increased [3H]thymidine incorporation indicating a role of CC chemokine receptor 3 in epidermal proliferation and differentiation. These data demonstrate that CC chemokine receptor 3 is expressed not only on hematopoietic cells but also on keratinocytes as nonhematopoietic cells with ectodermal origin. Therefore, the identification of CC chemokine receptor 3 on epidermal keratinocytes may indicate a role for CC chemokine receptor 3 and its ligands in skin physiology and pathophysiology.