Increased numbers of eosinophils are found in parasitic infections, autoimmune diseases and allergic diseases such as allergic asthma. They are activated by distinct cytokines and chemokines leading to the immigration in the inflamed tissue and mediate tissue damage by releasing reactive oxygen species. Here, the effect of the recently cloned CC chemokine human eotaxin was investigated for its ability to affect different eosinophil effector functions and compared to the CC chemokines MCP-3 and RANTES. Human eotaxin induced chemotaxis of human eosinophils in a dose-dependent manner. The range of efficacy of the CC chemokines compared to the well-known chemotaxin C5a was eotaxin = RANTES > MCP-3 = C5a. In addition, eotaxin induced rapid and transient actin polymerization, a prerequisite for cell migration, in eosinophils in the same range of efficacy as observed for chemotaxis. To investigate whether eotaxin was able to activate the respiratory burst of eosinophils, release of reactive oxygen species was measured by lucigenin-dependent chemiluminescence. Eotaxin induced production of significantly high amounts of reactive oxygen species at a concentration between 10 ng/ml and 500 ng/ml. Surprisingly, the effect of eotaxin was comparable to the well-known eosinophil activator C5a. The range of efficacy of the CC chemokines compared to C5a in the activation of the respiratory burst was eotaxin = C5a > MCP-3 > RANTES. Production of reactive oxygen species was inhibited by pertussis toxin, staurosporin, genestein and wortmannin. Furthermore, eotaxin induced transient increases in intracellular calcium concentration ([Ca2+]i) in human eosinophils. Therefore, pertussis toxin-sensitive Gi-proteins, protein kinase C, tyrosine kinase, phosphatidylinositol-3-kinase and transient increases in [Ca2+]i are involved in the signal transduction of eosinophils following stimulation with eotaxin. In summary, this study reveals the importance of the CC chemokine eotaxin as a potent activator of the respiratory burst, actin polymerization and chemotaxis. Eotaxin, therefore, plays an important role not only by attracting eosinophils to the site of inflammation but also by damaging tissue by its capacity to induce the release of reactive oxygen species.

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.Chemokines are chemotactic proteins that play a central role in immune and inflammatory responses by the attraction and activation of leukocytes. They can be divided into two major classes on the basis of the arrangement of the amino acid cysteine in the amino-terminal region: the CXC and CC chemokines, and two minor subclasses, each comprising a single member, the C and CX3C subclasses (1-3). Initially, it was generally accepted that the chemokine subclasses differ in their biological activity to stimulate different kinds of leukocytes, so that CXC chemokines are mediators of acute inflammation through neutrophil activation while the CC chemokines mediate chronic inflammation by attracting leukocytes such as eosinophils, monocytes, lymphocytes, basophils, and dendritic cells. However, this paradigm has recently been shown to have exceptions; for example, CXCR3 is expressed on activated T cells (4) and neutrophils can be activated by CC chemokines following stimulation with interferon-␥ (5).Chemokines mediate their effects by binding to cell-surface receptors that belong to the seven-transmembrane domain G protein-coupled receptor superfamily (1). More recently, chemokine receptors have been subject to intense scrutiny following the discovery that several of them are co-receptors for HIV 1 cell entr...

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.

As many new biologically active chemokines have been cloned exploring the genomic DNA sequence database in the vicinity of already known chemokine sequences without demonstrating their natural origin, it is important to transfer findings from in vitro experiments with chemokines into the in vivo situation. With respect to eosinophils and fibroblasts that play an important part in the pathogenesis of allergic and autoimmune diseases, the role of the recently discovered members of the eotaxin family, eotaxin-2 and eotaxin-3, is not really understood. In order to elucidate the origin and biologic potency of the eotaxin family this study was performed. Conventional reverse transcription-polymerase chain reaction analysis was suitable to detect mRNA for eotaxin and eotaxin-3 but not for eotaxin-2 in dermal fibroblasts. In contrast to conventional reverse transcription-polymerase chain reaction, LightCycler analysis revealed that dermal fibroblasts constitutively expressed mRNA not only for eotaxin and eotaxin-3 but also for eotaxin-2. Moreover, with this technique we investigated mRNA expression levels after stimulation of fibroblasts with interleukin-4 and interleukin-4 plus tumor necrosis factor-alpha: the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. To address the question of the efficacy of eotaxin-3, we compared its activity with eotaxin, eotaxin-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, and RANTES in different test systems for eosinophils. The efficacy of the CC chemokines at equimolar concentrations with respect to the chemotactic response of human eosinophils was eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4. The rank order of activity with respect to actin polymerization and release of toxic reactive oxygen species was eotaxin-3 = eotaxin = eotaxin-2 and eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic protein-4 = RANTES, respectively. This study indicated a distinct profile in expression levels of the members of the eotaxin family in dermal fibroblasts. Indeed, all three eotaxin ligands demonstrated activation of human eosinophils with similar efficacies for chemotaxis, cytoskeletal rearrangements, activation of Gi proteins and transients of [Ca2+]i, but a distinct profile of activity with respect to the binding to CCR3 and the release of toxic reactive oxygen species. These findings may help to understand further the role of CC chemokines in fibroblast/eosinophil activation, which is of interest particularly in allergic and autoimmune 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.

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.