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.

Whereas C5a is a well-established potent activator of eosinophils, the functional role of C3a in the activation of eosinophils is, so far, poorly understood. Here, the activation of human eosinophils stimulated with C3a was analyzed and compared to C5a activation. Flow-cytometrical measurements revealed that stimulation of eosinophils by C3a resulted in a transient elevation of the intracellular calcium concentration ([Ca2+]i) in a dose-dependent manner. In addition, the production of reactive oxygen radical species (ROS) of eosinophils after C3a and C5a stimulation was measured by lucigenin-dependent chemiluminescence and quantified by superoxide dismutase-inhibitable reduction of ferricytochrome C. Half maximal and maximal ROS production in response to C3a was observed at 50 ng/ml and 1000 ng/ml, respectively, whereas C3a-desArg was inactive. To ensure that C3a stimulation was not caused by contamination with C5a, monoclonal antibodies were used to demonstrate the specificity of C3a. The effect of C3a was completely abolished in the presence of monovalent antigen-binding fragments of a functionally blocking anti-C3a monoclonal antibody. In addition, blockade of the C5a receptor by the monoclonal anti-C5a receptor antibody S5/1 totally inhibited the C5a-evoked ROS production, whereas the C3a response in the presence of S5/1 was unaffected. Finally, desensitization experiments revealed a homologous desensitization of C3a after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. Furthermore, the C3a- and C5a-induced production of ROS of eosinophils was totally inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi-proteins). In summary, these results demonstrate that C3a is a potent activator for eosinophils initiating transient [Ca2+]i changes and production of reactive oxygen species. C3a therefore may play a part in the pathophysiology of diseases with eosinophil and complement activation.

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11(3–74). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.

The aim of this review is to give an overview of the role of chemokines, particularly ligands of the CC chemokine receptor CCR3, in allergic diseases and to show the new concept in the treatment of allergies using chemokine receptor antagonists. Allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis are characterized by a complex interaction of different cell types and mediators. Among this, Th2 cells, mast cells, basophils and eosinophils are found in the inflamed tissue due to the attraction of chemokines. Of all the known chemokine receptors, the chemokine receptor CCR3 seems to play the major role in allergic diseases which is supported by the detection of this receptor on the cell types mentioned above. Therefore, academic and industrial research focus on compounds to block this receptor. To date, certain chemokine receptor antagonists derived from peptides and small molecules exist to block the chemokine receptor CCR3. However, the in vivo data about these compounds and the mechanisms of receptor interaction are poorly understood, as yet. For the development of additional chemokine receptor antagonists, more details about the interaction between the ligands and their receptors are required. Therefore, additional studies will lead to the identification of novel CCR3 chemokine receptor antagonists, which can be therapeutically used in allergic asthma, allergic rhinitis, and atopic dermatitis.

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...