Daniela Kimmig

Characterization of Synthetic C3a Analog Peptides on Human Eosinophils in Comparison to the Native Complement Component C3a

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.

The CC chemokine receptor 3 CCR3 is functionally expressed on eosinophils but not on neutrophils.

The chemokine subclasses differ in their biological activity to stimulate different kinds of effector cells via distinct chemokine receptors. Controversial results about the expression of the CC chemokine receptor CCR3 on the surface of human neutrophils have been described. To find out whether eosinophil contamination might be responsible for these diverse observations, CCR3 expression on highly purified neutrophils and eosinophils was investigated. We enriched neutrophils from a heterogeneous granulocyte population with immunomagnetic beads coated with various anti‐CD52 monoclonal antibodies. This procedure was suitable to enrich neutrophils with a purity of up to 99.85%. Reverse transcriptase‐PCR revealed that CCR3 mRNA was not expressed by CD52‐negative selected neutrophils. In contrast to these cells, CCR3 mRNA could be detected in a heterogeneous granulocyte population and CD16‐negative selected eosinophils. In addition, spectrofluorometric measurement of intracellular calcium concentration ([Ca2+]i) demonstrated that CD52‐negative selected neutrophils did not show a transient [Ca2+]i increase following stimulation with the CCR3 ligand eotaxin, whereas the heterogeneous granulocyte population as well as eosinophils did respond. Therefore, previous studies demonstrating the expression of CCR3 on human neutrophils have to be re‐evaluated because CCR3 mRNA detection on human neutrophils due to contamination by mRNA from eosinophils could not be excluded.

Aminooxypentane-RANTES Induces CCR3 Activation and Internalization of CCR3 from the Surface of Human Eosinophils

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca2+]i) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37°C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.

748 Aminooxypentane-RANTES induces CCR3 activation and internalization of CCR3 fromthe surface of human eosinophils

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca(2+)](i)) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37 degrees C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.