title: Specific binding and chemotactic activity of mBD4 and its functional orthologue hBD2 to CCR6-expressing cells.
abstract: Beta-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG(1) to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these beta-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the beta-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the beta-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and beta-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the beta-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific beta-defensin receptor interactions.
authors.affiliation: Institute of Immunology, University of Regensburg, Regensburg D-93042, Germany.
authors.forename: Johann
authors.initials: J
authors.lastname: Röhrl
authors.forename: De
authors.initials: D
authors.lastname: Yang
authors.forename: Joost J
authors.initials: JJ
authors.lastname: Oppenheim
authors.forename: Thomas
authors.initials: T
authors.lastname: Hehlgans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D002460:Cell Line; D054418:Chemokine CCL20 / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis / Q000502:physiology*; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D054399:Receptors, CCR6 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; C493447:CCL20 protein, mouse; D054418:Chemokine CCL20; C106641:DEFB4A protein, human; C513975:Defb4 protein, mouse; D007136:Immunoglobulins; D054399:Receptors, CCR6; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins