SUMMARYThe mast cell is one of the major effector cells in inflammatory reactions and can be found in most tissues throughout the body. During inflammation, an increase in the number of mast cells in the local milieu occurs, and such accumulation requires directed migration of this cell population. As it has previously been reported that the human cathelicidin-derived antibacterial peptide, LL-37, stimulates the degranulation of mast cells, we hypothesized that LL-37 could be a mast cell chemotaxin. The present study shows that LL-37 is a potent chemotactic factor for mast cells. The chemotactic response was dose-dependent and bellshaped, reaching an optimal concentration of 5 mg/ml. In addition, checkerboard analysis showed that cell migration towards this peptide was chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125 I-labelled LL-37-derived peptide revealed that LL-37 has at least two classes of receptors, namely high-and low-affinity receptors, on mast cells. Furthermore, the competitive binding assay suggested that LL-37 is unlikely to utilize formyl peptide receptor-like 1 (FPRL1), a functional LL-37 receptor for neutrophil and monocyte migration, on mast cells. In addition, the treatment of cells with pertussis toxin and phospholipase C inhibitor, U-73122, inhibited LL-37-mediated migration, indicating that LL-37 induces mast cell chemotaxis through a Gi protein-phospholipase C signalling pathway. These results show that besides its antibacterial activities, LL-37 may have the potential to recruit mast cells to inflammation foci.

Antimicrobial peptides, human β‐defensins (hBD‐1/‐2), and LL‐37 (a peptide of human cathelicidin CAP18) are predominately expressed at epithelial tissues, where they participate in the innate host defense by killing invading microorganisms. In this study, to investigate the interactions between epithelial cell‐derived antimicrobial peptides and mast cells, we evaluated the effects ofhBD‐1/‐2 and LL‐37 on mast cell functions using rat peritoneal mast cells. hBD‐2 and LL‐37 but not hBD‐1 induced histamine release and intracellular Ca2+ mobilization, and hBD‐2 was more potent than LL‐37. Interestingly, histamine release and intracellular Ca2+ mobilization elicited by hBD‐2 and LL‐37 were markedly suppressed by BAPTA‐AM (an intracellular Ca2+ chelating agent), pertussis toxin and U‐73122 (a phospholipase C inhibitor). In addition, among the peptides examined, only hBD‐2 significantly induced PGD2 production, which was abolished by indomethacin (cyclooxygenase‐1/‐2 inhibitor) but not NS‐398 (cyclooxygenase‐2 inhibitor), suggesting that hBD‐2‐induced PGD2 production is mediated by cyclooxygenase‐1. Likewise, the PGD2 production was suppressed by pertussis toxin and U‐73122. These observations suggest that hBD‐2 and LL‐37 stimulate mast cells to mobilize intracellular Ca2+ and release histamine or generate PGD2 in a G protein‐phospholipase C‐dependent manner. Thus, hBD‐2 and LL‐37 may have modulatory effects on inflammatory reactions.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (α-/β-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14+ murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-α mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14+ cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14+ cells (peritoneal macrophages) and suppressed LPS-induced TNF-α expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.

CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-aminoacid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18 104-140. A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18 104-140 and a more active truncated fragment, CAP18 104-135 , were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18 104-140 may have therapeutic utility for conditions associated with elevated concentrations of LPS.

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