Besides their microbicidal functions, human beta-defensins (hBD) and LL-37 activate different immune and inflammatory cells, and their expression is enhanced in inflamed skin and cutaneous wound sites. To protect against pathogens, the skin produces antimicrobial peptides including hBDs and LL-37. Therefore, the aim of our study was to investigate whether hBDs participate in cutaneous inflammation and wound healing by inducing keratinocyte migration, proliferation, and production of proinflammatory cytokines/chemokines. We found that hBD-2, -3, and -4 but not hBD-1 stimulated human keratinocytes to increase their gene expression and protein production of IL-6, IL-10, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-3alpha, and RANTES. This stimulatory effect was markedly suppressed by pertussis toxin and U-73122, inhibitors for G protein and phospholipase C, respectively. We also demonstrated that hBDs elicited intracellular Ca2+ mobilization, and increased keratinocyte migration, and proliferation. In addition, these peptides induced phosphorylation of EGFR, signal transducer and activator of transcription (STAT)1, and STAT3, which are intracellular signaling molecules involved in keratinocyte migration and proliferation. In our study, inhibition of these molecules significantly reduced hBD-mediated keratinocyte migration and proliferation. In conclusion, this study provides evidence that human antimicrobial peptides may be involved in skin immunity through stimulating cytokine/chemokine production, and participate in wound healing by promoting keratinocyte migration and proliferation.
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.
In addition to their microbiocidal properties, human β-defensins (hBDs) and cathelicidin LL-37 stimulate a number of mammalian cell activities, including migration, proliferation, and cytokine/chemokine production. Because hBDs and LL-37 cause mast cells to release pruritogens such as histamine and PGs, we hypothesized that these peptides would stimulate the secretion of a novel pruritogenic mediator IL-31, predominantly produced by T cells. hBDs and LL-37 enhanced IL-31 gene expression and IL-31 protein production and release in the human mast cell line LAD2, as well as in peripheral blood-derived cultured mast cells, suggesting that mast cells are another source of IL-31. Moreover, the expression of IL-31 was elevated in psoriatic skin mast cells, and hBD-2–4 and LL-37, but not hBD-1, enhanced its expression in vivo in rat skin mast cells. hBDs and LL-37 also induced the release of other pruritogenic mediators, including IL-2, IL-4, IL-6, GM-CSF, nerve growth factor, PGE2, and leukotriene C4, and increased mRNA expression of substance P. hBD– and LL-37–mediated IL-31 production/release was markedly reduced by pertussis toxin and wortmannin, inhibitors of G-protein and PI3K, respectively. As evidenced by the inhibitory effects of MAPK-specific inhibitors, hBD-2–4 and LL-37 activated the phosphorylation of MAPKs p38, ERK, and JNK that were required for IL-31 production and release. The ability of hBDs and LL-37 to stimulate the production and release of IL-31 by human mast cells provides a novel mechanism by which skin-derived antimicrobial peptides/proteins may contribute to inflammatory reactions and suggests a central role of these peptides in the pathogenesis of skin disorders.
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