Summary: The 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 bell‐shaped, reaching an optimal concentration of 5 µg/ml. In addition, checkerboard analysis showed that cell migration towards this peptide was chemotactic rather than chemokinetic. Moreover, Scatchard analysis using ¹²⁵I‐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.

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. An antibacterial cathelicidin, human cationic antibacterial protein of 18 kDa/LL-37, not only exhibits potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also functions as a chemoattractant for immune cells, including neutrophils. During bacterial infections, the life span of neutrophils is regulated by various pathogen- and host-derived substances. In this study, to further evaluate the role of LL-37 in innate immunity, we investigated the action of LL-37 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, LL-37 dose dependently (0.01–5 μg/ml) suppressed neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/2, expression of Bcl-x_L (an antiapoptotic protein), and inhibition of caspase 3 activity. Interestingly, LL-37-induced suppression of neutrophil apoptosis was attenuated by the antagonists for formyl-peptide receptor-like 1 (FPRL1) and P2X₇ nucleotide receptor. Of importance, the agonists for FPRL1 and P2X₇ apparently suppressed neutrophil apoptosis. Collectively, these observations indicate that LL-37 cannot only kill bacteria, but also modulate (suppress) neutrophil apoptosis via the activation of FPRL1 and P2X₇ in bacterial infections. Suppression of neutrophil apoptosis results in the prolongation of their life span, and may be advantageous for host defense against bacterial invasion.

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.

Mast cells are known to accumulate at the sites of inflammation in response to chemoattractants generated in the local milieu. Since human β‐defensin‐2 (hBD‐2) is generated in several epithelial tissues where mast cells are present and because we have recently reported that this human antibacterial peptide induces mast cell degranulation, we thus hypothesized that hBD‐2 could be a mast cell chemotaxin. Here we report that hBD‐2 directly and specifically induces mast cell migration with an optimal concentration of 3 µg/ml. Checkerboard analysis showed that the migration was more chemotactic rather than chemokinetic. Moreover, Scatchard analysis using ¹²⁵I‐labeled hBD‐2 revealed that mast cells have at least two classes of receptors, high‐ and low‐affinity receptors, for this peptide. Moreover, the competitive binding assay suggested that hBD‐2 is unlikely to utilize CCR6, a functional receptor for hBD‐2‐mediated dendritic and T cell migration, on mast cells. In addition, treatment of mast cells with G protein inhibitor, pertussis toxin, and phospholipase C inhibitor, U‐73122, abolished the cell chemotaxis in response to hBD‐2, indicating that the G protein–phospholipase C signaling pathway is involved in hBD‐2‐induced mast cell activation. Thus, we suggest that hBD‐2, which was originally believed to be involved in innate host defense, may participate in the recruitment of mast cells to inflammation foci.