François Niyonsaba

A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis

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.

Human β-defensin-2 functions as a chemotactic agent for tumour necrosis factor-α-treated human neutrophils

Neutrophils are the effector cells in both innate and adaptive immunity, where they perform the functions of phagocytosis and killing of bacteria. They respond to a large number of chemoattractants, but their response to epithelial cell‐derived human β‐defensins (hBD) has not been investigated. Here we report that hBD‐2, but not hBD‐1, is a specific chemoattractant for tumour necrosis factor (TNF)‐α‐treated human neutrophils. The optimal concentration required for maximal chemotactic activity was 5 µg/ml. The effect of hBD‐2 on neutrophils was dependent on the G‐protein‐phospholipase C pathway, as demonstrated by inhibition by pertussis toxin and U‐73122. In addition, ligand‐receptor analysis indicated that the binding of hBD‐2 was markedly inhibited by macrophage inflammatory protein (MIP)‐3α, a specific and unique ligand for CCR6. Furthermore, anti‐CCR6 antibody could almost completely suppress the cell migration induced by hBD‐2, suggesting that hBD‐2 mainly utilizes CCR6 as a functional receptor. Thus, our finding that hBD‐2 is a potent chemoattractant for human neutrophils through specific receptors provides a novel mechanism by which this peptide contributes to the host defence system by recruiting neutrophils to inflammation/infection sites. This also suggests an important link between epithelial cell‐derived antibacterial peptides and neutrophils during infection or inflammation.

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells

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.

The Human β-Defensins (-1, -2, -3, -4) and Cathelicidin LL-37 Induce IL-18 Secretion through p38 and ERK MAPK Activation in Primary Human Keratinocytes

In addition to its physical barrier against invading microorganisms, the skin produces antimicrobial peptides, human β-defensins (hBDs) and cathelicidin LL-37, that participate in the innate host defense. Because IL-18 is produced by keratinocytes and involved in skin diseases in which hBDs and LL-37 are highly expressed, we hypothesized that these peptides would activate keratinocytes to secrete IL-18. We found that hBD-2, -3, and -4 and LL-37, but not hBD-1, activated normal human keratinocytes to secrete IL-18; this secretion reached peak strength at 3 h. In addition, the combination of peptides resulted in a synergistic effect on IL-18 secretion. We also revealed that hBD-2, -3, and -4 and LL-37 increased IL-18 mRNA expression, and that IL-18 secretion was more enhanced in keratinocytes differentiated in vitro with high Ca2+-containing medium. Furthermore, because IL-18 secretion induced by hBDs and LL-37 could not be suppressed by caspase-1 or caspase family inhibitors, and because these peptides failed to increase caspase-1 activity, we suggest that hBD- and LL-37-induced IL-18 secretion is probably via a caspase-1-independent pathway. To determine the molecular mechanism involved, we demonstrated that IL-18 secretion was through p38 and ERK1/2 MAPK pathways, because the inhibitors of p38 and ERK1/2, but not JNK, almost completely nullified IL-18 secretion. Moreover, hBD-2, -3, and -4 and LL-37 could induce the phosphorylation of p38 and ERK1/2, but not JNK. Thus, the ability of hBDs and LL-37 to induce IL-18 secretion by keratinocytes provides a new mechanism for these peptides in innate immunity and an understanding of their role in the pathogenesis of skin disorders.

Antimicrobial Peptides Human β-Defensins and Cathelicidin LL-37 Induce the Secretion of a Pruritogenic Cytokine IL-31 by Human Mast Cells

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.

Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

ABSTRACT Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L1 to S37) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K15 to V32) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q22, D26, and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating production of tumor necrosis factor alpha (TNF-α) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-α production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.

Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human α-defensins from neutrophils

Background  Psoriasis is characterized by epidermal infiltration of neutrophils that destroy invading microorganisms via a potent antimicrobial arsenal of oxidants and antimicrobial agents. In contrast to atopic dermatitis, psoriasis exhibits low levels of skin infections due to the presence of antimicrobial agents, including cathelicidin LL‐37. LL‐37 kills a broad spectrum of microbes, and activates neutrophil chemotaxis.

Antimicrobial peptides human β-defensin (hBD)-3 and hBD-4 activate mast cells and increase skin vascular permeability

Antimicrobial peptides human β‐defensins (hBD) are mainly produced by epithelia of several organs including skin, and participate in innate immunity by killing invading pathogens. Besides their microbicidal activities, hBD activate several inflammatory and immune cells. Since hBD are generated by tissues where mast cells are present, we hypothesized that these peptides could activate mast cells. In this study, we demonstrated that both hBD‐3 and hBD‐4 induced mast cell degranulation, prostaglandin D2 production, intracellular Ca2+ mobilization and chemotaxis. Furthermore, hBD‐3‐ and hBD‐4‐induced activation of mast cells was suppressed by pertussis toxin and U‐73122, inhibitors for G protein and phospholipase C, respectively. We further revealed that hBD‐3 and hBD‐4 increased vascular permeability in the skin, which was dependent on the presence of mast cells, because hBD‐3 and hBD‐4 failed to enhance vascular permeability in mast cell‐deficient Ws/Ws rats. We also demonstrated that hBD‐3 and hBD‐4 induced phosphorylation of MAPK p38 and ERK1/2, which were further required for hBD‐mediated mast cell activation, as evidenced by the inhibitory effects of p38 and ERK1/2 inhibitors on mast cell degranulation. Together, these findings suggest the key role of hBD in inflammatory responses by recruiting and activating mast cells, and increasing vascular permeability.

The extra domain A of fibronectin stimulates murine mast cells via Toll-like receptor 4

The activation of mast cells by extra domain A of fibronectin (FN‐EDA), an endogenous ligand of TLR4, and its contribution to the pathogenesis of rheumatoid arthritis (RA) in vivo were examined. FN‐EDA, but no other domain of the fibronectin fragment, III11 (FN‐III11) and III12 (FN‐III12), stimulated bone marrow‐derived murine mast cells (BMMCs) dose‐dependently to secret cytokines (TNF‐α, IL‐6, and IL‐1β), similar to the pattern produced by LPS. FN‐EDA‐induced cytokine production was mediated by TLR4, as cytokine production by FN‐EDA was absent in TLR4‐deficient (TLR4−/−) BMMCs. We examined the roles of TLR4‐mediated mast cell activation by this form of fibronectin fragment in the pathogenesis of RA in vivo. The injection of FN‐EDA, but not FN‐III11and FN‐III12, to joints resulted in joint swelling of mice in vivo. Genetically mast cell‐deficient WBB6F1‐W/Wv mice exhibited significantly less swelling and cytokine production compared with mast cell‐sufficient +/+ mice, suggesting that swelling and inflammatory cytokine production were partially dependent on tissue mast cells. Reduced swelling and cytokine production were recovered by the reconstitution of tissue mast cells by the injection of BMMCs from wild‐type mice but not from TLR4−/− mice. Altogether, these results suggest that the TLR4‐mediated activation of mast cells by endogenous ligand FN‐EDA might contribute to the pathogenesis of RA through proinflammatory cytokine production.

Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation

As effector cells in host defence, neutrophils actively destroy invading microorganisms via a potent antimicrobial arsenal composed of oxidants and antimicrobial peptides. Psoriasin, an Escherichia coli‐cidal antimicrobial protein, has been found to be overexpressed in psoriasis, a skin disease characterized by infiltration of neutrophils. In addition to its microbicidal activities and chemotaxis of neutrophils reported previously, we hypothesized that psoriasin might regulate other neutrophil functions such as cytokine and chemokine production, reactive oxygen species generation, and release of antimicrobial peptides. In the current study, we demonstrate that psoriasin activates neutrophils to produce a range of cytokines and chemokines including interleukin‐6 (IL‐6), IL‐8/CXCL8, tumour necrosis factor‐α, macrophage inflammatory protein‐1α (MIP‐1α)/CCL3, MIP‐1β/CCL4 and MIP‐3α/CCL20. Furthermore, psoriasin induces phosphorylation of mitogen‐activated protein kinase p38 and extracellular signal‐regulated kinase (ERK), but not c‐Jun N‐terminal kinase (JNK), both of which are required for the production of cytokines and chemokines as evidenced by the inhibitory effects of p38 and ERK inhibitors on psoriasin‐mediated neutrophil activation. Moreover, psoriasin stimulates the generation of reactive oxygen species from neutrophils, most likely via nicotinamide adenine dinucleotide phosphate oxidase activation. Finally, we demonstrate that psoriasin enhances messenger RNA expression of α‐defensins, termed human neutrophil peptides (HNP) 1 to 3, and induces their extracellular release. Besides its antimicrobial properties, therefore, psoriasin may contribute to innate immunity through enhancing neutrophil host defence functions at sites of inflammation or infection.