Michimasa Hirata

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.

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.

An Antimicrobial Cathelicidin Peptide, Human CAP18/LL-37, Suppresses Neutrophil Apoptosis via the Activation of Formyl-Peptide Receptor-Like 1 and P2X7

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-xL (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.

Complementary DNA sequence of rabbit CAP18—A unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).

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.

Anti-microbial activity of human CAP18 peptides

BACKGROUND CAP18 derived from rabbit leukocytes is a 142-amino acid protein recently demonstrated to have Lipopolysaccharide (LPS) binding and anti-microbial activity. The C-terminal 37 amino acids of rabbit CAP18 (CAP18(106-142) comprise the LPS-binding and anti-microbial domain. The homologous domain of human CAP18 (huCAP18(104-140) was identified from the recently cloned human CAP18 cDNA. OBJECTIVES To evaluate the antimicrobial activity of C-terminal peptides derived from human CAP18. STUDY DESIGN Prepare synthetic human CAP18(104-140) and study anti-microbial activity versus various gram-negative and gram-positive bacteria. RESULTS Synthetic human CAP18(104-140) has broad anti-microbial activity versus both gram-positive (IC50 = 2.5 micrograms/ml) and gram-negative bacteria (IC50 = 0.5-5 micrograms/ml). Susceptible strains include Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. A 32-amino acid peptide lacking five amino acids from the C-terminus of CAP18(104-140) has higher activity. Unlike previously characterized anti-microbial peptides derived from granulocyte proteins, CAP18(104-140) is active in serum. CONCLUSIONS Human CAP18(104-140) or a derivative peptide may have therapeutic potential for bacterial sepsis.

Antimicrobial activity of rabbit CAP18-derived peptides.

A cationic antimicrobial protein of 18 kDa (CAP18) was originally isolated from rabbit granulocytes by using as an assay the agglutination of Re-lipopolysaccharide-coated erythrocytes. The C-terminal 37 amino acids of CAP18 (CAP18(106-142)) make up the lipopolysaccharide-binding domain. Synthetic CAP18(106-142) has broad antimicrobial activity against both gram-positive (50% inhibitory concentration, 130 to 200 nM) and gram-negative (50% inhibitory concentration, 20 to 100 nM) bacteria. Susceptible strains include Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. Antimicrobial activity is highly dependent on peptide structure. Although a 32-amino-acid peptide resulting from the truncation of 5 amino acids from the C terminus of CAP18(106-142) is highly active, other fragments of CAP18(106-142), including CAP18(106-142) with a truncated N terminus, do not exhibit antimicrobial activity. Unlike previously characterized antimicrobial peptides derived from granulocyte proteins, CAP18(106-142) is active in serum. CAP18(106-142) or a derivative peptide may have therapeutic potential for bacterial sepsis. Images

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity

A glycoprotein (M r = 43 000) from horseshoe crab hemocytes with antimicrobial activity against Gram‐negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti‐human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2‐terminal signal sequence. The COOH‐terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH2‐terminal domain with weak sequence homology with part of the mammalian interleukin‐6 receptor α‐chain. Factor D is likely to have an important role in host defense mechanisms.

Epithelial cell-derived antibacterial peptides human beta-defensins and cathelicidin: multifunctional activities on mast cells.

Antibacterial peptides function as effectors for defense in innate immunity. In mammals, they are implicated in the barrier protection of epithelia where their expression can be induced during infection and inflammation. Over a dozen of antibacterial peptides have been identified in humans. Among these, defensins and cathelicidins have been well characterized. Two types of defensins (alpha- and beta-defensins) are recognized based on the presence of their conserved six cysteine residues, whereas cathelicidins are characterized by a homologous cathelin sequence in the pro-region and a variable antibacterial C-terminal sequence. Human beta-defensins and cathelicidin hCAP18/LL-37 are mainly expressed in epithelial tissues where mast cells are present. Here we review the structure of human beta-defensins and cathelicidin, and describe their multiple activities on mast cells to induce chemotaxis, degranulation and prostaglandin D(2) production, acting through receptors coupled to G-protein-phospholipase C pathway. Thus, in addition to their bactericidal activities, epithelial cell-derived antibacterial peptides may modulate the inflammatory responses by recruiting mast cells to inflammation foci and inducing the degranulation as well as prostaglandin production from this cell population.

Evaluation of the effect of human β-defensins on neutrophil apoptosis

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Antimicrobial human beta-defensins (hBDs) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, to further evaluate the role of hBDs in innate immunity, we investigated the action of hBD-1 to -4 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, hBD-3 most potently suppressed neutrophil apoptosis among hBD-1 to -4, accompanied with the down-regulation of truncated Bid (a pro-apoptotic protein), up-regulation of Bcl-x(L) (an anti-apoptotic protein) and inhibition of mitochondrial membrane potential change and caspase 3 activity. Furthermore, we revealed that neutrophils expressed CC chemokine receptor (CCR) 6, and the action of hBD-3 was completely abrogated by a neutralizing anti-CCR6 mAb. Collectively, these observations suggest that hBDs, especially hBD-3, can not only kill bacteria but also modulate (suppress) neutrophil apoptosis via the action on CCR6. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.