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-amino-acid 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.

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded beta-sheet structure. In humans, two classes of defensins can be found: alpha-defensins and beta-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form alpha-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL-37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an alpha-helical structure in a hydrophobic environment.

Antimicrobial peptides, human beta-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 of hBD-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 Ca(2+) mobilization, and hBD-2 was more potent than LL-37. Interestingly, histamine release and intracellular Ca(2+) mobilization elicited by hBD-2 and LL-37 were markedly suppressed by BAPTA-AM (an intracellular Ca(2+) chelating agent), pertussis toxin and U-73122 (a phospholipase C inhibitor). In addition, among the peptides examined, only hBD-2 significantly induced PGD(2) production, which was abolished by indomethacin (cyclooxygenase-1/-2 inhibitor) but not NS-398 (cyclooxygenase-2 inhibitor), suggesting that hBD-2-induced PGD(2) production is mediated by cyclooxygenase-1. Likewise, the PGD(2) production was suppressed by pertussis toxin and U-73122. These observations suggest that hBD-2 and LL-37 stimulate mast cells to mobilize intracellular Ca(2+) and release histamine or generate PGD(2) in a G protein-phospholipase C-dependent manner. Thus, hBD-2 and LL-37 may have modulatory effects on inflammatory reactions.

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 microg/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.

Schistosomes are the causative agents of schistosomiasis, one of the most prevalent and serious of the parasitic diseases that currently infects approximately 200 million people worldwide. Schistosome excretory/secretory (ES) proteins have been shown to play important roles in modulating mammalian host immune systems. In our current study, we performed a global proteomics identification of the ES proteins from adult worms of Schistosoma japonicum, one of the three major schistosome species. Our results unambiguously identified 101 proteins, including 53 putatively secreted proteins. By quantitative analysis, we revealed fatty acid-binding protein as a major constituent of the in vitro ES proteome. Strikingly the heat shock proteins HSP70s, HSP90, and HSP97 constituted the largest protein family in the ES proteome, implying a central role for these proteins in immunomodulation in the host-parasite relationship. Other important S. japonicum ES proteins included actins, 14-3-3, aminopeptidase, enolase, and glyceraldehyde-3-phosphate dehydrogenase, some of which have been considered as viable vaccine candidates and therapeutic targets. A comparison with previous studies suggests that 48.5% of S. japonicum ES proteins are common to other parasite ES products, indicating that the molecular mechanisms involved in evading the host immune response may be conserved across different parasites. Interestingly seven host proteins, including antimicrobial protein CAP18, immunoglobulins, and a complement component, were identified among in vitro S. japonicum ES products likely originating from the schistosome tegument or gut, indicating that host innate and acquired immune systems could defend against schistosome invasion. Our present study represents the first attempt at profiling S. japonicum ES proteins, provides an insight into host-parasite interactions, and establishes a resource for the development of diagnostic agents and vaccines for the control of schistosomiasis.

OBJECTIVE

Activated neutrophils extracellularly release antibacterial defensins and cathelicidins from the granules. In this study, to elucidate the interactions between defensins and cathelicidins in the extracellular environment, we evaluated the individual and synergistic actions of defensins and cathelicidins in the presence of physiological concentration of NaCl (150 mM).

METHODS

Antibacterial activities against Escherichia coli and Staphylococcus aureus were assessed using human and guinea pig defensins and cathelicidins. Furthermore, the effect of defensins and cathelicidins on membrane permeabilization was examined using E. coli ML-35p, as a target organism.

RESULTS

In the absence of NaCl, human defensin (HNP-1) and guinea pig defensins (GNCPs) exhibited the antibacterial activities in a dose-dependent manner (0.1-10 microg/ml); however, their activities were completely lost in the presence of 150 mM NaCl. In contrast, the antibacterial activities of human cathelicidin (CAP18/LL-37) and guinea pig cathelicidin (CAP11) were resistant to NaCl. Interestingly, HNP-1 and GNCPs synergized with CAP18/LL-37 and CAP11 to enhance the antibacterial activities against E. coli and S. aureus in the presence of 150 mM NaCl (p<0.05). Similarly, HNP-1 and GNCPs were synergistic with CAP18/LL-37 and CAP11 to potentiate the outer and inner membrane permeabilization of E. coli ML-35p (p<0.05).

CONCLUSIONS

Together these observations indicate that when extracellularly released from neutrophils, defensins cannot function as antibacterial molecules by themselves, but can synergistically work with cathelicidins to exert the antibacterial activity in the extracellular milieu by augmenting the membrane permeabilization of target cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-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-alpha 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-alpha 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 antimicrobial peptides human beta-defensin-1 (hBD1), hBD2, hBD3, and CAP18 expressed by keratinocytes have been implicated in mediation of the innate defense against bacterial infection. To gain insight into Staphylococcus aureus infection, the susceptibility of S. aureus, including methicillin-resistant S. aureus (MRSA), to these antimicrobial peptides was examined. Based on quantitative PCR, expression of hBD2 mRNA by human keratinocytes was significantly induced by contact with S. aureus, and expression of hBD3 and CAP18 mRNA was slightly induced, while hBD1 mRNA was constitutively expressed irrespective of the presence of S. aureus. Ten clinical S. aureus isolates, including five MRSA isolates, induced various levels of expression of hBD2, hBD3, and CAP18 mRNA by human kertinocytes. The activities of hBD3 and CAP18 against S. aureus were found to be greater than those of hBD1 and hBD2. A total of 44 S. aureus clinical isolates, including 22 MRSA strains, were tested for susceptibility to hBD3 and CAP18. Twelve (55%) and 13 (59%) of the MRSA strains exhibited more than 20% survival in the presence of hBD3 (1 microg/ml) and CAP18 (0.5 microg/ml), respectively. However, only three (13%) and two (9%) of the methicillin-sensitive S. aureus isolates exhibited more than 20% survival with hBD3 and CAP18, respectively, suggesting that MRSA is more resistant to these peptides. A synergistic antimicrobial effect between suboptimal doses of methicillin and either hBD3 or CAP18 was observed with 10 MRSA strains. Furthermore, of several genes associated with methicillin resistance, inactivation of the fmtC gene in MRSA strain COL increased susceptibility to the antimicrobial effect mediated by hBD3 or CAP18.

OBJECTIVE

Antimicrobial peptides are one of the factors involved in innate immunity. The susceptibility of periodontopathogenic and cariogenic bacteria to the major antimicrobial peptides produced by epithelia was investigated.

METHODS

Synthetic antimicrobial peptides of human beta-defensin-1 (hBD1), hBD2, hBD3 and LL37 (CAP18) were evaluated for their antimicrobial activity against oral bacteria. They included Actinobacillus actinomycetemcomitans (20 strains), Porphyromonas gingivalis (6), Prevotella intermedia (7), Fusobacterium nucleatum (7), Streptococcus mutans (5), Streptococcus sobrinus (5), Streptococcus salivarius (5), Streptococcus sanguis (4), Streptococcus mitis (2) and Lactobacillus casei (1).

RESULTS

Although the four peptides had bactericidal activity against all bacteria tested, the degree of antibacterial activity was variable against the different strains and species. The antibacterial activity of hBD1 was lower than that of the other peptides. Among the bacteria tested in this study, F. nucleatum was highly susceptible to hBD3 and LL37, and S. mutans was highly susceptible to hBD3. We measured the Zeta-potential, representing the net charge of whole bacteria, to study the relationship between susceptibility to cationic peptide and the net charge of the bacteria. Although we found some correlation in A. actinomycetemcomitans strains, we did not find a definite correlation with all the bacterial species.

CONCLUSIONS

These results indicate that beta-defensins and LL37 have versatile antibacterial activity against oral bacteria.

An association between moenomycin resistance and vancomycin intermediate resistance in Staphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as beta-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

BACKGROUND

Interest in airways inflammatory disease has increasingly focused on innate immunity. We investigated several components of innate immunity in induced sputum of patients with cystic fibrosis (CF), COPD, and asthma, and healthy control subjects.

METHODS

Twenty eight patients with mild CF lung disease (age>> or = 12 years; FEV1, 74 +/- 3% predicted [mean +/- SE]), 74 adults with COPD (FEV1, 55 +/- 2% of predicted), 34 adults with persistent asthma (FEV1, 66 +/- 2% of predicted), and 44 adult control subjects (FEV1, 85 +/- 1% of predicted) were studied while in stable clinical condition. Levels of sputum interleukin (IL)-8, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, human cationic antimicrobial protein 18 (CAP18), urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 were determined. Cell sources were investigated by flow cytometry and immunohistochemistry. Spirometry was performed prior to sputum induction.

RESULTS

CF patient sputum showed greatest increase in IL-8 compared to that of patients with COPD and asthma (which were also greater than control subjects), and elevated levels of TNF-alpha and IL-10 compared to other groups. There were no differences in IFN-gamma. CAP18 levels were elevated in CF and COPD patients compared to control subjects, while asthma patients had reduced CAP18 levels. uPA levels were similar but uPAR was elevated in CF and COPD patients more so than in asthma patients, while PAI-1 levels were elevated in all three disease groups. CAP18 localized to neutrophil secondary granules; neutrophils were also sources of IL-8 and PAI-1. CAP18 and PAI-1 negatively correlated with pulmonary function.

CONCLUSIONS

Induced-sputum innate immune factor levels discriminate inflammatory changes in CF, COPD, and asthma, suggesting potential roles in pathophysiology and as well as providing disease-specific biomarker patterns.

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

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 (L(1) to S(37)) 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 (K(15) to V(32)) 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 E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) 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-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha 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.

Cathelicidins are antimicrobial peptides from sheep (SMAP29 and SMAP34), rabbits (CAP11 and CAP18), rodents (CRAMP), and humans (FALL39, LL37, and h/CAP18). In a broth microdilution assay against nine ovine pathogens, SMAP29, SMAP34, mouse CRAMP, CAP18, CAP18(31), CAP18(28), CAP18(22), and CAP18(21a) were the most active, with MICs as low as 0.6 microg/ml. Other cathelicidins were less active. In lambs with pneumonia, 0.5 mg of SMAP29 reduced the concentration of bacteria in both bronchoalveolar lavage fluid and consolidated pulmonary tissues. Hence, the antimicrobial activity of SMAP29 suggests that it has applications in the treatment of respiratory tract infections.

Rabbit CAP18 (cationic antimicrobial protein, 18 kDa) is a leukocyte protein identified and purified using as an assay its capacity to bind and inhibit various activities of LPS. Oligonucleotide probes designed from the putative N-terminal protein sequence were used to obtain the corresponding cDNA from a rabbit bone marrow cDNA library. Examination of the cDNA sequence revealed that the protein fragment of the putative N-terminus was actually a 37-amino-acid C-terminal fragment. This fragment, designated CAP18(106-142), inhibits many activities of LPS. In the present studies, synthetic CAP18(106-142) is shown to: 1) bind to erythrocytes coated with diverse strains of LPS; 2) inhibit LPS-induced release of cytokines (TNF, IL-1, IL-6) and nitric oxide from macrophages; 3) inhibit LPS-induced LAL coagulation and 4) protect mice from LPS lethality. CAP18(106-142) may have therapeutic utility for conditions associated with elevated concentrations of LPS.

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to>> 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.

CAP18 (an 18-kDa cationic antimicrobial protein) is a granulocyte-derived protein that can bind lipopolysaccharide (LPS) and inhibit various activities of LPS in vitro. The present study examined the protective effect of a synthetic 27-amino-acid peptide (CAP18(109-135)) from the LPS-binding domain of CAP18 against antibiotic-induced endotoxin shock, using highly LPS-sensitive D-(+)-galactosamine (D-GalN)-sensitized C3H/HeN mice. The antibiotic-induced endotoxin (CAZ-endotoxin) was prepared from the culture filtrate of Pseudomonas aeruginosa PAO1 exposed to ceftazidime (CAZ). Injection of CAP18(109-135) protected the mice injected with LPS or CAZ-endotoxin from death and lowered their tumor necrosis factor (TNF) levels in serum in a dose-dependent manner. Treatment with CAZ caused death of the D-GalN-sensitized P. aeruginosa PAO-infected mice within 48 h, while injection with CAP18(109-135) rescued the mice from death. In the mice rescued from death by injection with CAP18(109-135), endotoxin levels in plasma and TNF production by liver tissues were decreased but the numbers of viable infecting bacteria in their blood were not decreased significantly and remained at the levels in CAZ-treated mice. These results indicate that CAP18(109-135) is capable of preventing antibiotic-induced endotoxic shock in mice with septicemia and that the effect is due to its LPS-neutralizing activity rather than to its antibacterial activity.

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).

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.

OBJECTIVE

To evaluate the antimicrobial activity of C-terminal peptides derived from human CAP18.

METHODS

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.

Cathelicidins are a family of antibacterial peptides. Human cathelicidin LL-37 inhibits the binding of endotoxin lipopolysaccharide (LPS) to CD14-positive cells and could ameliorate sepsis. The aim of this study was to observe the effect of LL-37 on sepsis in neonatal rats. Intraperitoneal injection (IPI) of LPS was used to create sepsis in suckling rats. Group 1 rats were given LPS with LL-37, group 2 rats were given LL-37 2 h after LPS, and group 3 rats were given LPS without LL-37. Control group rats were given isovolemic normal saline by IPI. Rats given LL-37 IPI were divided into seven subgroups. Following IPI, an overall assessment score (OAS) and rectal temperature (RT) were assessed hourly. Serum C-reactive protein (CRP) was also assessed at death or at sacrifice 10 h after IPI. All rats in group 3 died. For rats receiving lower doses of LL-37 in groups 1 and 2, mortality was decreased. No deaths occurred among those receiving higher doses of LL-37 in group 1; however, mortality increased in group 2. In group 1, OAS and RT deteriorated initially for those receiving lower doses of LL-37, then improved. OAS and RT did not deteriorate throughout the study in rats given higher doses of LL-37. In group 2 rats given higher doses of LL-37, OAS and RT were not significantly different from rats in group 3. CRP was significantly decreased in group 1 compared with group 3, and decreased in group 2 for lower doses only. We conclude that LL-37 may prevent sepsis and be useful in lower doses for treating sepsis. However, LL-37 appears to have adverse effects when used at higher doses for treating sepsis.

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.

Mammalian myeloid and epithelial cells express many antimicrobiotic peptides that contribute to innate host defense against invading microbes. In the present study, a 27-mer peptide of the C-terminal domain (hCAP18(109-135)) and analogs of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) were examined for tumoricidal activity. In vitro results showed that hCAP18(109-135) induced apoptosis in human oral squamous cell carcinoma (OSCC), SAS-H1 cells. The hCAP18(109-135) induced mitochondrial depolarization and apoptosis in SAS-H1 cells, but not in healthy human gingival fibroblasts (HGF) and human keratinocyte line HaCaT cells. Caspases were not activated during hCAP18(109-135)-induced apoptosis in SAS-H1 cells. The results indicate that hCAP18(109-135) may induce caspase-independent apoptosis in OSCC but not in normal cells. hCAP18(109-135) can therefore be a useful anti-tumor therapeutic agent in the treatment of OSCC.

Cationic antibacterial proteins (CAP) were purified from rabbit granulocytes, and the effects of CAP on lipopolysaccharide (LPS)-induced tissue factor generation by murine peritoneal macrophages and human blood monocytes were studied. CAP were purified from rabbit peritoneal leukocytes by using as an assay the agglutination of erythrocytes coated with Re-LPS. Two proteins with CAP activity, CAP18 (18 kDa) and CAP7 (7 kDa), were isolated by acid extraction, ethanol precipitation, affinity chromatography, gel filtration, and reverse-phase high-pressure liquid chromatography. On the basis of protein sequencing, CAP7 was identified as the C-terminal fragment of CAP18, designated CAP18(106-142). Various forms of LPS (S-LPS, Re-LPS, and lipid A) activate murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). Incubation of LPS for 18 h with partially purified CAP (heparin-Sepharose fraction) inhibited the capacity of LPS to induce tissue factor; however, purified CAP18 inhibited about 75% of the activity of S-LPS after 1 h of incubation. CAP more effectively inhibited S-LPS than Re-LPS or lipid A. Synthetic CAP18(106-142) inhibited LPS-induced tissue factor generation by murine macrophages. CAP18(106-142) has greater LPS-binding and LPS-neutralizing activities than CAP18. We hypothesize that CAP18 and the derivative peptide, CAP18(106-142), bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may have therapeutic potential for sepsis and endotoxin shock.