Human Cationic Antimicrobial Protein Research Articles

Application of a modern theoretical approach to the study of the interaction of KR-12 peptides derived from human cathelicidins with Cu(II) ions.

The human cationic antimicrobial protein (hCAP) corresponding to the overlapping sequences of 151-162 of hCAP named KR-12 peptide is the smallest portion of the only type of human Cathelicidin, which has been shown to be modifiable into a more effective antimicrobial. In this study, an in silico analysis, supported by potentiometric titration and isothermal titration calorimetry techniques, was performed to identify potential Cu(II) binding sites of KR-12. The analysis of the presented data at the given theoretical level (GFN2-xTB/ALPB) revealed which peptide chain fragments are involved in the most favourable KR-12-Cu(II) binding mode. Based on a quantum chemical approach, the most favourable coordination modes of Cu(II) to peptides are proposed together with the discussion of the chemical nature of the interactions. The presented results demonstrated that KR-12 interacts with metal ions mostly via the main chain's oxygen atoms; however, the two types of amino acids that are expected to be vital for the interaction of Cu(II) are D (aspartic acid) and R29 (arginine). It was demonstrated that in order to explain the complexity of the interaction process in peptide-metal ion systems, the use of theoretical methods is sometimes necessary to explain the details of the experimental results and provide an in-depth understanding of these dynamic systems.

Metal-Ion Interactions with Dodecapeptide Fragments of Human Cationic Antimicrobial Protein LL-37 [hCAP(134-170)

Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134–170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134–170)], namely, hCAP(134–145) (A1), hCAP(140–151) (A2), hCAP(146–157) (A3), hCAP(152–163) (A4), and hCAP(159–170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159–170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134–170).

Extending the vitamin D pathway to vitamin D3 and CYP27A1 in periodontal ligament cells.

In periodontal connective tissue cells, the vitamin D pathway has been elucidated, and vitamin D3 in the main storage form, 25-hydroxy vitamin D3 (25[OH]D3 ), and the functional form, 1,25-dihydroxy vitamin D3 (1,25[OH]2 D3 ), have been found to induce the expression of human cationic antimicrobial protein (hCAP-18)/LL-37. Moreover, synergistic effects between Toll-like receptor agonists and 25(OH)D3 have been reported. This research aimed at extending the vitamin D pathway to vitamin D3 and CYP27A1 in human periodontal ligament cells (hPDLCs) to further explore its function in periodontal inflammatory reaction. Vitamin D3 was used to stimulate hPDLCs in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Conversely, CYP27A1 RNA interference was performed to further validate the findings. The mRNA expression of hCAP-18 was determined with real-time polymerase chain reaction. Monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) were also detected. The cell supernatant levels of LL-37 were detected with enzyme-linked immunosorbent assay. Vitamin D3 significantly enhanced the generation of hCAP-18/LL-37. A combination of Pg-LPS and vitamin D3 significantly promoted hCAP-18/LL-37 expression. When the expression of CYP27A1 was knocked down with RNA interference, the induction of hCAP-18/LL-37 expression was significantly inhibited. Therefore, the mRNA levels of MCP-1 and IL-8 in hPDLCs were significantly decreased through the vitamin D pathway. The vitamin D pathway from vitamin D3 to hCAP-18/LL-37 exists in hPDLCs, and CYP27A1 might be involved in periodontal immune defense.

Association between Mycobacterium avium complex lung disease and serum vitamin D status, antimicrobial peptide levels, and bone mineral density.

Vitamin D maintains calcium balance and has immunomodulatory effects. Only few studies have revealed the relationship between vitamin D and its associated factors in Mycobacterium avium complex (MAC) infection. This study aimed to investigate the effects of MAC infection on serum vitamin D, human cationic antimicrobial protein 18, its C-terminal 37 amino acid fragment (hCAP18/LL-37) levels, and bone mineral density (BMD).We enrolled 58 patients with MAC lung disease and 15 control participants. Serum 25-hydroxyvitamin D and hCAP18/LL-37 levels were measured via enzyme-linked immunosorbent assay. Lastly, computed tomography scan density readings of the BMD of the thoracic and lumbar vertebral bones (Th4, Th7, Th10, and L1) were assessed.No significant differences in patient characteristics and serum vitamin D levels were observed. Patients with MAC lung disease had significantly low serum hCAP18/LL-37 levels (P = .049). Moreover, low BMD of the mean thoracic and lumbar vertebrae was observed (mean Th, P = .012; L1, P = .48, respectively). A higher prevalence of scoliosis (P = .031) was observed in the participants with low BMD compared with the control participants. Based on a multivariate analysis, patients with MAC lung disease had significantly lower body mass index [odds ratio (OR), 19.1; 95% confidence interval (CI), 2.0–419.0; P < .01] and vertebral BMD (OR, 12.4; 95% CI, 1.7–160.6; P = .012) than control participants.Serum hCAP18/LL-37 level and BMD were significantly decreased in patients with MAC lung disease without relation to serum vitamin D level. The vitamin D–independent pathway might affect the waning of antimicrobial peptides and decrease in BMD.

Preliminary investigation of the vitamin D pathway in periodontal connective tissue cells.

The vitamin D pathway, from toll-like receptor activation to human cationic antimicrobial protein (hCAP-18/LL-37) generation, has been identified in monocytes and keratinocytes. This study aimed to investigate the vitamin D pathway in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) and to provide preliminary evidence of its role in periodontal immune defense. Primary cultures of hGFs and hPDLCs were stimulated with 1,25-dihydroxy vitamin D3 and 25-hydroxy vitamin D3 , with or without Porphyromonas gingivalis lipopolysaccharide. CYP27B1 RNA interference and vitamin D receptor (VDR) antagonism were also used for reverse proof. The mRNA expression of hCAP-18/LL-37, VDR, interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 were detected using real-time polymerase chain reaction. The LL-37 concentrations were measured using enzyme-linked immunosorbent assay. In hGFs and hPDLCs, 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3 induced hCAP-18/LL-37 expression, which was further increased by Porphyromonas gingivalis lipopolysaccharide. If the function of CYP27B1 or VDR was blocked, the induction was significantly weakened. IL-8 and monocyte chemotactic protein-1 mRNA expression could be suppressed by the vitamin D pathway. These findings suggest that the vitamin D pathway exists in hGFs and hPDLCs and plays an important role in immune defense in periodontal soft tissues.

Detection and clinical application value of antimicrobial peptide hCAP18 in serum of colorectal carcinoma patients

Objectve To evaluate the serum level of antimicrobial peptide human cationic antimicrobial protein 18 (hCAP18) in colorectal patients and it auxiliary diagnosis and prognosis value. Methods Case-control study was used. The serum level of hCAP18 was measured by enzyme linked immunosorbent assay(ELISA) in 68 cases with colorectal patients of department of gastrointestinal surgery and 40 cases healthy people of department of physical examination from January 2014 to June 2015 in Tongji Hosptial of Tongji University. The concentrations of hCAP18 in serum of colorectal patients before and surgery were analyzed. Immunohistochemistry was used to detect hCAP18 expression in colorectal carcinoma. The effect of hCAP18 on colon carcinoma cell proliferation was detected by BrdU-ELISA and soft agar colony formation assay. The sensitivity and specificity of serum hCAP18 for the diagnosis of colorectal were evaluated using the receiver operating characteristic curves(ROC) . Date was analyzed by using the t-test and one-way analysis of variance. Results hCAP18 serum levels in colon cancer of stage Ⅰ, Ⅱ, Ⅲ and IV patients were (0.46±0.18)mg/L, (0.65±0.45)mg/L, (1.26±0.68)mg/L and (2.35±1.06)mg/L. Mean value was(1.16±0.88) mg/L, which was significantly higher than in normal people (0.19±0.07)mg/L(t=5.290, P<0.05). hCAP18 levels had significantly decreased in serum of colorectal patients after 30 d surgery compared to preoperative results[from (1.16±0.88)mg/L to(0.26±0.06)mg/L; t=3.971, P<0.05]. Immunohistochemistry results showed hCAP18 was high expression in colon cancer tissue compared with adjacent tissues; BrdU-ELISA assay results showed HCT116 and SW480 cell proliferation increased significantly after 0.05-1 mg/L of hCAP18 treatment; Soft agar clone formation experiment proved hCAP18 could significant enhance clone formation of HCT116 and SW480 colon cancer cell lines. The size of clonal cluster of HCT116 was increased from (145.40±35.20)μm to(370.80±32.65)μm(t=10.50, P<0.05)and SW480 was increased from (101.00±27.10)μm to(369.00±27.29)μm(t=15.58, P<0.05); The numbers of clonal cluster of HCT116 was increased from 8.50±2.30 to 42.80±6.60(t=3.945, P<0.05)and SW480 was increased from 6.20±1.70 to 46.00±7.20(t=4.775, P<0.05). ROC analysis of serum hCAP18 yielded an AUC(area under the ROC curve) of 0.93(95% CI=0.859-0.999)with 91.17% sensitivity and 80.00% specificity, which was higher than the CEA[0.78 (95% CI=0.699-0.933)]. Conclousions Detection of serum hCAP18 shows a good sensitivity and specificity for the auxiliary diagnosis of colon cancer. It is possible to be potential detection index for noninvasive diagnosis and monitoring progression of colon cancer. hCAP18 could promote the proliferation of colon cancer cells, it played an important role in the progression of colon cancer.(Chin J Lab Med, 2017, 40: 278-283) Key words: Antimicrobial cationic peptides; Tumor markers, biological; Colonic neoplasms; Cell proliferation

Impact of high-dose vitamin D3 on plasma free 25-hydroxyvitamin D concentrations and antimicrobial peptides in critically ill mechanically ventilated adults.

High-dose vitamin D3 increases plasma total 25-hydroxyvitamin D [25(OH)D] in critically ill, ventilated patients; however, to our knowledge, the effect on plasma levels of free (nonprotein-bound) 25(OH)D has not been investigated in critical illness. Moreover, the relationship of free 25(OH)D and the regulation of endogenous antimicrobial peptides (AMPs) remains unknown. The aims of this study were to determine in critically ill adults with respiratory failure the effect of previous high-dose regimens of vitamin D3 on free 25(OH)D concentrations, the relationship of free 25(OH)D with circulating cathelicidin (LL-37) and human beta-defensin-2 (hBD-2), and the associations between plasma levels of free 25(OH)D and these AMPs to alveolar macrophage phagocytosis function. In a double blind, randomized controlled trial, critically ill ventilator-dependent adults (N=30) received enteral vitamin D3 (250,000 or 500,000 IU total over 5d) or placebo. Plasma was obtained serially for concentrations of free 25(OH)D, LL-37, hBD-2, and expression of peripheral blood mononuclear cell human cationic antimicrobial protein (hCAP18) mRNA. Total 25(OH)D and LL-37 concentrations and alveolar macrophage phagocytosis were determined in bronchoalveolar lavage fluid. Plasma concentrations of free 25(OH)D over time were correlated with total 25(OH)D levels (r=0.82; P<0.001). The increase in free 25(OH)D was greater with the 500 000 IU vitamin D3 dose than with the lower dose. The percent change in mRNA expression of hCAP18 was positively associated with percent change in free 25(OH)D at days 7 and 14 (ρ=0.48; P=0.04 and ρ=0.59; P=0.03, respectively). Additionally, plasma LL-37 levels correlated with the percentage of alveolar macrophages exhibiting phagocytosis (ρ=0.51; P=0.04). The present study found a dose-related increase in plasma free-25(OH)D levels, which was associated with increasing circulating mRNA expression of hCAP18 over time. There were no correlations between changes in total and free 25(OH)D against plasma LL-37 and hBD-2 concentrations. Larger studies appear warranted to determine the impact of high-dose vitamin D3 administration on endogenous AMPs.

Expression and clinical application value of antimicrobial peptide hCAP18 in serum of NSCLC patients

Objective To evaluate the serum level of antimicrobial peptide human cationic antimicrobial protein 18 (hCAP18) in non-small cell lung cancer (NSCLC) patients and its auxiliary diagnosis and prognosis value. Methods Case-control study was used. The serum level of hCAP18 was measured by enzyme linked immunosorbent assay (ELISA) in 50 cases with NSCLC patients of department of thoracic surgery and 50 cases healthy people of department of physical examination from January 2011 to January 2012 in Tongji Hospital of Tongji University. The concentrations of hCAP18 in serum of NSCLC patients before and after surgery were analyzed. The sensitivity and specificity of serum hCAP18 for the diagnosis of NSCLC were evaluated using the receiver operating characteristic (ROC) curves. Data was analyzed by using the t-test and Log-rank test. Results Serum hCAP18 concentration in NSCLC patients (6 733±771.8) μg/L was significantly higher than in healthy controls (253±6.9) μg/L (t=8.396, P 390.0 μg/L (χ2=22.64, P<0.05). Conclusions Detection of serum hCAP18 shows a good sensitivity and specificity for the auxiliary diagnosis of NSCLC. It is possible to be a potential detection index for noninvasive diagnosis and monitoring progression of lung cancer.(Chin J Lab Med, 2016, 39: 595-598) Key words: Carcinoma, non-small-cell Lung; Cathelicidins; Tumor markers, biological; Prognosis

LL-37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection

The human cationic antimicrobial protein LL-37 is a multifunctional host defense peptide with a wide range of immunomodulatory activities. Previous work has shown that LL-37 exerts both pro- and anti-inflammatory effects. The role of mitochondria in the skin inflammatory effects of LL-37 has not been well studied. Therefore, our aim was to investigate the immunomodulatory effect of LL-37 in HaCaT cells and to delineate the underlying mechanisms related to mitochondrial function. Immunohistochemistry results from tissue microarrays showed strong cytoplasmic LL-37 staining in inflammatory cells in chronic dermatic inflammation. Using exogenous LL-37 stimulation and LL-37 knockdown and overexpression, LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL-1α and tumor necrosis factor-α (TNF-α), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 are dependent upon its ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. Furthermore, we observed that LL-37 enhances the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin can neutralize the protective effects of LL-37 on mitochondria. In conclusion, these results suggest that high LL-37 expression levels correlate with chronic skin inflammation; mitochondrial dysfunction occurs in HaCaT cells during inflammation; and LL-37 attenuates inflammatory impairment by stimulating mitochondrial biogenesis and protecting mitochondrial function, which are dependent upon mTOR signaling. These findings provide new insights into targeting mitochondria with LL-37 to prevent skin inflammatory reactions.

Anti-adenoviral Effects of Human Cationic Antimicrobial Protein-18/LL-37, an Antimicrobial Peptide, by Quantitative Polymerase Chain Reaction

PurposeAntimicrobial peptides have an important role in self-protection of the ocular surface. Human cationic antimicrobial protein (hCAP)-18 is a linear, α-helical peptide that consists of a conserved pro-sequence called a cathelin-like domain and a C-terminal peptide named LL-37. We investigated the in vitro anti-adenoviral activity of hCAP-18/LL-37 in several adenovirus types, inducing keratoconjunctivitis.MethodsA549 cells were used for viral cell culture, and human adenovirus (HAdV) types 3 (HAdV3, species B), 4 (species E), 8, 19a, and 37 (species D) were used. The cytotoxicity of LL-37 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay to obtain 50% cytotoxic concentration. After pretreatment of A549 cells with serial dilutions of LL-37 for 24 hours, adenovirus was cultured for seven days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction (PCR).ResultsThe 50% effective concentration of LL-37 obtained by real-time PCR ranged between 118 and 270 µM. LL-37 showed a significant inhibitory effect on adenoviral proliferation in all adenovirus types except HAdV4 in a dose-dependent manner.ConclusionsLL-37 has significant inhibitory activity against HAdV3, 8, and 19, which induce keratoconjunctivitis. These results indicate that hCAP-18/LL-37 may be a possible candidate for the treatment of HAdV keratoconjunctivitis.

Cathelicidins—Therapeutic antimicrobial and antitumor host defense peptides for oral diseases

The oral epithelium functions as a mechanical and protective barrier to resist bacterial infection. Several types of host defense peptides (HDPs), including defensins, cathelicidins, and histatins, may have important roles in innate host defense. HDPs, which are mostly cationic and have amphipathic structures, provide non-specific, rapid defense against invading pathogens. Human cationic antimicrobial protein 18 kDa (hCAP18/LL-37) is the only member of the cathelicidin family identified in humans thus far. Proteolytic processing releases LL-37 from its inactive precursor hCAP18/LL-37 to initiate its antimicrobial activity. As its name implies, LL-37 is made up of 37 amino acids. Various immune and epithelial cells secrete hCAP18/LL-37, and its level is altered in response to cariogenic, periodontal, congenital, inflammatory, and malignant diseases in the oral region. Human cathelicidin peptide, LL-37 exhibits antimicrobial activity against bacteria that cause oral pathological conditions, including cariogenic disease and periodontitis. Altered expression of hCAP18/LL-37 was observed in oral inflammatory lesions with and without microbial infection or oral cancer. Treatment with hCAP18/LL-37 may be useful in infectious, inflammatory, and cancerous diseases.

Urokinase‐type plasminogen activator system and human cationic antimicrobial protein 18 in serum and induced sputum of patients with chronic obstructive pulmonary disease

There is increasing evidence that the innate immune system plays an important role in the pathogenesis of COPD. The objective of this study was to quantify several innate immune biomarkers in serum and induced sputum of COPD patients, and healthy non-smokers and smokers. Serum and induced sputum levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator inhibitor (PAI-1) and human cationic antimicrobial protein 18 (CAP18) were measured by ELISA, in 13 patients with stage I or stage II COPD (COPD I + II), 15 patients with stage III or stage IV COPD (COPD III + IV), 18 healthy non-smokers and 14 healthy smokers. In addition, membrane-bound uPAR in peripheral blood and induced sputum was assessed by flow cytometry. Levels of uPAR, PAI-1 and CAP18 were elevated in induced sputum of COPD I + II and COPD III + IV patients, compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). uPAR, PAI-1 and CAP18 levels were significantly higher in COPD III + IV patients compared with COPD I + II patients (P < 0.05). The expression of uPAR on induced sputum neutrophils and macrophages was significantly higher in COPD patients compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). Sputum uPAR and CAP18 levels showed significant inverse correlations with FEV(1)% and 6MWD, and significant positive correlations with St. George's Respiratory Questionnaire scores. In COPD patients, increased induced sputum levels of uPAR, PAI-1 and CAP18 were associated with airflow limitation, health status and exercise tolerance, suggesting that these biomarkers may be implicated in the pathogenesis of COPD.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Ovarian cancers overexpress the antimicrobial protein hCAP‐18 and its derivative LL‐37 increases ovarian cancer cell proliferation and invasion

The role of the pro-inflammatory peptide, LL-37, and its pro-form, human cationic antimicrobial protein 18 (hCAP-18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL-37 functions as a chemoattractant, mitogen and pro-angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP-18/LL-37 in normal and cancerous ovarian tissue and to examine the effects of LL-37 on ovarian cancer cells. Expression of hCAP-18/LL-37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP-18/LL-37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial-derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP-18/LL-37. Treatment of ovarian cancer cell lines with recombinant LL-37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP-18/LL-37 is significantly overexpressed in ovarian tumors and suggest LL-37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro-inflammatory molecules and ovarian cancer progression.

Involvement of the Antimicrobial Peptide LL-37 in Human Atherosclerosis

Antimicrobial peptides are effector molecules of the innate immune system. To understand the function of vascular innate immunity in atherosclerosis, we investigated the role of LL-37, a cathelicidin antimicrobial peptide, in the disease process. Using real-time polymerase chain reaction, we found a 6-fold increase in human cationic antimicrobial protein 18/LL-37 transcript in human atherosclerotic lesions compared with normal arteries. Immunohistochemical analysis of atherosclerotic plaques showed that LL-37 was expressed mainly by macrophages and some endothelial cells. Western blot demonstrated existence of active LL-37 peptide and abundant proprotein in atheroma specimens. To understand the functional implication of LL-37 production in atherosclerosis, the transcription profile was assessed in endothelial cells treated with LL-37. Our data show that LL-37 induces expression of the adhesion molecule intercellular adhesion molecule-1 and the chemokine monocyte chemoattractant protein 1 in endothelial cells. Intriguingly, Chlamydia pneumoniae withstood the antimicrobial activity of LL-37 in vitro, although inflammatory response was induced on infection. LL-37 is produced in atherosclerotic lesions, where it may function as an immune modulator by activating adhesion molecule and chemokine expression, thus enhancing innate immunity in atherosclerosis.

LL-37 Protects Rats against Lethal Sepsis Caused by Gram-Negative Bacteria

We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 x 10(10) CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-alpha plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

Sputum Cathelicidin, Urokinase Plasminogen Activation System Components, and Cytokines Discriminate Cystic Fibrosis, COPD, and Asthma Inflammation

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. 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. 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. 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.

The human cathelicidin LL-37: a multifunctional peptide involved in infection and inflammation in the lung

Antimicrobial peptides play an important role in innate immunity of the lung by acting as effector molecules in host defence against inhaled pathogens. Various families of antimicrobial peptides have been identified, including the cathelicidins. Cathelicidins are characterized by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP-18), which is mainly expressed by neutrophils and epithelial cells. In addition to killing a broad spectrum of microorganisms, LL-37 was demonstrated to display various cellular activities related to inflammation including cytotoxicity to host cells, chemotaxis, epithelial cell activation, angiogenesis and epithelial wound repair. Focussing on this recent information, this review discusses the role of LL-37 in infection and inflammation in the lung. In addition, the importance of the fact that antimicrobial peptides such as LL-37 display a range of activities for the design and development of antimicrobial peptides for therapeutic use is discussed.

Human neutrophil-derived CAP37 inhibits lipopolysaccharide-induced activation in murine peritoneal macrophages

Human cationic antimicrobial protein, CAP37, is released from neutrophil granules during infection. CAP37 attracts monocytes, binds Gram-negative endotoxin (lipopolysaccharide, LPS), is bactericidal for a range of Gram-negative bacteria, and reduces mortality in murine polymicrobial sepsis. Here, we report that recombinant CAP37 specifically targets murine peritoneal macrophages. Under steady-state conditions, the bulk of cell-associated CAP37 was localized at the plasma membrane. However, a fraction of CAP37 gained access to the endocytic system, but did not accumulate in recycling endosomes or in the trans-Golgi network (TGN). Instead, CAP37 was internalized by fluid phase endocytosis and accumulated in a prelysosomal compartment. Macrophages that were preexposed to CAP37 exhibited diminished LPS responsiveness, as determined by analysis of c-Jun phosphorylation. Further examination showed that pretreatment with CAP37 reduced the ability of macrophages to bind LPS. Taken together, these observations demonstrate that prolonged exposure to CAP37 desensitizes macrophages to LPS and suggest that this protein plays a novel anti-inflammatory role in polymicrobial sepsis.

C-terminal domain of human CAP18 antimicrobial peptide induces apoptosis in oral squamous cell carcinoma SAS-H1 cells

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.