Cationic Antimicrobial Protein-18 Research Articles

Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.

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.

Investigation of human cationic antimicrobial protein-18 (hCAP-18), lactoferrin and CD163 as potential biomarkers for ovarian cancer

BackgroundEpithelial ovarian cancer is one of the leading causes of gynaecological cancer morbidity and mortality in women. Early stage ovarian cancer is usually asymptomatic, therefore, is often first diagnosed when it is widely disseminated. Currently available diagnostics lack the requisite sensitivity and specificity to be implemented as community-based screening tests. The identification of additional biomarkers may improve the diagnostic efficiency of multivariate index assays. The aims of this study were to characterise and compare the ovarian tissue immunohistochemical localisation and plasma concentrations of three putative ovarian cancer biomarkers: human cationic antimicrobial protein-18 (hCAP-18); lactoferrin; and CD163 in normal healthy women and women with ovarian cancer.MethodsIn this case–control cohort study, ovarian tissue and blood samples were obtained from 164 women (73 controls, including 28 women with benign pelvic masses; 91 cancer, including 21 women with borderline tumours). Localisation of each antigen within the ovary was assessed by immunohistochemistry and serum concentrations determined by ELISA assays.ResultsImmunoreactive (ir) hCAP-18 and lactoferrin were identified in epithelial cells, while CD163 was predominately localised in stromal cells. Tissue ir CD163 increased significantly (P<0.05) with disease grade. Median plasma concentrations of soluble (s)CD163 were significantly greater in the cases (3220 ng/ml) than in controls (2488 ng/ml) (P< 0.01). Median plasma concentrations of hCAP-18 and lactoferrin were not significantly different between cases and controls. The classification efficiency of each biomarker (as determined by the area under the receiver operator characteristic curve; AUC) was: 0.67± 0.04; 0.62 ± 0.08 and 0.51 ± 0.07 for sCD163, hCAP-18 and lactoferrin, respectively. When the 3 biomarkers were modelled using stochastic gradient boosted logistic regression, the AUC increased to 0.95 ± 0.03.ConclusionsThe data obtained in this study establishes the localisation and concentrations of CD163, hCAP-18, and lactoferrin in ovarian tumours and peripheral blood. Individually, the 3 biomarkers display only modest diagnostic efficiency as assessed by AUC. When combined in a multivariate index assay, however, diagnostic efficiency increases significantly. As such, the utility of the biomarker panel, as an aid in the diagnosis of cancer in symptomatic women, is worthy of further investigation in a larger phase 2 biomarker trial.

Peptidylarginine Deiminases Present in the Airways during Tobacco Smoking and Inflammation Can Citrullinate the Host Defense Peptide LL-37, Resulting in Altered Activities

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin‐1–Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1β, cathelicidin human cationic antimicrobial protein-18/LL-37 and β-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1β through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.

The antimicrobial peptide human cationic antimicrobial protein-18/cathelicidin LL-37 as a putative growth factor for malignant melanoma

Recent evidence suggests cathelicidin LL-37 to be a growth factor for various human cancers such as lung cancer, ovarian cancer and breast cancer. However, the effect of LL-37 against malignant skin cancer has not been reported. To investigate whether the human cathelicidin LL-37 is involved in the carcinogenesis of various skin tumours. Human cationic antimicrobial protein-18 (hCAP-18)/LL-37 production in several cell lines including HaCaT, a chronic myelogenous leukaemia (CML) cell line and various melanoma cell lines was examined using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Immunohistochemical analysis of melanoma, nonmelanoma skin cancer and precancerous and benign skin lesions was performed. After adding LL-37 to a melanoma cell line, tumour cell proliferation, migration and invasion were investigated. Human malignant melanoma cell lines overexpressed hCAP-18/LL-37 mRNA and peptide compared with HaCaT and CML cell lines. Immunohistochemistry showed that the peptide was strongly expressed in malignant melanoma and moderately expressed in squamous cell carcinoma, whereas basal cell carcinoma, precancerous lesions and seborrhoeic keratosis showed no or weak expression. LL-37 also stimulated melanoma cell proliferation, migration and invasion in vitro. Cathelicidin LL-37 was primarily expressed in human malignant skin cancer. LL-37 promoted melanoma cell proliferation, migration and invasion in vitro. We report that an increase in the level of LL-37 is associated with malignant skin tumours such as malignant melanoma. These results highlight the importance of LL-37 in the malignant tendency of skin tumours.

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.

Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

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 antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor.

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.

Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes.

Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.