Involvement of phospholipase D in regulating expression of anti-microbial peptide human beta-defensin-2

Human beta-defensin expression correlates with differentiation in oral epithelium, and calcium ion, an important regulator of epithelial differentiation, plays a critical role in regulation of human beta-defensin-2 (hBD-2) mRNA expression. Phospholipase D (PLD) also regulates epithelial differentiation. Therefore, we examined the role of PLD in hBD-2 up-regulation by cell wall extract of Fusobacterium nucleatum and phorbol 12-myristate 13-acetate (PMA), two known hBD-2 activators. We found that hBD-2 mRNA up-regulation in human gingival epithelial cells (HGECs) by these two activators was mediated by PLD activation and blocked by ethanol and 1-butanol, PLD inhibitors. PLD activity was induced by stimulation with these two activators, and phosphatidic acid (PA), a product generated from the PLD enzymatic activity, was detected in stimulated HGECs. Dioctanoyl PA commonly used for PA induced hBD-2 mRNA expression. mRNAs for PLD1 alpha and beta splice variants as well as PLD1 protein were constitutively expressed, whereas mRNA and protein for PLD2 were expressed at much lower levels than those for PLD1. Moreover, pre-treatment with (+/-)-propanolol, an inhibitor of phosphatidic acid phosphohydrolases that are the downstream signaling molecules in the PLD pathway, significantly blocked hBD-2 mRNA induction by PMA in a dose-dependent manner. In conclusion, these findings indicate the involvement of PLD activation in hBD-2 up-regulation in HGECs, which correlates with the state of epithelial differentiation.     

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.     

Production of β-Defensin Antimicrobial Peptides by the Oral Mucosa and Salivary Glands

beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms.     

Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells

Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a "zipping" mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions; immunohistochemistry and in situ hybridization

Human beta-defensin(hBD)-2, an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.     

Calprotectin expression by gingival epithelial cells

Calprotectin, a heterodimer of MRP8 and MRP14 with antimicrobial properties, is found in the cytosol of neutrophils, monocytes, and human gingival keratinocytes. During inflammation of the oral mucosa, the expression of immunoreactive calprotectin appears upregulated. Given the possible cell sources, we sought to learn if epithelial cells upregulate calprotectin in response to proinflammmatory agents. First, human gingival keratinocytes were maintained in primary culture until senescence. At each passage, cells were harvested and analyzed for quantitative expression of MRP8 and MRP14 subunit mRNA by RNase protection assays and calprotectin complex by enzyme-linked immunosorbent assay. Calprotectin expression was constitutive in the primary gingival keratinocytes, but calprotectin-specific mRNA and protein tended to increase as the cells neared senescence. To test whether calprotectin expression was inducible, immortalized gingival keratinocyte cultures were treated for 2 to 4 h with lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta). As a positive control for inducible expression, immortalized keratinocytes were incubated with phorbol myristate acetate (PMA) (50 ng/ml) for 24 h. Incubation with PMA stimulated increased expression of MRP8 and MRP14 mRNA within 2 h, peaking within 5 h. MRP8- and MRP14-specific mRNA expression by immortalized keratinocytes appeared to be unaffected by LPS or IL-1 beta. In contrast, LPS, IL-1 beta, and PMA each upregulated IL-8. These data show that calprotectin mRNA is expressed constitutively in cultured keratinocytes, while expression by immortalized cells appears to be independent of the exogenous proinflammatory agents LPS and IL-1 beta.     

Cetirizine counter-regulates interleukin-8 release from human epithelial cells (A549)

BACKGROUND: Cetirizine, a H1-receptor antagonist, exerts besides its well-known anti-allergic potential an array of anti-inflammatory activities. In particular epithelial cells activated in the presence of cetirizine showed a reduced ICAM-1 cell surface expression and a diminished release of sICAM-1. OBJECTIVE: We wondered whether cetirizine might influence the release of interleukin-8 (IL-8) from human epithelial cells activated with agonists distinct from histamine. METHODS: We used the human lung epithelial cell line A549 for our in vitro studies. IL-8 release was determined by IL-8 enzyme immunoassay, the intracellular staining for IL-8 and NF-kB was analysed by FACS analysis and IL-8 mRNA steady state level was studied by Northern blot analysis. Confluent epithelial cell monolayer were pre-incubated with cetirizine (0.01 -1.0 micromol/L) for 30 min and afterwards activated with pro-inflammatory cytokines (TNF-alpha IL-1beta, IL-6, IFN-gamma) or different agonists (PMA, NaF, respiratory syncytial virus [RSV]) for 24 h. RESULTS: Epithelial cells stimulated with TNF-alpha IL-1beta, PMA and RSV, respectively, showed a significantly increased release of IL-8. Pre-incubation with cetirizine diminished the IL-8 release from cells activated with TNF-alpha or PMA in a significant manner. The reduced IL-8 release coincided with a diminished percentage of cells expressing IL-8. Northern blot analysis revealed a reduced steady state level of IL-8 mRNA in cells pretreated with cetirizine and stimulated with TNF-alpha. Furthermore, a decreased amount of accessible DNA-binding sites of the nuclear factor kappa B (NF-kB) was determined by FACS analysis. CONCLUSIONS: These results suggest that cetirizine reduced the release of IL-8 from A549 cells stimulated with PMA and TNF-alpha, respectively, by lowering IL-8 gene expression. Therefore, cetirizine might exert anti-inflammatory effects beyond its H1-receptor antagonistic activity in the course of inflammatory respiratory tract disorders such as bronchial asthma and allergic rhinitis.     

乳杆菌细胞壁成分激活人阴道上皮细胞β-防御素-2表达机制研究

目的 探讨乳杆菌细胞肇成分(Lactobacillus cell wall extract,LCWE)对人阴道上皮细胞β-防御素-2的诱导作用及细胞内信号转导机制.方法 采用实时定量PCR和Western blot方法检测LCWE处理对人阴道上皮细胞(WZV-1)β-防御素-2表达的影响;Western blot方法检测LCWE处理后WZV-1细胞内NF-κB和p38MAPK信号通路活化情况;实时定量PCR和Western blot方法检测NF-κB抑制剂和p38MAPK抑制剂预处理WZV-1细胞对LCWE诱导β-防御素-2表达的影响.结果 LCWE可诱导WZV-1细胞β-防御素-2表达且存在着明显的时间效应和剂量依赖关系,50μg/mlLCWE处理细胞8 h对β-防御素-2的上调幅度最大,刺激0.5 h后可引起细胞内p38MAPK蛋白的磷酸化显著增加,1 h达到顶峰;刺激0.5 h后可引起细胞核内NF-κB含量显著增加(P<0.05),2 h达到顶峰.NF-κB抑制剂PDTC、p38MAPK抑制剂SB20380预处理WZV-1细胞,可明显抑制LCWE诱导WZV-1细胞β-防御素-2的表达.结论 LCWE具有激活NF-κB和p38MAPK信号通路诱导阴道-上皮细胞β-防御素-2的作用.

Abstract：

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.