Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Isolation and characterization of the cell‐surface polysaccharides of Porphyromonas gingivalis ATCC 53978

The cell‐surface carbohydrates of Porphyromonas gingivalis strain ATCC 53978 were isolated and partially characterized. Three separate polysaccharides were found to be present: an extracellular polysaccharide, capsular polysaccharide and lipopolysaccharide. The capsular polysaccharide, which had peculiar, gel‐like viscoelastic properties, was found to be comprised of mannuronic acid, glucuronic acid, galacturonic acid, galactose, and 2‐acetamido‐2‐deoxy‐ d ‐glucose in relative molar ratios of 0.6:0.9:0.5:0.5:1.0, respectively. The extracellular polysaccharide was found to contain mannose, rhamnose, glucose, galactose, and 2‐acetamido‐2‐deoxy‐ d ‐glucose in relative molar ratios of 13.5:1.4:1.0:2.0:1.0, respectively. The lipopolysaccharide was found to contain an O‐antigen with a regular tetrasaccharide repeat unit comprised of 4‐linked α‐ l ‐rhamnopyranosyl, 6‐linked α‐ d ‐glucopyranosyl, 3‐linked α‐ d ‐galactopyranosyl, and 4‐linked 2‐acetamido‐2‐deoxy‐β‐ d ‐glucopyranosyl residues in equimolar proportions.     

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.     

槲皮素对牙龈卟啉单胞菌脂多糖刺激的人牙龈成纤维细胞生物学行为的影响研究

目的研究槲皮素对牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g LPS)刺激的人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学行为的影响。方法采用DNA片段凝胶电泳、CCK-8法、细胞划痕法观察不同浓度槲皮素(10、20、50、100μmol/L)对体外培养HGFs的毒性作用,以及对HGFs增殖与迁移的影响。采用P.g LPS刺激HGFs来建立体外炎症刺激模型,通过流式细胞术、细胞活性氧(reactive oxygen species,ROS)检测、酶联免疫吸附试验(ELISA)和实时荧光定量PCR(qRT-PCR)进一步观察槲皮素对HGFs凋亡、ROS的产生及肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)和前列腺素E2(prostaglandin E2,PGE2)表达的影响。结果槲皮素对HGFs无毒性作用,且对HGFs的增殖无影响(P>0.05)。各组细胞迁移率总的比较,差异有统计学意义(F=9.973,P<0.05),在处理48 h后,20μmol/L槲皮素处理组细胞迁移率大于10、50μmol/L槲皮素处理组以及对照组,差异均有统计学意义(均P<0.05)。槲皮素对P.g LPS刺激的HGFs凋亡具有抑制作用,且其能够抑制并预防P.g LPS刺激的HGFs中ROS相对产生量增加现象(均P<0.05)。槲皮素处理显著抑制了P.g LPS诱导的TNF-α表达增加现象(P<0.05),而槲皮素处理组PGE2的表达水平与对照组比较,差异无统计学意义(P>0.05)。结论浓度为20μmol/L的槲皮素能够促进体外培养HGFs的迁移,且具有抗氧化、抗炎保护作用。     

Cytotoxicity of Porphyromonas gingivalis toward cultured human gingival fibroblasts

Direct Cytotoxicity of black-pigmented anaerobic rods was studied on the confluent monolayer of human gingival fibroblasts in vitro. Only strains of Porphyromonas gingivalis caused morphological alteration (cell-rounding) and notable depression of viability of fibroblasts. To determine the location of the Cytotoxicity, bacterial surface components, i.e., outer membrane, lipopolysaccharide, fimbriae and outer membrane vesicles were prepared from P. gingivalis and their cytotoxicity was assessed. Among these preparations, only outer membrane vesicles are supposed to have high affinity to human gingival fibroblasts, and the cytotoxicity of outer membrane vesicles was found to be much stronger than that of the other constituents. This cytotoxic factor seemed to consist largely of protein and to be associated with the enzyme activity of outer membrane vesicles. The effects of some protease inhibitors and L-cysteine on the cytotoxicity of outer membrane vesicles suggest that the mechanism of cell-rounding is different from that of cell death.     

Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis

We previosly reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.     

Surface components of Porphyromonas gingivalis

Background and Objective:  Research on Porphyromonas gingivalis , a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis .
Material and Methods:  Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa .
Results:  Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly.
Conclusion:  Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.     

Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein,Omp28

We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28‐kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET‐24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti‐whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti‐rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains.     

Identification and characterization of B‐cell epitopes of a 53‐kDa outer membrane protein from Porphyromonas gingivalis

We have previously reported that Porphyromonas gingivalis FDC 381 possesses a 53‐kDa protein antigen (Ag53) on its outer membrane that evokes a strong humoral immune response in many patients with periodontal disease and that the humoral immune responses to Ag53 differ greatly among patients. To understand how the individual humoral immune system against Ag53 was determined, the regions of Ag53 recognized by specific antibody (B‐cell epitopes) and dominant subclasses of serum immunoglobulin G (IgG) against major B‐cell epitopes were examined by enzyme‐linked immunosorbent assay. This study used sera from six patients with periodontitis, which all reacted strongly with sonic extracts of P. gingivalis 381 and with purified Ag53, and sera from six periodontally healthy children, which did not react with either sonic extracts of P. gingivalis 381 or Ag53. The epitopes were identified using synthetic 5‐residue overlapping decapeptides covering the entire Ag53. Thirteen of 89 synthetic decapeptides showed a strong reaction with sera from the periodontal patients, but no reaction with those from the healthy children. Four peptides of 13 exerted different immune responses among patients. Furthermore, restriction analyses of the highly antigenic regions revealed that three sequences, RAAIRAS, YYLQ and MSPARR, were identified as major B‐cell epitopes. Additionally, these epitopes were recognized mainly by the IgG2 isotype. These data suggest that the difference of B‐cell epitopes might influence individual differences in antibody titer against Ag53 and also that the epitopes recognized commonly by multiple antibodies are quite valuable for peptide vaccine development against P. gingivalis infection.     

Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein,Omp28

We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains.     

不同fimA基因型牙龈卟啉单胞菌对牙龈成纤维细胞基质金属蛋白酶表达的影响

目的 观察不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对人牙龈成纤维细胞基质金属蛋白酶(matrix metalloproteinases,MMP)表达水平的影响,探讨fimA基因型与Pg致病力之间的关系.方法 Pg ATCC 33277(Ⅰ型)、WCSP115(Ⅱ型)、WCSP1.5(Ⅲ型)、W83(Ⅳ型)分别与牙龈成纤维细胞在标准条件下共同孵育(对照组为达尔伯克氏改良伊格尔培养基),于孵育后1、3、6和12 h收集细胞和培养上清液,应用实时荧光定量反转录聚合酶链反应(RT-PCR)和ELISA法分别检测牙龈成纤维细胞MMP-1、MMP-2的mRNA及蛋白的动态表达.结果 与对照组相比,Pg刺激下牙龈成纤维细胞MMP-1、MMP-2的mRNA和蛋白水平表达量均明显上调(P<0.01);其中Ⅱ型fimA型Pg的刺激作用强于其他各型,不同时间点MMP-1 mRNA相对表达量及蛋白分泌水平分别为(28.88±3.12)～(231.01±24.99)、(1.35±0.17)～(3.08±1.20);MMP-2 mRNA相对表达量及蛋白分泌水平分别为(20.42±2.21)～(188.34±37.37)、(2.57±0.76)～(18.08±1.15),与其他各型相比差异有统计学意义(P<0.05);Ⅲ型Pg的刺激作用较弱,不同时间点MMP-1 mRNA相对表达量及蛋白分泌水平分别为[(5.11±0.55)～(72.84±8.84)]μg/L、[(0.68±0.13)～(1.46±0.94)]μg/L;MMP-2 mRNA相对表达量及蛋白分泌水平分别为[(4.55±0.55)～(25.75±3.12)]μg/L、[(2.28±0.93)～(11.22±2.46)]μg/L,与其他各型相比差异有统计学意义(P<0.05).结论 Pg可以诱导牙龈成纤维细胞过表达MMP,fimA基因型与Pg的毒力作用相关,fimA型可能为Pg致病力差异的基因基础.     

牙龈卟啉单胞菌脂多糖对人牙周膜成纤维细胞胶原吞噬作用的影响

目的研究牙周病致病菌牙龈卟啉单胞菌脂多糖（LPS）对体外培养的人牙周膜成纤维细胞（hPDLF）胶原吞噬作用的影响。方法将不同质量浓度的LPS加入体外培养的hPDLF 48 h后,采用荧光定位术和流式细胞技术检测hPDLF胶原吞噬率的变化。结果LPS导致hPDLF胶原吞噬率显著增加（P<0.05）。结论牙龈卟啉单胞菌脂多糖具有促进hPDLF吞噬胶原的作用,可能是牙周组织破坏机制之一。     

Strong cytotoxicity to human gingival fibroblasts by Porphyromonas gingivalis ATCC 33277

The aim of the present study was to analyze the cytotoxicity of some bacterial species associated with periodontal diseases. The specificity of cytotoxicity was estimated against cells of various origin and from different individuals. The reference bacteria were Actinobacillus actinomycetemcoinitans, Porphyromonas gingivalis and Fusobacterium nucleatum . These bacteria were cultured for 24 h in liquid media and the supernatants were used in cytotoxicity assays. The target cells used were human gingival fibroblasts (GF). dermal fibroblasts (K4), gingival epithelial cells (E) and HeLa-cells (HeLa). These cells were exposed at 4 h or 24 h, respectively, to various concentrations of culture supernatants from the selected bacteria. The influence on the viability and metabolism of the cells were estimated quantitatively as increase in neutral red uptake and lactic acid production. Growth medium supernatants of P. gingivalis 33277 were strongly cytotoxic to gingival fibroblasts after 24 h incubation, compared to supernatants of P. gingivalis 381 or W 50, A. actinomycetemcoinitans or F. nucleatum cultures. The toxic effect of P. gingivalis 33277 decreased drastically after heat inactivation, which indicates effects of proteins. By adding anti-sera the cytotoxicity of P. gingivalis 33277 could be dose dependently neutralized, which was not the case when supernatants of A. actinomycetemcoinitans was tested. Target cells of epithelial origin did not show increased cytotoxicity to P. gingivalis 33277 . The results of the present study strengthen the hypothesis that P. gingivalis remains as a suspect causative key component in periodontal diseases.     

Isolation and characterization of the cell-surface polysaccharides of Porphyromonas gingivalis ATCC 53978

The cell-surface carbohydrates of Porphyromonas gingivalis strain ATCC 53978 were isolated and partially characterized. Three separate polysaccharides were found to be present: an extracellular polysaccharide, capsular polysaccharide and lipopolysaccharide. The capsular polysaccharide, which had peculiar, gel-like viscoelastic properties, was found to be comprised of mannuronic acid, glucuronic acid, galacturonic acid, galactose, and 2-acetamido-2-deoxy-D-glucose in relative molar ratios of 0.6:0.9:0.5:0.5:1.0, respectively. The extracellular polysaccharide was found to contain mannose, rhamnose, glucose, galactose, and 2-acetamido-2-deoxy-D-glucose in relative molar ratios of 13.5:1.4:1.0:2.0:1.0, respectively. The lipopolysaccharide was found to contain an O-antigen with a regular tetrasaccharide repeat unit comprised of 4-linked alpha-L-rhamnopyranosyl, 6-linked alpha-D-glucopyranosyl, 3-linked alpha-D-galactopyranosyl, and 4-linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues in equimolar proportions.     

Effects of Porphyromonas gingivalis on human gingival fibroblasts from healthy and inflamed tissues

Background and Objective:  This study compared the ability of human gingival fibroblasts (HGFs) isolated from healthy and inflamed gingival tissues to degrade collagen in the presence and absence of Porphyromonas gingivalis supernatant.
Material and Methods:  Human gingival fibroblasts were cultured from explants of 21 healthy and 21 inflamed periodontal tissues. The HGFs that grew out of the explants were seeded in the center of six-well plates coated with collagen in the presence and absence of 10% P. gingivalis supernatant. An inflamed and a healthy cell line were also evaluated with Arg-gingipain. After 6 days, Coomassie Blue was used to visualize the collagen cleavage.
Results:  The collagen was totally cleaved in 12 (aggressive) of the 21 cell lines isolated from the inflamed tissues in the presence of P. gingivalis . The remaining nine cell lines (non-aggressive) cleaved only the collagen underneath the cell colonies in the presence of P. gingivalis . Of the healthy tissues, five (aggressive) of the 21 cell lines cleaved all the collagen and 16 cell lines (non-aggressive) only cleaved the collagen underneath the cell colonies in the presence of P. gingivalis . All the collagen was cleaved by an aggressive cell line and only the collagen underneath the cell colonies was cleaved by a non-aggressive cell line in the presence of Arg-gingipain.
Conclusion:  The collagen in the wells was more readily cleaved by the inflamed than by the healthy cell lines, and the difference was statistically significant ( p  = 0.0278). Arg-gingipain gave identical results to the P. gingivalis supernatant.     

Transcutaneous immunization with an outer membrane protein of Porphyromonas gingivalis without adjuvant elicits marked antibody responses

BACKGROUND/AIMS: We have previously reported that specific immunoglobulin G (IgG) antibodies induced by transcutaneous immunization (TCI) with a 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis, with cholera toxin (CT) as adjuvant, inhibited coaggregation by P. gingivalis. In this study, we further pursue the potential of the 40k-OMP as a transcutaneous vaccine. METHODS/RESULTS: TCI of rats administered 40k-OMP elicited significant 40k-OMP-specific serum IgG and IgA, as well as salivary IgG antibody titers. Importantly, these antibody responses were induced without adjuvant. Thus, both serum and saliva antibody titers induced by TCI with the 40k-OMP alone were identical to those of 40k-OMP plus cholera toxin as adjuvant. The serum antibody responses induced by 40k-OMP persisted for more than 140 days. On the other hand, salivary IgG anti-40k-OMP antibodies were gradually decreased. Analysis of antibody-forming cells (AFCs) confirmed the antibody titers by detecting high numbers of 40k-OMP-specific IgG AFCs in spleen and cervical lymph node. CONCLUSION: Since 40k-OMP-specific IgG inhibited the coaggregation of P. gingivalis with Streptococcus gordonii, and the hemagglutinin activity of P. gingivalis, TCI with the 40k-OMP may be important as an adjuvant-free immunogen for the prevention of chronic periodontitis.     

Infection of primary human gingival fibroblasts by Porphyromonas gingivalis and Prevotella intermedia

Adhesion and penetration of clinical isolates of Porphyromonas gingivalis and Prevotella intermedia in human gingival fibroblast monolayers were studied by transmission electron microscopy (TEM). Fibroblasts were cultured from biopsies of human healthy gingiva. Porphyromonas gingivalis and Prevotella intermedia were isolated from patients with periodontitis. Fibroblasts were incubated with microorganisms in an antibiotic-free medium for 24 h. Then cultures were washed to remove nonadherent bacteria. Consecutively, infected cultures were grown for another 24 h. Thereafter, the treated monolayers were prepared for TEM investigations. Internalized Porphyromonas gingivalis and Prevotella intermedia were visible after 24 h of incubation. Prevotella intermedia showed only division in cytoplasm of fibroblasts after 24 h and 48 h incubations. Infected fibroblasts revealed various morphological alterations such as extensive vacuolization and breakdown of mitochondria. These findings demonstrate that Porphyromonas gingivalis and Prevotella intermedia may invade human gingival fibroblasts and thus may damage these cells directly or due to the release of microbial cytotoxic components. Received: 15 October 1999 / Accepted: 19 November 1999     

Suppressive effect of the antimicrobial peptide LL-37 on expression of IL-6, IL-8 and CXCL10 induced by Porphyromonas gingivalis cells and extracts in human gingival fibroblasts

Porphyromonas gingivalis is a major periodontogenic bacterium and possesses immunostimulatory components, such as lipopolysaccharides (LPS) and fimbriae. The host antimicrobial peptide, LL-37, suppresses proinflammatory responses of immune cells but its effect on human gingival fibroblasts (HGFs) is not known. In this study, we assessed the effect of LL-37 on the proinflammatory responses of HGFs stimulated with P. gingivalis cells and their components. Live P. gingivalis cells did not induce proinflammatory responses of HGFs, and LL-37 did not alter these responses. However, LL-37 was able to suppress the killed P. gingivalis cell-induced secretion of interleukin (IL)-6 and IL-8. LL-37 also suppressed the expression of IL6, IL8, and CXCL10 genes that was induced by P. gingivalis components, including phenol-water extracts, lipid A, and fimbriae, and the induction of phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by P. gingivalis lipopolysaccharide (LPS). CAMP was found to be expressed in oral epithelial cells but not in HGFs, despite stimulation with P. gingivalis components. Therefore, LL-37 can exert a suppressive effect on P. gingivalis-induced proinflammatory responses of HGFs in a paracrine manner, suggesting that excess inflammatory responses to P. gingivalis in the gingival tissue are suppressed by LL-37 in vivo.     

Cytokine production induced by a 67‐kDa fimbrial protein from Porphyromonas gingivalis

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. P. gingivalis ATCC 33277 has two distinctly different fimbriae expressed on the cell surface. The 67‐kDa fimbriae differ in size and antigenicity from the earlier reported FimA, a major 41‐kDa fimbrial component of P. gingivalis. Expression of the 67‐kDa fimbriae on the cell surface of a fimA mutant was investigated by electron microscopy. The 67‐kDa fimbrial protein was purified from the fimA mutant by sonication, precipitation, and chromatography on a DEAE Sepharose CL‐6B column. The N‐terminal amino acid sequence of the 67‐kDa fimbrillin was distinct from that of the 41‐kDa fimbrillin. Moreover, we have found that the 67‐kDa fimbrial protein from P. gingivalis ATCC 33277 induced IL‐1α, IL‐β, IL‐6 and TNFα cytokine expression in mouse peritoneal macrophages. These results suggest that P. gingivalis 67‐kDa fimbriae may play a part in the inflammatory response during the development of periodontal diseases.