examine whether Porphyromonas gingivalis fimbriae, important structures involved in ... present study indicates that P. gingivalis fimbriae can induce gene ...
Vol. 60, No. 4
INFECTION AND IMMUNITY, Apr. 1992, p. 1544-1549 0019-9567/92/041544-06$02.00/0 Copyright © 1992, American Society for Microbiology
Porphyromonas gingivalis Fimbriae Induce Expression of the Neutrophil Chemotactic Factor KC Gene of Mouse Peritoneal Macrophages: Role of Protein Kinase C SHIGEMASA HANAZAWA,* YUKIO MURAKAMI, AKIRA TAKESHITA, HIDEKAZU KITAMI, KAZUZO OHTA, SHIGERU AMANO, AND SHIGEO KITANO Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan Received 17 September 1991/Accepted 23 January 1992
To account for infiltration of the periodontal tissues by neutrophils, the present study was undertaken to examine whether Porphyromonas gingivalis fimbriae, important structures involved in attachment of the bacteria to periodontal tissues, induce gene expression of the neutrophil chemoattractant KC in macrophages. The fimbriae induced expression of the KC gene of mouse peritoneal macrophages in a dose-dependent fashion. The peak of KC gene expression was observed as early as 1 h after initiation of the treatment. However, the gene expression was short lived, with the expression decreasing gradually after 6 h. A nuclear transcriptional assay showed that the fimbriae regulated the KC gene expression at a posttranscriptional level. We observed that the fimbria-induced KC gene expression was not regulated by endogenous or exogenous prostaglandin. Furthermore, forskolin, a potent activator of adenyl cyclase, and dibutyryl cyclic AMP were incapable of inducing KC gene expression of the peritoneal macrophages. H-8 and HA 1004, inhibitors of cyclic nucleotide-dependent protein kinases, had little effect on the fimbria-induced KC gene expression. On the other hand, the fimbria-induced KC gene expression was inhibited markedly by treatment with H-7, a potent inhibitor of protein kinase C. We also observed that phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, induced KC gene expression of peritoneal macrophages in a dose-dependent fashion. In addition, the fimbria-induced KC gene expression was suppressed in the peritoneal macrophages pretreated for 24 h with phorbol 12-myristate 13-acetate. These results suggest that the KC gene expression was mediated through activation of protein kinase C and not through that of cyclic nucleotide-dependent protein kinases. The present study indicates that P. gingivalis fimbriae can induce gene expression of the neutrophil chemotactic factor KC by macrophages via protein kinase C and suggests that this factor may be involved in infiltration of neutrophils into the periodontal tissues of adult periodontal patients.
stand the chemotactic mechanism(s) for infiltration of the cells into the periodontal tissues. The KC gene was cloned from mouse fibroblasts as one of the genes inducible by platelet-derived growth factor (PDGF) (9). PDGF is also known to induce the expression of the c-myc and c-fos protooncogenes. Since some studies (2, 10) have suggested that these oncogenes may play a functional role as mediators of the mitogenic response to PDGF and other growth factors, it had been assumed that the KC gene may also be involved in regulation of cell proliferation. However, some recent studies (23, 25) have demonstrated that products of the KC gene and the JE gene, another gene induced by PDGF, have cytokinelike properties. Watanabe et al. (29) recently showed that the amino acid sequences of the neutrophil chemoattractant CINC produced by a rat kidney epithelioid cell line have striking similarities to the sequence of the protein encoded by the KC gene, and they suggested that KC is the mouse counterpart of CINC. These findings indicate that KC is a novel neutrophil chemoattractant. Chemoattractants such as leukotriene B4, C5a, and platelet-activating factor have all been shown to be chemotactic for both neutrophils and monocytes or macrophages (4, 5). However, KC is a chemoattractant having high specificity for neutrophils. Therefore, in the present study, we examined the effect of P. gingivalis fimbriae on gene expression of the neutrophil chemotactic factor KC in mouse peritoneal macrophages. We demonstrate in this report that fimbriae strongly
Porphyromonas gingivalis fimbriae, important cell structures needed for attachment of the bacteria to periodontal tissues, may play an important role in triggering the patho-
genesis brought about by the organism. Our previous studies (15, 21) have indicated that human gingival fibroblasts spontaneously produce thymocyte-activating factor and that the fimbriae stimulate production of the activating factor by the gingival fibroblasts. In addition, we demonstrated more recently that fimbriae also induce gene expression and production of interleukin-1 of mouse peritoneal macrophages (16). However, the effects of the fimbriae on various functions of gingival fibroblasts, macrophages, and lymphocytes have not been defined in detail. Many investigators (1, 6, 12, 24, 27) have demonstrated that monocytes or macrophages and neutrophils have a functional role in the inflammatory response. Neutrophils can participate in the inflammatory process by the release of proteases and reactive oxygen. Recent studies (8, 11, 28) have also shown that neutrophils are able to synthesize a number of inflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-alpha. Since it has been shown that neutrophils are present in high proportions in gingivae of patients with periodontitis, the cells may contribute to inflammation and alveolar bone loss in periodontal disease. Therefore, it is very important to under-
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Corresponding author. 1544
VOL. 60, 1992
P. GINGIVALIS FIMBRIAE AND GENE EXPRESSION
induce gene expression of the KC chemotactic factor in macrophages, most probably via protein kinase C.
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(b) KC
MATERIALS AND METHODS Preparation of P. gingivalis fimbriae. P. gingivalis ATCC 33277 fimbriae were prepared and purified from cell washings by the method of Yoshimura et al. (30) as described previously (16). The purified preparation was observed on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a single band having a molecular mass of 43,000 Da. No significant contamination of lipopolysaccharides was observed in the purified sample according to some experimental proofs described in previous reports (15, 16). Protein content of the fimbriae was measured by the method of Bradford (3). Preparation of mouse peritoneal macrophages. BALB/c mice, 6 to 10 weeks of age, were injected intraperitoneally with 3 ml of thioglycolate medium (Difco Laboratories, Detroit, Mich.). The peritoneal exudate cells were harvested by washing the peritoneal cavity with RPMI 1640 containing heparin (50 U/ml) on day 3 after the injection. The peritoneal exudate cells (2 x 10 cells) were inoculated into 90-mm Falcon plastic dishes, and then the peritoneal macrophages were prepared as described earlier (17). The prepared macrophages were treated for selected times with test samples. cDNA hybridization probe. Plasmids containing mouse KC cDNA sequences were provided by C. D. Stiles. In addition, plasmids with human 3-actin cDNA were obtained from JCRB, Tokyo, Japan. The methods used for plasmid preparation were described earlier (20). Preparation of RNA and Northern blot analysis. Total cellular RNA in macrophages prepared from peritoneal exudate cells was extracted by the guanidine isothiocyanate procedure (7). The RNA was subjected to 1% agarose electrophoresis and blotted onto a nylon membrane (MSI Magnagraph, Westboro, Mass.). The membranes were then baked and prehybridized for 18 h at 42°C in a mixture containing 50% formamide, 1% SDS, 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]), lx Denhardt's solution (0.02% Ficoll, 0.02% bovine serum albumin BSA, 0.02% polyvinylpyrolidone), 0.25 mg of denatured salmon sperm DNA per ml, and 50 mM sodium phosphate buffer (pH 6.5). Hybridization to primer-extended cDNA probes labeled with 5'-[a-32P]dCTP (Amersham Japan, Tokyo, Japan) for KC and ,-actin was carried out overnight at 42°C in fresh Denhardt's solution containing 10% dextran sulfate. After hybridization, the membranes were washed sequentially with 2 x SSC-0. 1% SDS, 1 x SSC, and 0.1x SSC, each at 55°C for 15 min. The membranes were dried and exposed to X-ray film (Eastman Kodak Co., Rochester, N.Y.) at -70°C. 3-Actin was used as an internal standard for the quantification of total mRNA on each lane of the gel. Nuclear transcriptional (run-on) assay. Macrophages prepared from thioglycolate-induced peritoneal exudate cells (5 x 107 cells) were treated with fimbriae (4 ,ug of protein per ml), and their nuclei were isolated as described previously (13). Transcription initiated in intact cells was allowed to proceed for 30 min at 30°C in the presence of 5'-[ot-32P]UTP, and the RNA was isolated and hybridized to slot-blotted cDNA probes (5 ,ug per slot). Blots were hybridized for 72 h and autoradiographed for 72 h. The 1-actin gene was utilized as an
internal standard.
Reagents. Indomethacin, prostaglandin (PG) E2 (PGE2), phorbol 12-myristate 13-acetate (PMA), forskolin, and N,2O-dibutyryl cyclic AMP (dBt-cAMP) were purchased from
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FIG. 1. Inducing effect of P. gingivalis fimbriae on KC gene expression of mouse peritoneal macrophages. (a) The cells from BALB/c mice were incubated with or without fimbriae (4 ILg of protein per ml), and total RNA was prepared at various times after the initiation of incubation. (b) The cells from BALB/c mice were incubated for 1 h with or without various concentrations of fimbriae, and then their total RNA was prepared. Northern blot analysis was performed with KC and 3-actin cDNAs used as probes. Exposure times to the X-ray film were 17 (a) and 6 (b) h.
Sigma Chemical Co., St. Louis, Mo. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine-dihydrochloride (H-7), N-[2-(methylamino)ethyl 5 isoquinolinesulfonamide dihydrochloride] (H-8), and N-(2-guanidineethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004) were obtained from Seikagaku Kougyo, Tokyo, Japan. -
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RESULTS
Inducing effect of P. gingivalis fimbriae on KC gene expression of mouse peritoneal macrophages. By using Northern blotting analysis, we assessed the effect of the fimbriae on the KC mRNA level of the peritoneal macrophages. Fig. 1 shows the results. A marked mRNA level was observed as early as 1 h after initiation of the fimbria treatment; however, this striking increase was short lived, for the level decreased gradually after 6 h. KC mRNA was clearly induced by fimbriae at 1 ,ug of protein per ml, and the induction was dose dependent. The level of 1-actin mRNA, an internal control for the quantification of total mRNA on the gel, was not affected by the fimbria treatment. P. gingivalis fimbriae regulate KC gene expression at a posttranscriptional level in mouse peritoneal macrophages. To ascertain whether the fimbriae increase the rate of transcription of the KC gene, we examined transcriptional activity by a transcriptional assay using nuclei isolated from the peritoneal macrophages treated with the fimbriae. As shown in Fig. 2, since the fimbriae did not stimulate the transcriptional activity of the KC gene, the fimbria-induced KC gene expression may be regulated at the posttranscriptional level. No significant change was detected in the level of 3-actin mRNA. P. gingivalis fimbria-induced KC gene expression in mouse peritoneal macrophages is mediated via protein kinase C. We then examined the second messenger for the fimbria-induced KC gene expression in the peritoneal macrophages. First, we tested the role of protein kinase C in KC gene expression. The cells were incubated with fimbriae (4 ,ug of protein per ml) in combination with H-7 (100 ,uM), a potent inhibitor of protein kinase C. After 1 h, the KC mRNA level in the cells was analyzed by the Northern blotting assay. H-7 markedly inhibited the fimbria-stimulated KC mRNA level in the cells (Fig. 3). Also, KC gene expression was induced by PMA, a
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INFECT. IMMUN.
HANAZAWA ET AL.
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potent activator of protein kinase C, in a dose-dependent fashion (Fig. 4). In another experiment, we examined the effect of pretreatment with PMA on fimbria-induced KC gene expression. The peritoneal macrophages were pretreated with PMA for 24 h before initiation of the fimbria treatment, and thereafter the cells were treated with the fimbriae. After 1 h, the KC gene mRNA level was measured. Figure 5 shows that PMA pretreatment clearly suppressed fimbria-induced KC gene expression. We then examined the effect of modulators of cyclic nucleotide-dependent protein kinases on KC gene expression. Since it is well known that PGE2 causes accumulation of cyclic AMP by activating adenyl cyclase and consequently induces protein kinase A-dependent phosphorylation, we examined the effect of PGE2 on KC gene expression in the fimbria-treated and untreated cells. Figure 6 shows the results. Since the fimbria-induced KC gene expression was not affected by indomethacin (10-6 M), a cyclooxygenase inhibitor, endogenous PG is not involved in the induction of the gene expression. The fimbria-induced KC mRNA level was not affected by the addition of PGE2 at a concentration
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of 100 ng/ml, and exogenous PGE2 (100 ng/ml) was incapable of inducing the KC gene expression in the cells. Furthermore, the induction of KC gene expression was not affected by H-8 or HA 1004, inhibitors of cyclic nucleotide-dependent protein kinases (Fig. 7). Finally, the effects of forskolin, an activator of adenyl cyclase, and dBt-cAMP on the KC gene expression were examined. Neither reagent was capable of inducing the gene expression in the cells, and fimbria-induced KC gene expression was not affected by either treatment (Fig. 8). These results suggest that the fimbriae may induce KC gene expression of mouse peritoneal macrophages via protein kinase C and not by activation of cyclic nucleotidedependent protein kinases.
DISCUSSION Some investigators (1, 6, 12, 24, 27) have demonstrated the infiltration of neutrophils and macrophages into periodontal tissues in adult periodontal disease, a chronic inflamma-
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