CpG motifs in Porphyromonas gingivalis DNA stimulate interleukin-6 expression in human gingival fibroblasts

Abstract

We suggest here that Porphyromonas gingivalis DNA may function as a virulence factor in periodontal disease through expression of inflammatory cytokine. The bacterial DNA markedly stimulated in a dose-dependent manner interleukin-6 (IL-6) production by human gingival fibroblasts. The stimulatory action was eliminated by treatment with DNase but not RNase. The stimulatory effect was not observed in the fibroblasts treated with eucaryotic DNAs. The bacterial DNA also stimulated in dose- and treatment time-dependent manners the expression of the IL-6 gene in the cells. In addition, the stimulatory effect was eliminated when the DNA was methylated with CpG motif methylase. Interestingly, a 30-base synthetic oligonucleotide containing the palindromic motif GACGTC could stimulate expression of the IL-6 gene and production of its protein in the cells. Furthermore, the synthetic oligonucleotide-induced expression of this cytokine gene was blocked by pyrrolidine dithiocarbamate and N-acetyl-L-cystine, potent inhibitors of transcriptional factor NF-kappaB. Gel mobility shift assay showed increased binding of NF-kappaB to its consensus sequence in the synthetic oligonucleotide-treated cells. Also, using specific antibody against p50 and p65, which compose NF-kappaB, we showed the consensus sequence-binding proteins to be NF-kappaB. These results are the first to demonstrate that the internal CpG motifs in P. gingivalis DNA stimulate IL-6 expression in human gingival fibroblasts via stimulation of NF-kappaB.

FIG. 1

Stimulatory effect of P. gingivalis DNA on IL-6 production in human gingival fibroblasts. Human gingival fibroblasts were cultured in Falcon 24-well culture plates until subconfluent monolayers had formed, then washed and incubated in serum-free α-MEM, and washed again 24 h later. (A) The cells were incubated in the absence or presence of 100 μg of P. gingivalis DNA or eucaryotic DNA per ml. The culture supernatants were harvested 24 h later, and then IL-6 was measured with an ELISA kit. (B) Confluent monolayers were cultured in serum-free α-MEM supplemented or not with various doses of P. gingivalis 33277 DNA. The culture supernatant was harvested 24 h later and then measured for IL-6 with the ELISA kit as described in Materials and Methods. The results are expressed as means ± standard deviations for triplicate cultures. Cont., control.

FIG. 2

P. gingivalis DNA stimulates expression of the IL-6 gene in human gingival fibroblasts. Human gingival fibroblasts were cultured in Falcon 10-cm-diameter culture dishes until subconfluent monolayers had formed, then washed and incubated in serum-free α-MEM, and washed again 24 h later. (A) The cells were treated or not with P. gingivalis 33277 DNA at 100 μg/ml for the times indicated, and then total RNA was prepared. (B) Confluent monolayers were treated or not with P. gingivalis 33277 DNA at various doses, and then total RNA was prepared at 6 h after the initiation of treatment. Northern blot analysis was performed with mouse IL-6 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.

FIG. 3

P. gingivalis DNA-stimulated IL-6 expression in the fibroblasts is eliminated by DNase treatment. Confluent fibroblast monolayers were treated or not with P. gingivalis 33277 DNA (100 μg/ml) that had been pretreated or not with DNase (10 U) or RNase (10 μg). (A) The culture media were harvested at 24 h after the initiation of DNA treatment and then measured for IL-6 by use of the ELISA kit. The results are expressed as means ± standard deviations for triplicate cultures. (B) Total RNA was prepared at 6 h after the initiation of DNA treatment. Northern blot analysis was performed with mouse IL-6 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.

FIG. 4

Involvement of palindromic internal CpG motifs in P. gingivalis DNA-induced IL-6 expression in human gingival fibroblasts. Confluent monolayers were incubated in the absence or presence of P. gingivalis 33277 DNA (100 μg/ml) that had been pretreated or not with CpG methylase. (A) The culture media were harvested at 24 h after the initiation of DNA treatment and then measured for IL-6 with the ELISA kit. The results are expressed as means ± standard deviations for triplicate cultures. (B) Total RNA was prepared at 6 h after the initiation of DNA treatment. Northern blot analysis was performed with mouse IL-6 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.

FIG. 5

Synthetic palindromic internal CpG motifs stimulate also IL-6 expression in human gingival fibroblasts. Confluent monolayers of fibroblasts were incubated in the absence or presence of various doses of 30-mer synthetic oligonucleotides (GAC-30, AGT-30, and GAG-30) as described in Materials and Methods. (A) The culture media were harvested at 24 h after the initiation of the oligonucleotide treatment and then measured for IL-6 by use of the ELISA kit. The results are expressed as means ± standard deviations for triplicate cultures. (B) Total RNA was prepared at 6 h after the initiation of treatment. Northern blot analysis was performed with mouse IL-6 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.

FIG. 6

Involvement of transcriptional factor NF-κB in synthetic palindromic CpG motif-stimulated expression of the IL-6 gene in human gingival fibroblasts. Confluent fibroblast monolayers were treated or not with various doses of PDTC or NAC for 1 h. Then GAC-30 at 200 μg/ml was added, and total RNA was prepared 6 h later. Northern blot analysis was performed with mouse IL-6 and β-actin cDNAs used as probes. An identical experiment independently performed gave similar results.

FIG. 7

Synthetic palindromic CpG motifs stimulate NF-κB binding to its consensus sequence in human gingival fibroblasts. Confluent monolayers were incubated in the absence or presence of GAC-30 at 200 μg/ml, and the nuclear proteins were prepared 6 h later. (A) Gel mobility shift assay was performed with ³²P-labeled oligonucleotide containing the NF-κB consensus sequence. Unlabeled oligonucleotide was used as a competitor. The arrow indicates the position of the DNA and nuclear protein complexes. (B) The nuclear proteins were incubated together with anti-p50 antibody or anti-p65 antibody. Shifted bands of p50 and p65 are indicated by arrows α and β, respectively. An identical experiment independently performed gave similar results.