Interleukin (IL)-1 and Porphyromonas gingivalis lipopolysaccharide stimulation of IL-6 production by fibroblasts derived from healthy or periodontally diseased human gingival tissue.

BACKGROUND: Human gingival fibroblasts (HGF) play a key role in tissue repair and destruction. These cells express low levels of interleukin (IL)-6 constitutively and increased levels after stimulation with lipopolysaccharide (LPS) or the cytokines IL-1 and tumor necrosis factor-alpha (TNF-alpha). However, little information is available on IL-6 production by fibroblasts derived from diseased tissue. The present study compared constitutive and induced IL-6 production by human gingival fibroblasts (HGF) derived from healthy and diseased periodontal tissue. We also evaluated whether IL-1 acted in a synergistic manner with LPS in inducing IL-6 production by HGF and whether LPS acted via CD14. METHODS: HGF derived from healthy and diseased tissue and foreskin fibroblasts were grown to confluency, photographed, and counted, and their constitutive IL-6 production was quantitated by ELISA. Healthy and diseased HGF were also pretreated with IL-1alpha, followed by incubation with Porphyromonas gingivalis or Escherichia coli LPS. Culture supernatants were assessed for IL-6 protein by ELISA and cell lysates for mRNA by reverse transcription-polymerase chain reaction (RT-PCR). In order to determine if LPS stimulation was mediated through the LPS receptor, CD14, surface receptors on HGF were assessed by flow cytometry and the total RNA CD14 mRNA was assessed. RESULTS: Higher quantities of IL-6 were produced by the diseased HGF before and after stimulation than by the healthy HGF. Pretreatment with IL-1alpha followed by LPS stimulation of the healthy and diseased HGF cell lines resulted in an additive effect on IL-6 production. Pretreatment with IL-1alpha followed by a second incubation with the same stimulant produced higher amounts of IL-6 than cultures incubated with LPS alone or following IL-1alpha pretreatment. Similar amounts of IL-6 mRNA were present in unstimulated HGF from either diseased or healthy tissue and in those incubated with IL-1alpha only. After incubation with IL-1alpha and LPS, diseased HGF produced slightly more mRNA than healthy HGF. CD14 was not expressed by healthy or diseased HGF even after stimulation with either P. gingivalis or E. coli LPS. CD14 message was also undetectable. CONCLUSIONS: Taken together, these results indicate heterogeneity among gingival fibroblasts and an additive effect of IL-1 and P. gingivalis LPS on IL-6 production by HGF. Furthermore, the LPS effect on HGF was not mediated by CD14.

Effect of lipopolysaccharide and inflammatory cytokines on interleukin-6 production by healthy human gingival fibroblasts.

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.

Effect of in vitro passage of healthy human gingival fibroblasts on cellular morphology and cytokine expression.

Cytokines have been implicated in the regulation of antibody and inflammatory responses, but their role in periodontal diseases has not been elucidated. In the present study, cytokine production by human gingival fibroblasts (HGF) following in vitro passage was assessed in order to determine the basal levels of cytokine message and protein and to determine if the cellular morphology and the profile of cytokines produced differed with passage. The HGF cell line F-CL was established by explantation from non-inflamed gingival tissue, and cells from passages 1-10 were studied. The number of cells was determined in confluent cultures and cell morphology was examined by light microscopy. Fibroblasts from confluent cultures were examined for cytokine mRNA by reverse transcription-polymerase chain reaction and culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay. The morphology of F-CL fibroblasts in passages 1-4 was normal, while fibroblasts in passages 5-10 were larger. In general, the number of cells decreased from early to late passage. Fibroblasts from passages 1-10 contained message for interleukin-1 beta, -6 and -8, but not for interleukin-1 alpha or tumour necrosis factor-alpha. Interleukin-6 was detected in culture supernatants of F-CL fibroblasts by the enzyme immunoassay and its level decreased with increasing passage.