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-1 alpha, or IL-1 beta. 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 mi) by both HGF cell lines, Although TNF-alpha stimulated IL-6 production by HGF, >10-fold-larger amounts were induced with IL-1 alpha and IL-1 beta. Furthermore, the addition of both IL-1 alpha and TNF-ol 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-1 alpha and IL-1 beta, and 1,400-fold by IL-1 alpha plus TNF-alpha. IL-1 alpha and TNF-alpha alone upregulate IL-6 production in a dose-and time-dependent fashion, The addition of IL-1 alpha and TNF-a 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 mi was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.