EFFECT OF LIPOPOLYSACCHARIDE AND INFLAMMATORY CYTOKINES ON INTERLEUKIN-6 PRODUCTION BY HEALTHY-HUMAN GINGIVAL FIBROBLASTS

Cytokines are hormone-like proteins which mediate and regulate inflamm atory and immune responses, The purpose of this study was to investiga te the effect of lipopolysaccharide (LPS) and inflammatory cytokines o n regulation of interleukin-6 (IL-6) production by human gingival fibr oblasts (HGF), The HGF cell lines used in this study, H-CL and F-CL, w ere established by the explant technique from healthy gingival tissue, Cultured cells were grown to confluency and incubated with various co ncentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TN F-alpha), IL-1 alpha, or IL-1 beta. Culture supernatants were collecte d at various times and assessed for IL-6 production by enzyme-linked i mmunosorbent assay. Total RNA was isolated from the harvested cells an d used to assess levels of IL-6 mRNA by the RNase protection assay, Bo th 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 cult ured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines s ynergistically induced IL-6 production by HGF, IL-6 message in culture d 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 alph a and TNF-alpha alone upregulate IL-6 production in a dose-and time-de pendent 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 incubat ion and when greater than 10 pg of this combination per mi was used. O ur studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.