Synergistic effect of interleukin-1 beta and tumor necrosis factor alpha on interleukin-8 gene expression in synovial fibroblasts. Evidence that interleukin-8 is the major neutrophil-activating chemokine released in response to monokine activation

Abstract

Objective: To investigate both the involvement of chemokines in general and the relative importance of specific chemokines in rheumatoid arthritis (RA), we characterized the effect of the monokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) on the synthesis of neutrophil-activating factors by synovial fibroblasts isolated from the joints of patients with RA.

Methods: Neutrophil-stimulating activity was assessed by determining intracellular calcium mobilization. IL-8 synthesis and secretion was assessed by specific enzyme-linked immunosorbent assay, and IL-8 messenger RNA (mRNA) levels were determined by Northern blot.

Results: Treatment of synovial fibroblasts with IL-1 beta and TNF alpha resulted in the production of an activity which induced intracellular calcium mobilization in peripheral blood neutrophils. The 2 monokines combined had a synergistic effect on the release of the neutrophil-stimulating activity. The effect of the 2 monokines required gene transcription and translation, and closely mimicked the pattern of IL-8 secretion induced in these cells by the monokines. We confirmed that the majority of the neutrophil-stimulating activity was IL-8 by 3 different approaches: cross-desensitization experiments with IL-8, melanoma growth-stimulatory activity, and neutrophil-activating peptide 2, stimulation of calcium mobilization in cells transfected with the IL-8 receptor complementary DNA, and inhibition of the activity following pretreatment of the supernatants with an anti-IL-8 antibody. TNF alpha and IL-1 beta induced a time- and dose-dependent release of immunoreactive IL-8. A synergistic effect of TNF alpha and IL-1 beta was also observed for both IL-8 production and accumulation of IL-8 mRNA.

Conclusion: These results indicate that the monokines TNF alpha and IL-1 beta synergistically activate IL-8 expression and protein secretion by synovial fibroblasts, and that under these conditions, IL-8 appears to be the major neutrophil-activating factor released.