Tumor necrosis factor alpha and interferon gamma synergistically induce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-kB-like binding sites of the interleukin 8 gene.

Interleukin 8 (IL-8) is a novel cytokine which possesses neutrophil chemotactic and activating activities in addition to chemotactic activity for basophils and T lymphocytes. It has been shown that IL-8 is produced by a variety of human somatic cells including monocytes/macrophages, dermal fibroblasts, vascular endothelial cells, keratinocytes, mesangeal cells, and several types of tumor cell lines. We have examined here whether or not human gastric cancer cell lines produce IL-8 in vitro. The production of IL-8 protein was detected by enzyme-linked immunosorbent assay in the culture supernatants derived from eight of nine human gastric cancer cell lines stimulated with either interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), or TNF alpha plus interferon gamma (IFN gamma). In some of the gastric cancer cell lines such as MKN 45 and KATO, TNF alpha plus IFN gamma synergistically induced the production of IL-8. In MKN 45 cells, synergistic increase of the steady state level of IL-8 mRNA by TNF alpha plus IFN gamma was not inhibited by cycloheximide treatment. Scatchard analysis revealed that IFN gamma changed neither the number nor the affinity constant of TNF alpha binding sites on a gastric cancer cell line, suggesting that the synergism was a post-receptor event. Furthermore, synergistic induction of chloramphenicol acetyltransferase activity by TNF alpha plus IFN gamma was observed in MKN 45 that were transiently transfected with chimeric chloramphenicol acetyltransferase reporter genes driven by the transcriptional regulatory region of human IL-8 gene. Through the mutation of the regulatory region of the IL-8 gene, both AP-1- and NF-kB-like factor binding elements were presumed to be involved in conferring the responsiveness to TNF alpha plus IFN gamma. Moreover, gel retardation analyses revealed that TNF alpha and IFN gamma synergistically induced the binding of NF-kB like as well as AP-1 like proteins bound to these sites. These results indicated that IFN gamma synergistically enhanced TNF alpha-induced IL-8 production in a human gastric cancer cell line through synergistic activation of transcription factors without up-regulating TNF alpha receptor.