Am. J. Respir. Cell Mol. Biol., Volume 23, Number 1, July, 2000 103-111

Tumor Necrosis Factor- Enhances mRNA Expression and Secretion of Interleukin-6 in Cultured Human Airway Smooth Muscle Cells

Sue McKay, Stuart J. Hirst, Marion Bertrand-de Haas, Johan C. de Jongste, Henk C. Hoogsteden, Pramod R. Saxena, and Hari S. Sharma

Departments of Pharmacology and Paediatrics-Respiratory Medicine, Sophia Children's Hospital, Rotterdam; Department of Pulmonary Medicine, Erasmus University Medical Centre, Rotterdam, The Netherlands; and Department of Respiratory Medicine and Allergy, The Guy's, King's College and St. Thomas' Hospital Medical and Dental Schools, London, United Kingdom

Airway smooth muscle (ASM) is considered to be an end-target cell for the effects of mediators released during airway wall inflammation. Several reports suggest that activated ASM may be capable of generating various proinflammatory cytokines. We investigated the effects of tumor necrosis factor (TNF)-, a potent proinflammatory cytokine, on cultured human ASM cells by examining the expression and release of the cytokine interleukin (IL)-6, cell proliferation, and the expression pattern of c-fos and c-jun, two nuclear proto-oncogenes constituting the activator protein-1 transcription factor. Growth-arrested cell monolayers were stimulated with human recombinant TNF- in a concentration- and time-dependent manner. TNF- stimulated the expression of IL-6 messenger RNA (mRNA), which was detected after 15 min, reaching a maximum at 1 h. IL-6 protein was readily detected in ASM cell-conditioned medium after 2 h of TNF- stimulation. Protein levels increased in a time- and concentration-dependent manner. Release of IL-6 elicited by TNF- was significantly inhibited by dexamethasone, cycloheximide, and nordihydroguaiaretic acid (NDGA). TNF- did not alter DNA biosynthesis up to 48 h or cell numbers up to 120 h. Northern blot analysis of proto-oncogene expression revealed that c-fos and c-jun mRNA levels were elevated after 30 min of TNF- incubation with maximum levels at 1 h and 45 min, respectively. Expression of c-fos mRNA was downregulated by NDGA. Four hours of TNF- treatment resulted in translocation of c-jun immunofluorescence from the cytoplasm to the nucleus in human ASM cells. Our results suggest that despite the lack of a mitogenic response to TNF-, upregulation of primary response genes in human ASM cells may account for the induction of proinflammatory cytokines, such as IL-6, in human airways.

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