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We have previously shown that abdominal surgery (explorative laparotomy) reduces the ability of lipopolysaccharide (LPS)-triggered spleen macrophages to secrete TNF-α. In this study we characterize possible mechanisms which could be responsible for the reduction in splenic production of TNF-α. Post-operative and control (unoperated) rat splenocytes or enriched splenic macrophages were cultured with LPS. Steady-state levels of TNF-α mRNA were determined by Northern and slot blot analyses, and validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The amount of TNF-α protein was measured by Western blot analysis, and its biological activity was determined by the fibroblast L-929 cytotoxicity assay. Surgery induced a 12-fold inhibition in TNF-α activity (P < 0·02), caused up to two-fold reduction in the accumulation of TNF-α mRNA (P < 0·01), and suppressed TNF-α protein maturation into its 17-kD form in cellular extracts. Post-surgical spleen supernatants revealed mainly a band of a lower molecular weight (14 kD). Our data suggest a multilevel regulation of post-operative inhibition of TNF-α response to LPS, at the accumulation of mRNA, translational and secretory levels. We also suggest that the reduced bioactivity could be partially caused by a proteolytic cleavage of TNF-α. Since TNF-α is an important participant in immune responses, its reduced production and activity may be a central mechanism of post-operative immunosuppression.