Abdominal surgery reduces the ability of rat spleen cells to synthesize and secrete active tumour necrosis factor-alpha (TNF-alpha) by a multilevel regulation

Abstract

We have previously shown that abdominal surgery (explorative laparotomy) reduces the ability of lipopolysaccharide (LPS)-triggered spleen macrophages to secrete TNF-alpha. In this study we characterize possible mechanisms which could be responsible for the reduction in splenic production of TNF-alpha. Post-operative and control (unoperated) rat splenocytes or enriched splenic macrophages were cultured with LPS. Steady-state levels of TNF-alpha mRNA were determined by Northern and slot blot analyses, and validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The amount of TNF-alpha 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-alpha activity (P < 0.02), caused up to two-fold reduction in the accumulation of TNF-alpha mRNA (P < 0.01), and suppressed TNF-alpha 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-alpha 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-alpha. Since TNF-alpha is an important participant in immune responses, its reduced production and activity may be a central mechanism of post-operative immunosuppression.

Fig. 1

Biological activity of TNF-α in splenocytes stimulated with lipopolysaccharide (LPS), derived from control rats (□) and rats undergoing explorative laparotomy (•). L-929 TNF-α cytotoxicity test was performed on supernatants taken from splenocytes cultured with LPS (n = 4). Time was measured starting from the addition of LPS. The biological activity of TNF-α was markedly decreased after explorative laparotomy (*P < 0.02).

Fig. 2

Kinetics of TNF-α mRNA accumulation in isolated macrophages stimulated with lipopolysaccharide (LPS). (a) Northern blot analysis of TNF-α mRNA derived from control rats and from rats undergoing explorative laparotomy, and stimulated with LPS. (b) Densitometric analysis of the Northern blot, after normalization to β-actin. Time was measured starting from the addition of LPS. □, Control; ▪, laparotomy.

Fig. 3

Kinetics of TNF-α mRNA accumulation in isolated macrophages (a) and splenocytes (b) stimulated with lipopolysaccharide (LPS). Slot blot analysis of TNF-α mRNA in isolated macrophages (n = 3) and splenocytes (n = 3) derived from control rats (□) and rats undergoing explorative laparotomy (▪), and stimulated with LPS. The figure depicts mean densitometric values of the slot blots, after normalization to β-actin. Time was measured starting from the addition of LPS. The kinetics of mRNA accumulation in the two groups was similar, with a peak at 2 h. A reduction of up to 40% in TNF-α mRNA was observed after laparotomy during the peak period (at 1 or 2 h, *P < 0.05).

Fig. 4

Competitive polymerase chain reaction (PCR) determination of TNF-α mRNA accumulation in splenocytes after 2 h of stimulation with lipopolysaccharide (LPS). Fixed amounts of reverse transcribed RNA were co-amplified with two-fold serial dilutions of an internal standard. (a) A representative gel demonstrating the electrophoretic separation of the PCR products. NC, Negative control for TNF-α; M, molecular weight marker (φsX174 digested with HaeII). (b) Average level of TNF-α mRNA after 2 h of stimulation with LPS in the control and laparotomy groups (n = 3) (*P < 0.01).

Fig. 5

Western blot analysis of TNF-α. (a) A representative Western blot of cellular extracts (n = 5 in both groups) incubated with or without lipopolysaccharide (LPS) for 24 h. Splenocytes were derived from control and laparotomized rats, with or without stimulation with LPS. C, Control rats without LPS; CL, control rats with LPS; E, explorative laparotomy without LPS; EL, explorative laparotomy with LPS. A reduction in the different TNF-α protein forms is observed after laparotomy. (b) A representative Western blot of concentrated supernatants (n = 3 in each group). Secretion of TNF-α (17 kD) in control splenocytes is evident only after stimulation with LPS. Splenocytes after explorative laparotomy, with or without stimulation with LPS, show a band of a lower molecular weight (about 14 kD), probably resulting from a proteolytic cleavage of the mature TNF-α. EL, Explorative laparotomy with LPS; E, explorative laparotomy without LPS; CL, control with LPS; C, control without LPS; PC, positive control: 10 ng of rTNF-α were subjected to the same concentration procedure.