To investigate the induction of nitric oxide synthase type II (iNOS) in human peritoneal mesothelial cells (HPMC) using cytokines and bacterial lipopolysaccharide (LPS).
Design
Confluent monolayers of HPMC were exposed to cytokines [tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), interferon gamma (IFNγ)] or LPS, individually or in various double and triple combinations, for 24 – 72 hours. Concentrations of nitrate and nitrite in the media were quantified using the Griess reaction and used as indirect indices of nitric oxide (NO) production. The expression of iNOS was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.
Results
Neither single cytokines nor LPS was able to induce iNOS mRNA or NO production. Both double combinations of TNFα+ IFNγ and IL-1β + IFNγ were able to induce iNOS mRNA expression, but only TNFα + IFNγ induced significant NO production. The triple combination of TNFα + IFNγ + IL-1β induced even more NO production than TNFα + IFNγ. There was no constitutive NO synthase type III (eNOS) expression in HPMC.
Conclusions
Certain combinations of cytokines could stimulate cultured HPMC to produce NO, and HPMC might be a source of intraperitoneal NO production during peritonitis.
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