Nitric oxide production in peritoneal macrophages from peritoneal dialysis patients with bacterial peritonitis

Abstract

Nitric oxide (NO) is produced by various cell types, and it is an important mediator in many biological processes, including macrophage-mediated cellular host defense. The relevance and amount of NO production in peritonitis during peritoneal dialysis (PD) treatment is still not clear. We studied whether human peritoneal macrophages (PMΦ) isolated from healthy PD patients or PD patients with peritonitis showed different spontaneous or lipopolysaccharide (LPS)/interferon gamma (IFN-γ)-induced NO production (LPS, 1 ng/mL-10 μg/mL; IFN-γ, 10-1000 U/mL; incubation between 6-48 hours; measured by Griess reagent). Results were compared with human blood monocytes (HBM) isolated from buffy coats. Inducible nitric oxide synthetase (iNOS) mRNA expression was looked for in PMΦ by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, plasma (P) and peritoneal dialysate effluent (D) nitrite concentrations were measured in vivo. The dialysate-to-plasma ratio (D/P) of nitrite concentration was inverse in the case of peritonitis compared to infection-free patients (peritonitis D/P = 1.3, non peritonitis D/P = 0.4; p < 0.01). PMΦ from peritonitis patients produced higher amounts of NO than did those from infection-free patients (0.040 ± 0.044 nmol per microgram cell protein versus 0.018 ± 0.015 nmol per microgram cell protein, p < 0.05). NO release could not be further enhanced by stimulation with LPS plus IFN-γ (1 ng/mL, 250 U/mL, respectively). However, NO production in PMΦ from infection-free patients increased during in vitro stimulation (0.044 ± 0.031 nmol per microgram cell protein versus 0.018 ± 0.015 nmol per microgram cell protein, p < 0.01). An increase of iNOS mRNA expression could be demonstrated by RT-PCR. Blood monocytes from healthy donors also increased NO release during cytokine stimulation (0.032 ± 0.015 nmol per microgram cell protein versus 0.019 ± 0.009 nmol per microgram cell protein, p < 0.05). Our results indicate that significant amounts of NO are released intraperitoneally in the case of bacterial peritonitis. PMΦ represent a site of NO production, though the absolute amounts released in vitro are only moderate. NO production can be induced in PMΦ and HBM by LPS/IFN-γ stimulation in vitro.