BACKGROUND: Nitric oxide (NO) is known to be an important mediator of macrophage cytotoxicity. NO in macrophages is generated via the inducible nitric oxide synthases (iNOS). Macrophage dysfunction is an important contributory factor for the increased incidence of infections in uraemia. Recently, we identified phenylacetic acid (PAA) as a novel uraemic toxin in patients on regular haemodialysis. PAA inhibits iNOS expression. In the present study, we investigated the impact of PAA on macrophage function. METHODS: RAW 264.7 cells were stimulated by LPS/ IFN-gamma in the absence and presence of PAA. iNOS mRNA was determined by real-time PCR, iNOS protein was examined by western blotting and the NO degradation product, nitrite, by Griess assay. Macrophage phagocytosis was assessed by FACS and fluorescence microscopy. Further we quantified the cytotoxicity against intracellular bacteria (Salmonella typhimurium) by a macrophage-killing assay. ELISA and Bioplex protein array system was used for the investigation of iNOS second messenger pathways (NF-kappaB, ERK1/2, JNK and p38MAPK). iNOS mRNA half-lifetime in the presence or absence of PAA was determined by real-time PCR. RESULTS: PAA significantly inhibits iNOS mRNA induction in RAW 264.7 cells by LPS/IFN-gamma [6 h: LPS/IFN-gamma-stimulation: 100%; LPS/IFN-gamma-stimulation/PAA (1 mM): 68 +/- 7%] at concentrations comparable to those of patients on chronic haemodialysis. iNOS protein expression and nitrite formation in RAW 264.7 cells were significantly inhibited by PAA. iNOS mRNA half-lifetime was not affected by PAA. The phagocytic activity of RAW 264.7 was not significantly affected by PAA, whereas the cytotoxicity against intracellular bacteria was significantly reduced. Analysis of the iNOS signal transduction pathways provided evidence that activation of the mitogen-activated kinases ERK1/2 and JNK is significantly blocked by PAA, whereas activation of p38MAPK is unaffected. The NF-kappaB pathway was not affected by PAA. CONCLUSIONS: The present findings show that the uraemic toxin PAA has inhibitory effects on macrophage-killing function, which are mediated by inhibitory effects on transcriptional iNOS regulation. iNOS inhibition by PAA might affect immunoregulatory processes and could play a role in aggravation of immunodeficiency of patients with end-stage renal disease.
BACKGROUND:Nitric oxide (NO) is known to be an important mediator of macrophage cytotoxicity. NO in macrophages is generated via the inducible nitric oxide synthases (iNOS). Macrophage dysfunction is an important contributory factor for the increased incidence of infections in uraemia. Recently, we identified phenylacetic acid (PAA) as a novel uraemic toxin in patients on regular haemodialysis. PAA inhibits iNOS expression. In the present study, we investigated the impact of PAA on macrophage function. METHODS: RAW 264.7 cells were stimulated by LPS/ IFN-gamma in the absence and presence of PAA. iNOS mRNA was determined by real-time PCR, iNOS protein was examined by western blotting and the NO degradation product, nitrite, by Griess assay. Macrophage phagocytosis was assessed by FACS and fluorescence microscopy. Further we quantified the cytotoxicity against intracellular bacteria (Salmonella typhimurium) by a macrophage-killing assay. ELISA and Bioplex protein array system was used for the investigation of iNOS second messenger pathways (NF-kappaB, ERK1/2, JNK and p38MAPK). iNOS mRNA half-lifetime in the presence or absence of PAA was determined by real-time PCR. RESULTS:PAA significantly inhibits iNOS mRNA induction in RAW 264.7 cells by LPS/IFN-gamma [6 h: LPS/IFN-gamma-stimulation: 100%; LPS/IFN-gamma-stimulation/PAA (1 mM): 68 +/- 7%] at concentrations comparable to those of patients on chronic haemodialysis. iNOS protein expression and nitrite formation in RAW 264.7 cells were significantly inhibited by PAA. iNOS mRNA half-lifetime was not affected by PAA. The phagocytic activity of RAW 264.7 was not significantly affected by PAA, whereas the cytotoxicity against intracellular bacteria was significantly reduced. Analysis of the iNOS signal transduction pathways provided evidence that activation of the mitogen-activated kinases ERK1/2 and JNK is significantly blocked by PAA, whereas activation of p38MAPK is unaffected. The NF-kappaB pathway was not affected by PAA. CONCLUSIONS: The present findings show that the uraemic toxin PAA has inhibitory effects on macrophage-killing function, which are mediated by inhibitory effects on transcriptional iNOS regulation. iNOS inhibition by PAA might affect immunoregulatory processes and could play a role in aggravation of immunodeficiency of patients with end-stage renal disease.
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