Anti-oxidants reverse uraemia-induced down-regulation of mitochondrial membrane potential and interleukin-10 production.
BACKGROUND
The anti-inflammatory cytokine, interleukin-10 (IL-10), has potent immunomodulatory effects. We hypothesized that previously reported defective synthesis of IL-10 by immunocompetent cells exposed to a uraemic milieu may be due to impaired mitochondrial membrane potential (MMP).


METHODS
The human promonocytic THP-1 cell line was differentiated to monocytes and incubated with pooled control or uraemic plasma with and without catalase or N-acetyl L-cysteine (NAC). Basal hydrogen peroxide (H(2)O(2)) production was measured by flow cytometry. To measure MMP, cells were stained with rhodamine 123 (Rh123) and the uptake of Rh123 assessed by flow cytometry. To assess the relative contribution of the NADPH oxidase and mitochondrial electron transport chain (ELT) to endotoxin (ET)-stimulated IL-10 production among monocytic cells, cells were incubated with and without a selective NADPH oxidase inhibitor, apocynin and mitochondrial ELT inhibitors, diphenyliodinium and rotenone, washed and ET-stimulated IL-10 production was measured. In other experiments, cells were incubated with pooled control or uraemic plasma in the presence or absence of antioxidants followed by overnight incubation with ET. IL-10 production by monocytes in the cell supernatant was then quantified.


RESULTS
Basal H(2)O(2) production was significantly higher among differentiated THP-1 cells exposed to uraemic plasma compared with normal plasma (180.57 +/- 10.24 vs. 41.57 +/- 8.98 MCI; P = 0.02). Uraemic plasma also down regulated MMP (4.60 +/- 1.28 vs. 8.00 +/- 1.59 MCI with normal plasma; P = 0.03). Both diphenyliodinium and rotenone, selective inhibitors of the mitochondrial ELT, inhibited ET-stimulated IL-10 production. In contrast, apocynin, a selective NADPH oxidase inhibitor, did not inhibit ET-stimulated IL-10 production. Further, ET-stimulated IL-10 production by cells incubated with uraemic plasma was significantly lower when compared to cells exposed to normal plasma. Pre-incubation with catalase and NAC restored uraemia-induced down regulation of MMP. In addition, ET-stimulated IL-10 production by cells incubated with uraemic plasma was also restored by both catalase and NAC.


CONCLUSIONS
Our observations suggest that ET-stimulated IL-10 synthesis by monocytic cells is mitochondrial ELT-dependent and NADPH oxidase-independent. Monocytic cells exposed to a uraemic environment exhibit higher basal ROS production, lower MMP, and impaired ET-stimulated IL-10 synthesis. Anti-oxidants restore MMP and up-regulate ET-stimulated IL-10 synthesis.
Perianayagam
M C
MC
Division of Nephrology, Department of Medicine, Caritas St. Elizabeth Medical Center, 750 Washington Street, Boston, MA 02111, USA.
Morena
M
M
Jaber
B L
BL
Balakrishnan
V S
VS
D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D015536:Down-Regulation; D004731:Endotoxins / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D019255:NADPH Oxidases / Q000494:pharmacology; D014511:Uremia / Q000378:metabolism*
D000975:Antioxidants; D004731:Endotoxins; D016753:Interleukin-10; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases