Regulation of TNF-α by 1α,25-dihydroxyvitamin D₃ in human macrophages from CAPD patients

Background: We have previously reported that 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] accumulates in the dialysis fluid of uremic patients treated by continuous ambulatory peritoneal dialysis (CAPD). It has been reported that this metabolite regulates the production of cytokines by monocytes/macrophages. Since tumor necrosis factor-α (TNF-α) initiates an inflammatory cascade during peritonitis, the aim of the present study was to investigate the effect of 1α,25(OH)₂D₃ on the production of TNF-α by human peritoneal macrophages (HPMs). Methods: HPMs were obtained from patients on CAPD. Cells were incubated with various concentrations of 1α,25(OH)₂D₃, 1α,24(S) dihydroxyvitamin D₂ [1α,24(S)(OH)₂D₂] or 25-hydroxyvitamin D₃ (25-OH-D₃) for 16 hours. This was followed by lipopolysaccharide (LPS; 1 μg/mL) incubation for 2.5 to 6 hours. TNF-α protein production was determined by enzyme-linked immunosorbent assay. TNF-α mRNA was assayed by the reverse transcriptase-polymerase chain reaction procedure, using internal synthetic mRNA standards for quantitative results. Results: Incubation of HPMs with 1α,25(OH)₂D₃ prior to stimulation with LPS dose dependently inhibited the expression of TNF-α on both mRNA and protein levels. Similar results were obtained with the less calcemic vitamin D₂ analogue 1α,24(S)(OH)₂D₂. Incubation of HPMs with 25-OH-D₃ also revealed a down-regulation of TNF-α expression. Since this down-regulatory effect was blocked by ketoconazole, it is likely that this effect was caused by the conversion of 25-OH-D₃ into 1α,25(OH)₂D₃ by HPMs. Conclusions: 1α,25(OH)₂D₃ has a potent inhibitory effect on the production of TNF-α by LPS-activated HPMs. We hypothesize that 1α,25 (OH)₂D₃ may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory response of the peritoneum, will moderate tissue damage during peritonitis.