Recent studies have demonstrated increased levels of IL-6 in the peritoneal cavity during CAPD peritonitis. The current investigation was initiated (i) to examine the human peritoneal mesothelial cell (HPMC) as a possible source of this secreted IL-6 and (ii) to characterize the released product and examine its regulation by other cytokines. Unstimulated HPMC under growth arrested conditions released IL-6 in a time dependent manner. After 24-hour HPMC IL-6 release (mean +/- SEM, N = 13) (expressed as pg/micrograms cell protein) was 1.67 +/- 0.33. Stimulation of HPMC with IL-1 beta or TNF alpha resulted in a time (increasing up to 48 hr) and dose dependent IL-6 generation. After 24 hours the levels induced by IL-1 beta and TNF alpha (both at 1000 pg/ml) were (mean +/- SEM, N = 13) 19.08 +/- 2.98 and 6.62 +/- 1.72, respectively. Stimulation with combinations of IL-1 beta and TNF alpha resulted in additive increases in IL-6 release. This release could be inhibited by co-incubation with anti-IL-1 beta and/or anti-TNF alpha antibodies. The level of released HPMC IL-6 measured by immunometric assay (ELISA) correlated directly with that detected in the 7TD1 IL-6 bioassay (r = 0.63; P < 0.001). Western blot analysis of concentrated HPMC supernatants using specific anti-IL-6 antibody demonstrated immunoreactive bands at 23 and 28 Kd following IL-1 beta or TNF alpha treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
The present study compares the effects of lactate and bicarbonate buffered PDF on human neutrophil (PMN) and human peritoneal mesothelial cell (HPMC) viability and function. Acute exposure of PMN to lactate buffered PDF at pH 5.5 (CAPD 2, 1.5% and CAPD 3, 4.25% glucose) resulted in significant reductions in cellular ATP levels, the phagocytosis of serum treated zymosan (STZ) and respiratory burst activation (CL). Exposure of PMN to bicarbonate buffered PDF (BIC 20, 1.5% glucose and BIC 30, 4.25% glucose both at pH 7.2) had no significant effect on cell viability or the CL response. Phagocytosis was, however, depressed significantly more following exposure to BIC 30 than BIC 20. PMN cellular ATP levels and phagocytosis were significantly better in cells exposed to BIC 30 than to CAPD 3 at pH 7.4 (P = 0.043 for both). Pre-exposure of HPMC to CAPD 2, CAPD 3 or BIC 30 for 30 minutes resulted in a significant reduction in cellular ATP content compared to control medium. Pre-exposure to BIC 20 did not result in a reduction in HPMC ATP levels. HPMC synthesis of IL-6 was unaffected by 15 or 30 minutes pre-exposure to BIC 20 or BIC 30, in contrast pre-exposure to CAPD 2 or CAPD 3 for 15 or 30 minutes resulted in a significant reduction in stimulated IL-6 synthesis (24.5 +/- 3.01 and 32.3 +/- 5.0 vs. 43.9 +/- 10 pg/microgram cell protein in M199, N = 6; P = 0.02). Neutralization of the pH of CAPD 2 and CAPD 3 resulted in normalization of HPMC IL-6 secretion. Analysis of IL-6 mRNA expression in control, BIC 20 and 30 pre-treated HPMC subsequently stimulated with IL-1 beta revealed no differences in the expression of the IL-6 specific 465 base pair transcripts. The improved cellular function in bicarbonate buffered PDF indicates potentially improved host defence status and preservation of the peritoneal membrane in CAPD patients.
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