The Effect of Advanced Glycation End-Products and Aminoguanidine on Tnfα Production by Rat Peritoneal Macrophages

Abstract

To evaluate the effect of advanced glycation end-products (AGEs) and the inhibitor of their formation, aminoguanidine, on tumor necrosis factor-alpha (TNFalpha) production (as a functional marker) by rat peritoneal macrophages (PMphi). Charles River rats underwent a daily intraperitoneal injection of peritoneal dialysis solution [(PDS), 4.25 g/dL dextrose; Dialine, Travenol, Ashdod, Israel] for a 2-month period (group E). Another group of rats was subjected to the same protocol with the addition of 25 mg/kg aminoguanidine (group A). Three control groups were utilized: (1) rats that were injected daily with aminoguanidine only (group AO), (2) rats that were injected with Dulbecco's phosphate-buffered saline (group D), and (3) rats in which no intervention was carried out (group C). After 2 months, PMphi were isolated from rat peritoneal effluent and their TNFalpha production measured by ELISA in cell-free culture supernatants, in both the basal state and after 24-hour stimulation with lipopolysaccharide (LPS). The concentrations of AGEs in peritoneal effluent were assayed and correlated to TNFalpha levels. PMphi obtained from normal rats were then incubated for 24 hours with (1) the peritoneal effluent of each of the above respective groups, with or without LPS; (2) increasing concentrations of AGEs (0-250 microg/mL); and (3) increasing concentrations of aminoguanidine (0-7.5 mg/mL), and TNFalpha secretion again determined. After 2 months of daily intraperitoneal injection of PDS, in the basal state, TNFalpha production was significantly higher in PMphi isolated from the peritoneal effluent groups (groups E, A, and AO) compared to controls (group C). Following LPS stimulation, a further increase in TNFalpha secretion was seen, with a significantly greater response in group AO versus groups E, A, and D. Effluent AGEs were markedly elevated only in group E. No correlation was found between TNFalpha secretion by these PMphi and the concentration of AGEs. On incubation with the respective peritoneal effluents (groups E, A, and AO), in both the basal and stimulated state, TNFalpha production by PMphi from normal rats was significantly enhanced compared to group C. Incubation with increasing concentrations of AGEs or aminoguanidine resulted in an increase of TNFalpha secretion by these PMphi. Following intermittent intraperitoneal administration of glucose-based PDS, rat PMphi are chronically activated, as evidenced by increased basal TNFalpha secretion. The peritoneal effluent of such treated animals is capable of stimulating TNFalpha production by normal rat PMphi. These data suggest that glucose-based PDS acts as a primer of PMphi, which retain their ability to further stimulation by LPS. Although, in vitro, AGEs promote TNFalpha secretion by normal rat PMphi, in vivo, their influence is probably modulated by other factors. Aminoguanidine has a specific inducing effect on rat PMphi, independent of glucose-based PDS.