Macrophages selectively stimulate ecto-5′-nucleotidase activity of cultured mesangial cells

Abstract

Because rat peritoneal macrophages exhibit a particular binding capacity to rat glomerular mesangial cells in vitro, we have studied the effect of macrophages on two plasma membrane enzymes, ecto-5'-nucleotidase and ecto-Mg2+ ATPase, or rat cultured mesangial cells. A marked increase of ecto-5'-nucleotidase activity was observed in cocultures of rat mesangial cells and peritoneal macrophages. In addition, macrophage-conditioned medium (MCM) produced a dose-dependent increase of mesangial cell ecto-5'-nucleotidase activity. In contrast, ecto-Mg2+ ATPase activity was unaffected. The effect of MCM on ecto-5'-nucleotidase activity was apparent after 24 hours and increased with time. Reversion was obtained by MCM withdrawal. Stimulation of ecto-5'-nucleotidase activity by MCM was inhibited by cycloheximide, which suggests that protein synthesis was required. Induction of enzyme activity by MCM depended in part on the presence of extracellular adenosine. The macrophage-released factor responsible for this effect was non-dialysable, heat-stable at 56 degrees C for 30 minutes but heat-inactivated at 100 degrees C for five minutes, inactivated in the presence of trypsin or protease V8, and adsorbed on charcoal. Its apparent molecular weight estimated by gel chromatography was close to 20 kD. MCM from resident macrophages was as potent as MCM from thioglycollate-elicited or zymosan-stimulated macrophages. This stimulatory effect was specific of macrophages since it was absent in the culture medium of rat fibroblasts or mouse thymocytes but present in that of mouse macrophages. These results suggest that peritoneal macrophages synthesize a factor which selectively stimulates ecto-5'-nucleotidase activity of glomerular mesangial cells.

Effect of Polyamines on Mesangial Cell Ecto-5′-Nucleotidase and Ecto-ATPase Activity

“…Recently evidence is presented that polyamine protein conjugates, which accumulate in uremic patients, inhibit colony-forming units erythroid proliferation, contributing to the onset of uremic anemia [7]. Increased activity of ecto-ATPase and ecto-5′-nucleotidase by spermine and spermidine could result in an increased production of adenosine, a powerful local mediator, interesting in respect to a role of mesangial cells in inflammatory processes and, possibly, erythropoiesis [4, 8]. …”

“…Ectoenzyme activities were determined on cells in culture as described [4, 5]. For 5′-nucleotidase activity determination the incubation medium contained 30 m M Tris-HCl buffer, pH 7.4, 130 m M NaCl, 5 m M MgCl and 5.5 m M glucose [4]. Assay was started upon addition of 3 m M AMP and incubation was performed at 37°C during 2–15 min in order to maintain conditions of zero order reaction.…”

“…Mesangial cells were isolated from rat kidneys and cultured as described previously [4]. Polyamines were added to subcultured mesangial cells in concentrations from 0.04 to 10 µg/ml and incubated for 48 h. As determined by the trypan blue exclusion method and lactate dehydrogenase release into the culture medium, spermine was cytotoxic to mesangial cells in concentrations greater than 1 µg/ ml, spermidine in concentrations greater than 5 µg/ml, and putrescine was not toxic even in the concentration of 10 µg/ml.…”

“…Polyamines were added to subcultured mesangial cells in concentrations from 0.04 to 10 µg/ml and incubated for 48 h. As determined by the trypan blue exclusion method and lactate dehydrogenase release into the culture medium, spermine was cytotoxic to mesangial cells in concentrations greater than 1 µg/ ml, spermidine in concentrations greater than 5 µg/ml, and putrescine was not toxic even in the concentration of 10 µg/ml. Ectoenzyme activities were determined on cells in culture as described [4, 5]. For 5′-nucleotidase activity determination the incubation medium contained 30 m M Tris-HCl buffer, pH 7.4, 130 m M NaCl, 5 m M MgCl and 5.5 m M glucose [4].…”

Increased Expression of Glomerular Mesangial Cell 5’-Nucleotidase in Membranous Nephropathy

“…Extracellular adenine nucleotides had a significant pro-inflammatory activity during anti-Thy1 glomerulonephritis, however, glomerular ATP/ADPase, which in concert with 5′-nucleotidase converts ATP/ADP, and AMP to anti-inflammatory adenosine had an anti-inflammatory role. Substances released by macrophages, such IL-1 and TNFα, produce an increase of the expression of ecto-5′-nucleotidase, but not ATPase, in renal mesangial cells in culture [3, 4]. The aim of this investigation was to determine tissue distribution of these two ectoenzymes in frozen specimens of human kidney biopsies of 15 patients with glomerulonephritis (GN).…”