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Macrophages selectively stimulate ecto-5'-nucleotidase activity of cultured mesangial cells
V Stefanovic1, V Savic, P Vlahovic
1INSERM 64, Hôpital Tenon, Paris, France.
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.
Insights
Rat peritoneal macrophages release a factor that significantly increases ecto-5'-nucleotidase activity in glomerular mesangial cells. This macrophage-derived factor selectively stimulates this enzyme, suggesting a novel intercellular communication pathway.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Rat peritoneal macrophages demonstrate binding capacity to rat glomerular mesangial cells in vitro.
- Understanding cell-cell interactions is crucial for kidney physiology and pathology.
- Plasma membrane enzymes play key roles in cellular functions and signaling.
Purpose of the Study:
- To investigate the effect of rat peritoneal macrophages on two plasma membrane enzymes in rat cultured mesangial cells: ecto-5'-nucleotidase and ecto-Mg2+ ATPase.
- To characterize the factor released by macrophages that influences these enzymes.
Main Methods:
- Co-culture of rat mesangial cells with peritoneal macrophages.
- Treatment of mesangial cells with macrophage-conditioned medium (MCM).
- Enzyme activity assays for ecto-5'-nucleotidase and ecto-Mg2+ ATPase.
- Characterization of the macrophage-released factor using biochemical methods (heat stability, protease sensitivity, gel chromatography).
Main Results:
- Co-culture and MCM significantly increased ecto-5'-nucleotidase activity in mesangial cells in a dose- and time-dependent manner.
- Ecto-Mg2+ ATPase activity remained unaffected.
- The stimulatory effect required protein synthesis and was partially dependent on extracellular adenosine.
- The active factor was non-dialysable, heat-stable (56°C), trypsin-sensitive, charcoal-adsorbable, with an estimated molecular weight of ~20 kD.
- The effect was specific to macrophages and not observed with fibroblasts or thymocytes.
Conclusions:
- Peritoneal macrophages synthesize and release a specific factor that selectively stimulates ecto-5'-nucleotidase activity in glomerular mesangial cells.
- This finding suggests a novel mechanism of intercellular communication between macrophages and mesangial cells, potentially impacting kidney function.
- The characterized factor represents a new target for studying macrophage-mesangial cell interactions in renal contexts.