THE IN VITRO EFFECTS OF MONOCYTE/MACROPHAGE SUPERNATANT FACTOR(S) ON CULTURED HUMAN GLOMERULAR CELLS
AuthorWagner, Carmen Lucia Machado
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PublisherThe University of Arizona.
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AbstractImmunological mediators are thought to be responsible for many of the pathophysiologic changes observed in human glomerulonephritis. Previous studies have shown the importance of immunological mediators such as immune-complexes, complement and neutrophils. Recently, a significant role for the monocyte/macrophage system in glomerular injury has been emphasized by several investigators. The present study was designed to investigate the effect of macrophage supernatant factor(s) on glomerular cell metabolism in an in vitro tissue culture system. Human glomerular cells are grown in vitro and were characterized by their phagocytic capacity and their morphologic characteristics. Light microscopy, scanning and transmission electron microscopy, and observation of their growth pattern revealed two basic cell types: an epithelial and a mesangial cell. Epithelial cells were represented by large (100-200 μ in length) and small (50-70 μ in length) flat polygonal cells. The larger epithelial cell was primarily seen in the initial outgrowth and was not easily maintained in culture. Therefore they were not used for the metabolic experiments. On the other hand, the smaller epithelial cell was maintained in culture for an average of 5 to 8 passages. The mesangial cells were medium-sized (75-120 μ in length), of variable morphology but mostly spindle-shaped and grew in a characteristic storiform pattern. Both cell types kept their morphologic appearance with subculturing and cryopreservation. Cultured glomerular cells were treated with dialyzed macrophage supernatants obtained from mouse or human peripheral blood monocytes. Undiluted or diluted macrophage supernatants were over-layed on glomerular cells cultured in 96-well flat-bottom microtiter plates. DNA, RNA and protein synthesis were evaluated by incorporation of radio-labelled precursors. Macrophage supernatants failed to stimulate DNA synthesis in epithelial cells as measured by incorporation of 3HTdR. The same macrophage supernatant did, however, significantly increase the uptake of 3HUdR and a 14-C amino acid mixture, indicating an increase in RNA and protein synthesis. The results with DNA metabolism are consistent with in vivo observations in that epithelial cells are not regarded as the intrinsic proliferating cell in the hypercellularity observed in glomerular injury. The stimulation of RNA and protein synthesis may be related to the in vivo thickening of the glomerular basement membrane. In addition, it may be related to the production of molecules which may directly or indirectly affect endothelial or mesangial cells and/or affect the local charges in the glomeruli which are known to be important in permeselectivity of the capillary wall. In the case of mesangial cells, exposure to macrophage supernatants led to a significant increase in DNA snythesis as measured by the increase in uptake of 3HTdR. No stimulation was seen in RNA and protein synthesis as measured by the radioactive label technique. The increase in DNA synthesis correlates with in vivo observations of mesangial cell proliferation in glomerular injury. The factor(s) in the macrophage supernatant which affect the metabolism of glomerular cells in vitro is non-dialyzable and denatured by freezing and thawing. In addition, preliminary results indicate that the activity stimulating RNA and protein synthesis (epithelial cells) is insensitive to heat treatment while the one affecting DNA synthesis (mesangial cells) seems to be sensitive to heat treatment. Neither was shown to be species specific since both human and mouse macrophage supernatant induced the same changes in glomerular cell metabolism. The results of this investigation suggest that both cell types are selectively affected by a factor(s) present in the macrophage supernatant. It is likely that more than one factor is responsible for the metabolic changes observed. The possibility that this factor(s) may be identical with macrophage factors previously described in other cell systems cannot be ruled out at this time. These in vitro observations further support an active role for the monocyte/macrophage system in glomerular injury in experimental and clinical glomerulonephritis.
Degree ProgramGraduate College
Molecular and Medical Microbiology