Hematopoietic colony stimulatory factor formation by murine mesangial cells: gene expression and biological activity.

This study examined the ability of mesangial cells to synthesize colony-stimulating factors (CSF), cytoregulatory peptides associated with the differentiation and proliferation of hematopoietic cells. Conditioned media obtained from SV-40 transformed murine mesangial cells stimulated the growth of murine bone marrow progenitor cells of the myeloid series. Differential analysis of these cells showed the presence of both macrophages and granulocytes. Cellular identification of bone marrow colonies stimulated in response to mesangial cell conditioned media was examined by flow cytometric analysis and revealed the presence of F4/80 antigen positive macrophages (67%) and Gran-1 antigen positive granulocytes (21%). Neutralizing antibodies to macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not antibody to interleukin-3 (IL-3), or stem cell factor (SCF) significantly inhibited the growth of the progenitor cells induced by mesangial cell conditioned media. Utilizing Northern blot analysis, murine mesangial cells expressed mRNA transcripts for M-CSF, GM-CSF, and granulocyte colony-stimulating factor (G-CSF). Further studies were performed to determine optimal incubation conditions for mesangial cell CSF gene expression. These studies revealed that both GM-CSF and G-CSF mRNA were maximally expressed at early time points (4 and 8 h of incubation), while M-CSF mRNA expression remained unchanged during the incubation of mesangial cells from 4-48 h. Incubation of mesangial cells with various concentrations of fetal bovine serum (FBS, 0.5-15%) markedly increased the mRNA expression of M-CSF, GM-CSF and G-CSF in a dose-dependent manner. These studies indicated that transformed murine mesangial cells are able to synthesize and secrete biologically active CSF that are associated with the migration and proliferation of circulating mononuclear cells in the glomerulus. Furthermore, observations regarding the role of duration of incubation and the media concentration of FBS on mesangial cell CSF mRNA expression may provide useful data to understand the optimal conditions for studies that examine the gene expression of basal or inducible CSF in mesangial cells.

Detection of interleukin 2 receptors on murine lymphocytes using fluorescent interleukin 2.

Murine interleukin 2 receptors found on freshly isolated and on in vitro activated lymphocytes were identified using a fluorescent interleukin 2 (IL2F). Three percent of freshly isolated small thymocytes bound the IL2F; these cells appeared to be dual CD4 and CD8 positive cells. Ten percent of the larger thymocytes also bound the IL2F; phenotypically, these cells were more heterogenous in their CD4/CD8 composition than the small IL2F+ thymocytes. Freshly isolated splenocytes bound more IL2F than did the thymocytes. Twenty-four percent of the small splenocytes were IL2F+ and they were mostly B220+ cells. Half of the larger splenocytes were IL2 receptor positive and these cells consisted of B and T cells. Using mitogen stimulated splenocytes, three times as many LPS stimulated B220+ blasts bound the fluorescent IL2 than freshly isolated large B220+ cells; this level of IL2F binding was maintained for four days. Of the Con A blasts, more CD8+ cells (30%) bound IL2F than did CD4+ blasts (19%); these cells maintained this level of IL2F binding for only three days. The IL2F binding could be completely inhibited by excess unlabeled IL2 and could be inhibited by 92% using a monoclonal antibody directed against the IL2 binding region of the IL2 alpha receptor, indicating that IL2F can bind to both IL2 alpha and IL2 beta receptors.