Flow cytometric analysis of recombinant murine GM-CSF (rmuGM-CSF) induced changes in the distribution of specific cell populations in vivo.

The changes in the distribution of granulocytes, monocytes, and lymphocytes in various tissue compartments following subcutaneous (SC) administration of recombinant murine GM-CSF (rmuGM-CSF) in vivo was determined by flow cytometry in time course studies. Balb/c mice were given single, daily SC injections of 1 or 4 micrograms of rmuGM-CSF for 10 days. Flow cytometric analysis was performed on bone marrow (BMC), peritoneal exudate (PEC), and peripheral blood (PBC) cell preparations from mice treated for 1, 3, and 10 days. Dual fluorescence was employed to gate on leukocytes (T200+) and analyze for Ig+, Thy 1.2+, MAC+, and 8C5+ (granulocytes) cells. The analyses indicated that SC-rmuGM-CSF increased the percentage of 8C5+ cells in PBC after 1 day of treatment. However, significant changes in the cell composition of PEC and BMC were not observed until day 10 of treatment and included increases in 8C5+ cells and the myeloid cell population, respectively. Side scatter analysis (cell density) of PBC and PEC indicated that the percentage of the granulocytic cell population increased significantly in rmuGM-CSF treated mice. The changes observed in PEC and BMC appeared to be dose-related whereas those observed in PBC were not. These data clearly demonstrate the utility of flow cytometric analyses for detecting selective effects of cytokines on cell populations that are involved in host defense mechanisms.

Inhibition of graft-vs-host induced immunodeficiency with immunosuppressive therapy.

The pathologic features of the acute graft-vs-host disease occurring in unirradiated (C57Bl/6 X A/J)F1 mice injected intravenously with lymphocytes from the C57Bl/6 parent are similar to those reported for other parental----F1 hybrid combinations. When stimulated in culture with concanavalin A, lipopolysaccharide or alloantigen, spleen cells from B6AF1 mice that had been injected 11 days previously with B6 lymphocytes exhibited proliferative responses that were drastically reduced in comparison to the responses of spleen cells from F1 hosts injected with syngeneic lymphocytes. IL2 production in GVH spleen cell cultures was also diminished. Proliferative responses and IL2 production were partially restored in mice given immunosuppressive therapy with azathioprine, cyclosporin A or Sch 24937 a drug whose inhibitory effects on cellular and humoral immune responses in mice have recently been described. Phenotypic analyses by flow cytometry of the GVH splenocyte population indicated that the most consistent change in the GVH spleen was the appearance of an Lyt2+ L3T4+ T cell subset which in the majority of experiments was accompanied by an increase in cells expressing only the Lyt2 antigen. Both subpopulations were reduced in mice that had recovered immunological responsiveness following immunosuppressive therapy. The results suggest that in this GVH model the development of an immunodeficient state is directly related to the induction of an active T suppressor cell population and that such cells are effectively eliminated from the splenocyte population following treatment with some immunosuppressive drugs.

Inhibition of collagen II arthritis by simultaneous administration of concanavalin A and other substances with antigen emulsion.

Several pharmacological agents, some of which are known to have effects on the immune system, decrease the incidence of collagen II-induced arthritis when added to the antigen emulsion. Concanavalin A, which has been reported to exert suppressive effects on the immune system in vivo, consistently reduced the immune response to the collagen antigen. These effects were dose and time dependent. The suppressive effects of pokeweed mitogen, tilorone and carrageenan on anti-collagen II responses were somewhat variable. Suppressive activity could be observed with concanavalin A and levamisole when the drugs were injected at a site distant from the collagen emulsion. These studies indicate that local administration of drugs is an effective approach for demonstrating the activity of some agents that may alter the course of collagen II disease through an effect on the immune system.