concanavalin-a and gusperimus

The aim of this study was to develop a new quantitative method for measuring in vitro the effects of T-cell immunosuppressive drugs by flow cytometry. Rat whole blood samples were stimulated with the T-cell mitogen succinylated concanavalin A in the presence or absence of different drugs. After 3 days, the expression of CD25 and CD8alpha in mitogen-stimulated CD4(+) cells increased 10- to 20-fold as measured by flow cytometry. Drug efficacy and potency was calculated based on dose-response curves of the drug-mediated decrease in CD4(+)/CD8alpha(+)/CD25(+) cells. The expression of CD8alpha in mitogen-stimulated CD4(+) cells was blocked completely by calcineurin inhibitors (cyclosporine A and FK-506), and partially by rapamycin and SDZ-RAD. The IC(50) (50% inhibitory concentration) values obtained were (mean+/-S.E.): 99.5+/-16.6 nM for cyclosporine A, 10.4+/-1.3 nM for FK-506, 1.8+/-0.7 nM for rapamycin, and 6.4+/-1.1 nM for SDZ-RAD. Our results show, for the first time, that CD8alpha, used as an activation antigen, is a sensitive marker for monitoring T-cell immunosuppression.

Topics: Animals; Biomarkers; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; Concanavalin A; Cyclosporine; Everolimus; Fingolimod Hydrochloride; Flow Cytometry; Guanidines; Immunosuppressive Agents; Mitogens; Propylene Glycols; Rats; Rats, Inbred Lew; Sirolimus; Sphingosine; Tacrolimus

Experimental data show that relatively low concentrations of 15-deoxyspergualin (DSG) inhibit the induction of cytotoxic T lymphocytes (CTL) and the generation of antibody-producing cells. Considerably higher concentrations of DSG are required to inhibit proliferative responses. In this in vitro study, the effects of DSG on CTL induction, on proliferative responses induced by different stimuli, and on the production of interleukins IL-1, IL-2 and IL-6 and IFN-gamma (gamma-interferon) were assessed and compared with the effects of CsA (cyclosporine A) and/or FK506. We confirmed the suppressive action of DSG on the generation of CTL. Quite unexpectedly, however, we found that, although DSG did not affect the proliferative response to allogeneic lymphocytes or a superantigen, it did inhibit proliferation of peripheral blood leucocytes (PBL) stimulated with Staphyloccus aureus. DSG was active even when added on day 2 of in vitro culture, suggesting that DSG does not inhibit early events. The fraction of CD3+ lymphoblasts and the CD4/CD8 ratio was lower in cells stimulated by S. aureus in the presence of DSG, showing a selective effect on CD3+CD4+ responder T lymphocytes. The proportion of IL-2 receptor (CD25) positive cells was also reduced by DSG treatment. Moreover, we found that DSG inhibited the proliferation induced by PHA (phytohaemagglutinin) but not by Con A (concanavalin A). This effect of DSG was time-dependent, since PHA induced proliferation was not affected until day 4 after stimulation, and indicated that DSG may inhibit proliferation induced via a CD2- but not via a CD3-mediated pathway. DSG did not influence the production of IL-2 or IFN-gamma or the lipopolysaccharide induced production of IL-2 or IL-6. In contrast, the production of IL-6 was inhibited when cells were stimulated by allogeneic lymphocytes, S. aureus, PHA or Con A. This suggested to us that the DSG-suppressed IL-6 production could be the basis for the other observed effects. We tried to mimic the DSG effects with antibodies and indeed found that the IL-6 specific antibodies had similar effects. Furthermore, recombinant IL-6 completely overcame the suppressive effects of DSG on S. aureus and PHA induced proliferation, whereas addition of IL-6 to DSG treated PBL only partly restored the cytotoxic activity of lymphoblasts induced by allogeneic cells. Thus, the inhibitory effect of DSG on de novo synthesis of IL-6 could explain some of its immunosuppressive effects, but additional

Topics: Antibodies, Monoclonal; Antigens, CD; B-Lymphocytes; CD3 Complex; CD4-CD8 Ratio; Cells, Cultured; Concanavalin A; Guanidines; Humans; Immunosuppressive Agents; Interleukin-2; Interleukin-6; Lipopolysaccharides; Lymphocyte Activation; Phytohemagglutinins; Receptors, Interleukin; Receptors, Interleukin-6; Staphylococcus aureus; T-Lymphocytes, Cytotoxic; Tacrolimus

Topics: Amino Acid Oxidoreductases; Animals; Concanavalin A; Diabetes Mellitus, Type 1; Enzyme Induction; Guanidines; Immunosuppressive Agents; Islets of Langerhans; Islets of Langerhans Transplantation; Lymphocyte Activation; Macrophages; Nitric Oxide; Nitric Oxide Synthase; Polymerase Chain Reaction; Rats; Rats, Inbred BB; Rats, Inbred F344; RNA, Messenger; Spleen; T-Lymphocytes

Deoxyspergualin has strong immunosuppressive activity in animals. However, it shows less in vitro immunosuppressive activity at the therapeutic concentration used for in vivo administration. Recently, we reported that there are some technical problems with in vitro experiments. In this report, the effects of spergualins were examined under in vitro conditions which excluded these problems, and compared with cyclosporine A (CYA). Spergualins have suppressive effects on mixed lymphocyte response (MLR) and cytotoxic T lymphocyte induction. Furthermore, interleukin-2 (IL-2) induced proliferation of concanavalin A blasts and CTLL-2 were inhibited at low concentration. However, spergualins have little effect in the early stage of MLR or the mitogen response. These results suggest that spergualins act on the proliferation and differentiation of T cells which respond to growth factors, such as IL-2.

Topics: Animals; Antibiotics, Antineoplastic; Concanavalin A; Cyclosporins; Cytotoxicity Tests, Immunologic; Guanidines; Immunosuppression Therapy; Immunosuppressive Agents; Interleukin-2; Kinetics; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; T-Lymphocytes; T-Lymphocytes, Cytotoxic