Inhibition of equine mononuclear cell proliferation and leukotriene B4 synthesis by a specific 5-lipoxygenase inhibitor, A-63162.

The lipoxygenase metabolites of arachidonic acid have an important role in lymphocyte activation. We used a specific 5-lipoxygenase inhibitor, A-63162, to examine the role of 5-lipoxygenase (5-LO) in equine blood mononuclear cell (BMC) proliferation and leukotriene B4 (LTB4) synthesis after stimulation with mitogen (phytohemagglutinin, PHA) or calcium ionophore (A23187). The A-63162 inhibited PHA-induced equine BMC proliferation and, at the same concentration, also inhibited A23187-induced LTB4 synthesis. The presence of exogenous interleukin 2 (IL-2) or the cyclooxygenase inhibitor indomethacin, failed to reverse the immunosuppression caused by A-63162. Further, we found that A-63162, at the concentration that inhibited BMC proliferation and LTB4 synthesis, had no effect on BMC viability. The addition of the specific protein kinase C inhibitor, H-7, did not inhibit A23187-induced LTB4 synthesis. Results indicate that 5-lipoxygenase metabolites may have an important role in equine lymphocyte activation and that protein kinase C has no role in regulating LTB4 production after A23187 stimulation.

In vitro inhibition of 5-lipoxygenase metabolite, leukotriene B4, in bovine mononuclear cells by bovine viral diarrhea virus.

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-, PHA plus phorbol-12-myristate-13 acetate (PMA)- or PHA plus calcium ionophore (A23187)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation. Further, BVD-virus inhibited A23187-stimulated leukotriene B4 (LTB4) synthesis into the culture supernatants. Presence of exogenous LTB4 failed to reverse the BVD virus-induced immunosuppression. Our results suggest that BVD virus-induced immunosuppression is due to a factor that may be necessary to induce LTB4 synthesis for normal mononuclear cell proliferation.

Genistein, a selective protein tyrosine kinase inhibitor, inhibits interleukin-2 and leukotriene B4 production from human mononuclear cells.

In this study, genistein, a selective protein tyrosine kinase (PTK) inhibitor, inhibited peripheral blood mononuclear cell (PBMC) proliferation and interleukin-2 production from cultures that were stimulated with phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA) plus A23187, or PHA plus PMA, and genistein effectively blocked the PHA plus IL-2-induced PBMC proliferation. Further, we also found that genistein inhibited LTB4 production from A23187-stimulated cultures whereas H-7, a PKC inhibitor, had no effect on LTB4 production. Our results suggest that PTK may be necessary for the synthesis of LTB4.

Evidence that genistein, a protein-tyrosine kinase inhibitor, inhibits CD28 monoclonal-antibody-stimulated human T cell proliferation.

In the present investigation, we compared the immunosuppressive effects of genistein and CsA on anti-CD28 stimulated human T cell proliferation, IL-2 production, and IL-2R expression. Genistein, an isoflavanoid compound, is a specific protein tyrosine kinase inhibitor and inhibited the PMA plus anti-CD28 stimulated T cell proliferation. In contrast, proliferation of T cells stimulated with PMA plus anti-CD28 is resistant to the inhibitory effects of CsA. Similar results were obtained with IL-2 synthesis and IL-2R expression. PHA plus anti-CD28 or PMA plus anti-CD28-induced IL-2 synthesis was inhibited by genistein, and CsA, though it inhibited the PHA plus PMA-stimulated IL-2 synthesis, failed to have any effect on PMA plus anti-CD28-induced IL-2 synthesis. Genistein at the concentration that inhibited T cell proliferation and IL-2 synthesis also showed significant inhibitory effects on PMA plus anti-CD28 stimulated IL-2R expression while CsA had no effect on IL-2R from these cultures. Our data suggest that genistein is a powerful immunosuppressive agent, with no toxic effects on T cells, and has the potential for use in the prophylaxis and treatment of allograft rejection. Since genistein blocks the CsA-resistant pathway of T cell proliferation, the combined usage of these two agents may provide better immunosuppressive effect and a lesser degree of CsA-induced nephrotoxicity.

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7.

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.

Inhibition of phytohemagglutinin-stimulated bovine mononuclear cell proliferation, interleukin-2 production and protein kinase C activity by a protein kinase C inhibitor, H-7.

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent and selective inhibitor of protein kinase C (PKC), inhibited PHA-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation, interleukin-2 (IL-2) production, and cytosolic PKC activity without affecting the cell viabilities. Presence of exogenous cytokines, such as purified human IL-2 or recombinant bovine IL-2 (rbovIL-2), reversed the H-7 inhibitory effects on PHA-stimulated PBMC proliferation. We conclude that the PKC enzyme plays an important role as a second messenger in bovine PBMC proliferation in the early stages of cell activation.

In vitro interactions of cytokines and bovine viral diarrhea virus in phytohemagglutinin-stimulated bovine mononuclear cells.

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation and bovine interleukin-2 (IL-2) production. In the controls, the heat-inactivated BVD virus was not capable of suppressing the PHA-stimulated PBMC proliferation. Presence of exogenous cytokines, such as purified human IL-2, recombinant bovine interleukin-1 (rbovIL-1), recombinant bovine IL-2, and recombinant human IL-6 failed to reverse the BVD virus-induced immunosuppression. Also, we found that the BVD virus inhibited PHA and IL-2 induced proliferation of bovine PBMC in the early and late stages of activation. In summary, our data suggest that BVD virus induced immunosuppression was not due to destruction of the PBMC but may be inhibiting one or more of the important intracellular enzymes that may regulate PBMC proliferation.