Exogenous thymosin beta4 prevents apoptosis in human intervertebral annulus cells in vitro.

Loss of cells in the human disc due to programmed cell death (apoptosis) is a major factor in the aging and degenerating human intervertebral disc. Our objective here was to determine if thymosin beta(4) (TB4), a small, multifunctional 5 kDa protein with diverse activities, might block apoptosis in human annulus cells cultured in monolayer or three-dimensional (3D) culture. Apoptosis was induced in vitro using hydrogen peroxide or serum starvation. Annulus cells were processed for identification of apoptotic cells using the TUNEL method. The percentage of apoptotic cells was determined by cell counts. Annulus cells also were treated with TB4 for determination of proliferation, and proteoglycan production was assessed using cell titer and 1,2 dimethylmethylamine (DMB) assays and histological staining. A significant reduction in disc cell apoptosis occurred after TB4 treatment. The percentage of cells undergoing apoptosis decreased significantly in TB4 treated cells in both apoptosis induction designs. TB4 exposure did not alter proteoglycan production as assessed by either DMB measurement or histological staining. Our results indicate the need for further studies of the anti-apoptotic effect of TB4 and suggest that TB4 may have therapeutic application in future biological therapies for disc degeneration.

A prostaglandin-dependent immunoregulatory mechanism activated by in vivo administration of antithymocyte globulin.

The mechanism responsible for the profound and enduring immunosuppressive action of antithymocyte globulin (ATG) is not well understood. In a primate model, we found that a 5-day rabbit ATG (RATG treatment course activates a prostaglandin (PG)-dependent suppressor cell mechanism that persists for several months. Groups of normal rhesus monkey skin allograft recipients, kidney allograft recipients, or nontransplant control animals received either RATG or no immunosuppression. The peripheral blood mononuclear cell (PBMC) population was longitudinally monitored for (1) percentage of total T cells, helper cells, and suppressor/cytotoxic T cell subsets with the monoclonal antibodies Leu-5, OKT4, and CKT8, respectively, and (2) phytohemagglutinin (PHA)-induced lymphocyte proliferative responses (LPR). The nonimmunosuppressed control groups showed no significant changes in any of these parameters. In contrast, PBMCs from all RATG-treated monkeys developed a persistent imbalance in the ratio of OKT4+ and OKT8+ subsets. Their PBMCs became unresponsive to PHA and remained unresponsive (less than 20% of control level) for at least 3 months, although total T cell counts recovered within 2 to 3 weeks after cessation of RATG. Addition of PG synthetase inhibitors indomethacin. RO-20-5720, and tolmetin caused a significant, dose-dependent recovery of LPR that was completely inhibited by exogenous PGE2 at 1 X 10(-8) M. PBMCs from RATG-treated monkeys caused a dose-dependent suppression of the normal PHA response, and this suppressor cell activity was blocked by indomethacin. PHA responses of nonimmunosuppressed control groups were not increased significantly in the presence of PG synthetase inhibitors, were less sensitive to suppression by PGE2, and did not exhibit suppressor cell activity. These data suggest that the prolonged depression in LPR after RATG treatment is due to an active PG-dependent suppressor cell mechanism and provide a new concept to explain the immunosuppressive action of RATG.

Suppressor cells in rhesus monkeys treated with antithymocyte globulin.

Treatment of rhesus monkey skin allograft recipients with a brief course of rabbit antithymocyte globulin (ATG) produces an enduring immunosuppressive effect on the cellular immune system. Despite early recovery of circulating T cells, in vitro mitogen-induced lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) remain abnormally depressed for months. In this study we show that depressed mitogen-induced lymphoproliferative responses in these animals are attributable to regulatory effects of adherent PBMCs. Removal of the adherent fraction of PBMCs on Sephadex G-10 produced a significant restoration of the mitogen-induced lymphoproliferative responses in ATG-treated monkeys. Addition of the prostaglandin synthetase inhibitor indomethacin to cultures of unfractionated PBMCs from these animals also caused a significant recovery of the lymphoproliferative response. Indomethacin did not enhance the response of control animals or the response of the nonadherent PBMC fraction of ATG-treated animals. These data suggest that a prostaglandin-dependent mechanism is involved in the suppressive action of the adherent cells. PBMCs from ATG-treated monkeys cocultured with normal cryopreserved autologous cells induced a dose-dependent suppression in the response to both concanavalin A (Con A) and phytohemagglutinin (PHA). The suppressive activity depended upon the adherent cell fraction and was found to be resistant to low-dose gamma irradiation. These data indicate that administration of rabbit ATG induces nonspecific suppressor cells in the rhesus monkey. Preliminary characterization studies suggest the involvement of suppressor monocytes. The possible role of this suppressor cell system in the immunosuppressive action of rabbit ATG is discussed.