Jose Quintans

Bcl-2 and Bcl-2-related proteins in apoptosis regulation.

In this review we have discussed the importance of Bcl-2 and related proteins in the regulation of apoptotic cell death in mammalian systems. It is clear that Bcl-2 plays a critical role in controlling many forms of PCD. Bcl-2 seems to have particular significance in lymphocyte development and the function of the immune system. We have also discussed the increasing size of the newly identified Bcl-2 family. There are a number of Bcl-2 homologues in human, murine, avian, nematode, and viral systems. The evolutionary conservation of the function of the Bcl-2 homologues, reinforces the importance of PCD in all complex organisms. Some of these bcl-2-like genes function as agonists and others as antagonists. Despite the seemingly universal importance of Bcl-2, it is unable to prevent PCD in all systems. In addition, we have described a role for other Bcl-2 family members in systems in which Bcl-2 is ineffective and supplied a potential rationale for the large number of genes involved in the regulation of PCD. Identification and functional analysis of the Bcl-2 family members reveals the complex nature of cell death regulation. As we begin to appreciate the significance of PCD in the control of development and homeostasis, its regulation at the molecular level is becoming better understood. Bcl-2 has long been the only known intracellular regulator of the PCD pathway(s), although its ability to prevent apoptosis is not universal. We now know that bcl-2 is only one member of an evolutionary conserved family of genes which display different patterns of expression as well as function. At least two family members, Bcl-xs and Bax, act in opposition to Bcl-2. The discovery of these new family members, including those with Bcl-2-like function and antagonists, should help clear up the discrepancies seen in Bcl-2s ability to protect cells from PCD. In doing so, we will be able to further define the pathways associated with cell death signaling. The study of these family members, as well as the non-related genes of the PCD pathways (ced-3, ced-4, ice) should lead us to understanding of how cells of multicellular organisms make decisions to die.

Apoptosis in B lymphocytes:the WEHI-231 perspective

In this review we summarize recent work on the molecular basis of apoptosis in the murine B cell lymphoma WEHI‐231. WEHI‐231 cells undergo apoptosis in response to antigen receptor cross‐linking with anti‐Ig reagents. Death is specifically triggered via surface IgM (sIgM); cross‐linking sIgD, Ia or FcR has no effect. Apoptosis is preceded by growth arrest in the Go/G1 phase of the cell cycle and may not occur in all currently available WEHI‐231 sublines. The continuous passage of WEHI‐231 cells in different laboratories has yielded variants that differ greatly in their response to anti‐Ig treatment because apoptotic cells tend to be negatively selected in culture. Resistant and susceptible variants undergo growth arrest in response to anti‐Ig but only susceptible cells go on to die by apoptosis. Cells resistant to anti‐Ig have intact apoptotic machinery as indicated by their susceptibility to dexamethasone, irradiation and other treatments. However, anti‐Ig‐resistant cells are also resistant to apoptosis induced by the immunosuppressants cyclosporin A, FK‐506 and rapamycin. We discuss the experimental evidence indicating that the apoptotic machinery in WEHI‐231 cells is pre‐activated but under constant negative regulation by short‐lived protein inhibitors. Inhibition is removed by a mediator released in response to anti‐Ig treatment in susceptible sublines. The mediator of death is the sphingosine derivative, ceramide, presumably produced by membrane sphingomyelinases activated by anti‐Ig. A hypothetical model on how ceramide kills WEHI‐231 is presented.

Cross-Talk between Ceramide and PKC Activity in the Control of Apoptosis in WEHI-231

WEHI-231, a murine B-cell lymphoma, readily undergoes programmed cell death following surface immunoglobulin (Ig) cross-linking [1]. Ceramide has been shown to induce apoptosis in WEHI-231 following its exposure to anti-lg antibodies, dexamethasone, and irradiation [2]. Recently, Haimovitz-Friedman et al. have demonstrated in endothelial cells that PMA not only prevented ceramide mediated apoptosis, but inhibited the generation of ceramide following irradiation [3]. In this paper we use highly specific PKC inhibitors to explore the connection between PKC activity, ceramide signaling and apoptosis. Both chelerythrine chloride and calphostin C triggered rapid apoptosis in WEHI-231 and acted in synergy with exogenous ceramide to induce apoptosis. Detailed studies of chelerythrines mechanism of action revealed that 30 minutes following addition of 10 microM chelerythrine, sphingomyelin and phosphatidylcholine (PC) mass decreased confirming our previous findings of neutral, but not acidic, sphingomyelinase activation following treatment with PKC inhibitors [4]. The novel observation that inhibition of PKC isoforms present in WEHI-231 leads to a rapid rise in cellular ceramide as a results of sphingomyelin hydrolysis further suggests an antagonistic relationship between PKC activity and ceramide in the signaling events preceding apoptosis.

BETA -CELL FUNCTION IN MICE INJECTED WITH MONONUCLEAR SPLENOCYTES FROM MULTIPLE-DOSE STREPTOZOTOCIN DIABETIC MICE

Abstract Multiple low doses of streptozotocin (mid sz 40 mg/kg/day, five consecutive days) induce autoimmune diabetes in mice. The aim of the present work was to study β-cell function in mice injected with splenocytes from mld-sz diabetic mice. Mononuclear splenocytes (MS) from control or diabetic donors were injected into syngeneic C57BL/6J healthy mice (5 × 107 MS, Ip). MS from diabetic donors did not produce basal hyperglycemia, but they induced abnormal ip glucose tolerance in recipient mice. These “diabetic” MS were also preferentially trapped by the recipients pancreas. Perifused pancreas from mice injected with MS from mid sz-diabetic donors showed a diminished first and second phase of glucose-induced insulin secretion after 15 days of the cell injection. At this time, pancreatic insulin content among MS reciplents did not differ from that found in controls or diabetic donors. Diabetic MS treated with Mitomycin C prior to transfer did not inhibit insulin secretion in recipient mice. Injection of MS from mice made diabetic by a single high sz dose (200 mg/kg) did not induce any alterations of the insulin secretion in recipients. There is enough evidence when using athymic and euthymic (BALB/c nu/nu and +/nu) mice to suggest that proliferation of the injected splenocytes enhanced the progression to the diabetic state, and that both donor and recipient T lymphocytes played an important part in this progression. The results suggest that injection of MS from mid sz-diabetic mice interfere with glucose-stimulated insulin secretion in recipient mice and provide a basis for the study of the mechanisms involved in the onset and modulation of autoimmune pancreatic aggression.

Production of IL-2 and IFN by TH2 clones

We have studied the production of IL-2, IL-4 and IFN-gamma by a panel of CD4+ clones produced in our laboratory. The clones were classified as TH1 and TH2 because of their ability to secrete IL-2 or IL-4, respectively, following stimulation with APC + Ag and by their characteristic proliferative responses to exogenous IL-2 or IL-4. Some of the TH2 clones, all of which happened to be autoreactive, produced IL-2 and one of these, as well as one antigen-reactive TH2 clone, also secreted IFN-gamma following stimulation with immobilized anti-CD3 mAb. IL-2 production by TH2 cells required higher concentrations of anti-CD3 mAb than IL-4 production. Thus, the TH2 clones seem to be heterogeneous. We designate the IL-2/IL-4 secretors as TH2B and those making IL-4 as TH2A clones.

Failure of CBA/N mice to respond to thymus-dependent and thymus-Independent phosphorylcholine antigens

Abstract CBA/N mice have an X-linked defect of B lymphocyte differentiation. We have studied their responsiveness to different antigenic forms of the phosphorylcholine (PC) epitope which induce IgM responses of restricted heterogeneity. Although previous work had emphasized a defect of T.I. responses in CBA/N we found that both T.D. and T.I. anti-PC responses are severely depressed in CBA/N, in spite of the use of bacterial adjuvants or repeated immunizations; however, with certain immunization protocols we could detect in CBA/N direct and indirect PFC whose specificity was not limited to PC but included the PC-protein bridge. Transfer experiments involving normal or irradiated immunodeficient F 1 hybrid males as recipients of normal spleen or bone marrow cells failed to implicate suppressive mechanisms in the unresponsiveness to PC antigens. Our results indicate that the defect in antigen-induced humoral responses in CBA/N mice is not simply related to the thymus-dependence of the antigen used but rather a consequence of selective maturational defects of subpopulations of B lymphocytes and of subsets within each subpopulation.

Cellular pathways for rejection of class-I-MHC-disparate skin and tumor allografts

We have investigated the relative roles of the Lyt-2+ and L3T4+ T lymphocyte subsets in rejectio of class-I-MHC-antigen-disparate skin and tumor allografts. To deplete T cells in vivo, rar anti-Lyt-2 or anti-L3T4 monoclonal antibodies (mAb) were administered to adult-thymectomized (ATX) recipient mice prior to transplantation. BALB/c (H-2d) recipient mice jejected the Ia- Sarcoma I (Sa1) (H-2a) tissue culture-derived tumor after depletion of the L3T4+ T cell subset in vivo. In contrast, depletion of the Lyt-2+ T cell subset permitted lethal tumor growth in all recipient mice. To determine the role of particular T cell subsets in rejection of Ld class-I-MHC-antigen-disparate allografts, BALB/c skin was transplantd to BALB/c-H-24m2 recipient mice. Skin grafts were rjeected by control mice with a mean survival time (MST) of 14.5 days. The MST of skin grafts for mice treated with anti-L3T4 mAb was 16.6 days. In contrast, administration of anti-Lyt-2 mAb alone (MST = >47 days) ro together with anti-L3T4 mAb (MST = >50 days) caused prolonged or indefinite graft survival in all recipient mice. Depletion of specific T cell subsets was confirmed by flow cytometric analysis and by analysis of T cell function in vitro. These results suggest that Lyt-2+ T lymphocytes are essential for rejection of class-I-MHC-disparate allografts; indirect presentation of alloantigen to L3T4+ T cells may not be necessary for rejection.

Studies on phosphorylcholine-specific T cell idiotypes and idiotype-specific immunity

Abstract Because of the restricted heterogeneity of idiotypes generated in the course of antiphosphorylcholine (PC) ∗ immune responses and the availability of idiotype-specific probes, we studied idiotypic and anti-idiotypic murine T cells in the PC system. PC-specific helper activity was assayed in mice expressing varying levels of humoral HOPC-8 (H8) idiotype and inhibited with both conventional anti-H8 antiserum produced in A/He mice and a monoclonal anti-H8 reagent obtained from a hybridoma. Our results using the conventional anti-H8 indicate that T cell receptors bearing H8 idiotypic determinants are produced in the absence of circulating H8 antibody, and that H8 idiotype expression by T and B cells in the same mouse may not show parallel expression of idiotypes. The monospecific anti-H8 Ig could suppress H8-positive B cell responses but was incapable of inhibiting PC-specific T cells. The implications of these findings are discussed. Furthermore, we demonstrate that immunization with purified anti-PC idiotype generates T cells which regulate the proliferation of idiotype-bearing myeloma cells.

Immunity and inflammation: the cosmic view.

This paper presents an overview of the evolution of defence reactions in multicellular animal life. The co‐evolution of hosts and pathogens provides the context to describe the major features of defence reactions and the countermeasures they evoke in their targets. Three major types of solutions to the riddle of self‐non‐self discrimination are discussed briefly: non‐clonal recognition mediated by lectins, the preferential accumulation of C3 in microbial surfaces and vertebrate clonal immunity. Vertebrate immunity is described as a specialized type of inflammation against infectious agents that evolved in response to countermeasures successfully used by intracellular pathogens against non‐specific defences.

Ontogeny of murine B-cell responses to thymus-lndependent trinitrophenyl antigens

Abstract The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro . When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP- Brucella ) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP- Brucella . Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP- Brucella . Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP- Brucella was observed in vitro . PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic ( nu/nu ) BALB/c mice and their normal ( nu /+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.