A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes.

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

6-Methylprednisolone does not impair anti-thymocyte globulin (ATG) immunosuppressive activity in non-human primates.

BACKGROUND: Induction treatments with anti-thymocyte globulin (ATG) in solid organ transplantation may enhance the efficacy of maintenance immunosuppressive therapy. Since ATG can trigger Fas (CD95) mediated T cell apoptosis, a process antagonized in vitro by corticosteroids, an important issue is whether corticosteroids could interfere with T cell depleting and immunosuppressive activities of ATG. METHODS: MHC mismatched skin allografts were performed on cynomolgus and rhesus monkeys treated with ATG (20 mg/kg) associated or not with 6-methylprednisolone (10 mg/kg). RESULTS: There was no difference between the two immunosuppressive regimens as regards the intensity and duration of peripheral T lymphocyte depletion and the appearance of anti-ATG antibodies. Skin graft survival was increased in monkeys treated with 6-methylprednisolone as compared with ATG alone. CONCLUSIONS: In vivo, corticosteroids do not interfere with ATG ability to induce massive T cell depletion and to delay skin allograft rejection in non-human primates.

Mechanisms involved in antithymocyte globulin immunosuppressive activity in a nonhuman primate model.

BACKGROUND: The mechanisms of action of polyclonal antithymocyte globulins (ATGs) are still poorly understood and the selection of doses used in different clinical applications (prevention or treatment of acute rejection in organ allografts, treatment of graft-versus-host disease, or conditioning for allogeneic stem cell transplantation) remains empirical. Low T-cell counts are usually achieved in peripheral blood during ATG treatment but the extent of T-cell depletion in lymphoid tissues is unknown. METHODS: Experiments were conducted in cynomolgus monkeys using Thymoglobuline at low (1 mg/kg), high (5 mg/kg), and very high (20 mg/kg) doses. RESULTS: ATG treatment induced a dose-dependent lymphocytopenia in the blood and a dose-dependent T-cell depletion in spleen and lymph nodes but not in the thymus, indicating a limited access of ATG to this organ. T-cell apoptosis in peripheral lymphoid tissues was the main mechanism of depletion. Remaining T cells in peripheral lymphoid organs were coated by antibodies and had down-modulated surface expression of CD2, CD3, CD4, and CD8 molecules, whereas their responsiveness in mixed leukocyte reaction was impaired. The survival of MHC-mismatched skin and heart allografts was prolonged in a dose-dependent fashion, despite the occurrence of a strong anti-ATG antibody response resulting in the rapid clearance of circulating ATGs. CONCLUSION: The results indicate that T-cell depletion is achieved rapidly and primarily in peripheral lymphoid tissues at high ATG dosage. Short ATG treatments could therefore be clinically evaluated when major peripheral T-cell depletion is required.

Apoptosis of superantigen-activated T cells induced by mycophenolate mofetil treatment.

BACKGROUND: Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA), is a potent immunosuppressive agent used in clinical organ transplantation. MPA preferentially inhibits the type II isoform of inosine monophosphate dehydrogenase, depletes GTP, suppresses transfer of mannose and fucose to glycoproteins, and prevents lymphocyte proliferation in vivo. Whether MMF can also delete activated T cells in vivo by triggering an apoptotic signal was addressed in this study. To this end we analyzed the activity of MMF in mice injected with the bacterial superantigen staphylococcal enterotoxin B (SEB). Superantigens bind to MHC class II molecules without requirement for processing, and activate subsets of CD4+ and CD8+ T cells whose T cell receptor beta chains express Vbeta family-specific homologous sequences. This model that shares several features with direct allorecognition has the unique advantage of allowing a precise monitoring of activated T cells. METHODS: BALB/c mice treated with MMF (100 mg/kg/ day) or vehicle were injected with SEB. Serum cytokines, CD4+ and CD8+ Vbeta8+ cells were monitored in blood and lymphoid tissues, and apoptosis was determined by externalization of membrane phosphatidyl serine, double strand DNA breaks, and expression of B220 antigen by Vbeta8+ cells. RESULTS: MMF treatment decreased tumor necrosis factor alpha, interferon gamma, and interleukin-10 secretion induced by SEB. It did not modify other early activation events (blast transformation, CD69 and CD25 expression) but completely inhibited SEB-induced expansion of Vbeta8+ cells by inducing apoptosis of SEB-reactive T cells. A similar effect was observed in CD95-ligand-deficient mice. Repeated SEB injections associated with MMF resulted in a marked decrease of CD8+ Vbeta8+ T cells. SEB-induced increase of Vbeta8+ thymocytes was not prevented by MMF treatment. CONCLUSION: Results obtained in this in vivo model suggest that MMF treatment may induce deletion of activated peripheral T cells and decrease early cytokine responses.

Inhibition of thymidine synthesis by folate analogues induces a Fas-Fas ligand-independent deletion of superantigen-reactive peripheral T cells.

Methotrexate (MTX), a folate antagonist with multiple enzymatic targets, is used in the treatment of malignancies as well as in autoimmune and chronic inflammatory diseases, and ZD1694 (tomudex), a water-soluble quinazoline specific inhibitor of thymidylate synthase (TS), is used in the treatment of adenocarcinomas. In this study, we investigated the effects of these folate analogues on superantigen (SAg)-reactive peripheral T cells in vivo. In BALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokine secretion, IL-2R (CD25) expression and early deletion of a fraction of SEB-reactive V(beta)8(+) T cells were not impaired by either MTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTX and tomudex prevented V(beta)8-selective T cell expansion and accelerated their peripheral elimination. Administration of thymidine (500 mg/kg/12 h) completely abrogated this effect, indicating that inhibition of TS but not that of other folate-dependent enzymes was the main mechanism involved. Furthermore, a marked increase of apoptotic cells restricted to the V(beta)8(+) T cell subset indicated that proliferation inhibition was associated with apoptosis. In contrast with peripheral V(beta)8(+) T cell deletion, MTX and tomudex did not prevent the increase of V(beta)8(+) thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lpr mice further demonstrated that deletion of V(beta)8(+) T cells induced by folate analogues was independent of Fas-Fas ligand interaction. Our results provide evidence that folate analogues may selectively delete dividing peripheral T cells through TS inhibition, but do not interfere with other events triggered by SAg.

[Mechanisms of autoimmunity and therapeutic implications]. / Mécanismes de l'auto-immunité et implications thérapeutiques.

Autoimmune diseases include systemic or tissue-specific disorders induced by antibodies or CD8+ cytotoxic T lymphocytes and(or) CD4+ T lymphocytes that produce pro-inflammatory type-1 cytokines (gamma interferon and tumour necrosis factor). Some may be triggered by infectious agents. Search for predisposition genes is rapidly progressing. Disease-associated autoantibodies production requires T-B cell co-operation. Current research is focused on the characterisation of peptides processed from autoantigens and that of T lymphocytes involved in the initiation of the process. Those studies might eventually lead to immuno-intervention based on antigen administration aiming at the restoration of natural tolerance.

Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury.

BACKGROUND: CD11/CD18 beta(2) integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF). METHODS: To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion. RESULTS: After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry. CONCLUSION: These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.

The clinical usefulness of the measurement of cytokines.

The utilization of accurate and sensitive methods for the measurement of cytokines in body fluids is prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokine quantitation. Bioassays historically preceded immunoassays, which are now very popular, but there is a need for standardization. Nevertheless, due to the local effects of cytokines, the study of their blood levels is of limited value for an understanding of the pathophysiology of these mediators. This explains the development of alternative approaches to assess the ability of cells to produce cytokines. These include the Enzyme-Linked Immuno Spot Assay (ELISPOT), the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction, appear to be complementary tools for a better understanding of the biology of cytokines. Selected examples of possible clinical applications related to infectious diseases, cancer, autoimmune diseases, allergy, transplantation and preclinical evaluation of drugs and biotechnology products are given.

Sequential histological and immunohistochemical study of the skin of the first human hand allograft.

BACKGROUND: On September 1998, the first human hand allograft was successfully performed in Lyon. METHODS: A 48-year-old white man who had suffered accidental amputation of the arm in 1984, received a forearm and hand allograft from a 42-year-old white male cadaveric heart-beating donor. Immunosuppressive therapy included prednisone, mycophenolate mofetil, FK506, and antithymocyte globulins. Sequential skin biopsies were taken from the grafted limb and examined (immuno)histologically to detect a possible graft rejection and to evaluate the structural integrity of the skin of the allograft. RESULTS: The skin showed histologically a normal appearance, except on days 57 and 63, when a mononuclear perivascular cell infiltrate was observed in the dermis; this appeared concomitantly with erythematous lesions of the skin that developed after a slight decrease of the immunosuppressive treatment. These changes were considered as signs of graft rejection, and were reversed by an increase of the immunosuppressive treatment. No skin necrosis was seen at any time. Immunohistochemically, the main cell types of the skin were present throughout the study. From day 77 onward the epidermis of the grafted hand harbored some epidermal Langerhans cells of recipient's origin. CONCLUSION: This study shows that the skin of the hand allograft maintains overall a normal histological structure and contains most essential cell types, including cells of recipient origin, such as Langerhans cells. Furthermore, it shows that in this system of composite tissue transplantation, skin biopsies may reveal a starting graft rejection, before the appearance of clinically obvious lesions.

Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

The search for disease susceptibility genes. Medical and ethical problems of predictive medicine.

Thanks to major progress in genetics, a great number of disease susceptibility genes have been characterized. However, the concept of genetic defect is challenged by the elusive boundary between genetic diseases and allelic polymorphism which represents a major adaptative response to the diversity of environmental pressures. The detection of disease susceptibility alleles should be performed only if appropriate prevention can be initiated. Otherwise predictive medicine together with insufficient protection of the person's rights against the use of genetic markers may prove to be socially and psychologically detrimental. Ethical and social issues of predictive medicine definitely deserve to be fully debated.

In vivo effects of monoclonal antibodies against rat beta(2) integrins on kidney ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (IR) involves adhesion of leukocytes to the activated endothelium, leading to tissue damage. CD11/CD18 beta(2) integrins interact with their ligands on endothelial cells and may therefore represent a therapeutic target for the prevention of IR. We investigated the effects of three monoclonal antibodies (mAbs) that recognize epitopes of heavy or light chain of the beta(2) integrins on IR in kidneys. METHODS: Uninephrectomized Fischer rats were subjected to 45 or 60 min of renal ischemia, treated with intravenously anti-beta(2) integrin monoclonal antibodies (anti-CD11a, anti-CD11b, and anti-CD18) 5 min prior to reperfusion, and compared to a nontreated group. Serum creatinine, blood urea nitrogen (BUN), and kidney histopathological damages were assessed at 1, 2, and 7 days after ischemia. RESULTS: After 45 and 60 min of ischemia, serum creatinine and BUN were significantly higher in the control than in animals treated with anti-CD11a and anti-CD18 at 24 and 48 h. Administration of anti-CD11b had a beneficial effect on renal function after 45 min but not after 60 min of ischemia. Histologic and immunostaining studies demonstrated mild tubular necrosis and less leukocyte infiltration in the anti-CD11a- and anti-CD18-treated groups compared to the control group. CONCLUSION: These results indicate that selected antibodies to CD11a/CD18 may decrease kidney IR injury when administered prior to reperfusion.