Klaus-Michael Debatin

REGULATION OF APOPTOSIS IN THE IMMUNE SYSTEM

Apoptosis in T and B lymphocytes is involved in all fundamental processes in the immune system. It is a mechanism to regulate the course of an immune response and to establish immunological memory as well as central and peripheral tolerance. Apoptosis in lymphocytes is regulated by gene products that induce or block this process. Elucidating the molecular basis for sensitivity and resistance towards induction of apoptosis is the key to the understanding of the development of the immune system, basic immune reactions and the pathogenesis of autoimmune diseases, AIDS and cancer.

Cytotoxic drugs and the CD95 pathway.

Cytotoxic drugs commonly used in chemotherapy of leukemia and solid tumors have been shown to primarily act by inducing apoptosis in sensitive target cells. Apoptosis may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas). Treatment with anticancer drugs such as doxorubicin, methotrexate, cytarabine, etoposide and cisplatin at therapeutic concentrations leads to induction of CD95-ligand (CD95-L). CD95-L can mediate cell death in an autocrine/paracrine manner by crosslinking CD95 receptor (CD95). Interfering with CD95-ligand/receptor interaction by antagonistic antibodies to the receptor reduces sensitivity to drug-mediated apoptosis in some cell systems. In addition, treatment with cytotoxic drugs may result in upregulation of CD95, thereby increasing the sensitivity to the CD95 death signal. Apoptosis depends on activation of caspases. Deficient activation of the CD95 system was found in drug-resistant cells. In addition, CD95-resistant and doxorubicin-resistant cells displayed cross-resistance for induction of cell death. Thus, intact apoptosis pathways such as the CD95 system may play a role in determining sensitivity or resistance of tumor cells to chemotherapy.

MycN and IFNγ cooperate in apoptosis of human neuroblastoma cells

Neuroblastomas undergo spontaneous regression at an unusually high rate. The mechanisms are not clear, but apoptosis may be involved. A large proportion of neuroblastomas is characterized by amplification of MYCN. Using human neuroblastoma cells harbouring tetracycline controlled expression of MYCN we have analysed the role of the MycN protein and IFNγ in cell death decision. Neither conditional expression of MYCN nor treatment with IFNγ alone was sufficient to trigger cell death. However, when acting in concert MycN and IFNγ efficiently triggered cell death, which was accompanied by DNA fragmentation and required caspase activity, two hallmarks of apoptosis. MycN and IFNγ may cooperate along at least two different pathways. First, IFNγ increased the CD95 cell surface expression while MycN enhanced the cellular susceptibility for the CD95 mediated death signal. Second, IFNγ treatment induced expression of BAK mRNA while MycN and IFNγ in combination increased the amount of Bax protein, another activator of apoptosis, without a concomitant increase in BAX mRNA. MycN also increased cell death in response to TRAIL and TNFα, suggesting that enforced MYCN expression in general increases the susceptibility of neuroblastoma cells towards a variety of death stimuli.

Induction of apoptosis by IFNγ in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit.

The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferonγ (IFNγ) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNγ through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNγ-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNγ stimulation, (iii) was at least partially inhibited by using blocking F(ab′)2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNγ are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNγ may have a significant potential in the therapy of neuroblastoma in vivo.

Anti-CD95 (APO-1/Fas) autoantibodies and T cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected children.

Advanced stages of HIV-1-infection are characterized by progressive CD4+ T cell depletion. Peripheral T cells from HIV-1+ donors show accelerated apoptosis in vitro. The CD95 (APO-1/Fas) receptor/ligand system is involved in this process. To further study deregulation of the CD95 system in peripheral T cells during HIV-1-infection, we measured CD95-expression on CD4+ and CD8+ T cells together with serum levels of soluble CD95 (sCD95) and anti-CD95 autoantibodies in HIV-1+ children and healthy controls. Anti-CD95 levels in HIV-1+ children were significantly elevated when compared to uninfected controls, whereas serum levels of sCD95 were not different. In HIV-1+ children, CD95-expression on CD4+ and CD8+ T cells increased with age. A strong correlation between depletion of CD4+ cells in vivo and increase in CD95-expression on CD4+ T cells was observed. In contrast, such a correlation was not found for CD8+ T cells. A negative correlation between anti-CD95 autoantibody levels and CD4+ T cell counts, that was predicted by multiple linear regression analysis of pooled data, was found in individual patients observed longitudinally by repeated measurements. Since anti-CD95 autoantibodies isolated from HIV-infected adults have previously been shown to induce apoptosis of sensitive target cells in vitro, we speculate that the interaction of these antibodes with CD95-positive and CD95-sensitive T cells in vivo might be involved in progressive T cell loss during HIV-1-infection.

Apoptosis and Cancer Therapy

Head of Department: K-M. Debatin Professors: C. Beltinger, G. Lahr, M. Wabitsch Group Leaders/Postdocs: D. Fabricius, P. Fischer-Posovszky, L.H. Meyer, G. Strauss, M-A. Westhoff PhD Students: A. Bangert, A. Bender, S. Demir, C. Dorneburg, E. Enlund, S. Enzenmüller, N. Hartmann, N. Hasan, C. Jennewein, S. Karl, M. Keuper, V. Klinkosch, S. Löder, I. Mader, J.J. Meßmann, I. Naumann, S. Saxena, D. Stadel, T. Unterkircher, K. Vellanki, L. Wagner, I. Zagotta, V. Zoller Study Programme Experimental Medicine Students: R. Blossey, L. Breckerbohm, L. Christner, S. Hasslacher, M. Herrmann, M. Linn, B. Mandel, S. Nagel, B. Nussbaum, V. Panitz, J. Philipp, V. Schwar, F. Seyfried, S. Stroh, J. Stursberg, S. Ulrich, F. Zirngibl Additional Members of Thesis Advisory Committees: P. Agostinis (Leuven), B. Baumann (Ulm), A. Bürkle (Konstanz), C. Classen (Rostock), C. Friesen (Ulm), S. Fulda (Frankfurt), I. Jeremias (Neuherberg), H.A. Kestler (Ulm), T. Kietzmann (Oulu), P. Lovat (Newcastle), M. Lutz (Würzburg), O. Micheau (Dijon), T. Seufferlein (Ulm), S. Stilgenbauer (Ulm), G. te Kronnie (Padua), I. Wernstedt Asterholm (Dallas), L. Wiesmüller (Ulm), R. Zwacka (Galway) Department of Pediatrics and Adolescent Medicine

Decreased sensitivity of drug-resistant cells towards T cell cytotoxicity

Killing of target cells by cytotoxic T cells is mediated by induction of apoptosis requiring functional death pathways. Kill is mediated either by the CD95 or the perforin/granzyme pathway. We found that SH-EP neuroblastoma cells are preferentially killed via CD95, while in the T leukemia cell line CEM CD95 and perforin/granzyme are involved. In both types of cell lines, cells resistant to CD95- and drug-induced apoptosis are cross-resistant to cytotoxic T cell kill. Resistant cells show decreased apoptosis and deficient activation of caspases indicated by decreased cleavage of the prototype caspase substrate PARP. Preincubation with the caspase inhibitor zVAD-fmk strongly decreased LAK cell kill in sensitive cells. Although parental CEM cells could be sensitized for LAK kill by preincubation with doxorubicin, resistance could not be reverted in doxorubicin or CD95 resistant CEM cells. These data demonstrate the crossresistance in induction of apoptosis by different cytotoxic regimens in tumor cells and may have implications for the immunotherapy of tumors in which apoptosis resistance was induced by previous chemotherapy.

Activation of macrophages by lymphokines from T-cell clones: Evidence for different macrophage-activating factors

The data reported in this paper demonstrate that macrophage-activating factors (MAFs) are a heterogeneous group of T-cell-derived lymphokines. Two long-term T-cell clones, Cl 96 and PK 7.1.2E8, were potent sources of MAFs (MAF96 and MAF7.1.2E8). These MAFs could be distinguished by differential activation of macrophages. Activation of resident murine macrophages with MAF7.1.2E8 enhanced RNA and glycoprotein synthesis, hexosemonophosphate shunt (HMPS) activity, release of oxygen metabolites (O-2 and H2O2), pinocytosis and tumor cytostasis, whereas no effect on schistosomula killing and tumor cytolysis could be observed. In contrast, MAF96 enhanced glycoprotein synthesis, HMPS activity, release of oxygen metabolites and prostaglandin E, schistosomula killing, and tumor cytostasis and cytolysis, while RNA synthesis and pinocytosis were decreased. These findings show that MAFs from both T-cell clones share some properties but markedly differ in others. In addition, the macrophage-activating properties of MAF96 but not of MAF7.1.2E8 could selectively be inhibited by a rabbit anti-lymphokine antiserum. This demonstrates a serological difference between MAF activities from both clones. Although at optimal concns both MAFs were active in the absence of lipopolysaccharide (LPS), the activity of suboptimal doses of MAF96 but not of MAF7.1.2E8 could be enhanced by LPS. These findings show that different MAFs from T-cell clones may be useful to clarify molecular mechanisms of macrophage activation.

The Secretion of Lysosomal Enzymes by Human Polymorphonuclear Leucocytes (PMN) and Its Modulation by Serum Complement

When engaged in phagocytosis PMN release considerable amounts of their lysosomal constituents. This “regurgitation during feeding” is considered to be caused by passive loss out of incompletely closed phagosomes (1). However, there is an obvious discrepancy between the assumption, that the enzymes are lost during the phagocytic process and the important intraphagosomal function they are thought to have in the destruction of ingested microbes. In this study we investigated the release of lysozyme, myeloperoxidase (MPO) and protease activity at pH=5,5 in relation to the ingestion of E.coli. These enzymes are considered to have important bactericidal as well as digestive functions.

The Role of the Cell Membrane in the Killing Mechanism of Polymorphonuclear Leucocytes (PMN)

It is generally assumed that the killing of bacteria by leucocytes is performed exclusively intracellularly. However, the observation of a complement augmented cellular bactericidal mechanism raised doubts in that assumption (1). Complement activity is labile and transient and its intracellular action after phagocytosis therefore hard to imagine. Further studies revealed that the subsequent stages of phagocytosis were affected differently by complement activity. The attachment of bacteria to the cell membrane was particularly augmented but, surprisingly, the consecutive ingestion was not (2). This observation raised the question as to whether serum complement can act on the even later intracellular events, i.e. intracellular killing.