Biology of the Cell 91 (1999) 535-565 557

CONNECTWG THE BCR TO THE APOPTOTlC PATHWAY IN NORMAL HUMAN MATURE B CELLS: A MOLECUlAR AND PHYSIOLOGICAL PARADOX BERARD Marion, MONDIERE Paul, HENNINO Ana. CASAMAYOR Montserrat and DEFRANCE Thierry

INSERM U404, Lyon, France

In the lymphoid system the size of lymphccytic pools (homewtatic control) and the reactivity of the cells towards self molecules (tolerance control) must be tightly regulated. Both horneostasis and tolerance mechanisms involve induction of apoptosis that can be either the consequence of a 105s of survival signals or the &g&ring of surface receptors such as Fas or the antigen (Ag) receptor. The aim of the wesent work was to identifv the mechanisms underlvina the dual abilitv of the Aa ieceptor (BCR) to mediate bbth survival and apop& &Is in mature-B cells. Wi and’others have previously demonstrated a role for @R-mediated apoptosis of eerminal centre GC) B cells in the maintenance of tolerance in the B cell &npartment. We thei further studied the susceptibility of normal mature human B cells towards this process and demonstrated that: i) BCR-induced apeptosis is not an exclusive feature bf GC B cells but applies to all mature B cells, ii) susceptibility to BCR-induced death reauires prior activation (throurrh CD40 or the BCR) and entrv of

‘ . ”

the cells into S phase of the cell cycle. These characteristics are evocative of the’so called Activation-Induced Cell Death process that has already been described for T cells and which could contribute to maintain homeostasis of the B cell compartment during an immune response. At the beginning of the response, the BCR would mediate presurvival signals allowing the activation and proliferation of Ag-specific B cells. On the contrary, at the peak of the response the BCR would switch towards a proapoptotic fun&xl and thus contribute to fix an upper limit to B cell expansion. We hypothesize that the size of this expanded B cell pool would depend on trophic factors (such as IL4 for GC B cells) that can counteract the death promoting effect of the BCR but which are present in limiting amounts in the organism so as to limit the number of rescued B cells. The second part of our work aimed at dissecting the molecular pathway connecting the BCR to the death program. We demonstrated that the BCR death pathway in normal mature B czlls consecutively involves a caspase- independent and a caspase-dependent phase taking place upstream and downstream of the mitochondria respectively. Signaling from the BCR to the mitochondria involves the BCR-induced synthesis of protein mediators. This study highlights for the first time in activated nbrmal human B cells that connection of the -Kg to the cas~ase effecters of the awototic cell death is ensured bv the mitcchmldria. Finallv. . . mitochondrialdepen,~ent activation of caspase 9 contributes to initiate the caspase cascade that involves caspase 3KPP32 activation.

THE TRANSCRIPTION FACTOR C-REL BLOCKS PROLIFERATWN AND PROTECTS FROM TNFa-INDUCED APUPTOSlS VlA THE UP4?EGULATlON OF THE SAME TARGET: THE MANGANESE SUPEROXIDE DISMUTASE BERNARD Da&, QUATANNENS Brlgltte’, BEGUE AgnBs’, VANDENBUNDER Bernard’ and ABBADIE Connne’

EP5601 et UMR85262 CNRWnstitut Pasteur de LilWJniversit~ LilleP, lnsbtut de Wologre de We, 1 rue Calmelte. BP447, 59021 Lille Cedex. France

c-Rel belongs to the NF-rB family of transcription factors which are critical regulators of many genes implicated in inflammation, immunity, adhesion, differentiation, proliferation and apoptosis. To improve the comprehension of the function of c-Rel on proliferation and apoptosis, we have analyzed the effects of its overexpression in HeLa cells. After transient transfection of a bicistronic GFP-c-Rel expression vector, GFP- positive cells were checked for the expression, localization and activity of c-Rel. C-Rel overexpression caused a dramatic arrest of proliferation, and at the same time, protects from TNF a-induced apoptosis.

By RT-PCR, western-blot and immunofluorescence, we have shown that an antioxidant enzyme, the manganese-containing superoxide dismutase (MnSOD) is induced in c-Rel-expressing cells. This induction occurs through a region of the intron 2 containing a KB site. The inhibition of the MnSOD induction by antisense oligonucleotides partly reverses the cell- cycle arrest as well as the TNFa-resistance induced by c-Rel, hence demonstrating that c-Rel acts on both apoptosis and proliferation through the activation of a common effector gene, the MnSOD.

MnSOD eliminates the toxic superoxide anion (02.) by dismutating it to hydrogen peroxide (HZ02). Therefore, c-Rel-expressing cells will accumulate H,O,. We have some evidence that this accumulation leads to cell-cycle arrest, hence explaining why c-Rel is able to stop proliferation. On the other hand, it was shown that the toxicity of TNFa relies on its ability to produce 0,. . . Therefore, the c-Rel-induction of MnSOD would enable the cells to eliminate more efficiently the Oz. produced by TNFa, hence explaining why c-Rel is also able to protect from TNFainduced apoptosis.

APOPTOSlS ON THE ADRENAL CORTEX OF THE AGED RAT AFTER DEXAMETHASONE ADMlNlSTRATlON ALMEIDA Hennque, MAGALtiES Maria C. and MAGALH&S Manuel

lnstituto de Hjstologfa e Embnologla, Faculdade de Medicma do Porto, 4200 Port0 and lnstituto de Biologia Molecular e Celular da Universidade do Port0 flBMCJ, 4150 Porte, Portugal

The cells of the adrenal cortex of the rat show an age-related decreased secretory ability, both in unstimulated conditions and after ACTH injection, and, in addition, they tend to accumulate lipofuscin (Almeida et al., (1998), Me&. Aping Develop., 105:1-18; Almeida et nl., (1998), Aging tile, 1: 254-263). During studies on the effects of ACTH suppression by dexamethasone, we investigated the occurrence of apoptosis in such cells.

Male Wistar rats (n=2), aged 2, 12 and 24 months, with free access to water and laboratory diet, were injected i.m. with 4 mg/Kg of dexamethasone phosphate for three consecutive days. At the fourth day, the animals were sacrificed by decapitation, the adrenals were removed, fixed in buffered formaline and paraffin embedded following usual procedures. Sections were processed for the TUNEL technique (Gavrielli et al., (1992), J Cell Biol., 119: 493-501) employing a TdT-FragEL kit (Oncogene Res. Products) according to the manufacturer ‘s instructions, excepting the final staining which was made with haematoxylin.

The general structure of the adrenal cortex was conserved in all three zones. In the zona reticularis and deeper layers of the zona fasciculata, the majority of the nuclei displayed the usual rounded shape at all ages. However, some nuclei appeared shrunken, and were intensely labeled with a precipitate of diaminobenzidine. In the zona glomerulosa cells no precipitate was evidenced.

In randomly selected sections, a total of 1000-2000 nuclei per animal were analyzed. The percentage of labeled nuclei had an average of 3.84% at 2 months, 2.79% at 12 months and 2.77% at 24 months.

These results show a slight decrease in apoptotic late events in aged adrenals suggesting that aged cells have a different ability to withstand the lack of ACTH tropic effect, probably as a result of their decreased functional ability.

ANTHRACYCLINES TRIGGER APOPTOSlS OF BOTH GO/G1 AND CYCUNG PERlPHERAL BLOOD LYMPHOCYTES AND INDUCE MASSWE DELETION OF MATURE T AND B CELLS FERRARO Carole ‘, QLIEMENEUR Laurence’, PRIGEM Annie-France*, TAVERNE Catherine’. REVILLARD JeanPierre’ and BONNEFOY-BERARDNathalie’

‘Laboratory of Immunology, INSERM U503 UCBL, Hdpital E. Hernot, 69437 Lyon, France. ‘Laboratory of biochem&ry and pharmacology, INSERM U352, IN.Wlyw. 69621 Villeurbanne Cedex, France

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both non-activated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and -ical nuclear alterations reached a maximum after 48h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and campthotecin) and antimetabolites (methotrexate, 5-fluorouracile) which induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the GO/G1 phases of the cell cycle. In agreement with in vitro data, a single intraperitoneal injection of daunorubicin or doxorubicin in BALB/c mice induced T and B cell depletion in spleen, lymph nodes and to a lesser extend in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies as well as the p55 TNF-R-Ig fusion protein, did not inhibit drug-induced apoptosis. Level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in non-activated lymphocytes. However antioxidants such as N- acetyl-L-cysteine or gluthatione did not prevent apoptosis. Activation of caspase-3 following daunorubicin or doxorubicin treatment of either non- activated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide z-VAD-fmk. 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.

Lyon, 27-29 October 1999 Congress SBCF