SummaryIntraceilular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed ceil surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monodonal antibody was produced by immunizing mice with human CD95-transfected L ceils. Crosslinkiug of CD95 with DX2 resulted in the activation of a sphingomydinase (SMase) in promydocytic U937 ceils, as well as in other human tumor ceil lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomydin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labded SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hy&olysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to ceil-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be rdevant in defining the biochemical nature of intraceilular messengers leading to apoptotic cell death.

The early signals generated following cross‐linking of Fas/APO‐1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO‐1 cross‐linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine‐specific phospholipase C (PC‐PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC‐PLC inhibitor D609 blocked Fas/APO‐1‐induced aSMase activation, but not Fas/APO‐1‐induced neutral sphingomyelinase (nSMase) activation. Fas/APO‐1 cross‐linking resulted also in ERK‐2 activation and in phospholipase A2 (PLA2) induction, independently of the PC‐PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain‐defective Fas/APO‐1 splice isoform which blocks Fas/APO‐1 apoptotic signalling in a dominant negative fashion. Fas/APO‐1 cross‐linking in these clones fails to activate PC‐PLC and aSMase, while nSMase, ERK‐2 and PLA2 activates are induced. These results strongly suggest that a PC‐PLC/aSMase pathway contributes directly to the propagation of Fas/APO‐1‐generated apoptotic signal in lymphoid cells.

Background and aims: Epithelium derived interleukin (IL)-15 signalling via IL-15Ra is critical for the development, activation, and survival of intraepithelial lymphocytes (IEL). We aimed to better understand the IL-15 driven effects on IEL underlying mucosal damage and lymphomagenesis in coeliac disease (CD). Methods: Enterocytes, IEL, and lamina propria mononuclear cells (LPMC) were isolated from 46 patients with uncomplicated CD (25 untreated and 21 treated) and 22 controls. IL-15 and IL-15Ra expression were determined by immunoblotting. Secretion of IL-15, interferon c (IFN-c), tumour necrosis factor a (TNF-a), and granzyme B into cell culture supernatants was assessed by ELISA. The ability of IL-15 to regulate IEL proliferation, perforin/granzyme dependent cytotoxicity, and apoptosis was tested by adding different combinations of IL-15, IL-15 blocking antibody, or chloroquine to IEL cultured alone or with Caco-2 cells as target. IL-15 mucosal levels were also determined by ELISA in five patients with complicated CD (two ulcerative jejunoileites, one refractory sprue, and two enteropathy associated T cell lymphomas) tested for T cell receptor c chain clonality. Results: IL-15 was overexpressed in untreated CD enterocytes and LPMC, and in the mucosa of complicated CD patients and uncomplicated untreated CD patients, where its levels correlated with the degree of mucosal damage. Enterocytes from untreated, but not treated, CD patients and controls secreted IL-15. Untreated CD IEL, characterised by higher IL-15Ra expression, showed increased proliferation, production of IFN-c and TNF-a, and perforin/granzyme dependent cytotoxicity, and a decreased propensity to apoptosis in response to IL-15. Conclusions: Our findings suggest that IL-15 plays a crucial role in the generation of epithelial damage in active CD. Its promotion of IEL survival in CD may predispose to the emergence of T cell clonal proliferations. Blocking IL-15, by suppressing uncontrolled IEL activation and survival, has the potential to provide new therapeutic tools to prevent tissue damage and lymphomagenesis in CD.

Our results suggest that the anti-inflammatory effects of L. brevis could be attributed to the presence of AD which prevented nitric oxide generation. Our findings give further insights into the knowledge of the molecular basis of periodontitis and have a potential clinical significance, giving the experimental ground for a new innovative, simple and efficacious therapeutical approach of periodontal disease.

Glucocorticoid hormones (GCHs) regulate normal and neoplastic lymphocyte development by exerting antiproliferative and/or apoptotic effects. We have previously shown that dexamethasone (DEX)-activated thymocyte apoptosis requires a sequence of events including interaction with the glucocorticoid receptor (GR), phosphatidylinositol-specific phospholipase C (PI-PLC), and acidic sphingomyelinase (aSMase) activation. We analyzed the mechanisms of GCH-activated apoptosis by focusing on GR-associated Src kinase, cytochrome c release, and caspase-8, -9, and -3 activation. We show here that PI-PLC binds to GR-associated Src kinase, as indicated by coimmunoprecipitation experiments. Moreover, DEX treatment induces PI-PLC phosphorylation and activation. DEX-induced PI-PLC phosphorylation, activation, and apoptosis are inhibited by PP1, a Src kinase inhibitor, thus suggesting that Src-mediated PI-PLC activation is involved in DEX-induced apoptosis. Caspase-9, -8, and -3 activation and cytochrome c release can be detected 1 to 2 hours after DEX treatment. Caspase-9 inhibition does not counter cytochrome c release, caspase-8 and caspase-3 activation, and apoptosis. Caspase-8 inhibition counters cytochrome c release, caspase-9 and caspase-3 activation, and apoptosis, thus suggesting that caspase-8 inhibitor can directly inhibit caspase-9 and/or that DEX-

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Enterocyte apoptosis is increased in involved areas of Crohn's disease. This increase is not mediated by a Fas-Fas ligand mechanism or by an abnormal E-cadherin distribution. Increased matrix metalloproteinase-1 release from lamina propria mononuclear cells might be one of the possible mechanisms responsible for the increased enterocyte apoptosis in Crohn's disease.

Summal'yThe expression and function of CD69, a member of the natural killer cell gene complex family of signal transducing receptors, was investigated on human monocytes. CD69 was found expressed on all peripheral blood monocytes, as a 28-and 32-kD disulfide-linked dimer. Molecular crosslinking of CD69 receptors induced extracellular Ca z § influx, as revealed by flow cytometry. CD69 cross-linking resulted also in phospholipase A2 activation, as detected by in vivo arachidonic acid release measurement from intact cells and by direct in vitro measurement of enzymatic activity using radiolabeled phosphatidylcholine vesicles. Prostaglandin E 2 or, 6-keto-prostaglandin F lc~, and leukotriene B4 were detected by radioimmunoassay in supernatants from CD69-stimulated monocytes, suggesting the activation of both cyclooxygenase and lipoxygenase pathways after CD69 stimulation. CD69 cross-linking, moreover, was able to induce strong nitric oxide (NO) production from monocytes, as detected by accumulation of NO oxydixed derivatives, and cyclic GMP. It is important to note that NO generation was responsible for CD69-mediated increase in spontaneous cytotoxicity against L929 murine transformed fibroblast cell line and induction of redirected cytotoxicity towards P815 FctLII + routine mastocytoma cell line. These data indicate that CD69 can act as a potent stimulatory molecule on the surface of human peripheral blood monocytes.