Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

CD69 is expressed on platelets and mediates platelet activation and aggregation.

CD69, a surface dimer so far considered an early activation antigen restricted to lymphocytes, was found constitutively expressed on human platelets. Biochemical analysis revealed that platelet CD69 appears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad 55-65-kD band, in which three 55-, 60-, and 65-kD components were detectable when nonreduced, and as two 28- and 32-kD bands when reduced, corresponding to the two disulfide-linked chains of the dimer. It therefore closely resembles lymphoid CD69, although the resolution of the three bands under nonreducing conditions is not usually seen in lymphoid cells. Moreover, as CD69 expressed on activated lymphocytes and CD3bright thymocytes, both chains are constitutively phosphorylated. CD69 stimulation by anti-Leu-23 monoclonal antibodies induced platelet aggregation in a dose-dependent fashion. This effect was associated with Ca2+ influx and platelet degranulation, as revealed by adenosine triphosphate release. In addition, CD69 stimulation in platelets induced production of thromboxane B2 and PGE2, suggesting activation of arachidonic acid metabolism by cycloxygenase. As observed for CD69-mediated T cell activation, platelet activation through CD69 requires molecular crosslinking. These results suggest that CD69 may function as an activating molecule on platelets, as on lymphocytes, and point toward a more general role of this surface dimer in signal transduction.

Ceramide inhibits antigen uptake and presentation by dendritic cells.

Ceramides are intramembrane diffusible mediators involved in transducing signals originated from a variety of cell surface receptors. Different adaptive and differentiative cellular responses, including apoptotic cell death, use ceramide-mediated pathways as an essential part of the program. Here, we show that human dendritic cells respond to CD40 ligand, as well as to tumor necrosis factor-alpha and IL-1 beta, with intracellular ceramide accumulation, as they are induced to differentiate. Dendritic cells down-modulate their capacity to take up soluble antigens in response to exogenously added or endogenously produced ceramides. This is followed by an impairment in presenting soluble antigens to specific T cell clones, while cell viability and the capacity to stimulate allogeneic responses or to present immunogenic peptides is fully preserved. Thus, ceramide-mediated pathways initiated by different cytokines can actively modulate professional antigen-presenting cell function and antigen-specific immune responses.

Acidic sphingomyelinase (ASM) is necessary for fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells.

Ceramides deriving from sphingomyelin hydrolysis are important mediators of apoptotic signals originating from Fas (APO-1/CD95). However, definitive evidence for the role played by individual sphingomyelinases is still lacking. We have analyzed lymphoblastoid cell lines derived from patients affected by Niemann Pick disease (NPD), an autosomal recessive disorder caused by loss-of-function mutations within the acidic sphingomyelinase (ASM) gene. NPD lymphoblasts, which display normal neutral sphingomyelinase activity, fail to activate ASM in response to Fas cross-linking, unlike normal lymphoblasts. NPD lymphoblasts also fail to accumulate GD3 ganglioside, a downstream mediator of ceramide-induced cell death (De Maria, R., L. Lenti, F. Malisan, F. D'Agostino, B. Tomassini, A. Zeuner, M.R. Rippo, R. Testi. 1997. Science. 277:1652-1655), and display a substantially inefficient apoptosis after Fas cross-linking. Inefficient apoptosis is due to lack of ASM activity, because proximal signaling from Fas in NPD lymphoblasts is not impaired and apoptosis can be efficiently triggered by passing the ASM defect with exogenous ceramides. Moreover, mannose receptor-mediated transfer of ASM into NPD lymphoblasts rescues their ability to transiently activate ASM, accumulate GD3, and rapidly undergo apoptosis after Fas cross-linking. These results provide definitive genetic evidence for the role of ASM in the progression of apoptotic signals originating from Fas.

Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

Intracellular 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 cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-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 relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

Triggering of human monocyte activation through CD69, a member of the natural killer cell gene complex family of signal transducing receptors.

The 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 cross-linking of CD69 receptors induced extracellular Ca2+ 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 alpha, 6-keto-prostaglandin F 1 alpha, 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 FcRII+ murine mastocytoma cell line. These data indicate that CD69 can act as a potent stimulatory molecule on the surface of human peripheral blood monocytes.

Nitric oxide primes pancreatic beta cells for Fas-mediated destruction in insulin-dependent diabetes mellitus.

Fas is an apoptosis-inducing surface receptor involved in controlling tissue homeostasis and function at multiple sites. Here we show that beta cells from the pancreata of newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients express Fas and show extensive apoptosis among those cells located in proximity to Fas ligand-expressing T lymphocytes infiltrating the IDDM islets. Normal human pancreatic beta cells that do not constitutively express Fas, become strongly Fas positive after interleuken (IL)-1beta exposure, and are then susceptible to Fas-mediated apoptosis. NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, prevents IL-1beta-induced Fas expression, whereas the NO donors sodium nitroprusside and nitric oxide releasing compound (NOC)-18, induce functional Fas expression in normal pancreatic beta cells. These findings suggest that NO-mediated upregulation of Fas contributes to pancreatic beta cell damage in IDDM.

Membrane anchoring of a human IgG Fc receptor (CD16) determined by a single amino acid.

CD16 is a low-affinity immunoglobulin G (IgG) Fc receptor that is expressed on natural killer (NK) cells, granulocytes, activated macrophages, and some T lymphocytes. Two similar genes, CD16-I and CD16-II, encode membrane glycoproteins that are anchored by phosphatidylinositol (PI)-glycan and transmembrane polypeptides, respectively. The primary structural requirements for PI-linkage were examined by constructing a series of hybrid cDNA molecules. Although both cDNA's have an identical COOH-terminal hydrophobic segment, CD16-I has Ser203 whereas CD16-II has Phe203. Conversion of Phe to Ser in CD16-II permits expression of a PI-glycan-anchored glycoprotein, whereas conversion of Ser to Phe in CD16-I prevents PI-glycan linkage.

Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis.

Gangliosides participate in development and tissue differentiation. Cross-linking of the apoptosis-inducing CD95 protein (also called Fas or APO-1) in lymphoid and myeloid tumor cells triggered GD3 ganglioside synthesis and transient accumulation. CD95-induced GD3 accumulation depended on integral receptor "death domains" and on activation of a family of cysteine proteases called caspases. Cell-permeating ceramides, which are potent inducers of apoptosis, also triggered GD3 synthesis. GD3 disrupted mitochondrial transmembrane potential (DeltaPsim), and induced apoptosis, in a caspase-independent fashion. Transient overexpression of the GD3 synthase gene directly triggered apoptosis. Pharmacological inhibition of GD3 synthesis and exposure to GD3 synthase antisense oligodeoxynucleotides prevented CD95-induced apoptosis. Thus, GD3 ganglioside mediates the propagation of CD95-generated apoptotic signals in hematopoietic cells.

Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis.

The mechanisms responsible for thyrocyte destruction in Hashimoto's thyroiditis (HT) are poorly understood. Thyrocytes from HT glands, but not from nonautoimmune thyroids, expressed Fas. Interleukin-1beta (IL-1beta), abundantly produced in HT glands, induced Fas expression in normal thyrocytes, and cross-linking of Fas resulted in massive thyrocyte apoptosis. The ligand for Fas (FasL) was shown to be constitutively expressed both in normal and HT thyrocytes and was able to kill Fas-sensitive targets. Exposure to IL-1beta induced thyrocyte apoptosis, which was prevented by antibodies that block Fas, suggesting that IL-1beta-induced Fas expression serves as a limiting factor for thyrocyte destruction. Thus, Fas-FasL interactions among HT thyrocytes may contribute to clinical hypothyroidism.

Sphingomyelin breakdown and cell fate.

A growing number of cell-surface receptors are now being shown to generate signals that trigger the hydrolysis of sphingomyelin to release diffusible ceramides. Ceramides have been implicated as key mediators in signaling pathways, with outcomes as diverse as cell proliferation, differentiation, growth arrest and apoptosis. The response depends on cell type, whether the signal is integrated with other signals originating from the same receptor and on the subcellular location of sphingomyelin hydrolysis and ceramide release.

Acid reflux into the oesophagus does not influence exercise-induced airway narrowing in bronchial asthma.

OBJECTIVES: A few studies on small patient series have investigated the relationship between gastroesophageal reflux and bronchial responsiveness as expressed by exercise-induced bronchoconstriction (EIB), with non-conclusive results. The aim of this study was to evaluate whether the presence of acid in the oesophagus may influence EIB. METHODS: 45 patients with bronchial asthma underwent spirometry, exercise challenge on bicycle ergometer and 24 h oesophageal pH monitoring. Subjects with EIB (Forced expiratory volume in the first second (FEV1)) percentage decrease after exercise (DeltaFEV1) > or =15%, n = 28) were retested after a 2 week treatment course with omeprazole 40 mg/daily. Exercise at baseline was performed at the same time as oesophageal pH monitoring. RESULTS: In basal condition, there was no difference in FEV1, acid exposure time or number of refluxes measured during 24 h pH monitoring between patients with and without EIB. There was no relationship between spirometry results and DeltaFEV1 on one hand, and parameters of gastroesophageal reflux on the other. Nine patients with EIB (31.0%) and six patients without EIB (37.5%) had one or more episodes of GER during exercise challenge, without significant differences between the two groups. After gastric acid inhibition by omeprazole, DeltaFEV1 did not change significantly. CONCLUSIONS: The results indicate that acid in the oesophagus, or its short-term inhibition by proton pump inhibitors, has no influence on exercise-induced bronchoconstriction.

Silencing of SH-PTP2 defines a crucial role in the inactivation of epidermal growth factor receptor by 5-aminosalicylic acid in colon cancer cells.

Recent studies have suggested that 5-aminosalicylic acid (5-ASA) inhibits colorectal cancer (CRC) development. However, the mechanism underlying the antineoplastic effect of 5-ASA remains unknown. We here examined the effect of 5-ASA on epidermal growth factor receptor (EGFR) activation, a pathway that triggers mitogenic signals in CRC cells. We show that 5-ASA inhibits EGFR activation, through a mechanism that does not rely on CRC cell death induction. 5-ASA enhances the activity, but not expression, of phosphorylated (p)-EGFR-targeting phosphatases (PTPs), and treatment of cells with PTP inhibitors abrogates the 5-ASA-mediated EGFR dephosphorylation. Both SH-PTP1 and SH-PTP2 interact with EGFR upon 5-ASA treatment. However, knockdown of SH-PTP2 but not SH-PTP1 by small interference RNAs prevents the 5-ASA-induced EGFR dephosphorylation. Finally, we show that 5-ASA attenuates p-EGFR in ex vivo organ cultures of CRC explants. Data indicate that 5-ASA disrupts EGFR signalling by enhancing SH-PTP2 activity, and suggest a mechanism by which 5-ASA interferes with CRC growth.

Stimulated human lamina propria T cells manifest enhanced Fas-mediated apoptosis.

Lamina propria (LP) T cells respond poorly to a proliferative stimulus delivered via TCR/CD3 pathway, but retain considerable ability to respond to a stimulus delivered via CD2 costimulatory or accessory pathway. In the present study, we showed first that unstimulated LP T cells, as compared to unstimulated peripheral blood (PB) T cells, exhibit an increased level of apoptosis which is further increased following CD2 pathway stimulation, but not following via TCR/CD3 pathway stimulation. We next showed that IL-2 had a sparing effect on apoptosis of unstimulated LP T cells in that IL-2 decreased and anti-IL-2 increased apoptosis of these cells; in contrast, IL-2 had no effect on apoptosis of CD2-pathway stimulated cells. Finally, we showed that increased apoptosis of LP T cells induced by CD2-pathway stimulation is inhibited when Fas antigen is blocked by a nonstimulatory anti-Fas antibody. These studies suggest that LP T cells are characterized by increased susceptibility to Fas-mediated apoptosis most due to a downstream change in the Fas signaling pathway. Given that IFN-gamma secretion is significantly increased in LP T cells in which apoptosis is inhibited, this feature of LP T cells may represent a mechanism of regulating detrimental immune responses in the mucosal environment.

Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.

The expression and function of Fas (CD95/APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activation. Three color immunofluorescence and FACS analysis indicated that virtually all freshly isolated human gut lamina propria T lymphocytes (T-LPL) express Fas, together with the marker of progress activation CD45R0. A discrete fraction of freshly isolated T-LPL also constitutively expressed Fas ligand (FasL), perhaps as a result of recent in vivo activation. Importantly, whereas Fas cross-linking did not result in apoptosis induction in peripheral blood T lymphocytes (T-PBL), Fas was found to be fully effective in generating the apoptotic signal in T-LPL. This was associated with the activation of an acidic sphingomyelinase and with ceramide generation, early events known to be involved in Fas-mediated apoptotic signaling. By contrast, acidic sphingomyelinase activation and ceramide production were not detectable in T-PBL after Fas cross-linking. However C2-ceramide, a cell permeant synthetic analog of ceramide, could efficiently induce apoptosis in T-LPL and T-PBL when added exogenously. These data indicate that T-LPL constitutively express both Fas and FasL and that Fas cross-linking generates signals resulting in sphingomyelin hydrolysis and apoptosis, outlining a potential mechanism involved in intestinal tolerance. Moreover, they provide the first evidence of a role for ceramide-mediated pathways in normal immunoregulation.

Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.

Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

Lipid signaling in CD95-mediated apoptosis.

Ceramides play an important role mediating different cell responses such as proliferation, differentiation, growth arrest and apoptosis. They are released upon sphingomyelin hydrolysis which occurs after triggering of a number of cell surface receptors including CD95. Ceramide generation also regulates glycosphingolipid and ganglioside metabolism. In particular, ganglioside GD3 biosynthesis represents an important event for the progression of apoptotic signals generated by CD95 and mediated by ceramide in hematopoietic cells.

A simple method for the evaluation of receptor binding capacity of modified cytokines.

We have developed a flow cytometric method to evaluate the binding of interleukin-2 analogues to receptors. The method relies on competition for binding between a fluorescein-conjugated monoclonal antibody (MoAb) directed against the human interleukin-2 receptor alpha chain (fluorescein isothiocyanate (FITC) anti-IL-2R) and the test protein. IL-2R positive cells are incubated with FITC-anti-IL-2R MoAb in the presence of native IL-2 or IL-2 iodinated by either the chloramine-T or the lactoperoxidase-glucose-oxidase method. The binding of IL-2 is indicated by decreased fluorescence. This method is suitable for measuring the binding capacity of modified IL-2 molecules and avoids the need for radioactive tracers. It provides a simple and reproducible technique, which can be extended readily to the study of the receptor binding capacity of cytokines conjugated with toxins, drugs or other molecules.