Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal.

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 activities 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

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

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 crosslinking 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 B-4 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

SIGNALING IN LYMPHOCYTES-T - A POSSIBLE ACCESSORY PATHWAY INITIATED AT THE CD69 RECEPTOR

T cells interact with antigen presenting cells (APC) not only with their TCR/CD3 and CD4 or CD8 supercomplex, but also with a number of accessory receptors, many of which are able of independent signal transduction upon ligand crosslinking. Simultaneous signaling by accessory molecules is therefore likely to play a crucial role in the modulation and channeling of the response initiated at the TCR/CD3. Enhancement or interference mediated by second messengers on key intracellular relais may account for as different end responses as anergy and cell division, or for the activation of different sets of lymphokine genes. Defining the biochemical pathways of intracellular signaling from different receptors will provide the basic information to start to draw a temptative picture of how multiple signaling integrates in lymphocytes. Here we summarize the data about signals generated at the TCR/CD3, the main signal transducing apparatus in T cells, and CD69, a receptor expressed early during lymphocyte activation. While TCR/CD3 appears to utilize essentially a PI-PLC, CD69 preferentially activates a PLA2. CD69-mediated activation may therefore represent a model for alternative signaling in lymphocytes and prove useful in understanding potential interactions among signal transduction pathways generated by different receptors

Designing novel pH-sensitive non-phospholipid vesicle: Characterization and cell interaction

In this work, we report the preparation, the characterization and interaction with cells of novel pH-sensitive non-phospholipid vesicle formulations, from a non-ionic surfactant mixed with cholesterol (CHOL) and his derivative cholesteryl hemisuccinate (CHEMS), as pHsensitive molecule. This molecule, can destabilize the vesicle lipid bilayer when exposed to an acidic environment, with a subsequent release of vesicular content, enhancing the cytoplasmatic delivery of drugs to target cells. Vesicles were characterized by static and dynamic light scattering, in order to evaluate their dimensions, bilayer thickness and vesicle stability. Membrane permeability changes were determined by the release of entrapped hydroxypyrene-1,3,6-trisulfonic acid (HPTS). Also diphenylhesatriene (DPH) fluorescence anisotropy and potential measurements were used to evidence the pH sensitivity. Furthermore vesicles were characterized by means of electronic microscopy after freeze-fracture. The interaction of non-lipid vesicles containing different fluorescent dyes with Raw 264.7, mouse monocite macrophage, were analyzed by flow cytometric analysis. The obtained results indicate that the pH-sensitive vesicular structures show good plasma stability and relevant pH-sensitivity. Moreover this formulation was able to interact with target membranes (i.e. plasma or endosomal membrane) and to release the encapsulated material into the cytoplasm