Ruggero De Maria*, Monica Boivirant$, Maria Grazia Cifone@, Paola Roncaioli@, Michael Hahne§, Jurg Tschopp§, Francesco Pallone#, Angela Santoni^ and RobertoTesti*

*Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", 00133 Rome, Italy, ^Department of Experimental Medicine, University of Rome "La Sapienza", 00161 Rome, Italy, @Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy, §Institute of Biochemistry, University of Lausanne, Switzerland CH-1066, #Department of Experimental Medicine, University of Reggio Calabria, Reggio Calabria, Italy, and $Chair of Gastroenterology, University of Rome "La Sapienza", 00161 Rome, Italy.

Corresponding author: Roberto Testi

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 pregress 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 crosslinking 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 crosslinking. 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 crosslinking 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.