VEINCTR-N, an immunogenic epitope of Fas (CD95/Apo-I), and soluble Fas enhance T-cell apoptosis in vitro. II. Functional analysis and possible implications in HIV-1 disease.
BACKGROUND
Recent studies indicate that soluble Fas (sFas) may modulate T-cell apoptosis, since it inhibits Fas-ligand (Fas-L)-mediated cytotoxicity in vitro. Here, we explored whether the soluble receptor and its major immunogenic domain, namely VEINCTR-N, interfered with apoptosis of T cells from human immunodeficiency virus-type 1 (HIV-1)+ subjects showing serum elevations of both the soluble receptor and anti-Fas antibodies, and with that of several T-cell lines.


METHODS
Both proliferation and apoptosis extent of T cells from 16 HIV-1+ patients showing serum anti-VEINCTR-N immunoglobulin G (IgG) and 15 controls were tested after incubation with sFas and three 8-mer peptides of its first consensus sequence that included VEINCTR-N. Several cell lines were also investigated by flow cytometry for their expression of Ki-67, the APO2.7-related mitochondrial protein, and the annexin-V. In addition, we evaluated the expression of Fas-L and caspases FLICE, CPP32 and ICE either by flow cytometry, immunoblotting, and/or reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
Cell proliferation in cultures from both patients and controls was affected significantly by sFas and VEINCTR-N. However, a prevalent increase of the subdiploid DNA-containing cell population occurred within these cultures. Similarly, Jurkat, CEM cells, and a mouse WR19L transformant overexpressing native human Fas underwent prompt apoptosis, which was detected as enlargement of APO2.7-reactive and annexin-V-positive populations. By exploring the Fas pathway in Jurkat cells, we found that both apoptosis inducers acted through Fas, since Fas-L, as well as CPP32 and FLICE were activated. By contrast, ICE was up-regulated only in control cells treated with tumor necrosis factor alpha (TNFalpha).


CONCLUSIONS
These data suggest that the soluble molecular forms of Fas prime cell death in Fas-positive cells. Therefore, the shedding of high amounts of sFas in HIV- 1 disease is possibly entrusted with amplification of the death execution program by cells functionally exhausted and committed to die. It is conceivable that the appearance of anti-Fas antibodies reflects an attempt by the immune system to neutralize these effective forms of the receptor and its structurally degraded domains, such as VEINCTR-N.
Silvestris
F
F
Department of Biomedical Sciences and Human Oncology, University of Bari, Italy. f.silvestris@dimo.uniba.it
Cafforio
P
P
Tucci
M
M
Del Prete
A
A
Dammacco
F
F
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D015658:HIV Infections / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015497:HIV-1; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D019014:fas Receptor / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D016056:Immunodominant Epitopes; D008562:Membrane Glycoproteins; D019014:fas Receptor; D020169:Caspases; D002118:Calcium