Differential down‐regulation of CD95 or CD95L in chronically HIV‐infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT‐PCR

Abstract

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50 000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400-(basal) or 10-(induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.z 1999 Federation of European Biochemical Societies.

Mitochondrial Functionality and Mitochondrial DNA Content in Lymphocytes of Vertically Infected Human Immunodeficiency Virus–Positive Children with Highly Active Antiretroviral Therapy–Related Lipodystrophy

“…Because it is 20 bp shorter, it can be easily distinguished from wild-type DNA on agarose gel. This fragment was cloned into the pFASL plasmid that contains the competitor for CD95L [28], taking advantage of the consensus sequences for Eco RI and Xba I restriction enzymes present at both ends. The construct, named pMito, was extracted from Escherichia coli cells by use of a High Pure plasmid isolation kit, was quantified by spectrophotometer and limiting dilution analysis, and was stored at 220 C. This construct was used with QC-PCR to evaluate the number of copies of mtDNA or genomic DNA (gDNA) that are present in a sample, depending on the primers used in the PCR mix.…”

Mapping of Quantitative Trait Loci Determining NK Cell-Mediated Resistance to MHC Class I-Deficient Bone Marrow Grafts in Perforin-Deficient Mice

“…Regarding the housing conditions, we recently demonstrated involvement of the Fas/FasL pathway in resistance to BM grafts and that diminished BM rejection capacity during conventional housing conditions may be explained by down-regulation of NK cell FasL expression (21). This down-regulation could be induced by pathogens infecting the animals (22), since sentinel mice in our conventional animal facility occasionally carry viral infections, e.g., mouse hepatitis virus, mouse parvovirus, and sendai virus. In addition, the inhibitor cellular FLIP, overexpressed in susceptible hemopoietic stem cells, was shown to convey partial engraftment in B6 PKO mice, suggesting involvement of death receptors in BM rejection (23).…”

Perforin- and Fas-dependent mechanisms of natural killer cell-mediated rejection of incompatible bone marrow cell grafts

“…One explanation for the low levels of FasL on NK cells from mice housed in a conv colony is viral infections. Studies with lymphocytic cell lines have shown that FasL expression is down-regulated upon infection with HIV [23]. Interestingly, mice living in our conv colony have been diagnosed with several viral infections, such as mouse hepatitis virus and Sendai virus.…”