Opposite effects of IFN-gamma on CCR5 and CXCR4 expression and on entry of M- and T-tropic HIV in epithelial cells.

Abstract

HIV requires chemokine receptors as entry co-factors; several may be used, but CCR5 and CXCR4 are likely to be the physiologically relevant co-receptors used in vivo[1] by macrophage-tropic (M-tropic) and lymphocyte-tropic (T-tropic) strains, respectively. Early HIV isolates generally use CCR5, indicating that humans become infected mainly by M-tropic strains, even though donors may host both M-tropic, T-tropic or dual-tropic viruses [1]. The mechanism for this selection is unknown, but probably involves selective CCR5 expression. Epithelial cells are the first cells encountered by HIV during infection through sexual transmission and breastfeeding; they may replicate HIV both in vivo and in vitro (reviewed in Refs [2,3]), and spread internalized virus by transcytosis [4]. We studied CCR5 and CXCR4 modulation in vitro and the entry of HIV strains with different tropism into epithelial cells of cervical (HeLa-T4), colonic (HT-29) and bladder (T-24) origin, and in HUVE-cst endothelial cells. We used IFN-γ, because it is a major interplayer of inflammation and immune responses, and has been reported to have suppressive/enhancing effects on HIV [5,6]. CCR5 and CXCR4 RNA were evaluated after overnight incubation with/without IFN-γ. In Fig. 1a a representative semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) from HT-29 cells is reported;Fig. 1b shows the percentages of co-receptor transcript accumulation in IFN-γ-treated cells with respect to controls, as derived from semiquantitative RT–PCR with all the cell lines. Epithelial cells express both co-receptors; IFN-γ exerts opposite effects on co-receptor transcript accumulation: CCR5 is increased 2–18-fold, whereas CXCR4 is reduced 5.5–12.5-fold. HUVE-cst cells are CXCR4-negative; CCR5 is expressed and is IFN-γ sensitive (approximately 1.5 log increase). When dose–response experiments are performed (as shown in Fig. 1c for HT-29 cells), opposite dose–response curves are obtained for CCR5 and CXCR4 RNA. This dichotomy is also observed at the protein level, as detected by Western blot and flow cytometry evaluations (at 48 h HeLa-T4 cells gave the following immunofluorescence values: CCR5 27.2 versus 48.6% and CXCR4 65.5 versus 31.8%, for untreated versus IFN-γ-treated cultures).fig. 1.: CCR5 and CXCR4 expression in epithelial (HT-29, HeLa-T4 and T-24) and endothelial (HUVE.cst) cells exposed overnight to IFN-γ (1000 IU/ml) and dichotomic effects of IFN-γ on infection of HeLa-T4 cells by M-tropic HIV-BaL and T-tropic HIV-1MsaT strains. a. Representative semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) from HT-29 cells [8]. b. Percentages of co-receptor RNA in IFN-γ-treated cells with respect to controls, derived from semiquantitative RT–PCR with the four cell lines (mean of three experiments). c. Dose–response curves in HT-29 cells (IFN-γ 30–1000 IU/ml). d (left). Cell-associated p24 antigen, 1 h post-infection; (right) p24 antigen released 48 h post infection. e. gag and β-globin DNA sequences, 5 h post infection. (−) negative control; (+) positive control. Results similar to those of HIV-1MsaT were obtained with the T-tropic HIV-1RPdT isolate (not shown). Tropism characterization, stock preparations, evaluation of adsorption, entry and replication of M-tropic HIV-BaL and T-tropic HIV-1MSaT and HIV-1RPdT strains were as described [8].To verify whether IFN-γ modified cell sensitivity to HIV, cultures were infected with M-tropic HIV-BaL and T-tropic HIV-1MsaT and HIV-1RPdT strains [8]. In IFN-γ-treated HeLa-T4 cells there is preferential binding (Fig. 1d, left, cell-associated p24 antigen 1 h post-infection), entry (Fig. 1e, gag proviral DNA sequences at 5 h post infection) and yield (Fig. 1d, right, p24 antigen released 48 h post infection) of M-tropic HIV-BaL, with respect to the T-tropic strains, whose infection is reduced, paralleling IFN-γ modulation of co-receptor expression. This parallelism occurs in all the cells tested. The dichotomy is observed performing infectivity assays, calculating the virus bound/million cells, or seeding to a higher density the cultures to be treated with IFN-γ, to have similar cell densities at the time of infection (not shown). This report is the first to show: (i) the opposite effects of IFN-γ on CCR5 and CXCR4 in the same cells; (ii) that infection by biologically active HIV strains with different tropism varies accordingly; (iii) that epithelial cell co-receptors respond differently to IFN-γ, and favour the selective binding of M-tropic HIV strains. Published data reported either CCR5 increase by IFN-γ in cells of the monocyte lineage [7,9], or no effect, but concomitant CXCR4 downregulation [10]. Others showed CXCR4 reduction by IFN-γ in blood and bone marrow cells [11,12], with reduced activity of constructs containing sequences from T-tropic HIV strains. Our data suggest that co-receptor modulation also occurs at the epithelial level. Inflammatory stimuli may induce IFN-γ, whose differential regulation of CCR5 and CXCR4 may help explain the selection of M-tropic strains at the mucosal level. The data from HUVE.cst cells suggest that this may also occur at the endothelial level. Acknowledgements The authors thank Dr S. Bonfigli, Institute of Hematology and Endocrinology, University of Sassari, for helping with the flow cytometry determinations. Adriana Biolchini Sabrina Curreli Maria Ziccheddu Caterina Serra Antonina Dolei