X4 but not R5 HIV-1 infects and crosses Caco-2 or M cell monolayers. PCR analysis, with gag-specific primers, of DNA extracted from Caco-2 or M cell monolayers at day 9 and from CD4⁺ T cells (X4 targets) or monocytes (R5 targets) in the lower chamber of the Transwell devices. Target cells have been further cultivated for 7 days after exposure of the monolayers to HIV-1. An HIV-1 gag amplicon (115 bp) was detected in epithelial cells, whether or not converted, and in target CD4⁺ T cells as soon as 30 min after apical addition of X4 HIV-1 (a and b). No transcripts were detected in the Caco-2 or M cells and the target monocytes when exposed to R5 HIV-1 strains (c and d). The X4 HIV-1 amplicon observed in Caco-2 cells was not the result of de novo reverse transcription, because the virus was detected in the presence of 1 μg/ml zidovudine (e). A similar TCID₅₀ for X4 and R5 HIV-1 was applied. CD4⁺ T cells infected with X4 or R5 HIV-1 and monocytes infected with R5 HIV-1 served as positive control. The first two lanes in the right panel and the four lanes in the left panel are controls. One representative result of four independent experiments is shown.

Chemokine receptors specify HIV-1 strain specificity of infection. Confocal microscopy (x–y and x–z axes) of untransfected Caco-2 monolayers infected at 9 days with HxB₂ X4 (a), ADA R5 HIV-1 (b), or VSV as a positive control (c). On integration in the host cell, the virus produces the GFP protein. On day 8 postinfection the filters were examined for GFP expression. Caco-2 infected with X4 (a) and with VSV (c) were GFP-positive, whereas monolayers infected with R5 remained negative (b). In contrast CCR5-transfected Caco-2 monolayers when exposed to R5 HIV-1 were positive for GFP expression (d), indicating that R5 successfully infected the CCR5-transfected Caco-2 cells. Dotted white lines indicate the position of the filters. (Bar = 20 μm.)

Receptor-mediated transcytosis accounts for early HIV-1 penetration of the epithelial monolayers. (a Left) FITC-coupled beads added on the apical medium of both Caco-2 and M cell monolayers were transported within 1 h across converted but not across normal Caco-2 monolayers. Bars represent the average number of beads measured by cytofluorometry in the medium of the lower chamber from four independent experiments. Monolayers with low transepithelial resistance (TER), high ³H-inulin diffusion or high number of beads transported after the first 15 min of incubation were discarded. (a Right) The same monolayers used to monitor transcytosis were exposed apically to X4 or R5 HIV-1 and the underlying target CD4⁺ T cells or monocytes were recovered and tested for infection by PCR. No infection of target monocytes (0% infected samples) was observed in converted monolayers despite efficient transport of fluorescent beads. In contrast CD4⁺ T cells under normal monolayers that were totally impermeable to latex beads were infected. Mean values of four independent experiments (12 monolayers per experiment) expressed as percentage of infected samples (pool of mononuclear target cells underneath converted or not Caco-2 monolayers) with HIV-1. (b) HIV-1 p24 gag antigen was captured by ELISA in the basolateral medium, as soon as 30 min after apical infection of normal (n = 12) or converted Caco-2 (n = 12) monolayers, only when X4 but not R5 HIV-1 was added. Bars represent the average p24 gag concentration in picograms per milliliter in two independent experiments. Equal concentration of X4 and R5 HIV-1 was added apically. The maximum threshold of the detection system is 600 pg/ml; 10 pg of p24 correspond to 10⁵ particles.

Expression of HIV-1 receptors and coreceptors on Caco-2 monolayers and human ileal Peyer's patches. Confocal microscopy (x–y or x–z axes) of Caco-2 monolayers on day 9 labeled with human anti-GalCer (a), anti-CXCR4 (b), or anti-CCR5 (c) antibodies. GalCer (a) or CXCR4 (b) are expressed on the apical surface of the monolayers. CCR5 is absent (b). Dotted white lines indicate the position of the filters. (Bar = 50 μm.) Endoscopic view (d) of ileal Peyer's patches (PP) (white-yellow) were biopsied (square) and processed as described in Methods. (e) Low magnification of a Peyer's patch frozen section with typical follicles and their germinal centers (GC). The follicles are overlaid by the follicle-associated epithelium that is enriched in M cells (hematoxylin/eosin). (Bar = 100 μm.) (f) CCR5 labeling of the follicle-associated epithelium (Bar = 50 μm) showing labeling of the apical membrane of the enterocytes, better seen in g. (Bar = 20 μm.) (h) CXCR4 labeling of a serial section of (f). The FAE remains unlabeled, but as seen in (i), crypts (arrows) around the lymphoid follicle show strong apical labeling as already reported for colon crypts (21). (Bar = 50 μm.)

GalCer and chemokine coreceptors are required for HIV-1 infection of Caco-2 cells. Confocal microscopy (x–y and x–z axes) of Caco-2 monolayers on day 9 incubated for 1 h with HxB₂ X4 GFP-producing HIV-1 and isotype-matched control (a), anti-CXCR4 (c), or anti-GalCer (d) antibodies. Non-infected monolayers served as negative control (b). Infection was inhibited with anti-CXCR4 (c) or anti-GalCer (d) antibodies as reflected by the absence of GFP fluorescence when compared with control antibodies (a). Dotted white lines indicate the position of the filters. (Bar = 20 μm.)