Active and selective transcytosis of cell-free human immunodeficiency virus through a tight polarized monolayer of human endometrial cells

Abstract

We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.

FIG. 1

Transcytosis of cell-free and cell-associated HIV-1 through a tight monolayer of HEC-1 cells. (A) Kinetics of transcytosis of cell-free (full circles) and PBL-associated (open circles) HIV-1_NDK. Twenty nanograms of p24 (free virus) and 2 × 10⁶ infected PBL were deposited in the apical chamber of the transwell system. The results are expressed as the amount of p24 antigen recovered in the basolateral chamber as a function of time. (B) Temperature dependency of transcytosis. Transcytosis of free HIV-1_NDK through the HEC-1 cells monolayer was assessed at 37 and at 4°C by measuring the amount of p24 antigen in the basal chamber after 3 h of contact of cell-free virus (20 ng) with the apical membrane of HEC-1 cells. Results are expressed as means and standard deviations of three separate experiments.

FIG. 2

Detection of intracellular HIV-1 gp160 antigen (red) in transcytosed HEC-1 cells by immunoflorescence. The HEC-1 cells used in the transcytosis assays were washed, fixed with paraformaldehyde (4% in phosphate-buffered saline [PBS]) for 15 min, quenched of free aldehydes with 200 mM NH₄Cl in PBS, and permeabilized for 10 min with 0.5% of Triton X-100 in PBS. After being washed with PBS, cells were incubated for 1 h with human anti-gp160 IgG diluted in PBS buffer with 1% bovine serum albumin. Phycoerythrin-labeled F(ab′)2 goat anti-human IgG (Jackson Immunoresearch, West Grove, Pa.) was further added at a dilution of 1/10. The coverslips were mounted in Mowiol (Sigma, St. Louis, Mo.) and observed by confocal microscopy using a Leica microscope (Leica, Wetzlar, Germany). Magnification, ×630.

FIG. 3

Transcytosis of various isolates of HIV-1 through HEC-1 cells. (A) Transcytosis of cell-free HIV. (B) Transcytosis of cell-associated HIV. The viral strains that were used included the primary R5-tropic HIV-1_JRCSF (clade B) grown on PBL following stimulation with PHA and IL-2, the R5-tropic HIV-1_Bal (clade B) which was amplified in monocyte-derived macrophages of healthy donors, the laboratory-adapted R5X4-tropic HIV-1_Bang originating from a patient infected with clade A virus and further amplified in the Sup T1 T-cell line, the primary X4-tropic HIV-1_NDK (clade D) grown in PBL of healthy donors following stimulation with PHA and IL-2, and the laboratory-adapted X4-tropic HIV-1_Lai (clade B) amplified in U1 monocytic cells. Transcytosis was assessed as previously described (14). Filters were used when the resistivity of the monolayer had reached 200 Ω/cm² after 6 days. Free virus (20 ng/well) or HIV-1-infected cells (2 × 10⁶ cells) were added to the apical chamber of transwells. After 180 min at 37°C, transcytosis was quantified by measuring the p24 antigen in samples taken from the basolateral chamber by means of a capture enzyme-linked immunosorbent assay (DuPont de Nemours, Wilmington, Del.) (threshold of detection, 3 pg/ml). Results were expressed as a percentage of virus recovered in the basal chamber, calculated from the amount of HIV-1 applied in the apical chamber that represented 100%. The data are expressed as means and standard deviations for three independent experiments.

FIG. 4

Inhibition of transcytosis of free HIV-1_NDK through a tight HEC-1 epithelial barrier by anti-env and antireceptor antibodies. (A) Virus was incubated with serial amounts of purified polyclonal human antibodies to gp160 for 15 min at 37°C prior to being deposited in the apical chamber of the transwell system. (B) Cells were preincubated with 12G5 MAbs to CXCR4 (R & D Systems, Minneapolis, Minn.), and virus was incubated with rabbit polyclonal anti-Galcer antibodies (Sigma), d-(+)-mannose (Sigma), and N-acetylgalactosamine (Sigma). A positive control in the experiment was polyclonal IgG against gp160 purified from serum of HIV-1-seropositive individuals. The negative control was an irrelevant IgG purified from pooled serum of HIV-1-seronegative blood donors. The results are expressed as the percent inhibition of transcytosis and expressed as means ± standard deviations for four separate experiments.