Binding of LFA-1 (CD11a) to intercellular adhesion molecule 3 (ICAM-3; CD50) and ICAM-2 (CD102) triggers transmigration of human immunodeficiency virus type 1-infected monocytes through mucosal epithelial cells

Abstract

Transmigration of human immunodeficiency virus (HIV)-infected mononuclear cells through the genital mucosa is one of the possible mechanisms of sexual transmission of HIV. Here, we investigated the transmigration of cell-associated R5-tropic HIV type 1 (HIV-1) through a tight monolayer of human epithelial cells in vitro. We show that this process is dependent on an initial interaction between alphaLbeta2 integrin CD11a/CD18 on infected monocytic cells and intercellular adhesion molecule 2 (ICAM-2; CD102) and ICAM-3 (CD50) on the apical membrane of epithelial cells. The CD50 and CD102 ligands were overexpressed on epithelial cells when the cells were activated by proinflammatory cytokines in the cellular microenvironment. An accumulation of proviral DNA was found in the transmigrated cells, clearly reflecting the preferential transepithelial migration of HIV-1-infected cells under proinflammatory conditions. Our observations provide new insights supporting the hypothesis that HIV-infected mononuclear cells contained in genital secretions from infected individuals may cross the epithelial genital mucosa of an exposed receptive sexual partner, particularly under inflammatory conditions of damaged genital tissue. Understanding the fundamental aspects of the initial HIV entry process during sexual transmission remains a critical step for preventing human infection and developing further vaccinal strategies and virucidal agents.

FIG. 1.

Transmigration of HIV-infected MDM through a tight epithelial monolayer of HEC-1 cells. White bars, distribution of cells recovered in the lower chamber of the transwell system (expressed as percentages of the amount deposited in the upper chamber of the system) in nine separate experiments using MDM that had been infected with HIV-1 _Ba-L in vitro; hatched boxes, results obtained when transmigration assays were performed under proinflammatory conditions, i.e., in the presence of N. meningitidis LPS (5 ng/ml), IL-1β (2 ng/ml), and TNF-α (10 ng/ml). Top and bottom lines of bars, 75th and 25th percentiles; middle lines, arithmetic means; error bars, 90th and 10th percentiles.

FIG. 2.

Kinetics of transmigration of HIV-1_Ba-L-infected MDM (squares) and of uninfected MDM (circles) at 4 (open symbols) and 37°C (solid symbols). The experimental conditions were similar to those depicted in the legend of Fig. 1. The results are those of one representative experiment out of three obtained with cells from different donors.

FIG. 3.

Expression of CD11a by HIV-infected MDM. MDM were surface stained for the CD11a antigen and further stained intracellularly for the HIV-1 p24 antigen. (Left) Dot plot depicting the staining of uninfected MDM with the anti-CD11a MAb. Ninety-nine percent of uninfected cells expressed CD11a, with a mean fluorescence intensity of 11. (Right) Surface staining of infected cells with anti-CD11a (abscissa) and intracellular staining of the cells for HIV-1 p24 (ordinate). Two-thirds of the cells were positive for the intracellular p24 antigen and expressed CD11a with a mean fluorescence intensity of 16. Results are those from one representative experiment out of three. Vertical lines, background fluorescence of unstained cells.

FIG. 4.

Anti-CD11a MAb inhibits transmigration of HIV-1-infected MDM. MDM were preincubated with anti-CD11a (50 μg/ml) or with normal human IgG (intravenous Ig; 50 μg/ml) for 30 min at room temperature prior to being deposited in the upper chamber of the HEC-1 monolayer. The transmigration assay was performed under proinflammatory conditions as described for Fig. 1 and 2. Hatched bars, percentages of cells recovered in the lower chamber of the transwell system in nine separate experiments; white bar, background results obtained in the absence of proinflammatory conditions, i.e., in RPMI 1640 alone. Lines and error bars are as defined for Fig. 1.

FIG. 5.

Dose-response inhibition by anti-CD11a MAb of the transmigration of HIV-1-infected MDM. The experimental conditions were those depicted for Fig. 4, except that cells from only two donors were used. MDM were preincubated with the indicated amounts of anti-CD11a prior to interacting with HEC-1 cells.

FIG. 6.

Expression of CD50 (left), CD102 (middle), and CD54 (right) on the membrane of confluent HEC-1 cells, either unstimulated (top) or stimulated with N. meningitidis LPS (5 ng/ml), IL-1β (2 ng/ml), and TNF-α (10 ng/ml) (bottom). Relevant antibodies were conjugated with FITC, and cells were examined by laser confocal microscopy.

FIG. 7.

Inhibition of transmigration by preincubation of HEC-1 cells with anti-CD50, anti-CD54, and anti-CD102 MAb. The HEC-1 monolayer was preincubated with anti-CD50, anti-CD54, or anti-CD102 MAb (50 μg/ml) or with a mixture of anti-CD50 and anti-CD102 MAb for 30 min at room temperature prior to performing the transmigration assay under proinflammatory conditions. The results are expressed as in Fig. 4.

FIG. 8.

Presence of HIV proviral DNA in MDM recovered after transmigration through an epithelial cell monolayer. DNA was extracted from MDM recovered from the basal transwell chamber. The 219-bp amplicon of HIV was visualized after migration on an agarose gel of PCR-amplified proviral DNA. Lane 1, DNA extracted from transmigrated MDM; lane 2, DNA extracted from transmigrated HIV-infected MDM at 4°C; lane 3, DNA extracted from transmigrated HIV-infected MDM at 37°C in the absence of proinflammatory stimuli; lanes 4 to 6, DNA extracted from transmigrated HIV-infected MDM under conditions where HEC-1 cells had been preincubated with anti-CD102 (lane 4) or anti-CD50 (lane 5) or both MAb (lane 6) and the assay was performed under proinflammatory conditions. Results are those from one representative experiment out of three that were performed.

FIG. 9.

Intracellular IL-1β and TNF-α production by HEC-1 cells. Confluent HEC-1 cells were cultured alone (top) or in the presence of N. meningitidis LPS (5 ng/ml), IL-1β (2 ng/ml), and TNF-α (10 ng/ml) (bottom). Intracellular cytokine production was assessed using specific anticytokine fluorescent MAb. An isotypic control was used as a negative control. Cells were gated by forward and side scatter (SSC) parameters. At least 10,000 events were acquired for intracellular cytokine analysis. Results are presented as representative dot plots from one representative experiment out of three. Anti-TNF-α APC, allo-phycocyanin-labeled murine monoclonal anti-TNF-α.