Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay

Abstract

The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a quantitative assay, a new luciferase-based assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa-CD4-LTR-beta-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.

FIG. 1

NKI-L16 induces higher levels of HIV-1-mediated syncytium formation. J1.1 cells were incubated in the absence or presence of NKI-L16 (1 μg/ml) along with an equal number of uninfected cells, either Jurkat E6.1 (A) or 1G5 (B). Similar experiments were performed with a mixture of OM10.1 and 1G5 cells (C). After 16 h of incubation, cells were observed by light microscopy. Magnification, ×100.

FIG. 2

Induction of luciferase activity in a time-dependent fashion by the addition of J1.1 cells to HIV-1 LTR-driven luciferase-containing cell lines. Stably (1G5) (A) and transiently (Sup-T1 or Jurkat E6.1) (B and C) transfected cells were first incubated in the presence of J1.1 and lysed after 6, 12, or 24 h. Luciferase activity was read with a Dynex luminometer apparatus and is shown on a log₁₀ scale. The results are the mean ± standard deviation (SD) for samples studied in triplicate. RLU, relative light units.

FIG. 3

Several infected cell lines cocultured with 1G5 cells show increased luciferase activity over time. The 1G5 cell line was incubated along with the infected cell lines J1.1, H9/HIV-1_IIIB, H9/HIV-1_IIIRF, H9/HIV-1_IIIMN, and Jurkat-tat/PBS-WT and lysed after 6 h (A), 12 h (B), or 24 h (C). Luciferase activity was read with a Dynex luminometer apparatus. The results are the mean ± SD for samples studied in triplicate.

FIG. 4

Increases in HIV-1 LTR-driven luciferase activity are blocked by typical inhibitors of syncytium formation and are correlated with syncytium count. (A) Equal numbers of 1G5 and J1.1 cells were incubated along with the following inhibitors: anti-gp120 (20 μg/ml), sCD4 (20 μg/ml), or anti-CD4 antibodies (clones SIM.4 or SIM.2 at 20 μg/ml). The samples were lysed after a 12-h incubation. (B and C) Different concentrations of sCD4 (0.25 to 20 μg/ml) were also added, and after 12 h of incubation, the samples were immediately lysed for monitoring luciferase activity (B) or visual assessment of syncytium number and syncytium size (arbitrarily evaluated from +/− to ++++) was carried out (C). The results are shown as the mean ± SD for samples studied in triplicate.

FIG. 5

NKI-L16-mediated increase in syncytium formation measured by luciferase activity is optimal at a cell ratio of 1:1 and shows no sensitivity to AZT. (A) 1G5 cells were preincubated or not preincubated with NKI-L16 (1 μg/ml) for 30 min at 37°C. Different cell ratios of 1G5 to J1.1 cells were then incubated in a final volume of 200 μl and lysed after 12 h of incubation. (B) 1G5 cells were pretreated or not pretreated for 90 min at 37°C with AZT (1 μM). Afterward, half of each sample was incubated with NKI-L16 (1 μg/ml) for 30 min. Equal numbers of J1.1 cells were then added with or without sCD4 (20 μg/ml) or anti-CD4 SIM.2 antibodies (20 μg/ml). All the samples were lysed 12 h postincubation. In panel B, luciferase activity is shown on a log₁₀ scale. Luciferase activity was read with a Dynex luminometer apparatus. The results are shown as the mean ± SD for samples studied in triplicate.

FIG. 6

Enhancement of HIV-1-induced syncytium formation by the activated state of LFA-1 is also seen with monocytoid cells acutely infected with HIV-1. Untreated or NKI-L16-pretreated 1G5 cells were incubated for 12 or 24 h with an equal number of monocytoid U937 cells acutely infected with HIV-1. After lysis, the samples were measured for luciferase activity with a Dynex luminometer. The results are shown as the mean ± SD for samples studied in triplicate.

FIG. 7

The NKI-L16-induced increase of virus-mediated syncytium formation measured as luciferase activity is sensitive to blocking agents of the chemokine receptor CXCR4. Untreated or NKI-L16-treated 1G5 cells were mixed with the J1.1 cell line, and either anti-CXCR4 antibodies (A) or different concentrations of SDF-1 (5 and 12.5 μg/ml) (B) were added. SIM.2 (20 μg/ml) was also added for a set of samples (B). The samples were lysed 12 h after the start of the coincubation, and luciferase activity was evaluated with a Dynex luminometer. The results are shown as the mean ± SD for samples studied in triplicate.

FIG. 8

ICAM-1 and ICAM-2 are the main ligands mediating the NKI-L16-induced increase in HIV-1-dependent syncytium formation. (A) Untreated or NKI-L16-pretreated 1G5 cells were incubated with an equal cell number of J1.1 cells in addition to sCD4 (20 μg/ml), anti-CD4 SIM.2 (20 μg/ml), sICAM-1 (2.5 μg/ml), or anti-LFA-1 MEM-30 (20 μg/ml) antibodies. (B) FACS analysis was performed on J1.1 cells by using anti-ICAM-1 (clone RR1/1.1.1), anti-ICAM-2 (clone 92C12F), or anti-ICAM-3 (clone ICR-3.1) antibody and a fluorescein-conjugated goat anti-mouse IgG. Results are shown as an overlay with respect to the isotype-matched antibody control. (C) Untreated or NKI-L16-pretreated 1G5 cells were incubated with an equal number of J1.1 cells in the presence of anti-ICAM-1 (clone RR1/1.1.1), anti-ICAM-2 (clone 92C12F), anti-ICAM-3 (clone ICR-3.1), or anti-CD4 antibodies (clone SIM.2) (all used at 20 μg/ml). Luciferase samples were lysed 12 h after the start of the coincubation. Luciferase activity was evaluated with a Dynex luminometer. The results are shown as the mean ± SD of three independent measurements.

FIG. 9

The sensitivity of syncytium formation to sCD4 is the same in the presence or absence of NKI-L16. 1G5 cells were preincubated or not preincubated with NKI-L16 (1 μg/ml) for 30 min and then mixed with J1.1 cells in the presence of different concentrations of sCD4 (0.025 to 20 μg/ml) for 12 h. After lysis, the samples were measured for luciferase activity. The results are given as the percent inhibition by a given concentration of sCD4 with respect to the untreated samples and are representative of the mean of three different luciferase counts.

FIG. 10

NKI-L16 also induces an enhancement of HIV-1-dependent syncytium formation in PBMCs. Ficoll-Hypaque-isolated PBMCs were incubated in the absence (A) or presence (B) of NKI-L16 for 30 min before the addition of an equal number of J1.1 cells. After 36 h of incubation, light microscopy observation was performed and photographs were taken at ×100 magnification. Arrows point to syncytia.

FIG. 11

PBMCs infected with syncytium-inducing HIV-1 primary isolates are more prone to syncytium formation with NKI-L16-treated 1G5 cells. PBMCs infected for 7 days with two different syncytium-inducing HIV-1 primary isolates (92UG029 and 92UG021) were incubated with an equal number of 1G5 cells pretreated or not pretreated with NKI-L16. After 12 or 24 h of coculture, the cells were lysed and measured for luciferase activity with a Dynex luminometer. The results are shown as the mean ± SD of three independent measurements.