Macrophages transmit human immunodeficiency virus type 1 products to CD4-negative cells: involvement of matrix metalloproteinase 9

Abstract

It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.

FIG. 1.

Macrophage but not lymphocyte cell lines efficiently transfer HIV-1 products to cocultured epithelial cells. (A) FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8 T-cells cocultured with infected macrophages or lymphocytes. U937/HIV-1, D10, or 8E5 cells were cocultured with 293/CD8T cells for 16 h and then analyzed by FACS. As controls, the different cell types were analyzed alone (upper panels). (B) FACS analyses of HIV-1 Env gp120 and CD8 levels in 293/CD8T cells cocultured for 16 h with U937/HIV-1. (C) FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured for indicated times with U937/HIV-1. (D) 293/CD8T cells cocultured for 16 h with U937/HIV-1, separated from donor cells, and labeled with both anti-Gag and anti-Env gp120 MAbs upon cell permeabilization. Two sets of representative confocal microscope images are shown. The images in the lower panels are the results of computer-assisted analysis carried out by overlapping the images obtained with visible and fluorescent lights. Bars represent 10 μM. (E) FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured with U937/HIV-1, separated (transwell coculture) or not separated by a 0.4-μm microporous filter. (F) FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured with U937 cells previously infected with the VSV-G NL4-3/Nef_F12 HIV-1 variant or, as a control, with the wt counterpart. As additional controls, the different cell types were analyzed alone. (G) FACS analyses of either HIV-1 Env gp120 or ICAM-1 and CD8 levels in 293/CD8T cells cocultured for 16 h with U937/HIV-1. The amounts of cell-associated Env gp120 and ICAM-1 as measured by ELISA are also reported. The results are representative of three (A and G), four (B and F), two (C and E), or five (D) independent experiments. For single-cell-type cultures, the percentage of total events in each quadrant is reported, whereas each coculture plot shows the percentage of Gag-positive cells in the total number of CD8-positive cells. α, anti.

FIG. 2.

Analyses of additional donor/target cell types. (A) Primary macrophages efficiently act as donor cells. FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured with primary human macrophages infected with R5 ADA HIV-1 and in single-cell-type cultures. Results are representative of two independent experiments. (B) Primary activated CD4 lymphocytes do not transmit HIV-1 products to epithelial cells. FACS analyses of levels of HIV-1 Gag products in HIV-1 infected CD4 lymphocytes cocultivated with CFSE-labeled 293/CD8T cells or activated CD4 lymphocytes in the presence of 20 μM AZT for 16 h. Results are representative of experiments carried out with cells from two donors. (C) Both U87 astrocytes and EA-hy 926 endothelial cells but not primary CD8 lymphocytes act efficiently as target cells. FACS analyses for detection of HIV-1 Gag products and either CD8 (for CD8 lymphocytes) or CFSE (for both U87 and EA-hy 926 cells) in U937/HIV-1-based cocultures. 293/CD8T cells were used as target cell controls. Results are representative of five (for CD8 lymphocytes) or two (for both U87 and EA-hy 926 cells) independent experiments. For single-cell-type cultures, the percentage of total events in each quadrant is reported, whereas each coculture plot shows the percentage of Gag-positive cells in the total number of CD8- or CFSE-positive cells. PBL, peripheral blood lymphocyte; α, anti.

FIG. 3.

The presence of HIV-1 products in target cells does not rely on infection events. (A) AZT treatment does not influence HIV-1 Gag accumulation in target cells. FACS analyses of levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured for 16 h with U937/HIV-1 in the presence of 10 or 100 μM AZT (upper panels). As controls (Ctrl), human lymphoblastoid HIV-1-infected CEMss cells were treated with the same concentrations of AZT and analyzed for HIV-1 Gag expression 2 days later (lower panels). FSC, forward scatter. (B) Kinetics of HIV-1 Gag stability in target cells after separation from HIV-1-infected donor cells. FACS analyses of the levels of HIV-1 Gag/CD8 in 293/CD8T cells cocultured with U937/HIV-1 for 16 h and then separated from the donor cells. The target cells were analyzed immediately (time zero) or at the indicated times in culture after immunoselection. The results reported in both panels A and B are representative of two independent experiments. The percentages of Gag-positive cells in the total numbers of CD8-positive cells are shown in the coculture plots, whereas the percentages of HIV-1 Gag-expressing cells are shown in the lower plots of panel A. α, anti.

FIG. 4.

Gag and Env HIV-1 products accumulate and colocalize in donor cell-target cell contact zones. (A) Confocal microscope images of cocultures of 293/CD8T cells with U937/HIV-1 cells after labeling with both anti-CD8 (red fluorescence) and anti-Gag CAp24 (green fluorescence) MAbs. The same field was scanned at three section points. The arrow indicates a zone of high levels of accumulation of Gag products, seemingly corresponding to the contact area between two 293/CD8T cells and a U937/HIV-1 cell. (B) Fluorescence microscope images of 293/CD8T cells purified after 16 h of cocultivation with U937/HIV-1 cells and labeled for cell membrane-associated Env and intracellular Gag products in the presence of DAPI (4′,6′-diamidino-2-phenylindole). Arrows indicate the most evident zones of Gag-Env colocalization. (C and D) Fluorescence microscope images of 293/CD8T-U937/HIV-1 cocultures 16 h after the cultures were set. Cells were first labeled with Texas Red (C)- or FITC (D)-conjugated anti-CD8 MAbs, then permeabilized, and finally stained with DAPI and either FITC-conjugated anti-CAp24 MAb (C) or anti-Env gp120 human MAb followed by Texas Red-conjugated anti-human immunoglobulin G (D). Arrows indicate the zones of accumulation of HIV-1 products overlapping the cell-to-cell adhesion areas. For all panels, the bars represent 10 μM. α, anti.

FIG. 5.

Neither Env nor Nef is required for the transfer of HIV-1 products. FACS analyses of the levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured with U937 cells (upper panels) or 7-day-old MDMs (lower panels) previously infected with HIV-1 strain VSV-G wt, Δenv, or Δnef are shown. As negative-control target cells, PHA-activated CD8 lymphocytes from the same MDM donors were used. The percentage of Gag-expressing cells in the total number of CD8-positive cells is reported in each plot. Results are representative of two (for U937 cells) or four (for MDMs) independent experiments. PBLs, peripheral blood lymphocytes; α, anti.

FIG. 6.

Treatment with inhibitors of either HIV protease or MMPs blocks the transfer of HIV-1 products. FACS analyses of the levels of HIV-1 Gag (A) or Env gp120 (B) products in 293/CD8T cells cocultured for 16 h with U937/HIV-1 cells in the presence or absence (Ctrl) of 2 μM of the indicated PIs are shown. The percentage of Gag-positive cells in the total number of CD8 positive cells is indicated in each plot. Results are representative of six (for ritonavir) or three (for other PIs) independent experiments. (C) Percentages of HIV-1 Gag-positive 293/CD8T cells, as measured by FACS analysis, after 6 h of coculture with U937/HIV-1 cells in the presence or absence (Ctrl) of the indicated concentrations of either GM6001 or MMP IV are shown. Cocultures treated with 2 μM indinavir (IDV) were included as controls. The percentage of HIV-1-expressing U937/HIV-1 cells was 70 to 75% for all experiments. The means ± the standard deviations of the results of three independent experiments are shown. α, anti.

FIG. 7.

MMP-9 is involved in the transfer of HIV-1 products to target cells. (A) Zymograms of supernatants of either uninfected or HIV-1-infected cell lines. Supernatants from the indicated cells cultivated alone or in the presence of 293/CD8T cells (co-c) were analyzed for gelatinase activity. Ten microliters of serum-free supernatant from U937 cells treated for 2 days with 50 ng/ml of tetradecanoyl phorbol acetate was used as a positive control. The data are representative of two independent experiments. (B) FACS analyses of the levels of HIV-1 Gag in both CD4 lymphocyte and MDM-infected cultures. The percentages of HIV-1 Gag-positive cells are indicated. (C) Zymograms of the supernatants of uninfected or HIV-1-infected primary cells. Analyses were carried out on the supernatants from uninfected or HIV-1-infected CD4 primary lymphocytes or MDMs cultivated alone or in the presence of 293/CD8T cells (co-c). The data are representative of experiments carried out on cells from two healthy donors. (D) Semiquantitative analyses of the levels of MMP-9 released in the supernatants from equal numbers of either uninfected or HIV-1-infected MDMs. The data are representative of experiments carried out on cells from two healthy donors. In panels A, B, and D, MMP-2 and/or MMP-9 migration is indicated on the left, and molecular marker sizes are shown on the right. (E) FACS analyses of the levels of HIV-1 Gag products and CD8 in 293/CD8T cells cocultured with U937/HIV-1 in the presence of 5 μg/ml of an unspecific mouse immunoglobulin G or 2.5 or 5 μg/ml of anti-MMP-9-neutralizing MAb or (F) the indicated concentrations of sodium orthovanadate are shown. The percentage of Gag-positive cells in the total number of CD8-positive cells is shown in each plot. For both panels E and F, the results are representative of two independent experiments. PBLs, peripheral blood lymphocytes; α, anti.