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(Journal of Leukocyte Biology. 2001;69:405-413.)
© 2001 by Society for Leukocyte Biology

CD86 expression correlates with amounts of HIV produced by macrophages in vitro

Xiaoping Wang and Dorothy E. Lewis

Department of Immunology, Baylor College of Medicine, Houston, Texas

Correspondence: Dr. Dorothy E. Lewis, Baylor College of Medicine, One Baylor Plaza, Room BCMM-M929, Houston, TX 77030-3498. E-mail: dlewis{at}bcm.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Primary macrophages from different donors produce variable levels of HIV; however, the mechanisms are unclear. We tested whether variations in cell-surface or cell-cycle characteristics influenced HIV production. We found that greater basal proliferation of the macrophages prior to infection resulted in more arrested in G2M 3 days post-infection (r2=0.7, P<0.04). Likewise, the number of G2M-arrested macrophages correlated with p24 production (r2=0.78, P<0.02) and apoptosis (r2=0.67, P<0.05) later in the infection. Serum-starvation or reduction, which limit HIV spread, reduced G2M arrest and HIV amounts. Surprisingly, the amount of HIV produced correlated with expression levels of the costimulating ligand, CD86, but not with other important molecules, including class II, CD40, or CD54 (r2=0.96, P<0.0005). These data establish donor characteristics related to variable HIV production in vitro and suggest that altered expression of costimulatory ligands may influence HIV production in vivo.

Key Words: G2M arrest • apoptosis • p24 antigen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are critical cells in adaptive and specific immune responses because they are phagocytic cells, which make cytokines as well as processing antigens for presentation to antigen-specific T-cells [1 , 2 ]. Their infection by pathogens can have a profound impact on development or maintenance of immunity to the pathogen. Thus, human immunodeficiency virus (HIV) infection of macrophages results in altered cytokine expression as well as cell-surface molecules in vitro [3 4 5 6 7 ].

However, characterization of cell-surface molecule and cytokine secretion changes after HIV infection of macrophages in vitro is incomplete. Whereas, there is documented evidence that HIV infection up-regulates secretion of tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1, and IL-10 [3 4 5 6 7 8 ], there is also literature showing that IL-10, when added exogenously, suppresses HIV replication [9 , 10 ]. Likewise, changes in cell-surface characteristics after HIV infection have been variable for important cell-surface molecules for macrophage function, such as class II, CD40, CD54, CD80, or CD86 [6 , 11 , 12 ]. This variability could be a result of the cell populations used (monocytes or macrophages) or the condition of the various assays. Because these cell-surface molecules potentiate interactions of macrophages with T-cells, it is pertinent to study alterations in their expression levels after HIV infection.

Recent evidence suggests that altered ability to regulate CD86 expression on monocytes from some HIV+ patients lowers subsequent IL-2 production by T-cells [13 ]. Certain cytokines have been shown to modulate CD80 and CD86 expression in vitro so that Th2 cytokines (IL-4 and IL-10) down-regulate CD86 with a moderate increase in CD80. TNF-{alpha} decreases CD86 but has little effect on CD80 expression in human monocytes [14 , 15 ]. Interferon-{gamma} (IFN)-{gamma}, however, enhances CD80 and CD86 expression [14 ]. Because several studies suggest that TNF-{alpha} and IL-10 production is increased in HIV-infected persons, they could regulate expression of the costimulating molecules important for T-cell activation and development, including CD80 and CD86 [16 , 17 ]. Indeed, it was demonstrated recently that monocytes from HIV+ patients have increased expression of CD80 after overnight incubation compared with controls and have decreased resting CD86 expression, suggesting altered costimulating molecule regulation in vivo [13 ]. These data suggest that altered cytokine production by infected macrophages or by other cells in the HIV+ patient, which affect cells of the monocyte/macrophage lineage, may influence their subsequent costimulating abilities for lymphocytes.

HIV can infect nondividing cells such as macrophages because of unique nuclear localization sequences in several HIV proteins [18 19 20 21 22 23 24 ]. However, in T-cells, proliferation appears required for HIV viral particles to be made [25 , 26 ]. In macrophages, there is also evidence that proliferation is not required for DNA integration [27 ] but is required for particles to be made [28 ] with amounts of HIV produced by macrophages reduced compared with T-cells [29 ]. Two studies show that in T-cells [30 ] and in macrophages [31 ], progression to the G1b phase or G1/S phase of the cell cycle is necessary for HIV particle production. It is interesting that HIV is best able to complete reverse transcription in T-cells after CD28 costimulation, indicating a role for CD28-CD80, CD86 interactions for HIV infection [30 ]. In addition, there can be great variations in the amount of HIV produced by macrophages in vitro from 1–1000 ng/ml [32 ].

The present study examined monocyte-derived macrophages to determine mechanisms associated with variable HIV production. Our surprising results link HIV production by these cells to the basal division rate prior to infection and levels of costimulatory ligands on the macrophages after infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Conjugated antibodies
Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) to CD68 (clone kp1) was purchased from DAKO (Carpinteria, CA.). Phycoerythrin (PE)-conjugated mAb to CD86 (clone 2331) was purchased from Pharmingen (San Diego, CA). PE-conjugated mAb to CD83 was purchased from Beckman Coulter Immunotech (Westbrook, ME). Other Abs were also purchased from Pharmingen, including CD40, CD54, CD80, and DR. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was from R&D Systems (Minneapolis, MN). The p24 EIA kit was purchased from Beckman Coulter Immunotech.

Preparation of monocytes and macrophages
Monocytes were isolated from fresh leukocyte buffy coats obtained from the Gulf Coast Blood Bank [HIV-/hepatitis B virus (HBV-)] by Ficoll-Hypaque density centrifugation [33 ]. After washing three times with phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS), erythrocytes were lysed for 5 min at 37°C in NH4Cl buffer. The mononuclear cells were seeded in 6-well tissue-culture plates at 20 x 106/ml in RPMI 1640 medium containing 1% normal human serum. Monocytes were enriched using a 60-min adherence to culture plates. Nonadherent cells were removed, and the adherent cells were washed 2–3 times with PBS containing 2% FBS. The adherent population was composed principally of CD14+ monocytes (>80%) as determined by flow cytometry. The monocytes were cultured further in RPMI containing 10% FBS supplied with 10 ng/ml rhGM-CSF for 7 days to allow maturation [34 35 36 ]. During this time, the medium was changed every 2–3 days. The culture medium was removed completely at each time point, reducing the number of potential contaminating, nonadherent lymphocytes. After 7 days of culture in rhGM-CSF, the macrophages had increased forward-scatter, and 100% expressed the macrophage-surface marker, CD68, not CD83 or CD14 as determined by flow cytometry. After 7 days of culture, no GM-CSF nor any other cytokine was added to the cultures.

In vitro infection with HIV-1
After 7 days of in vitro culture, the mature, monocyte-derived macrophages were infected with the monocytotropic HIV-1 ADA strain (obtained from Howard Gendelman, M.D., University of Nebraska Medical Center) at a concentration of 20 ng/ml (titered by p24 assay) for 24 h. The culture medium was removed, and cells were washed three times with PBS. Fresh medium was added, and cultures were continued up to 14 days. Cells were monitored at different time points for viral production, cell-cycle arrest, and cell-surface molecule expression. Heat inactivation of HIV was carried out at 65°C for 2 h [37 ].

Serum-starvation
After growing the macrophages in rhGM-CSF for 7 days, mature macrophages were washed three times with Dulbecco’s modified Eagle’s medium (DMEM) without FBS and cultured for 24 h. After overnight infection, cells were washed three times with RPMI and cultured in medium containing 2% or 10% FBS as indicated in the text.

P24 assay
HIV p24 production was measured using a standardized EIA kit from Beckman Coulter Immunotech. Aliquots of cell-culture media were collected at different points and stored at -70°C until used. The p24 content from each sample was calculated according to the standard positive control provided in the kit.

Cell-surface antigen-staining
At the time of collection, the medium was removed, and cells were washed twice with PBS. To avoid potential loss, damage, or permeabilization of the HIV-infected cells, macrophages bound to the plastic dishes were incubated with ice-cold PBS containing 3 mM ethylenediaminetetraacetate (EDTA) for 3–5 min and gently scraped off the plate. Cells were washed once with PBS plus 2% FBS on ice as a blocking reagent and placed into tubes containing appropriate conjugated antibodies. The labeling was carried out on ice for 30 min. Excess antibody was washed away by adding 4 ml ice-cold PBS containing 2% FBS. After centrifugation, cells were fixed with 1% paraformaldhyde in PBS and stored at 4°C for flow cytometric analysis.

Determination of cell-cycle stage and apoptosis by propidium iodide (PI) staining
Cells were collected as described earlier. After washing, cells were resuspended in 200 µl PBS. While vortexing gently, 1 ml of 95% ethanol was added, and the fixation was carried out for at least 30 min at room temperature. After centrifugation, the supernatant was discarded, and cells were resuspended in 1 ml PBS containing 50 µg PI. RNase was added at a final concentration of 100 µg/ml, and the samples were incubated at 37°C for 30 min and then analyzed by flow cytometry. Gates based on the DNA content of the cell cycle in the uninfected sample were placed around areas separating the G0/G1, S, and G2M areas of the cell cycle. Apoptosis was measured by examining the subdiploid region to the left of the G0/G1 peak [38 ].

Flow cytometric analysis
All of the samples were analyzed on an XL flow cytometer (Beckman Coulter, Hialeah, FL). For purified populations, 5000 events gated on a viable light scatter region (LSC) were collected. After HIV infection for 12–14 days, the number of cells within the viable LSC decreased, and in some cases, fewer events were analyzed. The files were analyzed using XL-2 software. Fluorescence data are displayed as four-decade logarithmic histograms or dot plots for the immunofluorescence and linear histograms for the DNA/apoptosis analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIV-1 infection of primary macrophages results in G2M cell-cycle arrest, which leads to apoptosis
To investigate the mechanisms regulating variable HIV production by primary macrophages, we isolated monocytes from healthy donors to generate macrophages using GM-CSF and then infected them with the HIV-1 monocytotropic ADA strain. After 7 days of culture in the medium supplemented with 10 ng/ml rhGM-CSF, macrophages were mature, as determined by increased LSC and expression of the macrophage specific cell-surface marker, CD68, by flow cytometry. During the next 14 days of culture, with or without HIV infection, cell-cycle characteristics from each donor cell population were analyzed by PI-staining and flow cytometry. We also determined the percentage of apoptotic cells by measuring a subdiploid peak from the same DNA histogram. As shown in Figure 1 , increased percentages of cells arrested in the G2M phase of the cell cycle were observed in HIV-infected macrophages as has been observed with primary T-cells [33 , 39 ]. In the experiment in Figure 1 at 3 days post-infection, 12.3% of cells were arrested in the G2M phase of the cell cycle, whereas 15.1% and 20.4% cells were arrested in the G2M phase at 6 and 10 days post-infection, respectively. However, in uninfected cells from the same donor, only 6.3%, 7.8%, and 8.4% cells were in the G2M phase at the same time points. Figure 1 also shows that there was an increase in apoptosis, which occurred later than the G2M phase arrest. For example, the percentage of apoptotic cells increased from 9.8% at 3 days post-infection to 17.4% and 24.6% at 6 and 10 days after infection, respectively. However, in the uninfected cells, only 6.3%, 5.0%, and 2.7% of apoptotic cells were observed from the same donor at the same time points. Viable cell counts were also decreased significantly by 50% after 6 days of infection compared with uninfected cultures (unpublished results).



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  		Figure 1. HIV-1 infection causes G2M arrest in primary macrophages. Cells were collected at 3, 6, and 10 days after HIV-1 infection and stained with PI as described in Materials and Methods. Cell-cycle histograms from uninfected macrophages from donor 2 are shown in the top panels. HIV-1-infected macrophages are shown in the bottom panels. The two designated regions in each histogram indicate percentage of apoptotic cells (left) and those in the G2M phase (right).

 
The results of cell-cycle studies during HIV-1 infection from six individuals are summarized in Table 1 . Although cells from each donor had different basal proliferation rates, as indicated by the numbers of cells in the G2M phase prior to infection, every HIV-1 infection resulted in an increased number of cells in the G2M phase compared with the uninfected control culture from the same donor. The number of apoptotic cells also increased in the cultures from all six donors by day 12.


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  		Table 1. Cell-cycle characteristics from individual donors

 
Levels of G2M arrest and apoptosis correlate with amount of HIV p24 produced
To determine whether G2M arrest and apoptosis were associated with amounts of HIV produced by primary macrophages, we compared cell-cycle kinetics and HIV p24 production during a 14-day culture period (Table 2 ). Because the culture medium from each sample was changed completely every 2–3 days, the p24 value from each sample indicates the accumulation of viral proteins in the supernatant during the intervals. Results demonstrated that at early stages after viral infection, only a small amount of HIV was produced. At later stages post-infection, however, a dramatic increase in p24 production was observed, suggesting that the virus spread in the cultures. Because higher amounts of viral production were seen after a greater increase in G2M phase arrest, these results also suggest that HIV replication is favored by the accumulation of macrophages in the G2M phase. Whether all infected cells were also G2M-arrested was not determined in this study. However, statistical analysis showed a correlation between amounts of G2M arrest at 10 days post-infection (expressed as a percentage of the cell-cycle distribution) and p24 production at day 12 (r2=0.78, P<0.02). Thus, the level of cells arrested in G2M was linked to p24-increased levels in macrophages similar to T-cells [40 , 41 ].


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  		Table 2. HIV p24 production by macrophages (ng/ml)

 
Following the increased levels of viral production, which occurred after the G2M arrest, a greater number of cells also underwent apoptosis. Using donor 2 as an example, HIV p24 production increased from 5.1 ng at 3 days post-infection to 60.8 ng and 57.14 ng at 8 days and 12 days post-infection. Apoptotic cells were also increased proportionally from 6.2% at 3 days post-infection to 10.8% and 16.8% at 8 and 12 days after infection (Fig. 1 and Table 1 ). A statistical correlation between the amount of apoptosis and p24 production at 12 days post-infection was also observed (r2=0.67, P<0.05). However, whether both infected cells and uninfected cells were apoptotic was not determined.

Efficient initial infection of HIV in macrophages is associated with basal proliferation prior to infection
To investigate mechanisms of variable HIV production among different donors, we next compared the basal proliferation level of uninfected macrophages with the cell-cycle distributions of the same sample after HIV-1 infection. In Figure 2 , the average percentage of cells in the G2M phase from uninfected macrophages (5–6 time points) during the 14 days of incubation was used as a control and compared with the percent of cells in the G2M phase at 3–5, 6–8, or 12 days post-infection. The first donor shown in Figure 2 (donor 1) had an average of 2.7% ± 0.6% of the uninfected population in the G2M phase over the time course studied, and the amount of cells in the G2M phase after HIV infection was small, with 4.9%, 5.0%, and 9.4% of the infected population in the G2M phase at 3, 8, and 12 days post-infection, respectively. Conversely, the third donor demonstrated a greater basal proliferation level of uninfected cells, with an average of 7.62 ± 0.85 of the population in the G2M phase of the cell cycle. In HIV-infected cultures from the same person, 12.3%, 15.2%, and 20.4% of the cells were in the G2M phase at 3, 8, and 12 days post-infection, respectively. A statistical analysis of the basal proliferation level of macrophages prior to infection and the percentage of cells arrested at the G2M phase at 3 days post-infection show a good correlation (r2=0.70 and P<0.04). These results suggest that the amount of HIV-1 produced by macrophages is associated with the basal proliferation status of macrophages prior to infection.



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  		Figure 2. Levels of the G2M arrest early after infection correlate with basal proliferation levels. Cells were collected from cultured macrophages during the 14-day incubation period with or without HIV infection. The left bar from each donor represents the average percentage of cells in the G2M phase without HIV infection (n=3 time points). The percentage of cells in the G2M phase at 3/5, 6/8, and 10/12 days post-HIV infection is also shown.

 
Increased expression of CD86 correlates with increased basal proliferation rate
As viral production increased by day 8 post-infection, macrophages showed significant changes in morphology. We questioned whether such changes were associated with increased expression of activation or adhesion molecules, which are involved in regulatory interactions with T lymphocytes. We examined several cell-surface molecules important in interactions between antigen-presenting cells (APC) and lymphocytes, including CD54, CD40, and HLA-DR. These adhesion molecules were increased during the course of HIV infection from two- to tenfold compared with uninfected cells (unpublished results). However, there was no statistical correlation between increased expression levels of these molecules and levels of HIV produced.

Conversely, CD86, an important ligand for CD28 signaling during T-cell activation, was increased significantly in HIV-1-infected macrophages up to 20-fold 6 days post-infection compared with that of uninfected cells (Fig. 3A ). A similar increase also occurred in CD80 expression in two of two experiments (unpublished results). Although the basal expression level of CD86 from each individual varied prior to HIV infection, increased CD86 expression after infection occurred in cells from all six donors tested. The mean fluorescence intensity (MFI) and the percentages of CD68/CD86 (CD14/CD86 for donors 4–6)-positive cells were increased. Increased expression of CD86 on HIV-infected macrophages was maintained over the time course examined. However, the highest MFI occurred early, between 3 and 8 days after infection, parallel to the initial production of HIV but before significant levels of G2M arrest.



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  		Figure 3. Increased CD86 expression after HIV-1 infection correlates with cell-cycle arrest and apoptosis. (A) CD86 expression in uninfected and HIV-infected macrophages at 6 days post-infection expressed as percentage positive and MFI. The top panels are uninfected macrophages, whereas the histograms from HIV-1-infected macrophages are shown in the bottom panel. The MFI and percentages of CD68/CD86 (donors 1–3) or CD14/CD86 (donors 4–6) are indicated in each histogram. (B) Statistical analysis of the levels of CD86 expression with the numbers of cells in the G2M phase and apoptosis. The MFI levels of CD86 expression at 6 days after infection correlated with the amount of cell apoptosis 3 days post-infection (filled diamonds and line, r2=0.96, P<0.0005). The MFI of CD86 expression levels also correlated with increased numbers of cells in the G2M phase of the cell cycle at 8 days post-infection (empty squares and dashed line, r2=0.74, P<0.03).

 
Statistical analysis also showed a striking correlation between the numbers of apoptotic cells 3 days after infection and levels of CD86 expression at 6 days post-infection (Fig. 3B , empty squares and filled line, r2=0.96, P<0.0005). Levels of CD86 expression 6 days after infection also correlated with levels of G2M arrest 2 days later in the cultures (Fig. 3B , filled diamond and dashed line, r2=0.74, P<0.03).

Serum-starvation of macrophages results in delayed cell-cycle arrest, decreased p24 production, and reduced apoptosis
To further prove our hypothesis that amounts of HIV produced are dependent on the basal proliferation of macrophages prior to infection, we used serum-starvation to investigate whether limiting growth factors would block initial infection or merely reduce the kinetics and, therefore, the amount of HIV produced. After 7 days in culture with rhGM-CSF, macrophages were washed with DMEM without serum and incubated for 24 h in the same medium followed by an overnight infection with HIV as before. After the infection, medium was removed, and cells were washed and cultured in RPMI containing 2% (reduced) or 10% (normal) serum for 19 days. The cell-cycle status as well as p24 production were compared with infected cells that were maintained in 10% serum. Serum-starvation reduced the level of G2M arrest significantly in macrophages, as shown in Figure 4 . Levels of p24 were reduced, and viability was increased (unpublished results). However, the serum-starved cells were still infected because some p24 was detected later in the cultures. Therefore, viral spread, and not infection per se, was altered by serum-starvation.



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  		Figure 4. Serum-starvation results in less G2M arrest. The top panel is from uninfected cells 13 days (left) and 21 days (right) after culture (6 days and 12 days post-infection, respectively). The middle panel is untreated but HIV-infected cells. Pre-starved cells, which were cultured in 10% FBS, are shown in the bottom panels. The box indicates the percentage of cells in the G2M phase of the cell cycle.

 
We also examined expression levels of CD86 from pre-starved HIV+ macrophages, because if CD86-expression increase is a factor indicative of HIV-infection level, we expected less CD86 to be expressed on cells that were pre-starved or in reduced serum. As shown in Figure 5 , levels of CD86 expression from untreated HIV-infected macrophages were increased, as expected. By contrast, the level of CD86 expressed on pre-starved HIV+ macrophages was much reduced compared with untreated HIV-infected macrophages. Statistical analysis showed that in untreated, HIV-infected cells, the expression levels of CD86 (MFI) correlated with the number of cells arrested at the G2M phase and the amount of HIV-p24 produced (r2=0.604, P<0.001, and r2=0.905, P<0.001, respectively; unpublished results). Although CD86 expression levels from pre-starved HIV+ cells were reduced compared with untreated cells, there still was a correlation with G2M arrest and HIV p24 production (r2=0.634, P<0.03, and r2=0.959, P<0.0001, respectively; unpublished results).



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  		Figure 5. Serum-starvation results in less CD86 up-regulation after infection: CD86 expression levels as measured by MFI are plotted as a function of days post-infection for HIV-, HIV+, and cells that had been starved and then HIV-infected.

 
We also used heat-inactivated virus as a control for the infection assays to test for nonspecific effects. HIV-1 ADA strain virus was heated at 65°C for 2 h and then incubated with macrophages overnight. HIV p24 production and macrophage cell-cycle status were monitored. Results indicated that there was a 95% decrease in p24 production in the culture medium 15 days after infection, and cell-cycle studies also showed no increase in cell numbers in G2M phase, nor was there an increase in CD86 expression (unpublished results). These results indicate that inactivation of HIV-1 by heat treatment destroyed the ability of HIV to replicate in host cells and alter host-cell characteristics effectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Earlier work showed that HIV particle production by macrophages required proliferation because irradiation or mitomycin-C treatment prevented HIV production, although incomplete viral DNA synthesis was detected [28 ]. Another paper showed that productively infected HIV DNA-expressing cells are proliferating and nonproliferating, indicating that infection can occur at any stage of the cell cycle [27 ]. Rather, HIV particle production is facilitated by cellular proliferation; T-cells and macrophages require entry into G1b or the G1/S boundary to complete reverse transcription or DNA integration [30 , 31 ]. In this study, we show that G2M arrest is associated with increased HIV production and spread in the macrophage cultures. Optimal HIV production by macrophages also requires a basal level of proliferation prior to infection, and the more basal proliferation, the more HIV is subsequently produced. We do not interpret this to mean that more cells are necessarily infected but that more virus is made by proliferating cells. Increased cell proliferation may be inherent to the individual macrophage population or reflect their ability to respond to GM-CSF.

Our results with altering serum availability in the cultures also show that macrophage proliferation is associated with levels of HIV production. Serum-starved macrophages were capable of being infected in vitro during the starvation interval but never made appreciable amounts of HIV post-infection. Likewise, they did not demonstrate increased G2M arrest, increased apoptosis, or CD86 expression. These data support the idea that HIV infection might spread throughout the culture via up-regulation of CD86, cell-cycle arrest, and apoptosis.

Earlier studies with macrophage cell lines have suggested that HIV-1 infection in vitro results in variable expression levels of certain molecules that interact with T-cells, including major histocompatibility complex (MHC) class II, CD54, and costimulatory molecules such as CD86 [42 43 44 ]. Intercellular adhesion molecule 1 (ICAM-1, CD54) is expressed on APC normally and binds to the integrin, lymphocyte function-associated antigen-1 (LFA-1) on T lymphocytes [45 ]. In our studies, CD54 was increased about fivefold in the HIV-1-infected macrophages compared with uninfected cells, indicating that adherence to T-cells might be enhanced. The MHC class II molecule is expressed on dendritic cells, B-cells, monocytes, and macrophages, as well as activated T lymphocytes. Earlier studies have demonstrated that class II molecules bind CD4 on T-cells and hence are intimately involved in antigen-specific T-cell activation [46 ]. In this study, class II expression was also increased in HIV macrophages. CD40 is a costimulatory molecule, which is up-regulated after activation and expressed on APC, including B-cells, dendritic cell cultures, and macrophages [47 ]. CD40 interacts with CD40 ligand on activated T-cells, and costimulation via CD40-CD40L with antigen-receptor engagement facilitates T- and B-cell division. However, in the absence of a T- or B-cell antigen-specific signal, CD40-CD40L interaction can lead to apoptosis [48 ]. Although CD54, class II, and CD40 were all increased on macrophages after HIV infection, their expression levels did not correlate with levels of HIV produced.

By contrast, the expression of CD86, which is a ligand for CD28, correlated with amounts of HIV produced. The interaction of CD86 with CD28 provides important costimulatory signals for T-cells activated through the CD3/T-cell receptor (TCR) to promote T-cell proliferation and IL-2 production [49 , 50 ]. Without this costimulatory signal, CD3/TCR-stimulated lymphocytes undergo apoptosis or became anergic [51 , 52 ]. In HIV, there is evidence that CD28 is down-regulated on CD4+ and CD8+ lymphocytes, and this down-regulation is associated with reduced cytokine production, T-cell apoptosis, and eventually disease progression. Our previous study showed that increased lymphocyte apoptosis in HIV+ patients was associated with less CD28 expression on T-cells [54 ]. Abs to CD80 and CD86 or depletion of monocytes rescued CD8+CD28dim cells from apoptosis [55 ]. In the experiments demonstrated here, the expression of CD86 increased early after infection of macrophages in every donor (3–5 days). The timing of increase in CD86 expression coincided with the early G2M arrest but occurred before the increase in HIV production. The amounts of CD86 expression correlated directly with the amount of HIV in the cultures as well as the amount of G2M arrest. This result suggests that CD86 is not only a costimulating factor for T-cell activation but also an indicator of macrophage activation or maturation induced in the context of HIV infection. Indeed, for infected T-cells to complete reverse transcription, they require costimulation through CD28 [56 ]. This might be facilitated in vivo by APC, which have up-regulated the CD28 ligands CD80 and CD86. There is recent evidence showing that HIV infection of resting and stimulated T-cells up-regulates CD80 and CD86 expression in vitro [57 ]. The authors suggest a link between HIV and up-regulation of CD28 ligands similar to the results we show here for macrophages.

Alterations in the ability to regulate costimulatory ligands in HIV have been observed in vivo [58 ] and related to lowered IL-2 production in vitro [13 ]. Because in our experiments, the actual numbers of infected macrophages were not determined, but the increase of CD80 and CD86 expression occurred on the population as a whole, we think it is likely that CD86 is increased by cytokines produced by macrophages of only some of the cells after HIV infection. Given the results, the most likely candidate cytokines are that type I IFNs [59 ] are produced and secreted after infection, which then up-regulates CD80 and CD86 on the surface of infected and uninfected macrophages in the cultures. Consistent with this idea is recent evidence that type I IFNs mediate monocyte differentiation in vitro so that the cells become very efficient APC with up-regulation of CD80 and CD86 without increases in CD40 or CD54 [60 ]. Because HIV infection results in a selective IFN-{alpha} expression decrease [61 ], we suggest that IFN-ß may be responsible after HIV infection of macrophages.

Macrophages, which are targeted by HIV and act as a reservoir, are a major subpopulation of APC in the human immune system [62 ]. Our data indicate that the amount of HIV produced by macrophages is best reflected by levels of CD86 expression. The implications are that HIV production in vivo during early infection may be largely influenced by the activation environment of the host. Altered costimulating abilities of infected or cytokine-altered macrophages may enhance infection of CD4+ T-cells and/or increase apoptosis of activated CD8+ T-cells, which could contribute to HIV pathogenesis.


   ACKNOWLEDGEMENTS
 
This work was supported by NIAID grants (1 R01 AI36682) and CFAR (1 P30 AI36211). We thank Howard Gendelman, M.D., and P. Newman, Ph.D., for providing the HIV-1 ADA. We also gratefully acknowledge Mr. J. Scott for assistance with flow cytometry and members of the Lewis laboratory for helpful suggestions and technical support. Terry Saulsberry provided secretarial assistance.

Received June 28, 2000; revised November 13, 2000; accepted November 16, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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