HIV-1 productively infects 2 main groups of cells: CD4+ T cells and macrophages. Although most of the HIV-l RNA found in plasma is thought to come from productively infected CD4+ T cells,1-3 macrophages are an important secondary reservoir for HIV-1.4,5 Compared with HIV-1-infected CD4+ T cells, which have a half-life of 1 to 2 days, HIV-1-infected macrophages are relatively refractory to the cytopathic effects of HIV infection and can release virions for weeks after infection.4,6 Macrophages may be sites for continued HIV-1 replication during antiretroviral therapy, because many commonly used drugs fail to block HIV-1 replication in macrophages completely.7 Macrophages are also thought to be important reservoirs for HIV-1 in the central nervous system (in the form of microglial cells)8,9 and the lung (in the form of alveolar macrophages).10,11 Further support for the notion that macrophage are reservoirs for HIV-1 comes from a study of SHIV-infected macaques with high viral loads and extremely low CD4+ T-cell counts.12
The inability of many antiretroviral drugs to suppress HIV-1 replication completely in macrophages has sustained interest in the use of immunomodulatory therapies to restore macrophage function and enhance the ability of antiretroviral drugs to inhibit HIV-1 replication in macrophages. One immunomodulatory agent that has attracted the attention of HIV-1 researchers is the cytokine granulocyte-monocyte colony-stimulating factor (GM-CSF). Perno et al7 have reported that GM-CSF dramatically augments the ability of 3′-azido-2′3′-dideoxythymidine (AZT) to inhibit the growth of HIV-1 in monocyte-derived macrophages (MDMs), with the median inhibitory concentration (IC50) for AZT decreasing from 1000 mg/mL to 100 mg/mL in the presence of 10 ng/mL of GM-CSF. Meanwhile, Kedzierska et al13 have reported that GM-CSF dramatically increases the rate at which HIV-1-infected macrophages phagocytize the bacterium Mycobacterium avium, a result consistent with previous studies showing that GM-CSF increases the activity of uninfected macrophages against pathogens.14-19
In addition to studies investigating the ability of GM-CSF to increase the effectiveness of antiretroviral drugs and enhance antimicrobial activity in macrophages, GM-CSF has been investigated as an intrinsic inhibitor of HIV-1 replication in macrophages. The results of these investigations have been conflicting, however. Some studies have reported that GM-CSF enhances HIV-1 replication in macrophages.6,7,20,21 Other studies have reported that GM-CSF actually inhibits HIV-1 replication,22-25 whereas still others have reported no effect or mixed effects of GM-CSF on HIV-1 replication in macrophages.26,27 Researchers who commented on these discrepancies have speculated that conflicting results might be attributable to the use of different HIV-1 strains,23 different cells,23,24 different timings of GM-CSF addition in relation to viral infection,24 different GM-CSF concentrations,21 and different culture conditions.21,23 Kedzierska et al25 obtained equivalent results using MDMs from different donors, different HIV-1 strains, different GM-CSF concentrations, and different times of GM-CSF addition in relation to HIV-1 infection. This study is notable in that the investigators reported that GM-CSF significantly inhibited HIV-1 replication in MDMs from 26 of 30 donors (with statistically insignificant effects on MDMs from the other 4 donors). The explanation for different results from different studies has thus continued to be a mystery.
In reviewing this literature, we hypothesized that different groups may have obtained different results because GM-CSF has opposing effects on the viral life cycle parameters. For example, GM-CSF may decrease the ability of HIV-1 to get into macrophages by downregulating CCR5 expression,25 although increasing the rate at which infected macrophages release virions. We reasoned that if such a trade-off was occurring, GM-CSF could have different effects on viral replication depending on variables such as the inherent susceptibility of macrophages, virion density, and macrophage density. An initial set of these experiments in which some of these factors were varied did not support this “trade-off” model, however. Instead, we were struck by observations that the addition of GM-CSF can cause MDMs to degrade the medium rapidly (as evidenced by decreasing pH) unless the culture medium is replenished frequently. This prompted us to study the relation between cell density, medium pH, and viral replication (as measured by p24 production) systematically, using MDMs from different donors.
Isolation and Culturing of Monocyte-Derived Macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of HIV-1, HIV-2, and hepatitis B-seronegative anonymous donors (supplied by the American Red Cross Blood Services Pacific Northwest Region, Portland, OR) by Ficoll density gradient centrifugation (Lymphocyte Separation Medium, Cappel 50,494; MP Biomedicals, Aurora, OH). The PBMCs were resuspended in MDM medium: Iscove modified Dulbecco medium (IMDM; MediaTech, Herndon, VA) supplemented with 10% heat-inactivated human AB+ pooled serum (Irvine Scientific, Santa Ana, CA), 2 mM of l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Human monocytes were isolated from the PBMCs by plastic adherence in Primaria (BD Biosciences, Bedford, MA) flasks. The PBMCs were incubated for 2 hours at 37°C, nonadherent cells were removed, and adherent cells were cultured overnight (37°C, 5% CO2) in MDM medium. Nonadherent cells from the overnight incubation were removed, and adherent cells were detached on ice using cold phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid (EDTA, 0.5 mM). The detached adherent cells (monocytes or MDMs) were resuspended in MDM medium and plated onto 96-well or 24-well plates at 1.5 to 3 × 105 cells/cm2 (5-10 × 104 cells per well in 96-well plates) unless otherwise noted. Final monocyte preparations were >95% viable by trypan blue exclusion, >95% monocytes by morphology (Wright-Giemsa stain), and >90% CD14+ by immunofluorescent staining with anti-CD14 monoclonal antibody (Becton-Dickinson, Mountain View, CA) and flow cytometric analysis. Cultures were monitored daily for medium color and cell status. Except when otherwise noted, medium was replenished every 2 to 3 days. MDMs were cultured for 6 days to allow time for cell differentiation before infection with HIV-1. Culture reagents and culture supernatants were routinely screened and found to be endotoxin-free (<0.1 endotoxin units (EU)/mL, except for neat human AB+ serum, which was <5 EU/mL) by Limulus Amebocyte Lysate assay (LAL; Cambrex Bio Science, Walkersville, MD) and mycoplasma-free (MycoProbe Mycoplasma Detection Kit; R&D Systems, Minneapolis, MN).
Granulocyte-Monocyte Colony-Stimulating Factor Treatment
Freshly plated monocytes were cultured in the presence or absence of recombinant human GM-CSF obtained from PeproTech (Rocky Hill, NJ). Two additional sources of GM-CSF (R&D Systems and Biosource International, Camarillo, CA) were evaluated and found to have equivalent effects to the PeproTech GM-CSF used in all the experiments discussed in this article (data not shown). In our initial sets of experiments, MDMs were subject to 3 regimens of GM-CSF treatment or to no-GM-CSF controls. In our “continuous” treatments, GM-CSF was added at the time of transfer of monocytes to 96-well plates and was continued after HIV-1 infection until the end of the experiment. In “preinfection” treatments, GM-CSF was added to culture medium at time of the transfer of monocytes to 96-well plates and was discontinued at the time of viral infection. In “postinfection” treatments, GM-CSF was added to culture medium after the final wash phase during HIV-1 infection and was continued until the end of the experiment. In later experiments, however, we dropped the “postinfection” treatments after we observed few differences between postinfection treatment and controls in which no GM-CSF was added.
HIV-1 Virus Stock
HIV-1 isolates were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). HIV-1Ba-L4 and HIV-189.615 virus stocks were prepared in PBMCs as follows: PBMCs were stimulated for 72 hours with phytohemagglutinin (PHA; Murex HA16; Remel, Lenexa, KS), washed, and resuspended in Iscove medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO, Grand Island, NY) and 10 U/mL of interleukin (IL)-2 (Invitrogen, Carlsbad, CA). Cells were incubated with virus for 2 hours in a shaking water bath at 37°C and were then washed free of inoculum and resuspended to 106 cells/mL in Iscove medium supplemented with 10% heat-inactivated fetal calf serum and 10 U/mL of IL-2. Cultures were monitored for virus production using a p24 enzyme-linked immunosorbent assay (ELISA), and virus was harvested 7 to 14 days after infection when virus production was at its peak. The cell-free culture supernatants were passed through a 0.22-μm filter (Corning, Corning, NY), stored in 1.5-mL aliquots at −80°C, and thawed immediately before use. All stocks were evaluated for a 50% tissue culture infectious dose (TCID50)/mL28 and p24 (ng/mL). Unless otherwise stated, all results are with HIV-1Ba-L. In early experiments (described in the legend for Fig. 1A), we obtained similar results with HIV-1Ba-L and HIV-189.6. Because HIV-1Ba-L (CCR5 utilizing and highly monocytotropic) grows faster and to higher levels in most donors' MDMs, however, we focused our subsequent studies on HIV-1Ba-L. PHA-stimulated PBMCs of all cell donors in these studies were susceptible to HIV-1Ba-L, indicating that none of our cell donors were homozygous for a null CCR5 allele (Δ32). The HIV-1Ba-L stock used in these experiments contained 5 × 104 TCID50/mL on MDMs and 300 ng/mL of p24.
HIV-1 Infection of Monocyte-Derived Macrophages
MDMs cultured for 6 days in the presence or absence of GM-CSF were rinsed free of any residual nonadherent cells and infected overnight (∼16 hours) with 500 TCID50 units of HIV-1Ba-L (unless stated otherwise). Cells were rinsed 3 times with PBS to remove residual free virus and then fed with fresh MDM medium with or without GM-CSF (10 ng/mL unless stated otherwise). Cultures were observed daily for medium color and cell status. Medium was replenished every 2 to 3 days or as needed (unless stated otherwise). Culture supernatants were sampled before medium exchanges, and virus was quantified by a p24 ELISA.
Quantification of HIV-1 Replication
HIV-1 virus production was monitored by measuring the p24 in culture supernatants using an in-house double-antibody sandwich ELISA specific for HIV-1 p24. A standard curve was generated for each assay using a p24 standard obtained from the AIDS Vaccine Program at the National Cancer Institute (NCI)-Frederick Cancer Research and Development Center (Frederick, MD). We verified that p24 counts were not affected by substances in acidified medium by comparing p24 ELISA values from samples in which we spiked purified HIV-1 p24 into supernatants from spent acidified HIV-negative cultures or fresh medium.
Methods for Imaging
Cells were monitored for morphology, density, confluence, and measures of cell health by visual examination under ×10 to ×40 phase-contrast objectives on a Nikon Diaphot (Nikon, Melville, NY) or Zeiss Axiovert 200 microscope (Carl Zeiss Inc., Thornwood, NJ). Cell numbers and viability before plating were assessed by microscopic observation using a hemacytometer and trypan blue exclusion. Adherent cell numbers were determined by counting the number of cells in random 100-μm squared sections of the well surface as enlarged and photographed through the microscope using a ×10 objective and a Zeiss AxioCam MRm camera (Zeiss).
Determination of pH
The pH of the medium and culture supernatants was measured using an Orion Research Model 601A pH meter (Thermo Fisher Scientific, Waltham, MA) or Whatman type CS pH 6.0 to 8.1 pH indicator papers (Whatman International, Maidstone, England).
Effects of Granulocyte-Monocyte Colony-Stimulating Factor Concentration and Time of Addition of Granulocyte-Monocyte Colony-Stimulating Factor on HIV-1 Replication
In our first experiment, we treated cells from 6 donors with increasing doses of GM-CSF. GM-CSF enhanced HIV-1 replication in 5 of the 6 donors, and this occurred in a dose-dependent manner in 4 of the 5 responding donor MDM cultures in which medium remained within the optimal pH range of 6.5 to 7.2 (as indicated by yellow-orange to red-orange medium color) through the course of the experiment (see Fig. 1A; solid lines). In 1 of the 5 responding donors, viral production did not increase with the GM-CSF concentration. HIV-1 p24 dipped as GM-CSF was increased from 3 to 10 ng/mL (see Fig. 1A; dashed line); however, viral p24 was still higher at all 3 GM-CSF doses than in untreated controls. In the sixth donor, HIV-1 production declined with increasing GM-CSF concentration (see Fig. 1A; dotted line). In this case, the medium in the GM-CSF-treated wells precipitously turned lemon yellow (pH <6.2) between days 3 and 5 after infection, and the macrophages in these wells were more granulated than the no-GM-CSF controls. These cultures did not recover (no decrease in granules and no progressive medium acidification) during the days after medium replenishment. Adherent cell density and numbers decreased relative to the no-GM-CSF controls during the subsequent 7 to 10 days in culture. Therefore, we took the decreasing HIV-1 production to be an artifact of culture compromise by extreme medium acidification to pH <6.2.
We next determined the effect of time of addition and duration of GM-CSF on viral replication in cells from 7 donors. Viral replication was measured in cultures in which MDMs were (1) not treated with GM-CSF, (2) pretreated with GM-CSF (10 ng/mL) during the 6-day culture period before infection with HIV-1 (“preinfection treatment”), (3) treated with GM-CSF after infection with HIV-1 (“postinfection treatment”), and (4) treated with GM-CSF before and after infection with HIV-1 (“continuous treatment”). In 5 of the 7 donors, acidification of the MDM culture medium to less than pH 6.5 did not occur in any cultures and GM-CSF pretreatment or continuous treatment enhanced HIV-1Ba-L replication by comparable amounts in these donors. Postinfection treatment with GM-CSF, by contrast, was virtually indistinguishable from no treatment (see Fig. 1B). In 2 of the 7 donors, we observed a precipitous pH drop to less than pH 6.3 between days 10 and 12 in cultures treated with GM-CSF before infection. We observed lower p24 levels in the same GM-CSF-treated MDM cultures (data not shown).
Acidification of Medium to Less Than pH 6.3 Correlates With Granulocyte-Monocyte Colony-Stimulating Factor-Mediated Reduction in Viral Replication
In initial experiments involving 16 MDM donors, including the 13 described previously and 3 from other experiments (data not shown), we observed that GM-CSF enhanced HIV-1 replication in 10 donors and suppressed replication in 3. (Virus did not replicate to detectable levels in 2 MDM donors, and the cells failed to remain adherent in 1 donor). Most MDM cultures seeded at approximately 1.5 to 3 × 105 cells/cm2, progressively acidify the medium beginning on approximately day 5 in culture from a starting pH of 7.2 to between pH 6.8 and pH 6.5 over the course of the next 3 to 4 days (medium color changes from red orange to yellow orange) without any apparent cell crisis or effect on HIV expression. By comparison, the pH change in GM-CSF-treated MDM cultures from some donors is often markedly accelerated, with the pH dropping 0.5 to 1.0 per unit in <24 hours (medium color changes from red orange to yellow). We also noted this pattern of precipitous medium acidification around days 8 through 12 in cultures from donors in which the MDMs were >95% confluent by days 3 to 4 in culture, whether the cultures were treated with GM-CSF or not. As determined by microscopic observation and counting, GM-CSF treatment of monocytes did not increase the number of surviving adherent cells in most donor MDMs cultured in medium containing human sera.
This prompted us to review the available literature on GM-CSF effects on HIV-1 replication in MDMs carefully. We found no references to this acidification issue in any of the studies reporting that GM-CSF inhibits HIV-1 replication. We noticed that 2 research groups24,25 reporting GM-CSF suppression used cell densities >5 × 105 monocytes/cm2, however, compared with 1.5 to 3 × 105 monocytes/cm2 in our experiments. Furthermore, in these 2 studies, the medium (as best as we could tell from their methods) was not changed as often as our every 2- to 3-day schedule. This suggested that the apparent inhibition by GM-CSF in these studies could have been an artifact of excessive medium acidification attributable to high cell density combined with infrequent medium exchanges.
To explore the role of medium replenishment and acidification, we divided GM-CSF and control wells into “low-maintenance” wells in which 50% of the medium was replaced on day 5 (as done in the study by Kedzierska et al25) and “high-maintenance” wells in which >90% of the medium was replenished as often as needed (every 2-3 days) to prevent the medium from turning yellow. MDMs were seeded at 5.3 × 105 monocytes/cm2 (1 × 106 cells per well of a 24-well plate) and infected after 6 days in culture with 5000 TCID50 units of HIV-1Ba-L per well. By day 3 after infection, the medium in the low-maintenance GM-CSF-treated wells was already yellow orange (pH <6.5), and by day 5, the medium was extremely acidic (pH <6.1). These yellowed cultures contained many granulated cells; failed to recover after medium replenishment; and became highly vacuolated, with approximately 50% of the cells dead and detached by the end of the experiment on day 12. The medium in the low-maintenance untreated group and the high-maintenance GM-CSF-treated groups was orange yellow (pH ∼6.6) on day 5 after infection, whereas the medium in high-maintenance untreated wells was orange (pH ∼7.0). Consistent with our medium acidification hypothesis, GM-CSF increased viral production in the high-maintenance cultures but reduced viral production in the low-maintenance cultures (Fig. 2).
As a further test of our medium acidification hypothesis, we performed experiments on MDMs from 12 donors in which the number of monocytes seeded per well in a 96-well plate was intentionally varied (Table 1). In these cultures, >90% of the culture medium was replenished every 2 to 3 days. For cells grown at the lowest density, GM-CSF enhanced viral replication in 10 of 11 donor cells in which viral replication could be detected. On cells grown at the highest density, GM-CSF suppressed viral replication in 8 of 12 donors (P < 0.01 compared with the lowest density cultures by the Fisher exact test). Consistent with our earlier observations, pH fell to less than 6.1 (despite active culture maintenance every 2-3 days) in all the 8 high-density cultures in which GM-CSF suppressed viral replication. Representative graphs demonstrating the relation between cell density and the effect of GM-CSF on viral replication are shown in Figure 3.
The effect of GM-CSF on HIV-1 replication in macrophages has been controversial. Some groups have reported that this cytokine increases viral replication, whereas others have reported that it decreases viral replication. We demonstrate here that treatment of MDMs with GM-CSF during the first week of culture almost always enhances HIV-1 replication when cells are grown at low density. GM-CSF-mediated enhancement also occurs when MDMs are cultured at higher densities if the culture medium is replenished frequently enough to prevent cells from acidifying the culture medium. The only cultures in which GM-CSF reduced viral replication were cultures in which addition of GM-CSF accelerated acidification of the medium. The absence of any decrease in the infectivity of cell-free virus exposed to the pH 6 to 6.3 condition in acidified MDM cultures29 (our data not shown) suggests that acidification is harmful to cells, as opposed to virus. Our observation that cells left in acidified (and probably nutrient-depleted) conditions exhibit signs of stress such as granulation and, in extreme cases, premature detachment supports the hypothesis that low pH is deleterious to cells and to cells as viral hosts.
Our results thus contradict those of previous reports23-25 that GM-CSF inhibits HIV-1 replication on MDMs. Given the high cell densities or infrequent (or unspecified) feeding schedules in the articles by Di Marzio et al24 and Kedzierska et al,25 we suggest that medium depletion or acidification could have contributed to apparent inhibition in these 2 studies. Our conclusions are also inconsistent with those of Matsuda et al23 and Hammer et al.22 These latter studies differ from our study in important ways, however. Although Matsuda et al23 cultured their cells at low density, they compared GM-CSF-treated MDMs with M-CSF-treated MDMs. In the absence of a “no-cytokine” control, it is hard to assert with confidence that GM-CSF is inhibitory (as opposed to M-CSF being a better enhancer). This difference in design is likely explained by the fact that Matsuda et al23 cultured their cells in medium supplemented with fetal calf serum. We and others30 have observed that MDMs survive (remain attached and differentiate) in medium supplemented with 5% to 15% human serum but often do not survive in medium supplemented with 5% to 15% fetal calf serum unless M-CSF, GM-CSF, or human serum is added to the cultures. Our conclusions are also inconsistent with those of Hammer et al,22 although this comparison is even less straightforward, because they performed their studies with the U937 monocyte line, whose HIV-1 infection characteristics more closely resemble those of CD4+ T-cell lines.31
Our studies point to the following 4 factors that make deleterious pH drops easy to overlook:
1. The precipitous drops in pH needed to suppress HIV-1 replication (pH <6.2) do not usually occur during the first week of culture. Rapid overacidification of the culture medium occurs beginning approximately 8 to 12 days after cell plating, which is typically 2 to 6 days after HIV infection and GM-CSF withdrawal (in the case of pretreatment). When the pH does drop, however, it drops suddenly (eg, from 6.6 to <6.2 within a 16-hour period).
2. Transient drops in pH to <6.2 have irreversible effects on the ability of MDMs to support HIV replication. We have found that short periods (<24 hours) of sitting at <pH 6.2 are sufficient to suppress HIV replication in MDM cultures even after the medium is replenished. Once this has occured, the cells in these cultures do not continue to acidify the replenished medium rapidly; in extreme cases, they fail to acidify the medium at all (consistent with extreme acidification permanently damaging the cells).
3. Abrupt changes in pH occur only when monocytes are plated at higher cell densities.
4. Monocytes or MDMs from different donors vary significantly in their susceptibility to HIV-1 and in their culture characteristics, such as adherence, survival, differentiation, and morphology, as well as the rate at which they acidify culture medium. In some donors, replenishing the medium every 2 to 3 days was not sufficient to keep the pH within the optimal range of 6.8 ± 0.3. In these donors, it may be necessary to replenish culture medium every day. Such heterogeneities make this a difficult phenomenon to study.
Our results are consistent with the long-established role that GM-CSF has in promoting cell proliferation, survival, differentiation, and effector functions for cells in the macrophage and granulocyte lineages.32,33 GM-CSF engagement of its target receptor alters gene expression in cells by way of several pathways, including the JAK-STAT signaling cascade.34-39 Our observations that GM-CSF enhanced HIV-1 replication only if cells were treated before infection during the first 5 to 7 days in culture and that GM-CSF withdrawal after cell maturation had no effect reiterate the role that GM-CSF plays in monocyte differentiation. These time-related differences in GM-CSF effects may be related to changes in STAT5 activation and expression as monocytes differentiate into macrophage.40,41 HIV-1 inhibition of GM-CSF-induced STAT5A activation42 may further contribute to the lack of effect of GM-CSF on HIV-1 replication when GM-CSF is added after macrophage maturation and HIV infection. Our observation that GM-CSF-treated and control cultures contained comparable numbers of cells in most of our cultures suggests that enhanced HIV-1 replication and medium acidification are related to GM-CSF-induced increases in metabolic rate43 or changes in the activation state of the cells (or some subset of cells in the culture44). Although we cannot exclude the possibility that GM-CSF-differentiated macrophages release other soluble substances that modulate HIV-1 replication, accelerated differentiation and activation (and accompanying acidification) is the most parsimonious explanation for our results.
In this study, we have not investigated molecular mechanisms that cause activated or differentiated MDMs to be more permissive for viral replication. Recent work by Chiu et al45 and Stopak et al46 demonstrates a role for APOBEC3G, and future studies may benefit from further investigation of APOBEC3G (and JAK-STAT pathways). Studies of molecular changes can be complicated by variation in culture conditions and use of cells from different sources, however. The susceptibility of MDMs to HIV-1 infection and subsequent replication in vitro varies tremendously depending on the cell donor, cell differentiation status, and culture conditions such as vessel surface or serum additives.47-52 Our observations suggest that cell density and donor-specific variation in the ability of MDMs to acidify medium also contribute to this variability. Cell sources, serum sources, and culturing techniques thus need to be carefully controlled before one can optimally investigate molecular changes induced by GM-CSF and other cytokines. Descriptions of macrophage culturing techniques should include information on the frequency of feeding, the amount of medium replaced during each feeding, medium volume and pH (or medium color), and indicators of cell viability.
The authors thank Victor Swain and Ushnal Rao for excellent technical assistance in the laboratory.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
cell density; cytokines; granulocyte-monocyte colony-stimulating factor; HIV-1; macrophage; pH