American Journal of Pathology, Vol. 139, No. 4, October 1991 Copyright © American Association of Pathologist
Cytokine Influence on Simian Immunodeficiency Virus Replication Within Primary Macrophages TNF-ot, But Not GMCSF, Enhances Viral Replication on a Per-cell Basis Dennis G. Walsh, Christopher J. Horvath, Adel Hansen-Moosa, John J. MacKey, Prahbat K. Sehgal, Muthiah D. Daniel, Ronald C. Desrosiers, and Douglas J. Ringler From the Harvard Medical School, Departments of Pathology and Microbiology, New England Regional Primate Research Center, Southborough, Massachusetts
The control of HIV-1 or SIV replication within mac-
rophages is probably influenced by a variety of viral and cellular factors. Of the cellular factors; the authors have studied cytokine influence on SIV replication in vitro utilizing simian alveolar macrophages and uncloned SIVmacMTV, a macrophage-tropic variant The approach allowed quantification of viral replication on a per-cell basis. As reported for HIV-1 replication in macrophages, TNF-o significantly increased SlVproduction in these macrophage cultures. GMCSF also resulted in marked increases in SV gag protein in culture supernatants. However, after correcting for differences in total cell numbers and numbers of gag-containing cells in the treated and untreated cultures, GMCSF did not upregulate SlVproduction on a per-cell basis. IL-6 increased SlV replication little if at all but induced significantly greater cytopathic changes in the treated cultures compared with infecte4 untreated cultures In contrast, IFN-,y greatly decreased replication Our results for GMCSF, IFN-,y, and IL-6 are in contrast to previously published reports of cytokine control of HIV-1 growth in target cells, and they stress the importance of cell number analyses and the use of primary cultures in the study of lentiviral replication kinetics in
macrophages (Am JPathol 1991, 139:877-887)
Similar to other animal lentiviruses, HIV-1, the causative agent of AIDS, infects tissue macrophages or histogenetically related cell types. Therefore, follicular dendritic cells in lymph nodes,1'2 Langerhans cells in skin,3 macro-
phages in brain and spinal cord,' alveolar macrophages,7 and monocytes-10 have all been shown to be sites of HIV-1 replication in vivo. It has been shown that certain endogenous and exogenous factors can influence the regulation of HIV-1 in specific target cells.'1-14 Since tissue macrophages are abundant and represent a major target cell, agents that affect the efficiency of lentiviral replication in macrophages will probably influence the pathogenesis of disease in patients with AIDS. Control of replication of HIV-1 within macrophages is probably influenced by virally encoded as well as cellular factors. Of the cellular factors, cytokines have received significant attention because of their instrumental roles in normal immune function. As cytokines are involved in all arms of the immune response and can be localized to specific compartments in lymph nodes from patients infected with HIV-1,15 their ability to modulate virus expression is particularly noteworthy. A number of studies have shown that specific cytokines can upregulate viral expression in infected cells.121316-l18 However, we and others have noticed that certain cytokines can have a significant effect on viability and replicative potential of macrophages in vitro.9 19 These effects result in significant differences in cell number in these macrophage cultures over time. Therefore, studies that quantify virus in culture supernatants from macrophages exposed to exogenously added cytokines may not accurately reflect the effect of the agent on viral replication on a per-cell basis. In this study, we have taken into account cell numbers in quantifying simian immunodeficiency virus (SIV) production in primary rhesus monkey alveolar macrophage cultures. We have previously demonstrated that SIV has genetic, pathogenetic, and biologic properties in comSupported by Public Health Service grants A129855, A125644, and RR00168 from the National Institutes of Health. Accepted for publication June 5, 1991. Address reprint requests to Dr. Douglas J. Ringler, New England Regional Prmate Research Center, Box 9102, 1 Pine Hill Dr., Southborough, MA 01 772-9102.
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mon with HIV-1 (reviewed in 20). We find that on a per-cell basis, TNF-a increases viral replication, IFN--y decreases viral replication, and GMCSF and IL-6 have little or no effect. However, IL-6 induces profound cytopathic changes in these infected cultures.
Materials and Methods Propagation and Infection of Alveolar Macrophages Alveolar macrophages were obtained from normal rhesus macaques using bronchoalveolar lavage, the methods for which have been previously described.21 Cytologic examination of each lavage sample was performed before experimentation, and only normal lavage samples consisting of semipure populations of alveolar macrophages were used in these studies, as characterized previously.21 Animals for lavage were serologically negative for SIV, type D retroviruses, and simian foamy viruses. They were kept in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on the Care and Use of Animals of the Institute of Laboratory Animals Resources, National Research Council. Alveolar macrophages were maintained in vitro as we have described previously.2'22 Briefly, they were cultured in RPMI 1640 medium supplemented with 100 U/mI of penicillin, 100 ,ug/ml of streptomycin, 0.25 ,ug/ml of amphotericin B, 2.0 mM glutamine, 25 mM HEPES buffer, 2.5% HIV-negative human AB serum, and 10% fetal calf serum (Gibco Laboratories, Grand Island, NY). The final concentration of bacterial endotoxin (LPS) in the medium from the fetal calf serum for these experiments was < 6.0 pg/ml (Gibco Laboratories). Macrophages were cultured at a concentration of 0.5 x 1 06/ml in 25 cm2 plastic flasks (Nunc, Inc., Naperville, IL) for 3 days in the presence or absence of cytokines in an environment containing 5% CO2 at 37°C. On the third day, the cultures were infected with uncloned SlVmacMTV, a macrophage-tropic isolate of SIVmac251 that we have previously described.21'22 The cultures were infected by adding 2 x 103 TCID5Q of virus per 2 x 1 QS macrophages for 12 hours, after which the cells were scraped from the bottom of the flasks with a cell scraper (Nunc Inc.), washed in Hank's Balanced Salt Solution (HBSS)(Gibco Laboratories), resuspended in medium in the presence or absence of cytokine, and plated in 96-well flat-bottomed tissue culture plates (Becton Dickinson, Lincoln Park, NJ). Each well ultimately contained 1 x 105 macrophages in 200 ,ul of medium. Each variable (presence or absence of cytokines) was performed repeatedly in 3 to 11 wells. The media was changed in each well twice weekly by centrifugation of
the plates in a Sorvall Centrifuge to pellet nonadherent macrophages, carefully removing three-quarters of the supernatant, and replacing with fresh media. The supernatant from each well from an entire plate was harvested twice weekly and stored at - 800C for determination of SIV gag concentration. The desiccated plate was then wrapped in aluminum foil and kept at -200C for later determination of cell number and for immunohistochemical analyses. Because we have observed significant differences in the ability of individual rhesus monkeys' alveolar macrophages to support SIV replication to high titer in vitro, our data from cytokine-treated wells were, in all cases, compared with untreated wells that contained cells harvested simultaneously from the same animal.
Cell Number Analyses The number of cells from each well was calculated using a modified technique originally described by Remick et al.23 Briefly, the wells in the plate were fixed by adding 50 RI1 of 100% methanol per well for 15 minutes at room temperature. Subsequently, the methanol was removed and replaced with 50 p.1 of crystal violet (0.2% in 20% methanol) for 15 minutes at room temperature. The wells were then rinsed three times with distilled water using a Nunc microtiter plate washer. The plate was dried, and the absorbance at 630 nm was determined using a Dynatech plate reader. The number of cells was calculated from a standard curve generated with eight replicate wells that contained serial dilutions of rhesus alveolar macrophages and the same staining technique so that:
OD6w - 0.014 Cell Number = 8.43 x 10-6
SIV gag Concentration The concentration of SIV gag in the supernatants from the wells was determined using a commercially available solid-phase immunoassay (Coulter Corporation, Hialeah, FL). The cellfree supernatant from the wells was analyzed according to the directions from the manufacturer in triplicate using dilutions of 1:3, 1:10, and 1:100. The limit of detection of gag protein was determined for each assay and approximated 3.5 ng/ml in undiluted samples. Therefore, the limit of detection of supematants diluted 1:3 in this report was approximately 10.0 to 11.0 ng/ml. Supematants containing less than or equal to the limit of detection were arbitrarily assigned the value of the limit of detection for use in the data analyses.
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Immunohistochemistry After cell-number analysis, selected wells were used in an immunohistochemical procedure to determine the relative number of gag-containing cells in each well. This procedure was performed using techniques previously described.22.2F27 Briefly, the wells were rehydrated with 0.15 M phosphate-buffered saline (PBS) (pH 7.2) containing 0.2% gelatin. SIV gag protein was localized to macrophages in the wells by using an avidin-horseradish peroxidase reagent (ABC-Per)(Vector Laboratories, Burlingame, CA) and R1C7, a monoclonal antibody to SIV gag protein that has been described elsewhere.24 28 The cells were incubated overnight at 4°C with the monoclonal antibody, followed by 30-minute incubations of a biotinylated anti-mouse IgGl reagent (Vector Laboratories) and ABC-Per. These procedures removed any residual crystal violet staining that remained from the cell-number analysis. The immunohistochemical procedure was completed with the use of the chromogen diaminobenzidine (DAB), hydrogen peroxide, and sodium azide as we have described previously.27 Irrelevant mouse IgGl was used in one of the wells from each group as a negative control. Mayer's hematoxylin was used as a nuclear counterstain. The relative proportion of SIV gagcontaining cells in a group of wells was then determined by microscopy. In selected experiments, the mean number of gag + macrophages per well in a particular group was determined by multiplying the percentage of immunoreactive cells in a well by the total cell number/well.
Computation of SIV gag Production per Cell SIV gag concentrations in the supernatants were adjusted for the number of cells in the wells. The gag concentration from a particular well was divided by the average number of cells in the well over the time period between two successive harvest dates. The average cell number/well over the period was calculated as one half of the sum of the cell number/well at the time of harvest to the mean of the cell number/well within the same treatment group from the previous harvest.
and subsequently examined with a JEOL 1 00S transmission electron microscope.
Statistical Analyses Data were expressed as the mean of 3 to 11 samples (wells) ± 1 SEM. A t-test for differences between two means was used at a particular time point, and P values < 0.05 were considered significant.
Results TNF-a Upregulates SIV Replication in Alveolar Macrophages When simian alveolar macrophages were exposed to concentrations of human recombinant TNF-a (Sigma Chemical Co., St. Louis, MO) from 1.5 to 5.0 ng/ml (at 2.2 U/ng), there was no difference in cell numbers between treated and untreated cultures (Figure 1 A). However, TNF-a concentrations < 5.0 ng/ml elicited up to three to four times both the concentration of gag protein in the supernatant and amount of gag protein per cell than did cultures without TNF-a (Figure 1 B). The amount of LPS in the TNF-a preparation was < 120 pg LPS/,Lg of TNF-a. Therefore, the final concentration of LPS due to the TNF-a in the 2.5 ng/ml TNF-a medium was < 0.3 pg/ml. Similar levels of upregulation were observed at 2.5 and 1.5 ng/ml of TNF-a. This replication enhancement was observed whether TNF-a was added 3 days before or at the time of infection. Treatment with TNF-a did not affect time of peak viral production. Culture supernatants containing 5.0 ng/ ml of TNF-a did not have increased quantities of SIV gag protein compared with untreated cells, but the cells stimulated with this high dose of TNF-ot morphologically were granular and small. These results are consistent with the findings of other investigators who have demonstrated an upregulatory influence of exogenous TNF-a on HIV-1 replication in cultures of lymphoid cells or promonocytic cell lines or when used therapeutically in HIV-1 -infected
patients.'1 116,18,30,31
Interferon-y (IFN-y) Retards SIV Replication Electron Microscopy
in Macrophages
Alveolar macrophages infected with SIV were scraped from the bottom of flat-bottomed six-well plates and initially fixed in half-strength Karnovsky's fixative,29 washed in phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Ultrathin sections were cut and stained with uranyl acetate and lead citrate
It was previously reported12 that IFN-y upregulates HIV-1 replication in primary cultures of monocyte-derived macrophages if added 3 days before infection. For this reason, we compared the potential of IFN--y to influence the replicative potential of SIV in macrophages. Human recombinant IFN-y (Chemicon Co., Temecula, CA) was
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AJP Ocober 15991, Vol. 139, No. 4
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added to cultures at 300 U/ml 3 days before infection and continuously thereafter. The number of macrophages in the IFN-y-treated cultures was significantly greater than in the untreated wells (Figure 2A). The appearance of SIV gag in the supematants from these treated cultures was delayed by approximately 3 days, after which the concentration was either similar to or less than that seen in the untreated cultures (Figure 28). Therefore, when corrected for differences in cell number/well between the treated versus untreated cultures, the amounts of gag protein per cell in the cultures stimulated with IFN-y were significantly lower during either the entire length of culture in some experiments or, in others, at least until the last 4 to 5 days of the culture (Figure 2C).
5
Figure 1. Effects of TNF-a (1.5 ng/ml) on simian alveolar macrophage cultures infected with SIV Values represent means + 1 SEM (n = 3 to 6). A: Number of macrophages/well at time of plate harvest with (-0-) and without (-4*-) TNF-a. B: Relative amounts of SIV gag on a per cell basis for cultures with (-0-) and without (-*--) TNF-a. Superscipts at the time points indicate that the values are significantly different from the untreated control group. *P < 0.001, +P < 0.01.
'SF Does Not Upregulate SIV cation in Macrophages on a Per-cell been reported by a number of investigators that ,F upregulates HIV-1 replication in primary cultures man monocyte-derived macrophages and nocytic cell lines.12'13'17 However, we noticed that i recombinant GMCSF from two different sources tics Institute, Boston, MA and Amgen Biologicals, and Oaks, CA), when added to simian macro> cultures, significantly increased the number of phages in the cultures compared with the unJ cultures (Figure 3A). Therefore, quantification of
SIV Growth in Macrophages
881
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Figure 2. Effects of IFN-y (300 U/ml) on simian alveolar macrophage cultures infected with SI. Values represent means + 1 SEM (n = 3 to 8). SV-infected macrophages treated with IFN-y (-0--) are compared with untreated macrophages (-*-) in the same esperiment There are significantly more cells in the treated cultures (A); gag protein production in the supernatant is delayed by three days in these treated cultures (B), but gag production per cell is reduced in the treated culturesfor up to 17 days after inoculation (C). Superscripts at the time points indicate that the values are significantly different from the untreated control groups. *P < 0.001,
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virus in supematants from these cultures may not realistically reflect changes in the amount of virus produced by each infected cell. Accordingly, we tested the effects of GMCSF (5-100 U/ml) from both sources by adding it to simian macrophage cultures 3 days before infection with SIV and continuously thereafter. The amount of LPS in the
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Days after inoculation Figure 3. Effects of GMCSF (20 U/ml) on simian alveolar macrophage cultures infected with SIV Values represent means ± 1 SEM (n = 3 to 8). SNV-infected macrophages treated with GMCSF (-0-) are compared with untreated maacophages (-*--) in the same experiment. Macrophage numbers are markedly increased in GMCSF-treated cultures (A), and gag protein concentration in the superntant is increased in these treated cultures (B); however, gag production per cell ts similar in treated and untreated cultures (C). 7he difference between treated and untreated cultures at 21 days after inoculation (C) wAas not seen in all GMCSF experiments. Superscrpts at the time points indiate that the values are significantly different from the untreated control groups. *P < 0.001, 'P < 0.02, AP < 0.05.
GMCSF preparation from Amgen Biologicals was < 50 pg LPS/4 x 1 4 U of GMCSF. Accordingly, the final concentration of LPS from the 100 U/mI GMCSF preparation from Amgen Biologicals was < 0.125 pg/ml. Similarly, the
882 Walsh et al AJP October 1991, Vol. 139, No. 4
final concentration of LPS from the 100 U/ml preparation from Genetics Institute was < 0.05 pg/ml when tested using the Limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO). The amount of SIV gag protein in the supernatants from GMCSF-stimulated cultures at the time of peak production was approximately three to six times that from untreated cultures at all doses tested using both sources of GMCSF (Figure 3B). However, after adjusting for differences in cell number, GMCSF did not increase SIV virus production per cell at any of the doses tested (Figure 3C). The effect of GMCSF was not due to TNF-a release since neutralizing goat polyclonal antibodies to human TNF-a (R & D Systems, Minneapolis, MN), when added at three to five times the neutralizing dose50 (ND50), did not alter the amount of SIV gag protein in the supernatant or the overall kinetics at 20 U/ml of GMCSF (data not shown).
The Lack of Upregulation by GMCSF Is Not Accounted for by Reduced Numbers of Infected Macrophages Producing More Virus We next examined the effect of GMCSF on the numbers of gag-containing cells using immunohistochemistry. In the GMCSF-treated cultures, there were significantly more gag-containing cells than in the untreated cultures (Figure 4). Since GMCSF-treated cultures contained proportionally greater numbers of infected cells, the increased amount of gag protein detected in the supematants was not apparently due to increased production of virus from a limited number of infected cells.
IL-6 Induces Significantly Greater Cytopathic Changes in S/V-infected Macrophages Because it has been reported that IL-6 increases HIV-1 expression in human monocyte-derived macrophages,13 we studied the effect of IL-6 on SIV replication in simian macrophages. We added human recombinant IL-6 (Gibco Laboratories) at concentrations ranging from 3.1 to 100.0 ng/ml to macrophage cultures 3 days before infection with SIV and continuously thereafter. The LPS concentration of the IL-6 was < 20 pg/,ug of IL-6. Therefore, the final concentration of LPS due to the IL-6 in the 100.0 ng/ml medium was < 2.0 pg/ml. Concentrations > 6.25 ng/ml of IL-6 induced a number of changes in infected cells. IL-6-treated cultures showed earlier and greatly increased numbers of syncytia than did untreated cultures (Figure 5A, B). This effect was not seen in IL-6treated cultures that were not infected with SIV. SIV gag protein was not found in the supernatant until 2 to 3 days after the onset of syncytia formation in the IL-6-treated cultures. Although there were frequently more cells in the treated cultures before the detection of gag in the culture media (data not shown), once SIV gag protein appeared in the supernatants, the numbers of macrophages in the treated cultures were either similar or slightly lower than in the untreated cultures (Figure 6A). This finding suggests that the cytopathic changes and subsequent cell death in the IL-6-treated cultures resulted in reduced cell numbers compared with untreated cultures. Despite the greatly enhanced cytopathic effects in the IL-6-treated cultures, the amount of SIV gag per cell was only slightly greater than or equal to that in the untreated cultures (Figure 6B).
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Figure 5. Photomicrographs ofsimian alveolar macrophage culturesfour days after inoculation with SIVand as described in Materials and Methods. A: Cultures without IL-6 do not have any syncytial cells. B: Cultures containing IL-6 (50 ng/ml) and infected under otherwise identical conditions have numerous syncytial cells. A, B, X330.
IL-6 Does Not Increase Intracytoplasmic Viral Assembly and Budding Because there were significantly greater cytopathic changes in the IL-6treated cultures than in the untreated cultures despite comparable replication profiles, we investigated the possibility that IL-6 may alter viral assembly so that intracellar budding into membrane-bound cytoplasmic vacuoles predominates over extracellar budding. If so, IL-6 may upregulate SIV replication in these cultures without quantitative differences being detected in the supernatants. We explored this possibility by examining SIV-infected alveolar macrophages ultrastructurally with and without the presence of IL-6 (50.0 ng/ml). Macrophages were harvested for electron microscopy at 12 days after infection when the supematant from IL-6treated and control cultures contained 209 and 127 ng/ ml of SIV gag protein, respectively. Although there were significantly more syncytia in the IL--treated cultures, there was no detectable difference in the numbers of cells containing budding particles or the numbers of budding particles in representative cells between the two samples. In both cases, budding was predominantly
found intracellularly, although budding from the plasma membrane could occasionally be found. Cultures harvested before 12 days after inoculation had multiple syncytia in the IL--treated flasks, but no mature virions or budding could be found in either the treated or untreated samples.
Discussion In this report, we have presented evidence that the cytokines TNF-a and IFN--y affect SIV replication within primary cultures of mononuclear phagocytes, whereas IL-6 and GMCSF do not have potent viral regulatory influences on a per-cell basis. Since certain cytokines have cellular and growth regulatory properties in vitro that can affect cell viability, cell number, and subsequent quantity of extracellular virus, this study underscores the importance of considenng cell numbers when quantitating virus production in cell culture. Recombinant TNF-a greatly increased SIV replication within primary cultures of simian macrophages. This finding is consistent with previous studies using HIV-1 that
Walsh et al
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AJP October 1991, Vol 139, No. 4
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demonstrated the upregulatory influence of TNF-a on acutely infected primary cultures of macrophages and chronically infected cell lines. 11,13,16,17 Inasmuch as some patients with AIDS have elevated circulating levels of TNF-a32 and SIV-infected rhesus monkeys often have alveolar macrophages that secrete exaggerated levels of TNF-a on endotoxin challenge,33 the involvement of this molecule in the inductive events leading from persistent low-level virus expression to clinical disease seems likely. Furthermore, it has been established that TNF-a stimulates HIV-1 enhancer sequences in the LTR by activation of nuclear factor KB (NF-KB).18'30 As the SIV LTRs also contain NF-KB-like sequences,3'435it is likely that SIV can be stimulated in a similar fashion.
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Cytokine Influence on Simian Immunodeficiency Virus Replication Within Primary Macrophages TNF-ot, But Not GMCSF, Enhances Viral Replication on a Per-cell Basis Dennis G. Walsh, Christopher J. Horvath, Adel Hansen-Moosa, John J. MacKey, Prahbat K. Sehgal, Muthiah D. Daniel, Ronald C. Desrosiers, and Douglas J. Ringler From the Harvard Medical School, Departments of Pathology and Microbiology, New England Regional Primate Research Center, Southborough, Massachusetts
The control of HIV-1 or SIV replication within mac-
rophages is probably influenced by a variety of viral and cellular factors. Of the cellular factors; the authors have studied cytokine influence on SIV replication in vitro utilizing simian alveolar macrophages and uncloned SIVmacMTV, a macrophage-tropic variant The approach allowed quantification of viral replication on a per-cell basis. As reported for HIV-1 replication in macrophages, TNF-o significantly increased SlVproduction in these macrophage cultures. GMCSF also resulted in marked increases in SV gag protein in culture supernatants. However, after correcting for differences in total cell numbers and numbers of gag-containing cells in the treated and untreated cultures, GMCSF did not upregulate SlVproduction on a per-cell basis. IL-6 increased SlV replication little if at all but induced significantly greater cytopathic changes in the treated cultures compared with infecte4 untreated cultures In contrast, IFN-,y greatly decreased replication Our results for GMCSF, IFN-,y, and IL-6 are in contrast to previously published reports of cytokine control of HIV-1 growth in target cells, and they stress the importance of cell number analyses and the use of primary cultures in the study of lentiviral replication kinetics in
macrophages (Am JPathol 1991, 139:877-887)
Similar to other animal lentiviruses, HIV-1, the causative agent of AIDS, infects tissue macrophages or histogenetically related cell types. Therefore, follicular dendritic cells in lymph nodes,1'2 Langerhans cells in skin,3 macro-
phages in brain and spinal cord,' alveolar macrophages,7 and monocytes-10 have all been shown to be sites of HIV-1 replication in vivo. It has been shown that certain endogenous and exogenous factors can influence the regulation of HIV-1 in specific target cells.'1-14 Since tissue macrophages are abundant and represent a major target cell, agents that affect the efficiency of lentiviral replication in macrophages will probably influence the pathogenesis of disease in patients with AIDS. Control of replication of HIV-1 within macrophages is probably influenced by virally encoded as well as cellular factors. Of the cellular factors, cytokines have received significant attention because of their instrumental roles in normal immune function. As cytokines are involved in all arms of the immune response and can be localized to specific compartments in lymph nodes from patients infected with HIV-1,15 their ability to modulate virus expression is particularly noteworthy. A number of studies have shown that specific cytokines can upregulate viral expression in infected cells.121316-l18 However, we and others have noticed that certain cytokines can have a significant effect on viability and replicative potential of macrophages in vitro.9 19 These effects result in significant differences in cell number in these macrophage cultures over time. Therefore, studies that quantify virus in culture supernatants from macrophages exposed to exogenously added cytokines may not accurately reflect the effect of the agent on viral replication on a per-cell basis. In this study, we have taken into account cell numbers in quantifying simian immunodeficiency virus (SIV) production in primary rhesus monkey alveolar macrophage cultures. We have previously demonstrated that SIV has genetic, pathogenetic, and biologic properties in comSupported by Public Health Service grants A129855, A125644, and RR00168 from the National Institutes of Health. Accepted for publication June 5, 1991. Address reprint requests to Dr. Douglas J. Ringler, New England Regional Prmate Research Center, Box 9102, 1 Pine Hill Dr., Southborough, MA 01 772-9102.
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mon with HIV-1 (reviewed in 20). We find that on a per-cell basis, TNF-a increases viral replication, IFN--y decreases viral replication, and GMCSF and IL-6 have little or no effect. However, IL-6 induces profound cytopathic changes in these infected cultures.
Materials and Methods Propagation and Infection of Alveolar Macrophages Alveolar macrophages were obtained from normal rhesus macaques using bronchoalveolar lavage, the methods for which have been previously described.21 Cytologic examination of each lavage sample was performed before experimentation, and only normal lavage samples consisting of semipure populations of alveolar macrophages were used in these studies, as characterized previously.21 Animals for lavage were serologically negative for SIV, type D retroviruses, and simian foamy viruses. They were kept in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on the Care and Use of Animals of the Institute of Laboratory Animals Resources, National Research Council. Alveolar macrophages were maintained in vitro as we have described previously.2'22 Briefly, they were cultured in RPMI 1640 medium supplemented with 100 U/mI of penicillin, 100 ,ug/ml of streptomycin, 0.25 ,ug/ml of amphotericin B, 2.0 mM glutamine, 25 mM HEPES buffer, 2.5% HIV-negative human AB serum, and 10% fetal calf serum (Gibco Laboratories, Grand Island, NY). The final concentration of bacterial endotoxin (LPS) in the medium from the fetal calf serum for these experiments was < 6.0 pg/ml (Gibco Laboratories). Macrophages were cultured at a concentration of 0.5 x 1 06/ml in 25 cm2 plastic flasks (Nunc, Inc., Naperville, IL) for 3 days in the presence or absence of cytokines in an environment containing 5% CO2 at 37°C. On the third day, the cultures were infected with uncloned SlVmacMTV, a macrophage-tropic isolate of SIVmac251 that we have previously described.21'22 The cultures were infected by adding 2 x 103 TCID5Q of virus per 2 x 1 QS macrophages for 12 hours, after which the cells were scraped from the bottom of the flasks with a cell scraper (Nunc Inc.), washed in Hank's Balanced Salt Solution (HBSS)(Gibco Laboratories), resuspended in medium in the presence or absence of cytokine, and plated in 96-well flat-bottomed tissue culture plates (Becton Dickinson, Lincoln Park, NJ). Each well ultimately contained 1 x 105 macrophages in 200 ,ul of medium. Each variable (presence or absence of cytokines) was performed repeatedly in 3 to 11 wells. The media was changed in each well twice weekly by centrifugation of
the plates in a Sorvall Centrifuge to pellet nonadherent macrophages, carefully removing three-quarters of the supernatant, and replacing with fresh media. The supernatant from each well from an entire plate was harvested twice weekly and stored at - 800C for determination of SIV gag concentration. The desiccated plate was then wrapped in aluminum foil and kept at -200C for later determination of cell number and for immunohistochemical analyses. Because we have observed significant differences in the ability of individual rhesus monkeys' alveolar macrophages to support SIV replication to high titer in vitro, our data from cytokine-treated wells were, in all cases, compared with untreated wells that contained cells harvested simultaneously from the same animal.
Cell Number Analyses The number of cells from each well was calculated using a modified technique originally described by Remick et al.23 Briefly, the wells in the plate were fixed by adding 50 RI1 of 100% methanol per well for 15 minutes at room temperature. Subsequently, the methanol was removed and replaced with 50 p.1 of crystal violet (0.2% in 20% methanol) for 15 minutes at room temperature. The wells were then rinsed three times with distilled water using a Nunc microtiter plate washer. The plate was dried, and the absorbance at 630 nm was determined using a Dynatech plate reader. The number of cells was calculated from a standard curve generated with eight replicate wells that contained serial dilutions of rhesus alveolar macrophages and the same staining technique so that:
OD6w - 0.014 Cell Number = 8.43 x 10-6
SIV gag Concentration The concentration of SIV gag in the supernatants from the wells was determined using a commercially available solid-phase immunoassay (Coulter Corporation, Hialeah, FL). The cellfree supernatant from the wells was analyzed according to the directions from the manufacturer in triplicate using dilutions of 1:3, 1:10, and 1:100. The limit of detection of gag protein was determined for each assay and approximated 3.5 ng/ml in undiluted samples. Therefore, the limit of detection of supematants diluted 1:3 in this report was approximately 10.0 to 11.0 ng/ml. Supematants containing less than or equal to the limit of detection were arbitrarily assigned the value of the limit of detection for use in the data analyses.
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Immunohistochemistry After cell-number analysis, selected wells were used in an immunohistochemical procedure to determine the relative number of gag-containing cells in each well. This procedure was performed using techniques previously described.22.2F27 Briefly, the wells were rehydrated with 0.15 M phosphate-buffered saline (PBS) (pH 7.2) containing 0.2% gelatin. SIV gag protein was localized to macrophages in the wells by using an avidin-horseradish peroxidase reagent (ABC-Per)(Vector Laboratories, Burlingame, CA) and R1C7, a monoclonal antibody to SIV gag protein that has been described elsewhere.24 28 The cells were incubated overnight at 4°C with the monoclonal antibody, followed by 30-minute incubations of a biotinylated anti-mouse IgGl reagent (Vector Laboratories) and ABC-Per. These procedures removed any residual crystal violet staining that remained from the cell-number analysis. The immunohistochemical procedure was completed with the use of the chromogen diaminobenzidine (DAB), hydrogen peroxide, and sodium azide as we have described previously.27 Irrelevant mouse IgGl was used in one of the wells from each group as a negative control. Mayer's hematoxylin was used as a nuclear counterstain. The relative proportion of SIV gagcontaining cells in a group of wells was then determined by microscopy. In selected experiments, the mean number of gag + macrophages per well in a particular group was determined by multiplying the percentage of immunoreactive cells in a well by the total cell number/well.
Computation of SIV gag Production per Cell SIV gag concentrations in the supernatants were adjusted for the number of cells in the wells. The gag concentration from a particular well was divided by the average number of cells in the well over the time period between two successive harvest dates. The average cell number/well over the period was calculated as one half of the sum of the cell number/well at the time of harvest to the mean of the cell number/well within the same treatment group from the previous harvest.
and subsequently examined with a JEOL 1 00S transmission electron microscope.
Statistical Analyses Data were expressed as the mean of 3 to 11 samples (wells) ± 1 SEM. A t-test for differences between two means was used at a particular time point, and P values < 0.05 were considered significant.
Results TNF-a Upregulates SIV Replication in Alveolar Macrophages When simian alveolar macrophages were exposed to concentrations of human recombinant TNF-a (Sigma Chemical Co., St. Louis, MO) from 1.5 to 5.0 ng/ml (at 2.2 U/ng), there was no difference in cell numbers between treated and untreated cultures (Figure 1 A). However, TNF-a concentrations < 5.0 ng/ml elicited up to three to four times both the concentration of gag protein in the supernatant and amount of gag protein per cell than did cultures without TNF-a (Figure 1 B). The amount of LPS in the TNF-a preparation was < 120 pg LPS/,Lg of TNF-a. Therefore, the final concentration of LPS due to the TNF-a in the 2.5 ng/ml TNF-a medium was < 0.3 pg/ml. Similar levels of upregulation were observed at 2.5 and 1.5 ng/ml of TNF-a. This replication enhancement was observed whether TNF-a was added 3 days before or at the time of infection. Treatment with TNF-a did not affect time of peak viral production. Culture supernatants containing 5.0 ng/ ml of TNF-a did not have increased quantities of SIV gag protein compared with untreated cells, but the cells stimulated with this high dose of TNF-ot morphologically were granular and small. These results are consistent with the findings of other investigators who have demonstrated an upregulatory influence of exogenous TNF-a on HIV-1 replication in cultures of lymphoid cells or promonocytic cell lines or when used therapeutically in HIV-1 -infected
patients.'1 116,18,30,31
Interferon-y (IFN-y) Retards SIV Replication Electron Microscopy
in Macrophages
Alveolar macrophages infected with SIV were scraped from the bottom of flat-bottomed six-well plates and initially fixed in half-strength Karnovsky's fixative,29 washed in phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Ultrathin sections were cut and stained with uranyl acetate and lead citrate
It was previously reported12 that IFN-y upregulates HIV-1 replication in primary cultures of monocyte-derived macrophages if added 3 days before infection. For this reason, we compared the potential of IFN--y to influence the replicative potential of SIV in macrophages. Human recombinant IFN-y (Chemicon Co., Temecula, CA) was
880
Walsh et al
AJP Ocober 15991, Vol. 139, No. 4
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added to cultures at 300 U/ml 3 days before infection and continuously thereafter. The number of macrophages in the IFN-y-treated cultures was significantly greater than in the untreated wells (Figure 2A). The appearance of SIV gag in the supematants from these treated cultures was delayed by approximately 3 days, after which the concentration was either similar to or less than that seen in the untreated cultures (Figure 28). Therefore, when corrected for differences in cell number/well between the treated versus untreated cultures, the amounts of gag protein per cell in the cultures stimulated with IFN-y were significantly lower during either the entire length of culture in some experiments or, in others, at least until the last 4 to 5 days of the culture (Figure 2C).
5
Figure 1. Effects of TNF-a (1.5 ng/ml) on simian alveolar macrophage cultures infected with SIV Values represent means + 1 SEM (n = 3 to 6). A: Number of macrophages/well at time of plate harvest with (-0-) and without (-4*-) TNF-a. B: Relative amounts of SIV gag on a per cell basis for cultures with (-0-) and without (-*--) TNF-a. Superscipts at the time points indicate that the values are significantly different from the untreated control group. *P < 0.001, +P < 0.01.
'SF Does Not Upregulate SIV cation in Macrophages on a Per-cell been reported by a number of investigators that ,F upregulates HIV-1 replication in primary cultures man monocyte-derived macrophages and nocytic cell lines.12'13'17 However, we noticed that i recombinant GMCSF from two different sources tics Institute, Boston, MA and Amgen Biologicals, and Oaks, CA), when added to simian macro> cultures, significantly increased the number of phages in the cultures compared with the unJ cultures (Figure 3A). Therefore, quantification of
SIV Growth in Macrophages
881
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Figure 2. Effects of IFN-y (300 U/ml) on simian alveolar macrophage cultures infected with SI. Values represent means + 1 SEM (n = 3 to 8). SV-infected macrophages treated with IFN-y (-0--) are compared with untreated macrophages (-*-) in the same esperiment There are significantly more cells in the treated cultures (A); gag protein production in the supernatant is delayed by three days in these treated cultures (B), but gag production per cell is reduced in the treated culturesfor up to 17 days after inoculation (C). Superscripts at the time points indicate that the values are significantly different from the untreated control groups. *P < 0.001,
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virus in supematants from these cultures may not realistically reflect changes in the amount of virus produced by each infected cell. Accordingly, we tested the effects of GMCSF (5-100 U/ml) from both sources by adding it to simian macrophage cultures 3 days before infection with SIV and continuously thereafter. The amount of LPS in the
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Days after inoculation Figure 3. Effects of GMCSF (20 U/ml) on simian alveolar macrophage cultures infected with SIV Values represent means ± 1 SEM (n = 3 to 8). SNV-infected macrophages treated with GMCSF (-0-) are compared with untreated maacophages (-*--) in the same experiment. Macrophage numbers are markedly increased in GMCSF-treated cultures (A), and gag protein concentration in the superntant is increased in these treated cultures (B); however, gag production per cell ts similar in treated and untreated cultures (C). 7he difference between treated and untreated cultures at 21 days after inoculation (C) wAas not seen in all GMCSF experiments. Superscrpts at the time points indiate that the values are significantly different from the untreated control groups. *P < 0.001, 'P < 0.02, AP < 0.05.
GMCSF preparation from Amgen Biologicals was < 50 pg LPS/4 x 1 4 U of GMCSF. Accordingly, the final concentration of LPS from the 100 U/mI GMCSF preparation from Amgen Biologicals was < 0.125 pg/ml. Similarly, the
882 Walsh et al AJP October 1991, Vol. 139, No. 4
final concentration of LPS from the 100 U/ml preparation from Genetics Institute was < 0.05 pg/ml when tested using the Limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO). The amount of SIV gag protein in the supernatants from GMCSF-stimulated cultures at the time of peak production was approximately three to six times that from untreated cultures at all doses tested using both sources of GMCSF (Figure 3B). However, after adjusting for differences in cell number, GMCSF did not increase SIV virus production per cell at any of the doses tested (Figure 3C). The effect of GMCSF was not due to TNF-a release since neutralizing goat polyclonal antibodies to human TNF-a (R & D Systems, Minneapolis, MN), when added at three to five times the neutralizing dose50 (ND50), did not alter the amount of SIV gag protein in the supernatant or the overall kinetics at 20 U/ml of GMCSF (data not shown).
The Lack of Upregulation by GMCSF Is Not Accounted for by Reduced Numbers of Infected Macrophages Producing More Virus We next examined the effect of GMCSF on the numbers of gag-containing cells using immunohistochemistry. In the GMCSF-treated cultures, there were significantly more gag-containing cells than in the untreated cultures (Figure 4). Since GMCSF-treated cultures contained proportionally greater numbers of infected cells, the increased amount of gag protein detected in the supematants was not apparently due to increased production of virus from a limited number of infected cells.
IL-6 Induces Significantly Greater Cytopathic Changes in S/V-infected Macrophages Because it has been reported that IL-6 increases HIV-1 expression in human monocyte-derived macrophages,13 we studied the effect of IL-6 on SIV replication in simian macrophages. We added human recombinant IL-6 (Gibco Laboratories) at concentrations ranging from 3.1 to 100.0 ng/ml to macrophage cultures 3 days before infection with SIV and continuously thereafter. The LPS concentration of the IL-6 was < 20 pg/,ug of IL-6. Therefore, the final concentration of LPS due to the IL-6 in the 100.0 ng/ml medium was < 2.0 pg/ml. Concentrations > 6.25 ng/ml of IL-6 induced a number of changes in infected cells. IL-6-treated cultures showed earlier and greatly increased numbers of syncytia than did untreated cultures (Figure 5A, B). This effect was not seen in IL-6treated cultures that were not infected with SIV. SIV gag protein was not found in the supernatant until 2 to 3 days after the onset of syncytia formation in the IL-6-treated cultures. Although there were frequently more cells in the treated cultures before the detection of gag in the culture media (data not shown), once SIV gag protein appeared in the supernatants, the numbers of macrophages in the treated cultures were either similar or slightly lower than in the untreated cultures (Figure 6A). This finding suggests that the cytopathic changes and subsequent cell death in the IL-6-treated cultures resulted in reduced cell numbers compared with untreated cultures. Despite the greatly enhanced cytopathic effects in the IL-6-treated cultures, the amount of SIV gag per cell was only slightly greater than or equal to that in the untreated cultures (Figure 6B).
40
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sa t umrs.Loof gag-containing e-:gu simian ave alveoFigure A4. Naumbers lar macrophages in cultures with (-0-) and without (-0*-) GMCSF (20 U/ml) as detected by immunohistochemistry and calculated as described in Materials and Methods. Values represent means + 1 SEM (n = 6 to 8).
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Figure 5. Photomicrographs ofsimian alveolar macrophage culturesfour days after inoculation with SIVand as described in Materials and Methods. A: Cultures without IL-6 do not have any syncytial cells. B: Cultures containing IL-6 (50 ng/ml) and infected under otherwise identical conditions have numerous syncytial cells. A, B, X330.
IL-6 Does Not Increase Intracytoplasmic Viral Assembly and Budding Because there were significantly greater cytopathic changes in the IL-6treated cultures than in the untreated cultures despite comparable replication profiles, we investigated the possibility that IL-6 may alter viral assembly so that intracellar budding into membrane-bound cytoplasmic vacuoles predominates over extracellar budding. If so, IL-6 may upregulate SIV replication in these cultures without quantitative differences being detected in the supernatants. We explored this possibility by examining SIV-infected alveolar macrophages ultrastructurally with and without the presence of IL-6 (50.0 ng/ml). Macrophages were harvested for electron microscopy at 12 days after infection when the supematant from IL-6treated and control cultures contained 209 and 127 ng/ ml of SIV gag protein, respectively. Although there were significantly more syncytia in the IL--treated cultures, there was no detectable difference in the numbers of cells containing budding particles or the numbers of budding particles in representative cells between the two samples. In both cases, budding was predominantly
found intracellularly, although budding from the plasma membrane could occasionally be found. Cultures harvested before 12 days after inoculation had multiple syncytia in the IL--treated flasks, but no mature virions or budding could be found in either the treated or untreated samples.
Discussion In this report, we have presented evidence that the cytokines TNF-a and IFN--y affect SIV replication within primary cultures of mononuclear phagocytes, whereas IL-6 and GMCSF do not have potent viral regulatory influences on a per-cell basis. Since certain cytokines have cellular and growth regulatory properties in vitro that can affect cell viability, cell number, and subsequent quantity of extracellular virus, this study underscores the importance of considenng cell numbers when quantitating virus production in cell culture. Recombinant TNF-a greatly increased SIV replication within primary cultures of simian macrophages. This finding is consistent with previous studies using HIV-1 that
Walsh et al
884
AJP October 1991, Vol 139, No. 4
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demonstrated the upregulatory influence of TNF-a on acutely infected primary cultures of macrophages and chronically infected cell lines. 11,13,16,17 Inasmuch as some patients with AIDS have elevated circulating levels of TNF-a32 and SIV-infected rhesus monkeys often have alveolar macrophages that secrete exaggerated levels of TNF-a on endotoxin challenge,33 the involvement of this molecule in the inductive events leading from persistent low-level virus expression to clinical disease seems likely. Furthermore, it has been established that TNF-a stimulates HIV-1 enhancer sequences in the LTR by activation of nuclear factor KB (NF-KB).18'30 As the SIV LTRs also contain NF-KB-like sequences,3'435it is likely that SIV can be stimulated in a similar fashion.
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points indicate that the values are significantly differentffrom the untreated controlgroups. *P < 0.001, + P
