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Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido- 2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine

We have investigated the influence of granulocyte-macrophage CSF (GM- CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity of several 2'3'- dideoxynucleoside congeners of thymidine in these cells in vitro. We found that replication of both HTLV-IIIBa-L (a monocytotropic strain of HIV-1) and HTLV-IIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in culture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of 3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners: 2'3'-dideoxythymidine (ddT), 2'3'- dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU). Inhibition of viral replication in GM-CSF-exposed M/M was achieved with concentrations of AZT and related drugs, which were 10- 100 times lower than those inhibitory for HIV-1 in monocytes in the absence of GM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSF-exposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF. We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites of AZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio of AZT-5'- triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'- didehydrothymidine-5'-triphosphate/2'-deoxythymi dine- 5'-triphosphate is several-fold higher in GM-CSF-exposed M/M, and this may account for the enhanced activity of such drugs in these cells. Taken together, these findings suggest that GM-CSF increases HIV-1 replication in M/M, while at the same time enhancing the anti-HIV activity of AZT and related congeners in these cells. These results may have implications in exploring new therapeutic strategies in patients with severe HIV infection.

Human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), infects and replicates within several types ofhuman cells (1)(2)(3)(4). Although early studies of this retroviral pathogen focused on CD4' T lymphocytes as the principal target cell, there is increasing evidence that infection of cells belonging to the monocyte/macrophage (M/M)' lineage plays a crucial role in the pathogenesis and progression of this disease (2,5,6). In addition, since M/M may differ from T cells in their intracellular metabolism of drugs and their reaction to lymphokines, it is important that one consider the effect of antiretroviral therapeutic strategies on cells of the M/M lineage.
Our group has previously shown that 2'3'-dideoxynucleosides (ddN), a family of compounds lacking an hydroxy group at the 3' position ofthe sugar .noiety, are potent in vitro inhibitors ofHIV replication in both human T lymphocytes and M/M (7)(8)(9). In addition, several members ofthis family of drugs have been shown to have clinical activity when administered to patients with severe HIV infection (10)(11)(12). One of these compounds, 3'-azido-2'3'-dideoxythymidine (AZT) prolongs life and improves neurologic abnormalities (10,(12)(13)(14) in certain patients with AIDS . The use of AZT, however, is associated with bone marrow suppression and other toxic side effects (15), particularly in patients with established AIDS, so that a recent focus of investigation has been directed toward developing strategies to overcome this problem. One approach currently under investigation is the use of granulocyte-macrophage CSF (GM-CSF). GM-CSF is a cytokine that stimulates the maturation and differentiation of granulocyte and monocyte bone marrow precursor cells (16)(17)(18) . In addition, Hammer et al. (19,20) have suggested that GM-CSF can inhibit HIV replication in U937, a CD4+ cell line with certain monocytoid properties. However, Folks et al. (21) have found that GM-CSF may actually stimulate HIV replication in a subclone of U937.
Because of the interest in using GM-CSF in patients with HIVrelated disease and the conflicting results obtained in monocytoid cell lines, which may or may not be reliable biochemical models for normal M/M, we undertook an investigation of the in vitro effect of GM-CSF on HIV replication in purified human peripheral blood M/M, both as a single agent and in conjunction with dideoxynucleosides. Our results show that GM-CSF enhances HIV replication in fresh M/M by several hundred-fold, and moreover, that infection of such stimulated cells can take place-with lower concentrations of virus. In spite of this, the net potency of AZT and related dideoxythymidine analogues in inhibiting HIV infection is markedly increased in the presence of GM-CSF. This increase in activity appears to result from an enhanced cell entry ofdrug and production ofphosphorylated (activated) drugs in the face of minimal increases in the levels of the competing thymidine triphosphate. These results may have implications for the design of improved therapeutic strategies for patients with AIDS and related conditions.

Materials and Methods
Cells. Monocyte-enriched PMBC were obtained from healthy, HIVnegative donors using a cell separator (Fenwal C3000; Travenol Inc., Deerfield, IL). The PBMC were then further enriched for M/M by elutriation as described by Gerrard et al. (22). M/M obtained by elutriation followed by a 5-d culture were >95% nonspecific esterase positive (Technicon Instruments Corp., Tarrytown, NY), and >95% actively phagocytized 0.8-P. latex beads (Sigma Chemical Co., St. Louis, MO) . In addition, they were <1% E-rosette positive, and 50% OKT4A' (Ortho Diagnostic Systems Inc., Raritan, NJ) . Cell viability (as determined by trypan blue exclusion) was always >95%. In some experiments, H9, a T4' lymphocytic cell line, was used as control . Also, in certain experiments, enriched lymphocyte populations were obtained by elutriation followed by incubation on plastic dishes to remove adherent cells . These cells were <2°Io nonspecific esterase positive . It should be noted that the control M/M used in this study (i.e., elutriated cells cultured for 5 d before exposure to HIV) are different than the M/M populations that formed the core ofour previous paper on the effect of dideoxynucleosides on HIV infection in M/M (8) .
Drugs. AZT (Wellcome Research Laboratories, Research Triangle Park, NC), 2'3'-dideoxycytidine (ddC), 2'3'-dideoxythymidine (ddT), 2'3'-dideoxyadenosine (ddA), 2'3'-dideoxyinosine (ddI) (Pharmacia Fine Chemicals, Piscataway, NJ), 2'3'-dideoxy-2'3'-didehydrothymidine (D4T) (supplied by Developmental Therapeutics Program, DCT, NCI, Bethesda, MD), and 3'-azido-2'3'-dideoxyuridine (AZddU) (kind gift of Dr. R. F. Schinazi, VA Medical Center, Decatur, GA) were diluted in distilled water and kept at 4'C until used. The chemical structures ofAZT, AZddU, ddT, and D4T are depicted in Fig. 1 Antiviral Drug Assay. The tests of antiviral activity were performed with minor modifications of a previously published procedure (8) . Briefly, 105 purified elutriated M/M were seeded at day -5 in a 1-cm2 well of a 48-well plate (Costar, Cambridge, MA), and cultivated in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 20% heat-inactivated FCS (Hyclone Laboratories, Logan, UT), 2 MM L-glutamine, 50 U/ml penicillin, and 50 t~g/ml streptomycin (Gibco Laboratories) (complete medium) for 5 d in the presence or absence of 100 U/ml of GM-CSF Unless stated otherwise, cells were treated from day -5 with GM-CSF and then cultured in the continuous presence of this cytokine . Cells, viruses, and FCS were tested for mycoplasma contamination, and found negative. At day 0, M/M were preexposed for 20 min to various concentrations of drugs, and then challenged with HTLU IIIB,,-,. (80,000 cpm/ml reverse transcriptase) as previously described (8) . This is at least 10 times the minimum infective dose in 5-d cultured M/M not exposed to GM-CSF (see Results, Fig . 2 b) . Cells were kept at 37°C in a humidified atmosphere supplemented with 5% C02 . 2 d after infection (day +2) cells were extensively washed in order to remove excess virus, and cultivated under the same conditions with the same concentrations of drugs as before . Cells were then fed every 4-5 d . In some experiments, purified lymphocytes stimulated at day 0 with PHA, 1 :200 vol/vol (Gibco Laboratories), or H9 T cells were exposed to 500 viral particles/cell of HTLU-IIIB (500 times the minimum infective dose for ATH8 cells, a T4 cell clone used as virus infectivity control). 20 U/ml of human rIL-2 (AmGen Biological, Thousand Oaks, CA) plus 15% vol/vol of human natural IL-2 (ABI, Silver Spring, MD) were added to medium for lymphocytes (but not for other cells). Viral Detection. Viral production was assessed at days 2, 5, and 7 after viral challenge, and then every 7 d, using several different methods . (a) reverse transcriptase (RT) activity was evaluated using a previously described procedure (8,24). (b) HIV-p24 antigen production into the supernatant was assessed by a RIA and by ELISA (DuPont Co., Wilmington, DE). (c) Syncytia formation was evaluated at regular time points by visual inspection in an inverted microscope.
Drug toxicity was assessed in mock-infected cells cultivated for 14 d in presence of different concentrations ofddN, by trypan blue exclusion, and by phagocytic activity using 0.8-1.t latex beads .
Anabolic phosphorylation of dideoxynucleosides was analyzed using minor modifications of previously published procedures (25,26). Briefly, ddN metabolism was measured by incubating 5 x 106 cultivated M/M or H9 T cells, in the presence or absence of 100 U/ml of GM-CSF, in 12-well plates (Costar) . After 5 d of culture, cells were exposed to 10 uM ['H]AZT or 10 p,M [3H]D4T for 24 h in 4 ml of complete medium. Cells were then harvested by gentle scraping, counted, pelleted after three washings in cold RPMI, and immediately frozen in dry ice . Frozen cell pellets were extracted with 500 ill of60% (vol/vol) methanol, and the methanol extracts further heated at 95°C for 1.5 min. The extracts were clarified by centrifugation at 12,000 g for 6 min. 200-ttl aliquots were then loaded onto a radially compressed column of Partisil SAX and eluted with an ammonium phosphate gradient as previously described (24,25) . Endogenous deoxyribonucleoside-5'-triphosphate pools and nucleoside kinases were measured as previously described (27). Statistics.
The statistical significance ofthe difference of viral suppression by dideoxynucleosides in GM-CSFstimulated cultures vs. cultures without GM-CSF was assessed using the two-tailed student's t test.

Results
Fresh elutriated M/M cultured for 5 d before exposure to HTLVIIIBa-L were consistently found to be infected in these experiments (Fig. 2). In the absence of GM-CSF, viral production was detectable no earlier than day 7 and then increased, being detectable at least up to day 35 ( Fig. 2 A). It should be noted that the M/M population and the culture conditions used are critical variables in experiments such as these, since the degree of HIV infection and viral suppression by dideoxynucleosides may vary substantially depending on the exact M/M population and conditions (8). Culturing the elutriated M/M for 5 d before exposure to virus and drugs is thus an important variable that should be taken into consideration .
M/M exposed continuously to 100 U/ml GM-CSF showed a high titer of viral production (at least 10 times more than without this cytokine), consistently detectable by day 5. Viral production was sustained for at least 35 d. No substantial enhancement ofviral production was seen in most experiments using lower concentrations of GM-CSF, i.e., 1 and 10 U/ml (data not shown). Since GM-CSF induces proliferation of monocytes, we wondered whether the increase in viral production was simply due to the GM-CSFinduced increase in cell number. However, the number of cells 7 d after viral challenge ranged up to 20-fold higher for M/M exposed to GM-CSF as compared with unexposed cells, whereas the increase in p24 production at day 7 was -100-1,000-fold. (Table I and  . Thus, it is unlikely that the increase in cell number per se caused by GM-CSF accounts for the major enhancement of viral production observed in our studies. Syncytia formations were easily detected, starting from day 9, in GM-CSF M/M exposed to HTLV IIIBa-L ( Fig . 3) . At this time, multinucleated giant cells were seen in culture, in association with an inhibition of cell replication . From day 20, giant cells became pycnotic. However, they maintained their ability to produce a large amount of virus at least up to day 35 . In one experiment, we subjected HIVexposed GM-CSFstimulated M/M to three cycles of freezing and thawing to explore whether this would release additional viral particles that might be sequestered within intracytoplasmic vacuoles ; however no increase in HIV-p24 antigen was observed (data not shown) .
As shown in Fig. 2 a and in Table I, M/M treated with GM-CSF only before virus challenge (that is from day -5 to day 0), followed by extensive washing to remove this cytokine, initially gave rates of viral production comparable with those or Media Alone at Different Time Points and Exposed to HIV-1/HTLV-IIIea _L 105 fresh elutriated M/M were placed in culture at day -5 in 1 ml of complete medium, and treated with GM-CSF 1 .00 U/ml from day -5 to 28, from day -5 to day 0, or from day 0 to 28 . On day 0, M/M were exposed to saline or AZT for 20 min and challenged with HTLV-IIIB a -1 80,000 cpm RT/ml while maintaining AZT in the media . Viral replication was assessed by HIV-p24 antigen production in the supernatants . The level of infection is expressed in pg/ml (numbers in parentheses show the percent of viral suppression) . Results reported here represent a typical experiment . seen in cells continuously exposed to GM-CSF. After day 14, however, a substantial waning in viral production was seen, and from day 35 after viral challenge this level became similar to the viral yield obtained in GM-CSFunexposed M/M. Viral production in M/M treated with GM-CSF starting from day 0 was less enhanced than that observed with continuous treatment from day -5 . In some experiments, we infected blood-derived M/M that had been allowed to mature for 5 d on plastic dishes, were further purified by adherence, and then exposed to GM-CSF. GM-CSF did not have any substantial effect on HIV replication in these 5-d adherent M/M (data not shown), possibly because of the relatively low number of high-affinity GM-CSF receptors in mature macrophages (28,29). This is a topic for further research .
In similar experiments, we exposed elutriated M/M cultivated for 5 d in the presence or absence of GM-CSF to HTLV IIIB, a lymphocytotropic viral strain (Fig.  4). In agreement with our previous experience, in the absence of GM-CSF we obtained inconsistent viral replication using this strain ; productive infection was attained in only three of six experiments using M/M not exposed to GM-CSF, and in the productive cultures there was a delayed, lower peak of viral production, as compared with the results obtained using the monocytotropic strain HTLV IIIBa-l, . However, HTLVIIIB replication in M/M exposed to GM-CSF was consistent and easily detectable starting from 7 d after virus exposure, with a peak of viral production after 14-21 d of culture -20 times greater than the maximum level obtained without GM-CSF. We also tried to define the minimum infective dose in these cells. As shown in Fig. 4, we observed that in the absence ofGM-CSF, at least 100 times more   (Table I). Over 90% suppression ofviral replication was obtained with 1 AM AZT, and 23 % suppression was obtained with 0.1 AM. Interestingly, despite the substantial increase in HIV production induced in such cells by GM-CSF alone, AZT was much more potent in inhibiting viral replication in the presence of this cytokine (Fig. 5 A) ; >98% inhibition of viral replication was achieved with 0.1 AM AZT, and even with 0.01 AM AZT, N90% viral suppression was observed (this was not due to an artifact of increased cell death, a point we will address later in this article). This effect was not seen in fresh, monocyte-depleted lymphocytes; in these cells, 10 AM AZT completely Kinetics of HTLV-IIIB infection in M/M exposed to GM-CSF 100 U/ml at day -5, and challenged at day 0 with different multiplicity of infection of the lymphocytotropic strain HTLVIIIB . Open symbols represent M/M treated with GM-CSF from day -5 to day 30 . Closed symbols represent control M/M cultivated in media alone from day -5 to day 30 . (0/*) M/M exposed to 10,000 HTLV-IIIB particles/cell ; (0/") M/M exposed to 1,000 HTLV IIIB particles/cell; (0/0) M/M exposed to 100 HTLVIIIB particles/cell ; (O/") M/M exposed to 10 HTLVIIIB particles/cell . Shown here is a representative experiment in which some detectable infection with HTLVIIIB was attained in M/M not exposed to GM-CSF While substantial viral replication was consistently achieved in GM-CSF exposed M/M in all experiments, no detectable viral replication was obtained in three out of six experiments in M/M not exposed to GM-CSF.
inhibited HIV1/HTLVIIIB replication in the absence of GM-CSF, and neither stimulation of viral production nor enhancement of antiviral activity of AZT was seen with exposure of lymphocytes to 100 U/ml GM-CSF (data not shown) . This is in agreement with the results obtained by Walker and Burgess (28), in which no substantial amount of GM-CSF receptors could be detected on T lymphocytes.
To determine whether GM-CSF enhancement of antiviral activity in M/M was specific for AZT, or was also seen with other 2'3'-dideoxy analogues of thymidine, we tested the effect of GM-CSF on three AZT congeners, ddT, D4T, and AZddU. Although AZddU is in fact a uridine congener, it is phosphorylated by thymidine kinase (30) and in this sense acts as congener of AZT. We also studied three other 2'3'-dideoxynucleosides, ddC, ddA, and ddI, drugs with different bases and which are phosphorylated in human cells by different kinases compared with AZT. The results obtained with the monocytotropic strain HTLV IIIBa-L are shown in Fig. 5. With each of the AZT congeners, there was an enhancement of antiviral activity in the presence of GM-CSF (Fig. 5 A) . Consistent with our previous results, the ED5o for AZT in elutriated M/M cultured for 5 d was -0.1 AM, while it decreased down to between 0.001 and 0.01 AM when these M/M were exposed to GM-CSF. HIV activity in T cells in our hands (7). However, in the presence of GM-CSF, the ED50 was lowered to 0.1 j.M. In a similar manner, the ED5o of D4T was between 0.1 and 1 uM for untreated M/M, and between 0.01 and 0.1 AM for GM-CSFexposed M/M. Finally, AZddU was only partially effective in our hands in elutriated M/M after 21 d of culture, even at the highest concentrations tested (-60% protection at 100 AM after 21 d of culture). However, 10 pM AZddU induced complete viral suppression in GM-CSFtreated M/M, with an ED50 of 0.1-1 p.M. These data were confirmed in two different experiments by determination of RT activity in culture supernatants ; the results were substantially parallel to those obtained measuring HIV p24 antigen production (data not shown) . We also tested the ability of AZT and its congeners to inhibit syncytia formation. As shown in Fig. 3, low concentrations of AZT and ddT completely inhibited syncytia induced by HIV infection in GM-CSFexposed M/M; similar effects were seen with D4T and AZddU (data not shown) .
We asked whether GM-CSF had an effect on viral infection and/or on antiviral activity of AZT in H9, a T lymphocytic cell line. Neither increase of viral production nor enhancement of antiviral activity of AZT and related congeners was seen, in agreement with data obtained with AZT in fresh lymphocytes (data not shown) . Finally we exposed M/M to the lymphocytotropic strain HTLVIIIB in the presence or absence ofGM-CSF. The results, shown in Fig. 6, are substantially parallel to those obtained with the monocytotropic strain HTLVIIIB .-L, showing an overall potentiation of viral suppression by AZT and related congeners in M/M exposed to GM-CSF. Thus, the results indicated that AZT and each of the closely related congeners tested are 10 or more times more active at suppressing replication of two different strains of HIV in GM-CSFexposed M/M than in M/M not exposed to GM-CSF.
By contrast, when we tested the effect of GM-CSF on other dideoxynucleosides not related to AZT, we found that there appeared to be some reduction of anti-HTLV IIIBa-1, activity in fresh M/M in the presence of GM-CSF (Fig. 5 B) . Comparable results were obtained using the other HIV-1 strain, HTLV IIIB (Fig . 6). Our data do not distinguish whether this represented unchanged anti-HIV activity in the presence of an increased efficiency of viral replication, or alternatively, an actual reduction of anti-HIV activity. In either case, the results indicate that GM-CSF seems to enhance the anti-HIV effect of AZT and its congeners in M/M, while the effect of several other dideoxynucleosides is unchanged or possibly even reduced in M/M cultured with this cytokine .
To determine whether the enhanced antiviral activity of AZT and its congeners by GM-CSF might be due to an increased cell toxicity, we assessed this parameter by two different methods. In the absence ofGM-CSF, M/M failed to show any drugrelated toxicity, as assessed by trypan blue exclusion, at concentrations up to 100 AM AZT, 500 AM ddT, 10 AM D4T, or 100 AM AZddU (data not shown) . Also, no inhibition ofphagocytic activity was seen at the same concentrations . In the presence ofGM-CSF, phagocytosis was substantially unaffected by drugs (Table II). However, high concentrations of AZT and AZddU did lower the number of M/M exposed to GM-CSF in a dose-dependent fashion (Table II); in the case of AZT, a 50% reduction of viable M/M was obtained at 10 A.M. On the other hand, 1 AM AZT was devoid of cytotoxicity in monocytes exposed to GM-CSF, and this concentration is >100 times the ED5o described earlier under these conditions. Similarly, ddT and D4T did not substantially affect cell viability at concentrations up to 500 and 10 AM, respectively. Thus, while GM-CSF does have an effect on the toxicity of AZT and analogues at the same time as it increases their antiviral activity, >9570 HIV suppression by AZT analogues in GM-CSF treated M/M is obtained at concentrations 10-500 times (depending on different AZT analogues) lower than those necessary to induce such viral inhibition in HIV-exposed T lymphocytes; moreover, TABLE II Toxicity of Dideoxynucleosides in M/M Exposed to GM/CSF 10 5 fresh elutriated M/M exposed to 100 U/ml GM-CSF were cultivated for 14 d in 2 ml of complete medium in the presence of different concentrations of ddN . Viability was measured by trypan blue exclusion method, and phagocytosis was evaluated after a 1-h incubation with 0 .8 Et latex beads ; viable cells ingesting at least two beads were considered positive . in the case of AZT, little or no toxicity is observed at drug concentrations typically attained in vivo (1-3 uM) .
To evaluate some possible mechanisms responsible for the interaction of GM-CSF and AZT congeners in fresh M/M cultivated for 5 d, we studied the metabolism of AZT and D4T in M/M with or without GM-CSF. The results are summarized in Fig. 7 and in Table III . In the absence of GM-CSF, M/M had low levels of intracellular AZT 5'-triphosphate (AZTTP). As can be seen by the high AZT 5'-monophosphate/AZT5'-diphosphate (AZTMP/AZTDP) ratio in Fig. 7 A, GM-CSF does not eliminate the relative block at the level of thymidylate kinase that is characteristic for AZT metabolism. Exposure ofthe M/M to GM-CSF dramatically increased the intracellular levels ofboth parent AZT and its mono-, di-, and triphosphate anabolites . In particular, AZTTP level increased >15-fold. These effects were not seen in H9 T cell line exposed to GM-CSF. In part, the increase in AZTTP in M/M exposed to GM-CSF may be due to an increase in the cell entry of AZT, as evidenced by the higher intracellular levels ofAZT as parent compound. Also, GM-CSF may have an effect on the level of intracellular thymidine kinase . The activity ofthis enzyme, which catalyzes the initial phosphorylation of AZT and related congeners, and thymidine as well, is very low in M/M when compared with T cells (8,31). M/M exposed to GM-CSF had approximately a twofold increase in thymidine kinase compared with control M/M cultured without GM-CSF (data not shown). A similar increase in phosphorylation was obtained with D4T active metabolites ofD4T were barely detectable in M/M unexposed to GM-CSF, whereas large amounts of both . Analyses were carried out using radial compression columns of Partisil-10 SAX equilibrated and developed with 0.01 M ammonium phosphate, pH 3.6, for 15 min, followed by a linear gradient to 0.6 M ammonium phosphate, pH 3.8, over the next 25 min, and finally by a 15 min isocratic elution with 0.6 M ammonium phosphate, pH 3.8 . Sample volume : 200 pl . 1-min fractions were collected, and data are expressed as dpm/10 6 cells. Note that the ordinate utilizes a logarithmic rather than a linear scale because of the marked accumulation of AZT at the 5'-monophosphate level and the resulting disparity in peak heights between those for AZTMP and for AZTTP. D4T and its mono-, di-, and triphosphate moieties were detected in GM-CSFexposed M/M. Indeed, the levels in such cells were comparable with those seen in H9 T cells.
We also studied the endogenous 2'-deoxythymidine-5'-triphosphate (dTTP) pool, which competes with AZTTP and related congeners at the level of viral RT. We found that the concentration of dTTP was very low in this population of M/M, as previously reported (8) for M/M tested under different conditions, and dTTP increased less than twofold in M/M exposed to GM-CSF for 5-7 d (Table III) . In the current work, we observed that elutriated M/M cultured for 5 d in media alone have a very low level of endogenous dNTPs compared with the levels obtained in other M/M populations (8). Thus, GM-CSFexposed M/M have a substantially higher ratio ofAZTTP/dTTP than control M/M, a result that might explain the increased activity of AZT and related congeners in such cells.

Discussion
In this report, we have found that GM-CSF activates the replication of HIVI in fresh M/M cultured for 5 d, yet at the same time it potentiates the anti-HIV activity of AZT and related congeners in fresh M/M. Furthermore, we show that this effect on AZT may be accounted for by a preferential enhancement of AZT cell entry and subsequent phosphorylation induced by GM-CSR Similar considerations apply to other dideoxy analogues of thymidine.
We wish to stress that any specific clinical or therapeutic inference from our data should be made only with great caution at this time. Nevertheless, the infection of cells belonging to the M/M lineage is increasingly recognized as an important event in the pathogenesis of AIDS (2,5,32). M/M are currently believed to be one of the earliest cells infected after exposure to HIV (33), and may play an important role in spreading HIV throughout the immune system (34). In addition, monocyte- derived cells appear to be the primary target for HIV infection of the nervous system (2,5,35). Thus, factors that influence the degree of replication of HIV in M/M may influence the course of HIV infection in patients. While certain strains of HIV have been reported to replicate in peripheral blood monocytes in the absence of exogenous stimulation (2,8,32), we show here that stimulation of these cells by GM-CSF markedly enhances their capacity to permit replication of HIV. Indeed, even the lymphocytotropic strain of HIV1, HTLV IIIB, which in our hands does not replicate well in unstimulated M/M, replicates quite efficiently in M/M exposed to GM-CSF. It is conceivable that in our system, GM-CSF stimulates the production of host-cell transcriptional factors that in turn can potentiate viral transcription or posttranscriptional events (36). It is of note that another marrow growth factor, macrophage-CSF (M-CSF or CSF1) has been shown by Gendelman et al. (37) to enhance HIV production by M/M. In the case of CSFl-stimulated M/M, however, most of the HIV production was reported to be sequestered in vacuoles, while with GM-CSF, abundant virus was released into the media. Even though we failed to find evidence of an increase in p24 released into the media by disrupting GM-CSF-exposed M/M using three cycles of freeze-thawing, this would not completely exclude the possibility that GM-CSF induced a limited form of virus sequestration in intracytoplasmic vacuoles ; to specifically address this point, one would have to examine GM-CSFexposed HIV-infected M/M by EM.
It is of interest that GM-CSF treatment of target M/M potently enhances the replication of the lymphocytotropic strain HTLVIIIB in these cells; in this context GM-CSF has a net effect of converting a lymphocytotropic virus to a monocytotropic virus (Fig. 4). Thus, the available data from this and other studies suggest that GM-CSF is, if anything, an even more potent stimulus for HIV replication in M/M than CSF1 (37,38). GM-CSF is produced by a number of cells in vivo (39)(40)(41)(42)(43)(44)(45). As one such cell is the T lymphocyte (39,42,43), it is possible that GM-CSF might play a central role in the progression of HIV infection to AIDS . Antigenic stimulation ofT cells may lead to GM-CSF production, which may then lead to enhanced HIV replication in macrophages and, in turn, spread of virus to more T cells.
At the same time, GM-CSF markedly enhances the anti-HIV effect of AZT and closely related drugs in M/M. It appears to do so by substantially increasing intracellular AZTTP levels, while exerting only a slight effect on the levels of competing endogenous dTTP. To some degree, the enhanced phosphorylation of AZT may be due to a GM-CSFinduced increase in thymidine kinase (which not only catalyzes the initial phosphorylation ofthymidine and related ddN analogues, but also is reported to act on AZddU [30]). However, this does not appear to be the only mechanism responsible. M/M stimulated with GM-CSF and exposed to AZT have substantially higher levels of intracellular AZT as unphosphorylated compound than unstimulated M/M, suggesting that GM-CSF may also selectively enhance the cell entry of AZT and related drugs into M/M. It is worthwhile to point out that AZT and its normal counterpart, 2'-deoxythymidine, very likely permeate the cell membrane oflymphocytes and erythrocytes via different mechanisms (46) ; thus, lymphokines may have differential effects on the entry of different nucleosides. Also, in the case of the physiologic 2'-deoxynucleoside-5'-triphosphate, some ofthe endogenous thymidine pools may depend heavily on de novo synthesis from 2'-deoxyuridine-5'-mono-phosphate (dUMP), and be regulated by feedback mechanisms . The physiologic 2'-deoxynucleotide may therefore not be affected in precisely the same way as those arising from ddN, by changes in membrane transport or diffusion. Additional studies will be required to detail the mechanism(s) responsible for the effect of GM-CSF on the phosphorylation ofAZT and related analogues, and the mechanisms responsible for the reduced antiviral activity ofddN not related to AZT in GM-CSFexposed M/M.
We are presently in the process of evaluating how exposure of M/M to GM-CSF affects the metabolism of another dideoxypyrimidine, ddC, and a dideoxypurine, ddA, in order to delineate the basis for the differential effects observed on antiviral activity. Preliminary results indicate that by contrast to the >10-fold enhancement of AZTTP induced by GM-CSF, there is only a twofold enhancement of ddCTP. The competing physiologic nucleotide, 2'-deoxycytidine-5'-triphosphate (dCTP), however, was below the limit of detection of our assay in both of these cultured populations of M/M, and for this reason, we could not calculate the effect of GM-CSF on the ddCTP/dCTP ratio. Also, while ddATP and dATP were measurable in M/M exposed to GM-CSF, they were below the limit of detection in the media-control (unstimulated) M/M. Thus, while these preliminary results suggest that the increase in ddCTP induced by GM-CSF was substantially less than the increase ofAZTTP, and that the increase of ddATP was likely coupled with a substantial increase in the competing dATP, additional experiments will be needed to fully characterize the effects of GM-CSF on the intracellular biochemistry of these dideoxynucleosides. Nevertheless, we can speculate that the limited net activation of metabolism of such nonthymidine ddN is probably not sufficient to overcome the dramatic increase in viral yield induced by GM-CSF stimulation.
GM-CSF is currently being studied as a supplementary drug for the treatment of AIDS because of the possibility that it may counteract the marrow suppression induced by HIV infection or by AZT administration . The results reported here suggest that one may wish to closely monitor patients when using GM-CSF in the setting of HIVinfection since it might enhance viral replication. However, as also shown here, the simultaneous administration of GM-CSF with AZT, D4T, or related analogues may induce a potent antiviral effect in monocytes. Could such a combination also increase AZT toxicity in vivo in marrow blood-forming cells? The answer is not known; however, a recent study suggested that in contradistinction to our results obtained in M/M, GM-CSF might reduce the phosphorylation of AZT in bone marrow cells while increasing the levels of thymidine triphosphate (47). It is thus conceivable that GM-CSF may act to increase the overall therapeutic index of AZT and related thymidine analogues. The simultaneous administration of these two agents, therefore, may be worth exploring in patients with severe HIV infection.

Summary
We have investigated the influence of granulocyte-macrophage CSF (GM-CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity ofseveral 2'3'-dideoxynucleoside congeners ofthymidine in these cells in vitro. We found that replication of both HTLV IIIBa-L (a monocytotropic strain of HIV1) and HTLVIIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in cul-ture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners : 2'3'-dideoxythymidine (ddT), 2'3'-dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU) . Inhibition of viral replication in GM-CSF exposed M/M was achieved with concentrations ofAZT and related drugs, which were 10-100 times lower than those inhibitory for HIV-1 in monocytes in the absence ofGM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSFexposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites ofAZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio ofAZT 5'-triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'-didehydrothymidine-5'-triphosphate/2`-deoxythymidine-5'-triphosphate is several-fold higher in GM-CSFexposed M/M, and this may account for the enhanced activity of such drugs in these cells.
Taken together, these findings suggest that GM-CSF increases HIV I replication in M/M, while at the same time enhancing the anti-HIV activity ofAZT and related congeners in these cells . These results may have implications in exploring new therapeutic strategies in patients with severe HIV infection .