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JAIDS Journal of Acquired Immune Deficiency Syndromes:
doi: 10.1097/01.qai.0000214809.83218.88
Basis Science

Adverse Effects of Antiretroviral Drugs on HIV-1-Infected and -Uninfected Human Monocyte-Derived Macrophages

Azzam, Rula BSc(Hons)*†; Lal, Luxshimi BApp Sci, B Pharm*; Goh, Suet-Ling*; Kedzierska, Katherine PhD‡; Jaworowski, Anthony PhD*†; Naim, Eman BSc(Hons)*; Cherry, Catherine L. MBBS, FRACP*†§; Wesselingh, Steven L. BMBS, PhD, FRACP*†§; Mills, John BSc, MD, SACP, FIDSA, MASM*†; Crowe, Suzanne M. MBBS, FRACP, MD*†§

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From the *AIDS Pathogenesis and Clinical Research Program, Macfarlane Burnet Institute for Medical Research and Public Health; †Department of Medicine, Monash University; ‡Department of Microbiology and Immunology, The University of Melbourne; and §Infectious Disease Unit, Alfred Hospital, Melbourne, Australia.

Received for publication November 3,2005; accepted January 13, 2006.

Reprints: Prof Suzanne Crowe, AIDS Pathogenesis and Clinical Research Program, Macfarlane Burnet Institute, 85 Commercial Rd, Victoria, Australia 3004 (e-mail: crowe@burnet.edu.au).

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Abstract

Summary: Antiretroviral drugs approved for treatment of HIV-1 infection include nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). Use of these drugs in combinations (highly active antiretroviral therapy) has delayed disease progression. However, long-term therapy is associated with potentially serious adverse effects. NRTIs are thought to contribute to these adverse effects via depletion of mtDNA. Inasmuch as macrophages (major targets for HIV-1) are highly metabolically active with large numbers of mitochondria, we investigated the effects of NRTIs (didanosine, stavudine, lamivudine, and zidovudine) on the viability and function of HIV-1-infected and -uninfected human monocyte-derived macrophages (MDMs). We demonstrate that the combinations didanosine/stavudine and lamivudine/zidovudine decrease mtDNA content in MDMs, with HIV-1-infected MDMs displaying a greater reduction than uninfected cells. This decrease correlated with decreased complement-mediated phagocytosis (C'MP) by MDMs, a process dependent on mitochondrial function. Inasmuch as PIs have previously been reported to interact with cellular proteases and given that cellular proteases are involved in the phagocytic process, we investigated the effects of the PI indinavir on C'MP. We demonstrate that indinavir augments C'MP by uninfected MDMs, but not HIV-1-infected MDMs. This study provides additional understanding on the effects of commonly used antiretroviral drugs on cellular immune function.

The introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 infection has caused a significant decline in AIDS-related morbidity and mortality.1 Despite clinical benefits however, current antiretroviral therapy does not eradicate the virus.2-5 Consequently, life-long treatment is required to provide continual suppression of HIV-1 replication.

Long-term use of HAART can be associated with potentially severe adverse effects.6 A number of these are due to drug-related mitochondrial dysfunction or depletion.7,8 Nucleoside reverse transcriptase inhibitors (NRTIs), generally forming the backbone of HAART regimens, are phosphorylated by cellular kinases to their triphosphate form and compete with endogenous deoxynucleotide triphosphates for incorporation into proviral DNA. NRTIs can also act as substrates for human DNA polymerases,9 particularly polymerase γ that is indispensable for mtDNA synthesis (reviewed by Brinkman et al.10). NRTI-induced mitochondrial dysfunction is believed to occur as a result of mtDNA depletion and therefore impaired synthesis of mitochondrial enzymes that generate adenosine triphosphate by oxidative phosphorylation.7,8 This is thought to underpin adverse effects including peripheral neuropathy, lactic acidosis, myopathy, pancreatitis, and hepatic steatosis7,10-16 and has more recently been implicated in development of the lipodystrophy syndrome.17,18

Several studies have noted mtDNA depletion in adipose tissue samples,19-23 peripheral blood mononuclear cells (PBMCs)23,24, and muscle biopsy specimens25 from patients treated with NRTIs compared with untreated HIV-infected patients. Studies using Molt 4F cells (a human lymphoblastic cell line)26,27 have also demonstrated the ability of NRTIs to decrease mtDNA. However, the effect of NRTIs on the mtDNA content of cells of the macrophage lineage has not been well investigated.

Macrophages are major targets for HIV-1. After HIV-1 infection, macrophages display impaired effector mechanisms,28-36 contributing to the opportunistic infections at late stages of immune deficiency in HIV-infected individuals.37 Macrophages are extremely active metabolically,38 containing large numbers of mitochondria and, correspondingly, large amounts of mitochondrial enzymes and high rates of cellular respiration.39 These energy sources are necessary for their considerable phagocytic, migratory, and secretory functions and generation of the respiratory burst required for intracellular killing. In particular, phagocytosis by macrophages is an energy-requiring process,40 characterized by periods of intense metabolic activity41 with energy provided by adenosine triphosphate generated largely via oxidative phosphorylation.42

It is conceivable therefore that NRTIs may adversely affect macrophage functions via their effect on mtDNA. Antonelli et al43 have reported decreased intracellular killing and oxygen production by macrophages upon exposure to the NRTI zalcitabine (ddC), which correlated with decreased mtDNA in the same cells. De Simone et al44 reported that in vitro incubation of human monocytes with zidovudine resulted in impaired phagocytosis. However, these few prior studies have only investigated the effects of single antiretroviral drugs. In the age of combination therapy, a detailed investigation is required for the effects of combinations of antiretroviral drugs on macrophage mitochondria and relevant metabolic functions.

Protease inhibitors (PIs) were initially designed to be highly specific for the HIV aspartic protease, but interactions with human45 and fungal46 aspartic proteases have also been reported. Some endogenous aspartate proteases, such as cathepsins D and E, are considered to have an important role in macrophage function,45 and thus, it has been postulated that PIs may directly affect these cells. Reports on the effects of PIs on macrophage function are inconsistent. Indeed, the PI indinavir impairs phagocytosis of malaria-infected erythrocytes by human macrophages47 but increases phagocytosis of Cryptococcus neoformans.46 Thus, there is some evidence that PIs may directly alter macrophage phagocytic responses.

In this report, our aim was to investigate the effect of NRTIs and PIs, individually and in clinically relevant combinations, on the viability and function of HIV-1-infected and -uninfected human monocyte-derived macrophages (MDMs), with particular attention to the effect of these drugs on mtDNA and the consequences to the phagocytic response.

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MATERIALS AND METHODS

Isolation and Culture of Human Monocytes

Human peripheral blood monocytes were isolated from buffy coats seronegative for HIV, human T-cell lymphotropic virus, syphilis, and hepatitis B and C (supplied by the Red Cross Blood Bank, Melbourne) by Ficoll-Paque density gradient centrifugation and plastic adherence as previously described.48 For selected studies, monocytes were also isolated by counter-current elutriation. Briefly, monocytes were recovered from PBMCs by elutriation at 1500 × g, 4°C, using a centrifuge with 5.0 standard chamber (Beckman, Fullerson, CA), and collection of monocytes at a flow rate of 35 ml/min. Immediately after monocyte isolation, cell viability was greater than 95% as assessed by trypan blue exclusion, and purity was greater than 85% as determined by immunofluorescent staining with anti-CD14 monoclonal antibody (Becton Dickinson, Mountain View, CA) and flow cytometric analysis (FACS StarPlus flow cytometer; Becton Dickinson). Cells were cultured in Iscove's modified Dulbecco medium (GibcoBRL, Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated human AB+ serum (Sydney Blood Bank, Australia), 2 mmol/L l-glutamine (GibcoBRL) and 24 μg/mL gentamicin (Delta West, Bentley, WA, Australia). Monocytes were cultured in suspension in polytetrafluoroethylene (Teflon) pots (Savillex, Minnetonka, MN) at an initial concentration of 1 × 106 cells per milliliter as previously described.49

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HIV-1 Infection of Monocyte-derived Macrophages

The M-tropic strain HIV-1Ba-L (the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD) was amplified using uninfected PBMCs from 2 donors as previously described.48 Briefly, PBMCs stimulated with phytohemagglutinin (10 μg/mL, Murex Diagnostics, Dartford, UK) for 3 days were infected with HIV-1Ba-L, then cultured together in Iscove's medium containing 10% heat-inactivated human AB+ serum and 5% IL-2 (10 U/mL; Boehringer-Mannheim, Mannheim, Germany). Culture supernatants were collected on days 7, 10, and 13 after infection; stored at −70°C in small volumes; and thawed immediately before use.

Five days after isolation, MDMs were infected with HIV-1 for 4 hours at 37°C, 5% CO2, at a multiplicity of infection of 0.1 to 1.0 infectious particles per cell as previously described.48 Cells were then washed with calcium- and magnesium-free phosphate-buffered saline (PBS-CMF, GibcoBRL) and resuspended in fresh supplemented Iscove's medium. MDMs were then cultured for 7 days after infection either in suspension in Teflon pots or adherent to plastic in 96-well plates (Nalge Nunc International, Naperville, IL). Uninfected MDMs from the same donors were used as controls for each experiment.

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Quantification of HIV-1 Replication

HIV-1 replication in MDMs was measured by monitoring reverse transcriptase (RT) activity in culture supernatant using a micro-RT assay.30 Culture supernatant (10 μL) was added to 10 mL of 0.3% NP-40 in a 96-well plate and incubated at 4°C for 30 minutes. Subsequently, 40 μL RT mixture [50 mmol/L Tris pH 7.8, 7.5 mmol/L KCl, 5 mmol/L MgCl2, 2 mmol/L dithiothreitol (Sigma, St. Louis, MO), in distilled H2O up to 4 mL per plate], together with 5 μg/mL template primer p.An.dT12-18 (Pharmacia-Biotec, Australia) and 3 μCi 33P-dTTP (Amersham International, Buckinghamshire, UK), was added to each well, and the mixture was incubated for 4 hours at 37°C. The reaction products were spotted onto DE81 anion exchange paper (Whatman International, Maidstone, UK) and air dried. Dry filters were washed 8 times with 2 × SSC buffer (0.3 mol/L sodium chloride and 34 mmol/L sodium citrate; Merck, Victoria, Australia), rinsed twice in 95% ethanol, and dried. Meltilex scintillant (Wallax, Turku, Finland) was spotted onto the filter, and bound radioactivity was counted in the LKB micro betacounter (Wallax).

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Antiretroviral Drugs

Nucleoside reverse transcriptase inhibitors used were didanosine (ddI; Sigma), stavudine (d4T; Sigma), zidovudine (AZT; GlaxoSmithKline, Hertfordshire, UK), and lamivudine (3TC; GlaxoSmithKline). The PI utilized was indinavir (IDV; Merck & Co Inc, NJ) dissolved in dimethyl sulfoxide (DMSO). All drugs were used either individually or in double (ddI/d4T or 3TC/AZT) or triple (two NRTIs plus IDV: IDV/ddI/d4T or IDV/3TC/AZT) combinations. Drug concentrations tested ranged from 0.01 to 100 μmol/L, in serial 10-fold dilutions. Where DMSO was used, the final concentration did not exceed 0.5% in cell cultures.

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Assessment of Drug Toxicity on Monocyte-derived Macrophages

Cell membrane integrity was assessed by trypan blue exclusion (Sigma). Five days after isolation, MDMs were resuspended at a concentration of 1 × 106 cells per milliliter of supplemented Iscove's medium, cultured for 7 days in Teflon jars with the appropriate concentration of antiretroviral drugs. The trypan blue exclusion assay was performed on days 1, 2, 5, and 7 after addition of the drug, accounting for the usual decay in MDM numbers during culture in suspension.49

The effect of antiretroviral drugs on the levels of mitochondrial respiration was assessed using a cell proliferation kit (XTT)50 according to the manufacturer's instructions (Roche Diagnostics, Branchburg, NJ). This measures reduction in the electron acceptor XTT to a water-soluble formazan dye by the uncoupled mitochondrial electron transport chain. On day 5 postisolation, MDMs were plated onto 96-well flat-bottomed plates at 1 × 104 MDMs per well, and incubated with the appropriate drugs at the indicated concentrations for 7 days. A standard curve of MDMs, ranging from 1.5 × 103 to 1 × 105 MDMs per well, was prepared at the same time to provide a standard for comparison for each donor. The XTT assay was performed on day 7 postaddition of drug. The XTT labeling mixture was added to each well to a final concentration of 0.3 mg/mL and incubated for 4 hours at 37°C. Absorbance of the formazan product was measured using a spectrophotometric plate reader, with a test wavelength and a reference wavelength of 492 and 650 nm, respectively.

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Quantification of Mitochondrial DNA
DNA Extractions

On day 4 postisolation, monocytes were plated onto 6-well plates at 1 × 106 cells per milliliter of supplemented Iscove's medium. On day 5, MDMs were infected with HIVBa-L then cultured with the appropriate concentration of antiretroviral drugs. After 7 days of culture, total cellular DNA was extracted from MDMs (DNeasy tissue kit; Qiagen, Valencia, CA) according to manufacturer's instructions. Levels of mtDNA were quantified by real-time polymerase chain reaction (PCR) as previously described.51 To normalize for input DNA, a separate real-time PCR was carried out simultaneously to quantify a segment of the CCR5 gene.

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Complement-mediated Phagocytosis Assay

To measure the effect of antiretroviral drugs on the ability of MDMs to carry out a physiological function, a complement (C)-mediated phagocytosis assay was performed, as previously described.52 Briefly, on day 4 postisolation, MDMs were adhered onto 96-well plates at 5 × 104 cells per well at 37°C and infected with HIVBa-L after 24 hours, with drugs added immediately after infection. MDMs were cultured for a further 7 days in the presence of drugs, and the C-mediated phagocytosis assay was performed. Before the assay, MDMs were stimulated with phorbol-12-myristate-13-acetate (200 nmol/L; Sigma) for 10 minutes.

Target particles were opsonized immediately before the phagocytosis assay as previously described.52 Sheep red blood cells (sRBCs; ICN-Cappel, Aurora, OH) were washed 3 times in cold PBS, then opsonized for 60 min at 37°C with C5-deficient human serum as a source of human C components (Sigma). sRBCs prepared with heat-inactivated serum were used as controls. Opsonized sRBCs were then washed 3 times in PBS and resuspended at a concentration of 1 × 108 cells per milliliter. Phagocytic targets were added to MDMs at a ratio of 20 sRBCs to 1 MDM and centrifuged onto the adherent MDMs (100 × g, 5 minutes, 4°C). Cells were incubated with targets at 37°C for 60 minutes.

Internalized sRBCs were quantified using a colorimetric assay.53 Phagocytosis was terminated by washing each well with cold (4°C) PBS to remove unbound sRBCs. Adhered but nonphagocytosed sRBCs were lysed with 0.2% NaCl solution for 3 minutes, then removed by washing 3 times with prewarmed (37°C) Iscove's medium. MDMs and internalized sRBCs were lysed with 6 mol/L urea solution containing 0.2 mol/L Tris-HCl buffer (pH 7.4). The number of phagocytosed sRBCs was determined by measuring the amount of fluorene blue converted from 2,7-diaminofluorene (Sigma) by the pseudoperoxidase activity of hemoglobin. Absorbance was determined at 620 nm in an ELISA plate reader (Labsystems, Helsinki, Finland).

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Statistical Analysis

The significance of the effects of antiretroviral drugs on the viability and function of MDMs was assessed using the Kruskal-Wallis test. A probability of 0.05 was used to reject the null hypothesis.

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RESULTS

The Effect of Antiretroviral Drugs on MDM Viability

The NRTIs ddI, d4T, 3TC, and AZT and the PI IDV were added to MDMs 5 days after isolation, and viability of drug-exposed and control MDMs was assessed over 7 days in culture by the trypan blue exclusion assay. Incubation of MDMs with these antiretroviral drugs, individually and in double and triple combinations at concentrations ranging from 0.01 to 100 μmol/L, did not decrease MDM viability throughout the 7-day culture (Fig. 1 shows 0 and 100 μmol/L concentrations only). Appropriate vehicle controls were performed for all experiments and demonstrated no adverse effects.

Figure 1
Figure 1
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The Effect of Antiretroviral Drugs on Mitochondrial Respiration by MDMs

Antiretroviral drugs, either alone or in dual or triple combinations, were added to 1 × 104 adherent MDMs 5 days after isolation, and a standard curve obtained from MDMs cultured in the absence of drug was prepared as an internal control (ranging from 1.5 × 103 to 1 × 105 MDMs per well). Mitochondrial respiration by MDMs was assessed by XTT reduction assay after 7 days of incubation with the drugs and compared with that of control MDMs represented in the standard curve. Mitochondrial respiration by MDMs incubated for 7 days in the presence of individual NRTIs (data not shown) and double combinations of NRTIs or IDV (Fig. 2) were not altered in the XTT reduction assay, as compared with cells cultured in the absence of drugs. However, the triple combinations of IDV/ddI/d4T and IDV/3TC/AZT at 100 μmol/L decreased mitochondrial respiration to 0.69 ± 0.16 (P = 0.13) and 0.62 ± 0.12 (P = 0.002) relative to controls, respectively.

Figure 2
Figure 2
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In 8 of 20 experiments where XTT assay was performed, crystals were observed in cultures incubated with very high concentrations (100 μmol/L) of IDV. Mitochondrial respiration was abolished in cultures where these crystals were evident. Given that the effects of the crystal formation could not be distinguished from potential effects of IDV on mitochondrial respiration, all results from cultures where IDV crystals were visible were not included in the mean data presented. Crystal formation was not observed during any other experiments.

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The Effect of Antiretroviral Drugs on Mitochondrial DNA Content of MDMs

Monocyte-derived macrophages were infected with HIV-1 5 days after isolation and incubated with NRTIs immediately after infection for 7 days, followed by DNA extraction and quantitation of mtDNA by real-time PCR. There was no difference in the MDM mtDNA content between HIV-1-infected and -uninfected MDMs, as assessed by the mtDNA:CCR5 gene ratio (data not shown, n =19, P = 0.68). Individual NRTIs did not alter mtDNA content in MDMs (Fig. 3). However, the NRTI combinations ddI/d4T and 3TC/AZT both decreased mtDNA content in MDMs, with the decrease achieving significance in the HIV-1-infected MDMs at 100 μmol/L ddI/d4T (P = 0.04) and 10 and 100 μmol/L 3TC/AZT (P = 0.04 and P = 0.02, respectively).

Figure 3
Figure 3
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Viral replication in MDMs was inhibited by more than 85% by all antiretroviral drugs at a concentration of 10 μmol/L, and combinations of 3 drugs at 10 μmol/L inhibited viral replication by more than 99% (Table 1).

Table 1
Table 1
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The Effect of Antiretroviral Drugs on Phagocytosis by MDMs

To determine whether a reduction in mtDNA content by antiretroviral drugs results in altered MDM function, we assessed the ability of these cells to phagocytose C-opsonized erythrocytes. Adherent MDMs were infected with HIV-1Ba-L 5 days after isolation, antiretroviral drugs were added immediately after infection, and C-mediated phagocytosis was measured 7 days later (Fig. 4). Individually, ddI and d4T decreased phagocytosis by both HIV-1-infected and -uninfected MDMs in a concentration-dependent manner. Culture of MDMs with both ddI/d4T did not further decrease phagocytosis, suggesting no additive or synergistic effect of the 2 drugs. Neither 3TC nor AZT individually inhibited phagocytosis by MDMs. In combination, phagocytosis was inhibited by 3TC/AZT, although this reached significance only in uninfected MDMs. IDV (100 μmol/L) increased phagocytosis by uninfected MDMs by 33% ± 9% above control levels (n = 9). However, when IDV was combined with ddI/d4T, phagocytosis was decreased by an amount similar to that with the NRTIs alone. It is worth noting that, whereas phagocytosis by MDMs was decreased in the presence of 3TC/AZT, when IDV was added to this combination, phagocytosis was either normal (10 μmol/L) or improved (100 μmol/L).

Figure 4
Figure 4
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DISCUSSION

Our data show clearly that selected, commonly used NRTI combinations ddI/d4T and 3TC/AZT decrease mtDNA content in MDMs, with a much greater decrease observed in HIV-infected MDMs than in uninfected cells. HIV-1 infection alone did not alter mtDNA content in MDMs. Furthermore, both ddI/d4T and 3TC/AZT inhibited C-mediated phagocytosis by MDMs, with suppression also evident with ddI and d4T individually. The protease inhibitor IDV augmented C-mediated phagocytosis by uninfected cells. None of the drug combinations altered cell viability, even at concentrations well above those found in serum levels of treated patients.

Reported serum concentrations of antiretroviral drugs vary widely, likely because of differing treatment regimens, variation in absorption or binding to serum albumin, and possible drug interactions. Concentrations used in this study (0.1-100 μmol/L) are clinically attainable, as shown by antiretroviral drug levels in serum of patients receiving these drugs. Patients receiving a combined tablet of 150 mg of 3TC and 300 mg AZT were reported to have mean plasma concentrations of 8 ± 2.6 μmol/L for 3TC and 7.4 ± 3 μmol/L for AZT.54 Maximum serum concentration of ddI in one study was reported at 14 ± 5 μmol/L,55 whereas maximum serum concentration of d4T can range from 3.1-8.9 μmol/L.56 In a cohort of 11 children receiving IDV, the maximum plasma concentration of IDV ranged from 4.5 to 40.4 μmol/L.57

Nucleoside reverse transcriptase inhibitors display a hierarchy regarding their potency of inhibition of polymerase γ in vitro, with ddI and d4T typically showing potent inhibition of polymerase γ at therapeutic levels.26,58 ddI has been shown to efficiently decrease mtDNA synthesis in nerve growth factor primed PC-12 cells59 and PBMCs,23 whereas mtDNA depletion has been shown to be more pronounced in subcutaneous fat samples from patients treated with ddI or d4T than with other NRTIs.19,23,60 Clinically, the combination of ddI and d4T is now used less often because of high rates of adverse effects relating to mitochondrial dysfunction, including peripheral neuropathy, pancreatitis, and lipoatrophy.61-63 Our finding that ddI/d4T decreased mtDNA content in our model is consistent with these clinical observations. That this change occurred only at high concentrations of ddI/d4T may be explained, at least in part, by the fact that monocytes/macrophages do not express thymidine kinase 1, the rate-limiting enzyme in the activation of d4T.7 Therefore, the toxicity of d4T may be less marked in this tissue than in those where it is more efficiently activated.

Although 3TC and AZT are relatively weak inhibitors of mtDNA synthesis in vitro,58 this combination also resulted in decreased cellular mtDNA content in our study. 3TC and AZT have been shown to bind much less tightly to polymerase γ than do ddI or d4T,64 but their use can still be associated with toxicities including lipoatrophy, albeit at lower rates than seen with d4T.17 NRTIs affect tissues differentially, with both clinical and laboratory data demonstrating that AZT is a potent cause of mitochondrial toxicity in muscle25,65,66 and, to a lesser extent, in a lymphoblastic cell line,26 but has no effect on the neuronal PC-12 cell line59 or a human hepatoma cell line.67 Tissue-specific NRTI effects may be explained by the fact that NRTIs require intracellular phosphorylation by cellular kinases, and these enzymes vary in expression between different cell types.68-70 Levels of active and potentially toxic metabolites of NRTIs will therefore vary in different tissues, potentially explaining the tissue-specific side effects of these drugs and the observed effect of 3TC/AZT in our system.

It has been proposed that HIV-1 infection may decrease mtDNA independent of NRTIs, and some in vitro and in vivo studies support this idea,71-73 although the mechanism remains obscure (reviewed by Cherry and Wesselingh74). Our data suggest that HIV-1 infection did not alter mtDNA concentration in macrophages. However, the decrease in mtDNA in MDMs resulting from incubation with ddI/d4T and 3TC/AZT was more pronounced in the HIV-1-infected MDMs than in the uninfected cells from the same donors. This finding suggests that HIV-1 infection may not directly inhibit mitochondrial function in MDMs, but may render the cells more susceptible to NRTI toxicity. This is supported by several studies showing HIV-induced mitochondrial abnormalities in a T-lymphoblastoid cell line, a monocytoid cell line, an HIV-infected human T-cell line, and in lymphocytes from HIV-positive patients undergoing seroconversion, but not in control cell populations.75-77 Hence, our findings suggest that HIV-1 infection itself may predispose individuals to NRTI toxicity.

We demonstrated that ddI/d4T inhibits C-mediated phagocytosis by MDMs by approximately 60%, with less suppression by 3TC/AZT (approximately 30%). There was a concomitant reduction in mtDNA in the presence of the 2 combinations, suggesting that NRTIs may impair phagocytosis through their adverse effects on mitochondrial energy production. Very few studies have examined the effect of NRTIs on macrophage function. Antonelli et al43 described unaltered phagocytosis but decreased intracellular killing of Candida albicans by MDMs exposed to zalcitabine, attendant with reduced mtDNA. Early work characterizing phagocytosis showed that this process is energy dependent,40 and macrophages derive most of their energy for phagocytosis from glucose metabolism and oxidative phosphorylation by mitochondria. Thus, it is logical that a decrease in mtDNA might impair MDM phagocytic activity.

Given that ddI or d4T alone did not alter mtDNA content but decreased phagocytosis by MDMs, this suggests that other mechanisms may also underlie altered phagocytosis by MDMs exposed to NRTIs. NRTIs may alter mitochondrial bioenergetic function by mechanisms distinct from their effect on mtDNA.78 AZT inhibits electron transfer through respiratory enzyme complex I and affects the activity of complex I and III of the mitochondrial respiratory chain,65,79 whereas AZT-treated HIV-infected patients show impaired mitochondrial oxidative metabolism.80 In addition, decreased activity of enzymes of the mitochondrial respiratory chain in an HIV-infected patient displaying symptomatic hyperlactatemia has been reported.81 These studies suggest that NRTIs may alter mitochondrial function via several mechanisms, any one of which could underlie decreased phagocytic capacity seen in the present study. That mitochondrial respiration was not altered by MDMs incubated in the presence of dual NRTI combinations may be because of the redundancy of mitochondria within a cell, which sometimes requires a substantial loss before pathology is evident.82

Although IDV or combinations of 2 NRTIs did not alter mitochondrial respiration, a combination of IDV with 2 NRTIs decreased mitochondrial respiration. It was noted that IDV crystals sometimes formed at high concentrations (100 μmol/L) and that this was associated with abolition of respiration. IDV is a weak base that exhibits a pH-dependent solubility profile and has been shown to precipitate out of solution and form crystals under neutral to mildly alkaline conditions83 at concentrations similar to the maximum concentration used in this study. Clinically, this drug is associated with nephrolithiasis84-86 because of precipitation of this drug in urine at physiological pH and intratubular deposition of crystals, composed of IDV and IDV metabolites,85,87 in the kidneys (reviewed by Perazella88). It is possible therefore that, although IDV crystals were not apparent in some cultures, they may nevertheless have been present and inhibited mitochondrial respiration by MDMs incubated in triple combinations of antiretrovirals.

In contrast to the inhibitory effect of some NRTIs on C-mediated phagocytosis, IDV (100 μmol/L) slightly increased phagocytosis by uninfected MDMs. Immunomodulatory effects of IDV have previously been described. Blasi et al46 demonstrated increased phagocytosis of the opportunistic pathogen C. neoformans by microglia upon exposure to IDV. Several studies have shown activity of HIV-1 PIs toward cellular aspartic proteases, resulting in the modulation of leukocyte activation, antigen presentation and cytotoxic T-cell activity and dendritic cell function.89-91 Cellular proteases are important in immune cell function; calpains are involved in cytoskeletal signaling complexes and in anchoring integrin adhesion receptors to the contractile cytoskeleton,92 and cytoskeletal signaling and rearrangement are central to the process of phagocytosis. This suggests that IDV may directly interact with human proteases to augment phagocytosis. In addition, IDV seems to selectively enhance phagocytosis when combined with 3TC/AZT but not with ddI/d4T. This may be attributable to the higher toxicity of ddI/d4T compared with 3TC/AZT in macrophages. Whereas the immune-enhancing effects of IDV can possibly reverse the minimal inhibition of phagocytosis associated with 3TC/AZT, it seems unable to reverse the inhibition associated with ddI/d4T.

This study extends our knowledge by demonstrating that some NRTIs, both individually and in clinically relevant combinations, adversely affect mitochondrial content of MDMs and that this is associated with reduced phagocytic capacity of macrophages, a crucial function in host surveillance and immunity. These results have important implications for use of these antiretroviral drugs in immunocompromised individuals.

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ACKNOWLEDGMENTS

The authors gratefully acknowledge Merck & Co Inc for kindly supplying the compound indinavir for this study.

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Keywords:

antiretrovirals; HIV-1; macrophages; mitochondrial DNA; phagocytosis

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