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Keywords:

  • dementia;
  • glutamate;
  • glutaminase;
  • HIV-1;
  • macrophage;
  • neurotoxicity

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV-1 strains including ADA and DJV (macrophage tropic, M-tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M-tropic viruses. AZT, an inhibitor of HIV-1 replication, inhibited glutamate generation, demonstrating a linkage between HIV-1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6-diazo-5-oxo-l-norleucine, a glutaminase inhibitor. Supernatants collected from HIV-1-infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate-generating enzyme, such as phosphate-activated mitochondrial glutaminase (PMG) in MP-mediated glutamate production.

Abbreviations used
HAD

HIV-1 associated dementia

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HIV-1

human immunodeficiency virus type 1

L-DON

6-diazo-5-oxo-l-norleucine

M-CSF

macrophage colony-stimulating factor

MCM

macrophage-conditioned media

MDM

monocyte-derived macrophage

MP

mononuclear phagocyte

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

PMG

phosphate-activated mitochondrial glutaminase

PVDF

polyvinyldifluoridene

RP-HPLC

reverse phase high-performance liquid chromatography

Human immunodeficiency virus type 1 (HIV-1) infection and dysregulation of mononuclear phagocyte (MP, macrophages and microglia) immune function is thought to play an important role in the pathogenesis of HIV-1-associated dementia (HAD). A neuro-inflammatory disease that affects 7–15% of AIDS patients, HAD is characterized by neuronal dysfunction including synaptic damage, neuronal degeneration, and cell dropout (Navia et al. 1998; Everall et al. 1999; Rostasy et al. 2000). How infected macrophages and microglia mediate neuronal injury and cognitive dysfunction in HAD remains unclear. It has been hypothesized that MP induce neuronal injury through the production and release of soluble neurotoxic factors, such as glutamate (Giulian et al. 1993; Pulliam et al. 1994; Zink et al. 1999; Belmadani et al. 2001; Jiang et al. 2001).

The predominant excitatory neurotransmitter expressed within the mammalian CNS, glutamate mediates numerous physiological functions through activation of multiple receptors (Cutler and Dudzinski 1974; Fonnum 1984; Orrego and Villanueva 1993). However, high concentrations of extracellular glutamate can induce neuronal damage (Olney 1971; McCall et al. 1979; Choi 1988; Newcomb et al. 1997). It has been reported that HIV-1-infected patients have significantly higher plasma concentrations of glutamate as compared to uninfected controls (Droge et al. 1987; Ollenschlager et al. 1988). Although, HIV-1-infected macrophages appear to be an important cellular source of extracellular glutamate (Jiang et al. 2001), the mechanism by which HIV-1 infection regulates glutamate production remains to be elucidated.

Phosphate-activated mitochondrial glutaminase (PMG), which catalyzes the conversion of glutamine to glutamate, is the primary enzyme for the production of glutamate (Ward et al. 1983; Nicklas et al. 1987; Wurdig and Kugler 1991; Curthoys and Watford 1995) and is also the predominant glutamine-utilizing enzyme of the brain (Holcomb et al. 2000; Kvamme et al. 1982). Glutamine is an amino acid that is present in the brain extracellular fluid in high concentrations (Tossman et al. 1986) and provides an abundant substrate for PMG in vivo (Newcomb et al. 1997). It is thus hypothesized that increased glutamate production from HIV-1-infected MP occurs through the activation and production of PMG.

Isolation and culture of monocyte-derived macrophages (MDM)

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Human monocytes were recovered from peripheral blood mononuclear cells of HIV-1, -2, and hepatitis B seronegative donors after leukopheresis, and then purified by counter-current centrifugal elutriation. Monocytes were cultured as adherent monolayers (3.3 × 106 cells/well in 6-well plates; 1.1 × 106 cells/well in 24-well plates) and differentiated for 7 days in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% heat-inactivated pooled human serum, 50 µg/mL gentamicin (Sigma), 10 µg/mL ciprofloxacin (Sigma), 4 mm glutamine (Fisher, Pittsburgh, PA, USA), and macrophage colony-stimulating factor (M-CSF, 1000 U/mL highly purified recombinant human M-CSF; a generous epst from Genetics Institute, Inc., Cambridge, MA, USA). All tissue reagents were screened and found negative for endotoxin (< 10 pg/mL; Associates of Cape Cod, Inc., Woods Hole, MA, USA) and mycoplasma contamination (General-probe II; General-probe Inc., San Diego, CA, USA).

Infection and treatment of MDM

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Seven days after plating, MDM were infected with HIV-1ADA, HIV-1DJV, or HIV-189.6, at a multiplicity of infection of 0.1 virus/target cell. Viral stocks were screened for mycoplasma and endotoxin using hybridization and Limulus amebocyte lysate assays, respectively. In some experiments, MDM were pre-treated for 24 h before infection with 5 µm AZT, a retroviral replication inhibitor (Sigma) or 1 mm 6-diazo-5-oxo-l-norleucine (L-DON, Sigma), a glutaminase inhibitor. Culture medium was half-exchanged every 2 days. The samples treated with AZT or L-DON were re-treated when culture medium was exchanged. In some experiments, cells were treated at 7 days after infection with staurosporine (Calbiochem, San Diego, CA, USA), an inducer of apoptosis (Kovacs et al. 1999), for 24 h before the collection of supernatants. Cells were infected from 1 to 10 days and then treated with varying concentrations of glutamine (Fisher) ranging from 0 to 25 mm for 12–72 h at 37°C prior to collection of supernatants.

Measurement of reverse transcriptase activity

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Reverse transcriptase activity was determined in triplicate samples of cell culture fluids. For this assay 10 µL of supernatant was added to a reaction mixture of 0.05% Nonidet P-40, 10 µg/mL poly(A), 0.25 µg/mL oligo(dT), 5 mm dothiothreitol, 150 mm KCl, 15 mm MgCl2, and [3H]dTTP (NEN, Boston, MA, USA) in Tris-HCl buffer (pH 7.9) for 24 h at 37°C. Radiolabeled nucleotides were precipitated with cold 10% trichloroacetic acid and 95% ethanol in an automatic FiltermateTM Cell Harvester (Packard, Meridan, CT, USA) on 96-well unifilter GF/C plates (Packard). MicroScint-20 liquid scintillation cocktail (Packard) was added and radioactivity determined in a TopCount NXT liquid scintillation counter (Packard).

Cell viability assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Cell cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) mitochondrial dehydrogenase assay. Cells were incubated with a 1 : 10 dilution of MTT solution to cell media for 20 min at 37°C. The extent of MTT conversion to formazan by mitochondrial dehydrogenase was determined by measuring optical density at 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The ratio of optical density from treated cells to optical density from control cells reflected the percentage of surviving cells.

Analyses of glutamate and glutamine by RP-HPLC

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

RP-HPLC analysis was performed using an HP series II 1090 liquid chromatograph and HP1046A fluorescence detector (Agilent, Palo Alto, CA, USA). The concentrations of the working glutamate standard solutions (Sigma) were 0, 0.2, 0.5, 1, 2, 5, 10, 25, and 50 µm, respectively. The standard solutions were stored at −80°C and thawed prior to use. Each 5 µL sample or standard solution was derived with ortho-phthalaldehyde (Agilent, Palo Alto, CA, USA) by autosampler for fluorescence detection. For each experiment, 300 µL of sample was used and was mixed with equal volumes of 3% perchloric acid (Aldrich, Milwaukee, WI, USA). This mixture was then immediately neutralized with 14 µL saturated potassium carbonate (Aldrich). Samples were centrifuged at 14 000 rpm (18 600 g) for 15 min at 4°C. Samples were then injected into an RP-HPLC system. The experiments utilized 4.6 × 150 mm, 3.5 µm ZORBAX Eclipse AAA analytical columns (Agilent). Glutamate detection was monitored using a fluorescence detector with wavelengths of excitation at 340 nm and emission at 450 nm. A gradient elution program was optimized for glutamate measurement with a flow rate of 0.45–0.80 mL/min. This program began at 100% mobile phase A, containing 2.72 g/L sodium acetate tri-hydrate, 180 µL/L triethylamine, and 3 mL/L tetrahydrofuran (the solution was adjusted to pH 7.2 with 2% acetic acid before adding tetrahydrofuran). At 17 min, 60% mobile phase B was used containing 2.72 g/L sodium acetate tri-hydrate, 40% acetonitrile, and 40% methanol (the solution was adjusted to pH 7.2 by using 2% acetic acid before mixing in acetonitrile and methanol). Total analysis time was 30 min.

SYBR green real-time RT-PCR determination of glutaminase mRNA expression

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

TRIZOL reagent (Gibco, Grand Island, NY, USA) was used to extract total RNA from MDM that were pre-treated with or without 5 µm AZT and infected with HIV-1ADA for 3, 5, 7, or 10 days. Total RNA was purified by chloroform/ethanol extraction and treated with DNAse prior to amplification. Glutaminase primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA). One hundred nanograms of total RNA and 200 nm glutaminase sense primer (5′-GCTGTGCTCCATTGAAGTGACT-3′) and antisense primer (5′-GGGCAGAAACCACCATTAGC-3′) were utilized for amplification. GAPDH primers were purchased (Applied Biosystems) and used at 200 nm. Glutaminase and GAPDH were amplified using a one-step RT-PCR kit with a SYBR Green reporter (Applied Biosystems) according to the manufacturer's instructions. Real-time RT-PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). All reactions were carried out in triplicate and performed in a 50 µL volume. The reaction cycle parameters were an initial incubation at 50°C for 2 min, denaturation at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The SYBR green signal was continually monitored and the cycle to cross threshold determined for each sample. To confirm amplification specificity, the PCR products were subjected to melting temperature (Tm) dissociation curve analysis. In parallel, no amplification controls were run to rule out the presence of fluorescence contaminants in the sample or in the thermocycler heat block. A ratio of glutaminase to GAPDH was determined for each sample.

Detection of glutaminase by western blot

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Macrophage-conditioned media (MCM) from control and HIV-1-infected MDM were collected 7 days post-infection and cells were solubilized in 100 µL M-PER Mammalian Protein Extraction Buffer (Pierce, Rockford, IL, USA). MCM from multiple wells were centrifuged together at 100 000 × g for 50 min. The resulting pellets were resuspended in 200 µL Laemmli Sample Buffer. Lysate protein levels were quantified via BCA Protein Assay (Pierce). For western blot analysis, primary antibody (IgG) for phosphate-activated mitochondrial glutaminase (a generous epst from Norman Curthoys, Colorado State University, Fort Collins, CO, USA) was purified via fast protein, peptide and polynucleotide liquid chromatography (FPLC) methods following the protocol provided by the manufacturer (Amersham Piscataway, NJ, USA). Proteins from lysates and resuspended supernatant pellets were separated on a 10% Tris-HCl gel by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After electrophoretic transfer to polyvinyldifluoridene (PVDF) membranes (Millipore and Bio-Rad), proteins were treated with purified primary antibody overnight at 4°C followed by a horseradish peroxidase-linked secondary anti-rabbit antibody (1 : 5000 dilution; Cell Signaling Technology, Beverly, MA, USA). Antigen–antibody complexes were visualized by enhanced chemiluminescence western blotting on Hyperfilm ECL (Amersham). Gel and immunoblot images were quantitated using Nuceleovision software (Nucleotech, San Carlos, CA, USA).

HIV-1 infection enhances glutamate production in human MDM

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

To determine whether glutamate production is enhanced following HIV-1 infection, MCM was collected 3, 5, 7, and 10 days after infection with HIV-1ADA and the glutamate concentration was determined by RP-HPLC (Fig. 1). MCM from infected cells displayed higher rates of glutamate production than MCM from uninfected cells at all time points collected. Glutamate reached maximal levels at day 7 post-infection and then leveled off by day 10. To account for any donor variation, glutamate levels in supernatants from 10 human MDM donors were analyzed (Table 1). Based on these data, we proposed that a link exists between HIV-1ADA infection and glutamate production in MDM.

image

Figure 1. Effects of the anti-retroviral drug AZT on HIV-1-mediated glutamate production in MDM. Elutriated and M-CSF differentiated human MDM were pre-treated with 5 µm AZT and infected with HIV-1ADA after 7 days in culture. Cell culture supernatants were collected 3–10 days after initial infection. Cells were washed three times and incubated for an additional 24-h period in serum-free neurobasal media treated with AZT 5 µm after desired infection time. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC. Data are expressed as absolute concentration of glutamate (µm). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control; #denotes p < 0.01 in comparison with HIV-1ADA.

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Table 1.  Effects of HIV-1ADA infection after 7 days on glutamate concentrations (µm) in MDM cultures
DonorControlHIV-1ADA
  1. Each entry is representative of separate MDM donors and are the actual values found in each sample. The mean value (n = 3) ± SD for samples analyzed by RP-HPLC is given. The data were evaluated statistically by the analysis of variance (anova), followed by the Tukey-test for paired observations. *Statistically different from control (p < 0.05). †Statistically different from control (p < 0.01).

A6.80 ± 0.89496.20 ± 4.714
B10.88 ± 0.45269.28 ± 11.770*
C4.58 ± 0.358173.43 ± 4.126
D15.17 ± 2.56642.80 ± 7.593*
E9.32 ± 0.20929.65 ± 2.342
F15.19 ± 0.54147.28 ± 8.991*
G9.22 ± 0.48331.36 ± 1.770
H13.75 ± 0.58785.43 ± 5.947
I10.66 ± 0.56962.10 ± 3.528
J11.60 ± 1.768110.36 ± 9.275

To confirm whether the increase in glutamate production from MDM was indeed HIV-1 dependent, cells were infected in the presence of the anti-retroviral drug AZT. For this work, MDM were infected with HIV-1ADA following a 24- h pre-treatment with 5 µm AZT. Every 2 days after the initial infection, fresh AZT was added to the cultures. Supernatants were collected 3, 5, 7, and 10 days after infection and glutamate concentration was determined by RP-HPLC. Our results demonstrated that, at all time points, HIV-1-infected MDM pre-treated with AZT did not induce significant increases in glutamate production when compared to HIV-1-infected MDM alone (Fig. 1). These data suggest that the increase in glutamate production from MDM was a result of HIV-1 infection.

Viral strain effects on glutamate generation

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Having demonstrated that MDM infected with HIV-1ADA could enhance the production of glutamate, we next examined the effects of other HIV-1 viral strains. For this work, MDM were infected with HIV-1ADA, HIV-1DJV, and HIV-189.6 for 7 days. Our data showed that the amount of glutamate produced was dependent upon the viral strain used for infection (Fig. 2a). HIV-1ADA and HIV-1DJV both caused increases in glutamate production, and HIV-1ADA was the most effective at inducing an increase. HIV-189.6 had no significant effect on glutamate production as compared to uninfected controls.

image

Figure 2. Comparison of viral strain effects on glutamate production and cell viability. Human MDM were infected with HIV-1ADA, HIV-1DJV, and HIV-189.6 for 7 days. Cells were washed three times and incubated for an additional 24-h period in serum-free neurobasal media. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC (a). Data are expressed as absolute concentration of glutamate (µm). Cell viability was determined by MTT assay (b). Reverse-transcriptase activity of the different HIV strains is also represented (c). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control. (d) and (e) show the correlation of glutamate production to cell viability and to viral reverse transcriptase activity, respectively.

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To determine whether cell viability played a role in the observed viral strain differences in glutamate production, an MTT assay was performed following media collection (Fig. 2b). We observed a correlation (Fig. 2d, r2 = 0.9977) between cell viability and glutamate production in macrophages infected with different viral strains. HIV-1ADA induced the highest levels of glutamate production and caused the greatest decrease in cell viability. HIV-1DJV caused moderate increases in glutamate generation and caused a moderate decrease in cell viability. HIV-189.6, which did not significantly enhance glutamate production, caused only a slight decrease in cell viability as compared to control.

Having observed that cell viability diminished in response to infection with different viral strains, we next measured the level of productive infection using reverse transcriptase activity. HIV-1ADA, as expected, had the highest level of reverse-transcriptase activity (9424 ± 946 cpm). HIV-1DJV had the next highest level and HIV-189.6 showed only minimal reverse transcriptase activity (4982 ± 1337 and 574 ± 403 cpm, respectively) (Fig. 2c). The level of productive infection correlated with the amount of glutamate produced (Fig. 2e, r2 = 0.9887). Together, these data suggest that the level of HIV-1 infection and the subsequent decrease in cell viability correlates with the amount of glutamate produced by MDM cultures. Thus, MDM infected with a viral strain that induces high infectivity and subsequently causes a large decrease in cell viability, will also generate a high level of glutamate production.

Cell viability effects on glutamate production

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

To further determine whether there is a correlation between cell viability and glutamate production, MCM was collected 3, 5, 7, and 10 days after infection with HIV-1ADA and the glutamate concentration was determined by RP-HPLC (Fig. 3a). An MTT assay was also performed following media collection to determine cell viability (Fig. 3b). A correlation (Fig. 3c, r2 = 0.4772, p = 0.0129) between cell viability and glutamate production was observed in macrophages at different days after infection. The significant reduction in cell number at day 10, due to prolonged exposure to HIV-1, makes the glutamate production level-off at 10, creating a lower correlation efficiency. Based on this data, we proposed that a link exists between HIV-1ADA infection, cell viability, and glutamate production in MDM.

image

Figure 3. The effects of the duration of HIV-1 infection on glutamate production and cell viability. Human MDM were infected with HIV-1ADA for 3, 5, 7, and 10 days. After desired incubation time, cells were washed three times and incubated for an additional 24-h period in serum-free neurobasal media. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC (a). Data are expressed as absolute concentration of glutamate (µm). Cell viability was determined by MTT assay (b). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control. The correlation of glutamate production to cell viability is also shown (c).

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To determine if the correlation between cell viability and glutamate production in macrophages are HIV-1 specific, MDM were treated with different concentrations of staurosporine (0.1–10 µm), which is a caspase activator and inducer of apoptosis (Kovacs et al. 1999). MCM were collected after 24 h of treatment and the glutamate concentration was determined by RP-HPLC (Fig. 4a). An MTT assay was also performed following media collection (Fig. 4b). We observed a correlation (Fig. 4c, r2 = 0.5757, p = 0.0042) between cell viability and glutamate production in macrophages treated with different concentrations of staurosporine. As the concentration of staurosporine increased, there was a substantial decrease in cell viability, which correlated with how much glutamate is produced by macrophages. This result is similar to that seen in HIV-1-infected macrophages.

image

Figure 4. Staurosporine, an inducer of apoptosis, shows comparable trends in glutamate production and cell viability. Human MDM were infected with HIV-1ADA for 7 days. Cells were washed three times and treated with staurosporine with concentrations ranging from 0 to 10 µm and incubated for an additional 24-h period in serum-free neurobasal media. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC (a). Data are expressed as absolute concentration of glutamate (µm). Cell viability was determined by MTT assay (b). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control. The correlation of glutamate production to cell viability is shown in (c).

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Glutamine enhances glutamate production

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

As the release of glutamate from dying cells is unlikely to account for the high levels of glutamate we detected in the MCM from HIV-1-infected cells, we next explored other potential sources of glutamate. In vivo, glutamate is generated from glutamine by the enzyme PMG. We hypothesized that an up-regulation or release of this enzyme from HIV-1-infected MDM could be a possible source of the observed glutamate increase. For this work, glutamine-free media with or without the addition of 5 mm glutamine was added to control and HIV-1ADA-infected MDM. Supernatants were collected at 12, 24, 48, and 72 h following glutamine addition. Supernatants collected from HIV-1-infected cells cultured in glutamine-free media showed only a small increase in the production of glutamate when compared to uninfected cells (Fig. 5a). However, supernatants from HIV-1ADA-infected MDM incubated with media containing 5 mm glutamine showed a dramatic increase in glutamate production at all time points as compared to uninfected cells.

image

Figure 5. HIV-1-mediated glutamate production is dependent on the addition of glutamine. Human MDM were infected with HIV-1ADA for 7 days. Cells were washed three times and incubated for 12–72 h in serum-free neurobasal media with or without 5 mm glutamine (a). A dose–response curve was also generated after a 48 h treatment with 0–25 mm glutamine (b). In (c), the effects of a glutaminase inhibitor on HIV-1-mediated glutamate production in MDM was examined. Human MDM were pre-treated with 1 mm L-DON (a glutaminase inhibitor) and infected with HIV-1ADA after 7 days in culture. Cells were washed three times and incubated for an additional 24-h period in serum-free neurobasal media treated with 1 mm L-DON. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC. All data are expressed as absolute concentration of glutamate (µm). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control; #denotes p < 0.01 in comparison with HIV-1ADA.

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Next, we examined the dose-dependent effects of glutamine on glutamate production. For this work, control and HIV-1-infected MDM were treated with doses of glutamine ranging from 0 to 25 mm. After 48 h of glutamine treatment, supernatants were collected and the glutamate concentration was measured (Fig. 5b). Uninfected MDM showed only a small dose-dependent increase in glutamate production following glutamine addition. In contrast, MDM infected with HIV-1 showed a dramatic dose-dependent increase in glutamate production when treated with glutamine. Based on this data, we hypothesized that enhanced glutamate production in HIV-1-infected MDM is mediated, at least in part, by a glutamine-to-glutamate converting enzyme, such as PMG.

A glutaminase inhibitor blocks the generation of glutamate

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

To confirm that PMG was involved in the conversion of glutamine to glutamate in our HIV-1-infected cells, the glutaminase inhibitor, L-DON (1 mm), was used. Beginning 24 h prior to infection with HIV-1ADA, MDM were pre-treated with L-DON and then re-treated every 2 days thereafter. Media with 5 mm glutamine was added 24 h prior to supernatant collection on days 3, 5, 7, and 10 post-infection. MDM pre-treated with L-DON prior to HIV-1 infection produced less glutamate than HIV-1-infected MDM alone (Fig. 5c). Treatment with L-DON did not cause changes in cell viability (data not shown). This finding suggests that PMG may contribute to enhanced glutamate production in HIV-1-infected MDM.

HIV-1 infection of MDM does not alter PMG mRNA expression

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Next we determined whether HIV-1 infection enhances PMG expression in MDM. To test this, MDM were infected for 3, 5, 7, or 10 days with HIV-1ADA in the presence or absence of 5 µm AZT. Total RNA was collected in TRIZOL and purified by chloroform/ethanol extraction. Using primers for PMG and GAPDH (loading control), mRNA was amplified by real-time RT-PCR. A ratio of PMG to GAPDH was then determined for each sample. HIV-1 infection of MDM did not lead to enhanced PMG expression at any time point examined (Fig. 6).

image

Figure 6. Total PMG mRNA expression in HIV-1-infected MDM. Total RNA was extracted with TRIZOL from human MDM infected for 3, 5, 7, or 10 days with HIV-1ADA in the presence or absence of 5 µm AZT. PMG and GAPDH mRNA was amplified and visualized by real-time RT-PCR using SYBR Green and an ABI Prism 7000 Sequence Detection System. The ratio of PMG/GAPDH was determined. Each reaction was performed in triplicate.

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PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Although the total amount of PMG message did not increase, we hypothesized that HIV-1 infection might increase the amount of PMG that is released from MDM. To test this hypothesis, supernatants and cell lysates were collected from HIV-1ADA-infected and replicate uninfected MDM, 7 days post-infection. Supernatants from multiple wells were first concentrated through centrifugation (100 000 × g, 50 min). Following electrophoresis and transfer to PVDF membrane, the proteins were detected with a purified antibody to PMG. Two peptides with apparent molecular mass at ∼60 kDa and ∼50 kDa were identified (Fig. 7). Both anti-glutaminase immuno-reactive peptides were also recovered from media of both control and HIV-1-infected MDM. The amount of ∼60 kDa protein appears lower in HIV-1-infected MDM lysates as compared to uninfected MDM lysates, whereas the amount of ∼50 kDa appears the same as compared to uninfected MDM lysates (Fig. 7a). Notably, supernatants from HIV-1-infected MDM had 62.57% (n = 3, p < 0.05) and 49% higher levels (n = 3, p > 0.05) of ∼50 kDa and ∼60 kDa protein, respectively, when compared to uninfected MDM (Figs 7b and c).

image

Figure 7. PMG protein expression in cell lysates and supernatants from HIV-1-infected MDM. Human MDM were infected with HIV-1ADA for 7 days. Whole cell lysates and MDM conditioned media (MCM) were collected. Protein amounts were normalized for cell lysates prior to loading onto the gel. MCM from multiple wells were centrifuged together at 100 000 × g for 50 min. The resulting pellets were resuspended in 200 µL Laemmli Sample Buffer. PMG protein expression in cell lysates and concentrated MCM were determined by western blot. (a) and (b) show representative western blots of cell lysates (n = 3) and concentrated supernatants (n = 3), respectively. Crude rat brain homogenate (lane 1 in a and b) and recombinant PMG (rPMG, lane 5 in b) were used as positive controls. Concentrated culture media alone without incubating with MDM was used as a negative control (lane 4 in b). (c) shows the calculated percentage of control for all experiments. *Denotes p < 0.05 when compared to control.

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Glutamine enhances glutamate production in collected supernatants

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Next, we confirmed that higher concentrations of PMG exist in the supernatants of HIV-1-infected MDM. For this work, control and HIV-1ADA-infected MDM were washed 7 days after infection and glutamine-free media was added. After 24 h, supernatants were collected and treated with or without 5 mm glutamine. RP-HPLC was performed 12, 24, 48, and 72 h following glutamine treatment. Glutamine-free supernatants collected from HIV-1-infected cells showed only a small increase in the production of glutamate when compared to uninfected cells (Fig. 8a). However, supernatants from HIV-1ADA-infected MDM in which 5 mm glutamine had been added, showed a dramatic increase in glutamate production at all time points as compared to uninfected cells (Fig. 8a).

image

Figure 8. HIV-1-mediated glutamate production is dependent on the addition of glutamine. Human MDM were infected with HIV-1ADA after 7 days in culture. Seven days after infection, cells were washed three times and incubated for an additional 24-h period in glutamine-free and serum-free neurobasal media. Supernatants were collected and incubated for 12–72 h with or without 5 mm glutamine (a). A dose–response curve was also generated after a 48 h incubation of supernatants with 0–25 mm glutamine (b). Beginning 24 h prior to infection with HIV-1ADA, MDM were pre-treated with L-DON (0.1, 1, or 2.5 mm) and then re-treated every 2 days thereafter. At 7 days post-infection, cells were washed three times and serum-free, glutamine-free neurobasal media was added 24 h prior to supernatant collection. Collected glutamine-free conditioned media (MCM) were treated for 48 h with 5 mm glutamine (c). Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC. Data are expressed as absolute concentration of glutamate (µm). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control.

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We also examined the dose-dependent effects of glutamine on glutamate production at doses of glutamine ranging from 0 to 25 mm. Collected glutamine-free supernatants were treated for 48 h with glutamine before RP-HPLC was performed (Fig. 8b). Uninfected MDM supernatants showed only a small dose-dependent increase in glutamate production following glutamine addition. In contrast, supernatants from HIV-1-infected MDM showed a dramatic dose-dependent increase in glutamate production when treated with glutamine. Together, these data confirm that not only was glutaminase present in HIV-1-infected MDM supernatants, but also it could actively convert glutamine to glutamate.

To further confirm PMG activity in HIV-1-infected MDM supernatants, a glutaminase inhibitor, L-DON (1 mm), was used. Beginning 24 h prior to infection with HIV-1ADA, MDM were pre-treated with L-DON and then re-treated every 2 days thereafter. Media void of 5 mm glutamine was added 24 h prior to supernatant collection at 7 days post-infection. Collected glutamine-free supernatants were treated for 48 h with 5 mm glutamine before RP-HPLC was performed. Supernatants from MDM pre-treated with L-DON prior to HIV-1 infection produced less glutamate than HIV-1-infected MDM alone after the addition of glutamine (Fig. 8c). This result further confirms that activated glutaminase is present in HIV-1-infected MDM supernatants and could convert glutamine to glutamate.

Staurosporine treatment enhances glutamate production in collected supernatants

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

Next, we wished to confirm the presence of PMG in the supernatants of MDM damaged by staurosporine in comparison with HIV-1-infected MDM. Both staurosporine and HIV-1 infection have been shown to induce macrophages to produce increased amounts of glutamate while reducing cell viability in the process (Fig. 9b). For this work, control and HIV-1ADA-infected MDM were washed 7 days after infection and treated with and without 1 µm of staurosporine in glutamine-free media. After 24 h, supernatants were collected and treated with a dose range of glutamine from 0 to 25 mm. RP-HPLC was performed 48 h following glutamine treatment (Fig. 9a). Cell viability was determined using an MTT assay performed following the collection of supernatants (Fig. 9b). Although staurosporine induced more than 40% cell death of MDM, supernatants collected from staurosporine-damaged MDM only slightly increased the transduction of glutamine to glutamate. In contrast, supernatants from HIV-1-infected MDM significantly enhanced glutamate production from glutamine as compared with control and staurosporine-treated supernatants. Additionally, the highest levels of glutamine to glutamate production were observed with supernatants collected from HIV-1-infected MDM treated with staurosporine (Fig. 9a). There also was a dramatic decrease in cell viability in HIV-1-infected cells treated with staurosporine, which corresponds to increased production of glutamate (Fig. 9b). Together, these data confirm that HIV-1-mediated glutamate production, as a response to cell death, is, at least in part, through PMG release.

image

Figure 9. Glutamate production and cell viability in HIV-1-infected macrophages treated with an apoptosis inducer, staurosporine. Human MDM were infected with HIV-1ADA after 7 days in culture. At 7 days post-infection, control and HIV-1-infected cells were washed three times and treated with 1 µm staurosporine for an additional 24-h period in serum-free, glutamine-free neurobasal media. Following a 24 h incubation, MDM conditioned media (MCM) from the four conditions: control, staurosporine-treated, HIV-1 infected, and HIV-1 infected treated with staurosporine; were collected and incubated with 0–25 mm glutamine for an additional 24-h period. Samples were deproteinized with 3% perchloric acid and treated with saturated potassium carbonate. The concentration of glutamate in cell-free supernatants was determined by RP-HPLC. Data are expressed as absolute concentration of glutamate (µm) (a). Cell viability as determined by MTT assay is also shown (b). Results are expressed as average ± SD of triplicate samples and are representative of three independent experiments. *Denotes p < 0.01 in comparison to control; #denotes p < 0.01 in comparison to HIV-infected cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

In this report, we investigated the potential link between phosphate-activated mitochondrial glutaminase (PMG) expression and glutamate production in HIV-1-infected macrophages. Our results showed that HIV-1 infection of monocyte-derived macrophages (MDM) enhanced glutamate production, an effect that was blocked by treatment with the antiviral drug, AZT. Moreover, the increase in glutamate production observed was shown to be dependent upon the presence of glutamine and could be inhibited by treatment with the PMG inhibitor, L-DON. Notably, different viral strains of HIV-1 induced variable increases in glutamate production based on their level of infectivity and effect on cell viability, suggesting a role for HIV-1-mediated cell death in the production of glutamate. Moreover, levels of PMG were shown to be higher in the supernatants of HIV-1-infected MDM as compared to controls. These observations provide evidence that PMG may play a role in the HIV-1 dependent increase in glutamate production from MDM.

The importance of HIV-1-infected and immune-activated MP in HAD is widely recognized (Koenig et al. 1986; Glass et al. 1995; Wiley 1995; Strizki et al. 1996; Gendelman 1997; Nath and Geiger 1998). However, the mechanisms by which HIV-1 infection induces MP-mediated neurotoxicity are still largely unresolved. Based upon the currently understood neuropathogenesis of HIV-1 infection, it is hypothesized that secretory products from infected and activated MP contribute to neuronal compromise and ultimately neuronal dropout via apoptosis (Tornatore et al. 1991; Moses et al. 1993; Lipton and Gendelman 1995; Nath et al. 1995; Jiang et al. 2001; Zheng et al. 2001b; Gabuzda et al. 2002). Indeed, a variety of in vitro assay systems have demonstrated that macrophage secretory products can induce the over-activation of neuronal NMDA receptors and result in neuronal death. Our initial characterization of MP neurotoxic products suggested that an NMDA-like soluble factor, such as glutamate, was responsible for MP-mediated neuronal injury (Jiang et al. 2001; Zheng et al. 2001b). This observation was confirmed by calcium imaging analysis and neuronal antigen ELISA. This work showed that MK-801, an NMDA receptor channel blocker, partially inhibited neuronal damage caused by secretory products released from HIV-1-infected MDM. These observations suggest that glutamate-induced over-activation of neuronal NMDA receptors is one mechanism by which HIV-1-infected MP may induce neuronal injury in HAD (Jiang et al. 2001; Zheng et al. 2001b).

In this report, we have demonstrated that the increase in glutamate production from HIV-1-infected MDM might be mediated through the release of activated glutaminase. These findings suggest that the same infected macrophages that are secreting glutamate may also be producing activated forms of the conversion enzyme, PMG. Therefore, it is possible that PMG could contribute to excess glutamate production and macrophage-mediated neuronal destruction. Certainly, further studies are required to determine the relative contributions of both glutamate and PMG to neuronal injury during HIV-1 infection of the nervous system.

As the predominant glutamine-utilizing enzyme in the brain, PMG catalyzes the enzymatic conversion of glutamine to glutamate and ammonia (Ward et al. 1983; Nicklas et al. 1987; Curthoys and Watford 1995; Holcomb et al. 2000) and contributes to the glutamate neurotransmitter pool (Kvamme et al. 1982). Glutamine is present in the brain extracellular fluid in high concentrations (Tossman et al. 1986) and provides an abundant substrate for PMG in vivo (Curthoys and Watford 1995; Newcomb et al. 1997). It has been reported that the concentration of glutamine in the extracellular space between astrocytes and neuronal cells is around 0.25 mm (Hagberg et al. 1985). The concentration of glutamine ranges between 0.3 and 0.5 mm in the CSF and between 2 and 4 mm in the bulk nervous tissue (Newcomb et al. 1997). Certainly, an up-regulation in PMG within this brain microenvironment would affect the delicate balance between glutamine and glutamate levels. Previous research has suggested that PMG released from damaged neurons can accelerate cell death in surrounding neurons (Newcomb et al. 1997). Our results further extend these observations and suggest that HIV-1-infected macrophages could be another important source of PMG and play an important role in glutamate up-regulation.

Indeed, several lines of evidence presented within this report support this notion. First, the up-regulation of glutamate from HIV-1-infected MDM is glutamine dependent (Fig. 4). Second, it is possible to block the glutamate increase with the antiviral drug AZT and the PMG inhibitor L-DON (Figs 1, 5 and 8, respectively). Third, glutamate concentrations from HIV-1-infected MDM (between 30 and 170 µm from 10 human donors, Table 1) are higher than in uninfected MDM. This concentration range is neurotoxic and consistent with our previous report regarding direct neurotoxicity by HIV-1-infected MDM (Jiang et al. 2001; Zheng et al. 2001a,b). Fourth, the amount of PMG appears to be higher in supernatants from HIV-1-infected MDM than uninfected MDM (Fig. 7), although the total mRNA and protein expression of PMG does not increase (Figs 6 and 7, respectively). Interestingly, we observed that the ∼60 kDa glutaminase protein was decreased in cell lysates 7 days post-HIV-1 infection, whereas the ∼50 kDa glutaminase protein remained the same (Fig. 7). The exact mechanism by which PMG is regulated during HIV-1 infection awaits further investigation. It is important to note that there is a correlation between HIV-1 infection, decreased cell viability, and increased glutamate production in MDM (Figs 2d and e). HIV-1ADA caused the largest decrease in MDM cell viability and consequently the highest level of glutamate production. In contrast, HIV-189.6 did not induce cell death and induced only a minimal increase in glutamate production. This observation is consistent with the previously published finding that damaged neurons produce PMG, leading to increased extracellular concentrations of glutamate, and subsequent injury to surrounding neurons (Newcomb et al. 1997).

Interestingly, non-HIV-1-induced MDM cell death, by staurosporine, also caused the increases in glutamate production (Fig. 4a). However, although staurosporine induced similar or higher cell death than HIV-1 infection, the increases in glutamate production appears to be lower than HIV-1-infected MDM. Furthermore, the supernatants from staurosporine-damaged MDM only slightly increased the transduction of glutamine to glutamate. In contrast, HIV-1-infected MDM supernatants significantly enhanced glutamate production from glutamine, in vitro. Additionally, when HIV-1-infected MDM were treated with staurosporine, the highest levels of glutamine to glutamate production were observed when compared with supernatants from HIV-1-infected or staurosporine-treated MDM. It is possible that HIV-1 infection not only causes the production of glutamate and release of PMG through cell death, but also manages to increase the activated state of the PMG enzyme. This hypothesis certainly deserves further investigation. Furthermore, how macrophage activation affects this process also remains an important area of investigation. It is well known that HIV-1-infected and activated MP are associated with neuronal injury during HAD and HIV-1 encephalitis (Koenig et al. 1986; Glass et al. 1995; Wiley 1995; Strizki et al. 1996; Gendelman 1997; Nath and Geiger 1998; Power et al. 1998). How immune activation changes the fate of glutaminase during HAD would help to further our knowledge regarding the exact role of this enzyme in HIV-1 neuropathogenesis.

While we believe that glutamate and glutaminase are important players in MP-mediated neuronal injury during HAD, we acknowledge that HIV-1-infected and immune-activated MP are capable of producing a wide variety of other toxic factors. These factors include pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin-1 beta (Gelbard et al. 1993), arachidonic acid and its metabolites (Genis et al. 1992), platelet activating factor (Gelbard et al. 1994), quinolinic acid (Heyes et al. 1991), NTox (Giulian et al. 1996), nitric oxide (Adamson et al. 1996), and reactive oxygen species (Mollace et al. 2001). Ultimately, excitotoxic damage mediated by high concentrations of glutamate, alone or in combination with other MP-secreted neurotoxins, could be the primary cause of neuronal demise in HAD.

In summary, this report has demonstrated that HIV-1 infection leads to increased glutamate production by a PMG-dependent mechanism. Our data suggest that HIV-1 infection can reduce MDM viability causing a release of intracellular glutamate and PMG from damaged cells into the extracellular space. This extracellular PMG could mediate the conversion of extracellular glutamine to glutamate and lead to further increases in glutamate concentrations. By identifying PMG as an important mediator in HIV-1-induced glutamate production, we hope to provide a new therapeutic target for the treatment of HAD and provide important avenues to combat this devastating disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References

We kindly thank Drs Howard Gendelman (CNND) and Sandy Markey (NIMH) for scientific support and fruitful discussion. Hui Peng, Lisa Ryan, Clancy McNally, Nathan Erdmann, Yunlong Huang, My Hanh Che, Mary Robinson (Colorado State University), Barb Switzer and the UNMC Monoclonal Antibody Facility provided technical support for this work. Drs Kim Carlson and Tsuneya Ikezu provided critical reading of the manuscript. Julie Ditter, Robin Taylor, Nell Ingraham, and Theresa Grutel provided outstanding administrative and secretarial support. This work was supported in part by research grants by the National Institutes of Health: R01 NS 41858-01, P20 RR15635-01 and 1 P01 NS043985 (JZ).

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Isolation and culture of monocyte-derived macrophages (MDM)
  5. Infection and treatment of MDM
  6. Measurement of reverse transcriptase activity
  7. Cell viability assay
  8. Analyses of glutamate and glutamine by RP-HPLC
  9. SYBR green real-time RT-PCR determination of glutaminase mRNA expression
  10. Detection of glutaminase by western blot
  11. Statistical analysis
  12. Results
  13. HIV-1 infection enhances glutamate production in human MDM
  14. Viral strain effects on glutamate generation
  15. Cell viability effects on glutamate production
  16. Glutamine enhances glutamate production
  17. A glutaminase inhibitor blocks the generation of glutamate
  18. HIV-1 infection of MDM does not alter PMG mRNA expression
  19. PMG protein expression is increased in supernatants, but not cell lysates, from HIV-1-infected macrophages
  20. Glutamine enhances glutamate production in collected supernatants
  21. Staurosporine treatment enhances glutamate production in collected supernatants
  22. Discussion
  23. Acknowledgements
  24. References
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