Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dickie, P.
Right arrow Articles by Lee, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dickie, P.
Right arrow Articles by Lee, R.
(Journal of Leukocyte Biology. 2001;70:592-600.)
© 2001 by Society for Leukocyte Biology

A defect in HIV-1 transgenic murine macrophages results in deficient nitric oxide production

Peter Dickie, Amanda Roberts and Raymond Lee

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

Correspondence: Peter Dickie, 1-40 HMRC, University of Alberta, Edmonton, AB T6G 2S2, Canada. E-mail: peter.dickie{at}ualberta.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV transgenic mice bearing multiple copies of a noninfectious ({Delta}gag/pol) proviral DNA were tested for the systemic production of nitric oxide (NO). Serum levels of NO metabolites were reduced about 50% in HIV transgenic mice compared with nontransgenic sibling mice. This difference persisted when NO production was induced with peritoneal injections of bacterial endotoxin (LPS). Peritoneal inflammatory macrophages, but not resident peritoneal macrophages, derived from HIV-1 transgenic mice and activated in vitro with LPS and IFN-{gamma} (or tumor necrosis factor {alpha} and IFN-{gamma}) also produced about 50% less NO than did macrophages harvested from nontransgenic littermates. Isogenic, transgenic mice bearing mutated nef or vpr genes had normal serum levels of NO metabolites and their macrophages produced normal levels of NO when stimulated. An explanation for the reduced NO response of HIV[Vpr+Nef+] macrophages was not apparent from measured levels of iNOS expression, viral gene expression, or arginase activity in activated macrophages. Inhibition of nitric oxide synthase (NOS) isoforms with L-NAME or aminoguanidine blocked time-dependent increases in HIV gene expression in activated macrophages cultured ex vivo. Inhibition with L-NAME occurred despite high levels of NO generated by iNOS, and exogenously supplied NO induced HIV gene expression only weakly, suggesting that cNOS had the greater influence on proviral gene induction. This system is presented as a model of HIV-1 proviral gene expression and dysfunction in macrophages.

Key Words: transcription • arginase • TNF-{alpha} • NOS • Nef • Vpr


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) has dual functions as a cellular messenger or modulator and a potent antimicrobial-effector molecule. NO is derived from the catalytic conversion of L-arginine to L-citrulline. It is produced in low quantities by constitutively expressed NO synthases (NOS-I and -III) in controlling homeostatic processes such as neurotransmission and blood pressure [1 , 2 ]. NO produced by the inducible form of NOS (iNOS or NOS-II) appears in high quantities after cytokine activation and functions principally in an inflammatory, antimicrobial role as part of the host’s innate immune defenses [3 ]. This latter role has been amply demonstrated through the inhibition of iNOS activity with pharmacological inhibitors or in mice genetically deficient in iNOS, with the result that animals are acutely susceptible to infection with bacteria, parasites, and certain viruses [3 , 4 ]. The cytotoxicity of NO dictates its tight regulation. Metal- and thiol-containing proteins are the major N-nitrosation targets in NO-mediated signal transduction, whereas deoxyribonucleic acid and tyrosine residues are the principal targets of its toxic effects [5 ]. The inappropriate expression of NO has been associated with inflammatory diseases (e.g., arthritis, Cohn’s disease, graft-versus-host disease) and various autoimmune diseases [6 , 7 ]. Beyond cytokine regulation at the transcriptional level, iNOS activity is regulated by various posttranscriptional mechanisms including cofactor and substrate availability [1 , 8 ].

There are studies showing that the production of NO is elevated in human immunodeficiency virus (HIV)-1-infected patients [9 , 10 ], a phenomenon replicated in acute simian immunodeficiency virus (SIV)-infection models [11 ]. A similar induction of NO was observed in acute HIV infections of human cells in vitro [12 , 13 ]. Elevated levels of NO could have a variety of deleterious, immunologic effects contributing to AIDS, including the deactivation of lymphocytes, the induction of apoptosis, and the suppression of T helper cell type 1 (Th1) responses [14 15 16 ]. HIV Env protein alone can activate NO production in cultured monocytes, astrocytoma, and glial cells [17 18 19 ], and this may be critical to the development of AIDS dementia complex (ADC). In support of this hypothesis, recent studies have pointed to the coexistence of viral protein, iNOS, and macrophage/microglia in brain centers [20 , 21 ]. One neuropathologic effect of HIV gp120 coat protein may be the stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors that is mediated in part by NO [22 , 23 ]. The HIV protein Tat up-regulates nuclear factor (NF)-{kappa}B expression, a transactivator of the iNOS gene, and exogenous Tat has been shown to up-regulate NO production in microglial cells [24 ]. However, it remains uncertain whether NO production is elevated in HIV-infected cells [25 ] or whether infected cells induce NO production in bystander cells as demonstrated in macrophage-astrocyte co-cultures [26 ].

In other scenarios, an underproduction of NO has been associated with HIV infection. For example, the infection of monocytes/macrophages from normal individuals with Rhodococcus equi induced NO production, whereas the infection of cells derived from HIV-positive patients failed to induce NO [27 ]. In AIDS patients with toxoplasmic encephalitis, it was discovered that central nervous system (CNS)-related NO production was not elevated and possibly nonoptimal to control the recurrence of Toxoplasma gondii infection [28 ]. A similar observation was made in HIV-positive patients with pulmonary tuberculosis. In this case, patients had normal serum levels of NO metabolites, whereas HIV-negative patients with pulmonary tuberculosis displayed elevated levels of NO metabolites [29 ]. Reduced NO production was also apparent in a cohort of HIV-positive patients as determined from the concentration of NO metabolites in exhaled air [30 ]. Collectively, these observations suggest that HIV infection could result in inadequate NO responses that contribute to secondary infections.

NO has an established, anti-HIV effect, demonstrated in acute infections of human peripheral blood mononuclear cells (PBMCs) [31 ] and astrocytoma cells [32 ]. Numerous mechanisms for this exist, including the inhibition of HIV protease and reverse transcriptase [33 , 34 ] and the inactivation of NF-{kappa}B [35 ] by blocking the inactivation of I-{kappa}B, an inhibitor of NF-{kappa}B, or of DNA-binding by NF-{kappa}B [36 ]. It is interesting that there are at least two studies suggesting that NO may activate virus in chronically infected cultures. NO donor compounds stimulated virus replication in latently infected U1 macrophage cells [31 ], and the cross-linking of CD23 (Fc{varepsilon} RII), known to stimulate NO production, induced p24 production in a variant of U1 macrophages [37 ].

HIV-1 transgenic mice model a form of chronically, nonproductively infected cells [38 ] in which viral gene expression can be induced with microbial infection [39 ]. In animals carrying a noninfectious, proviral DNA (based on the clone pEVd1443 [40 ], deleted in gag and pol), inducible viral gene expression was supported by peritoneal macrophages [38 ]. Thus, uncomplicated by the influences of an active HIV infection and secondary infections, this model has enabled a study of the intrinsic effect of chronic, low-level HIV gene expression on NO metabolism in animals and in macrophages derived from them. In addition, this system has been used to examine the role of NO in the induction of HIV transcription. We discovered that NO production in response to cytokine and lipopolysaccharide (LPS) activation is significantly reduced in HIV transgenic mice, and this phenomenon is dependent on the HIV nef and vpr genes. We also provide experimental evidence for the NO-dependent induction of HIV gene expression in this system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HIV transgenic mice and ex vivo macrophage culture
We studied established lines of HIV proviral transgenic mice. The prototype FVB/N mouse line, HIVd1443[{Delta}Gag/Pol] or T26, bore a proviral DNA deleted in gag and pol, but the genes for Env and the six accessory gene products remained intact [38 , 40 ]. Variants of this line bearing mutations in nef (X5) or vpr (V13) were included [38 ]. We identified transgenic offspring in lines T26 and V13 on the basis of congenital cataracts that characterized HIV-positive mice expressing Nef [41 ]. Transgenic X5 offspring were identified by polymerase chain reaction amplification of the nef gene in tail DNA preparations [38 ]. The three lines were maintained in the hemizygous state with respect to the HIV transgene. They were housed in conventional holding facilities and treated in accordance with Canadian Council on Animal Care regulations. All animals used experimentally were between 7 and 12 weeks old.

Peritoneal macrophages were harvested from untreated animals (resident macrophages) or mice injected intraperitoneally (i.p.) with 1.5 ml 3% thioglycollate medium 4 days before harvesting (inflammatory macrophages). Peritoneal cavities were washed with 6 ml sterile, ice-cold, phosphate-buffered saline (PBS), and the cells were collected by centrifugation. Peritoneal exudate cells were washed twice with cold PBS and counted before resuspending in RPMI medium supplemented with 10% fetal calf serum, 1 mM glutamate, and ß-mercaptoethanol (4x10-5 M). Cells were plated in tissue culture-treated, 96-well plates at 2 x 105 cells per well, in 24-well dishes at 2 x 106 cells per well, or in 6-well dishes at 5 x 106 cells/well, depending on the experiment. Cells were incubated at 37°C, 5% CO2, for 3 h before washing 3x with warm PBS to remove nonadherent cells. Macrophage monolayers were stimulated with Escherichia coli LPS (serotype 055:B5, Sigma Chemical Co., St. Louis, MO) at 1 µg/ml, tumor necrosis factor-{alpha} (TNF-{alpha}; Life Technologies, Bethesda, MD) at 25 µg/ml, and interferon-{gamma} (IFN-{gamma}; Life Technologies) at 10 U/ml, or were left untreated.

Modulation of NO in mice and macrophage cultures
As a source of exogenous NO, the NO-donor, S-nitro-N-acetylpenicillamine (SNAP; Sigma), was added to the culture medium overlaying macrophage monolayers. SNAP was added at a concentration of 0.4 mM (from an 80-mM stock solution, in dimethyl sulfoxide) and replenished every 6 h. Control cultures received carrier alone. Two competitive inhibitors of NOS were used to block the endogenous generation of NO: L-NAME (NG-nitro-arginine-methyl ester; Sigma) and aminoguanidine (AG; Sigma). For in vivo experiments, LPS (200 µg/mouse) was administered i.p. in a volume of 0.1 ml. Control animals received carrier (saline) alone.

MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay
As a measure of cell viability and for the normalization of experimental data, the MTT assay of mitochondrial activity was applied. MTT (Sigma) was dissolved in PBS to 5 mg/ml and filtered. A 10-µl vol was added to macrophage monolayers in 96-well plates, and the plates were incubated at 37°C for 4 h. After fluid was withdrawn, the blue precipitate was dissolved in 100 µl acid-propanol (0.1 N HCl). Absorbance was read at 570 nm in a microplate reader.

Measurement of NO production and arginase activity
Screening for nitrate in mouse sera provided estimates of systemic NO production [42 ]. Briefly, mouse serum (100 µl of a 1/20 dilution in 0.2 M HEPES, pH 7.4) was incubated with 50 µl of 0.2 M HEPES and 50 µl of 5 x 109 Pseudomonas oleovorans diluted in phenol red-free DMEM (as a source of nitrate reductase) for 90 min at 37°C. After clarifying the sample by centrifugation, nitrite was measured by combining 100 µl sample and 100 µl Griess reagent [0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H3PO4] [43 ] in 96-well dishes and by reading the absorbance at 570 nm in an ELISA plate reader. To measure NO produced by macrophage monolayers, 100 µl culture medium was combined with 100 µl Griess reagent in micro-well plates. Nitrite concentrations were determined by comparisons with a standard curve of NaNO in the range of 1–100 µM.

To measure arginase activity in macrophage monolayers [44 ], cells were lysed with 100 µl 0.1% Triton X-100 added to wells of a 96-well microtiter plate seeded with 2 x 105 exudate cells. After 30 min on ice, 100 µl 25 mM Tris-HCl (pH 7.4) was added, 100 µl of this mixture was combined with 10 µl of 10 mM MnCl2, and the enzyme was activated by heating to 56°C for 10 min. The lysate was then mixed with 100 µl of 0.5 M arginine (pH 9.7) and incubated at 37°C for 60 min. The reaction was stopped with 900 µl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7). Then, 40 µl {alpha}-isonitrosopropriophenone (in 100% ethanol) was added, and the mixture was heated to 95°C for 30 min. Absorbance was measured at 570 nm. One unit of enzyme catalyzed the formation of 1 µmol urea per minute.

Statistical significance of data was calculated using a one-tailed test for a t distribution (Student’s t-test).

Northern and Western analyses
Total RNA was extracted using a guanidinium isothiocyanate procedure [45 ] from macrophage monolayers plated at an initial density of 2 x 106 peritoneal exudate cells/well in a 24-well plate. RNA (total well contents) was fractionated in 1% agarose (2.2 M formaldehyde) and transferred to nylon membranes (Nytran®, Schleicher and Schuell, Inc., Keene, NH). After UV cross-linking, HIV RNA was detected by hybridization with a 32P-labeled Nef cDNA and exposure to X-ray film. Quantitation was accomplished using NIH Image 1.61 software analysis of digitalized images. Blots were stripped and re-probed with a radio-labeled, human ß-actin probe (Clontech, Palo Alto, CA).

For immunoblot analyses, 5 x 106 plated cells were washed twice with PBS and lysed on ice for 30 min with 0.1% Triton X-100, 10 mM Tris•HCl (pH 7.4). Cell debris was removed by centrifugation in a benchtop microfuge at 4°C, and the clarified samples were measured for protein content using Bradford reagent (Bio-Rad, Hercules, CA). For the detection of iNOS, 1 µg protein was electrophoresed through 10% acrylamide, transferred to cellulose membranes, and subjected to immunoblotting using a monoclonal antibody for iNOS (Transduction Laboratories, Lexington, KY). Membranes were blocked with 5% nonfat dry milk (in PBS) and exposed for 1 h to anti-iNOS (1/1000 dilution). Washed membranes were subsequently treated with horseradish peroxidase-conjugated donkey anti-mouse immunoglobulin (Ig)G (1/2000). Immunoreactive peptides were visualized following reaction with a chemiluminescent substrate (Pierce, Rockford, IL) and exposure of blots to Hyperfilm (Amersham, Arlington Heights, IL). Pre-stained protein molecular-weight ladder was obtained from Life Technologies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced NO production in HIV mice and peritoneal macrophages
A general attribute of HIV-1 transgenic mice, in which viral gene expression is directed by the native HIV long-terminal repeat (LTR), is the capacity of peritoneal macrophages to support viral gene transcription [38 , 39 ]. It has been shown that peritoneal macrophages from two of the lines included in this study (T26 or wild-type HIVd1443 and X5 or HIVd1443[Nef-] transgenic mice) efficiently expressed viral transcripts of the multiply spliced class [38 ]. In the present study, we have included an isogenic line mutated in vpr (HIVd1443[Vpr-]), which we call line V13. Although otherwise vigorous and healthy, V13 transgenic mice were phenotypically marked by the presence of perinatal cataracts and mild runting (unpublished results), and V13 transgenic mice were distinguished by their failure to develop HIV-associated nephropathy characteristics of the two Vpr+ lines. In Figure 1 , the level of HIV transcription in V13 mouse peritoneal macrophages is compared with that of cells harvested from T26 and X5 mice. In resident peritoneal macrophages, Nef+ lines (T26 and V13) supported negligible levels of expression, whereas the Nef-deficient line (X5) expressed relatively well. In contrast, inflammatory macrophages from all three lines transcribed proviral HIV genes to a comparable level. On a per-cell basis, viral gene expression in inflammatory macrophages was similar to expression in X5 resident macrophages. This is not apparent in Fig. 1 because the level of ß-actin expression was greater in inflammatory macrophages compared with resident macrophages. A modest level of proviral gene induction could be generally achieved by stimulating macrophages with LPS (2–3x over unstimulated cultures depending on the line).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. HIV gene transcription in transgenic macrophages. Peritoneal exudate cells (resident or inflammatory) were plated at an initial density of 2 x 106 cells/well, and adherent cells were harvested 12 h after treatment as indicated. Total RNA was extracted, fractionated by gel electrophoresis, probed with a nef cDNA probe specific for HIV transcripts, and measured by densitometry. Measurements of HIV transcripts were normalized to actin transcripts, and levels in Vpr+ Nef- inflammatory macrophages were arbitrarily set at 1. As different actin expression levels characterized resident and inflammatory macrophages, relative RNA expression cannot be compared between the two populations as plotted (see text). Shown is the cumulative data of three experiments with macrophages pooled from at least three transgenic mice per group. Error bars represent one standard deviation. Three HIV transgenic lines defined by their HIVd1443 proviral transgene are included: T26, Vpr+Nef+; X5, Vpr+Nef-; and V13, Vpr-Nef+.

 
The capacity of peritoneal macrophages from HIV mice to produce NO following activation with endotoxin (LPS) and/or IFN-{gamma} was determined by assaying culture medium for nitrite, a NO metabolite, 24 h after stimulation (Fig. 2 ). With inflammatory macrophages specifically, NO production was reduced in HIV transgenic (T26) mice when stimulated with LPS or with LPS plus IFN-{gamma}. Normalization of NO production in T26 macrophages to that by control (nontransgenic FVB/N) macrophages in six independent experiments confirmed the significance of the reduced capacity of T26 macrophages to generate NO (P<0.01). Subnormal NO production was dependent on the nef and vpr genes because mutations in either gene restored NO production to normal, if not greater-than-normal, levels in the macrophages of proviral HIV mouse lines X5 and V13. This defect was specific for inflammatory macrophages because NO production was normal in HIV-positive, resident macrophage populations. The reduced capacity of wild-type HIV macrophages to produce NO in response to activation had in vivo relevance. We observed that serum nitrate levels in untreated animals were reduced significantly (about 50%; P<0.025) in wild-type HIV mice relative to nontransgenic siblings and mutant HIV transgenic lines (Fig. 3 ). When NO production was induced in vivo with i.p. injections of bacterial LPS, NO metabolite levels increased rapidly in all mice (by 8 h; Fig. 3 ). However, lower levels of NO production were observed again in treated, wild-type HIV animals (P<0.025) but not in the mutant lines.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. NO production in HIV transgenic macrophages. Adherent peritoneal exudate cells (inflammatory or resident) were treated with E. coli LPS (1 µg/ml) and/or IFN-{gamma} (10 U/ml), and culture fluids were assayed in triplicate for the presence of nitrite as a measure of NO production after 24 h. Results shown are the cumulative data of six experiments with macrophages pooled from at least three mice per group. Transgenic macrophages are compared with similarly treated cells from nontransgenic, sibling mice (FVB/N).

 


View larger version (34K):
[in this window]
[in a new window]
  		Figure 3. NO production in HIV transgenic mice. Serum was collected from groups of at least six mice (equal numbers of males and females) representing nontransgenic, sibling controls (FVB/N) and the three HIV transgenic lines. Serum nitrates/nitrites were measured in triplicate 8 h after the i.p. injection of E. coli LPS (200 µg/mouse). Untreated animals received saline alone. Average measurements of the six mice are shown.

 
Induction of iNOS and arginase in macrophage cultures
We explored possible mechanisms for the apparent down-regulation of NO production in wild-type HIV transgenic macrophages. iNOS is trancriptionally up-regulated in response to LPS and IFN-{gamma} [1 ], and levels of iNOS protein induced by LPS/IFN-{gamma} stimulation were measured by Western analysis (Fig. 4 ). In three experiments, using elicited peritoneal macrophages pooled from three to five mice, iNOS expression was undetected in untreated cultures. Upon stimulation, iNOS protein was visible in all macrophage cultures, but the level of iNOS in wild-type (T26) murine macrophages was comparable with nontransgenic cultures (Fig. 4) . The induction of iNOS in the mutant HIV lines was also near normal. Thus, the reduced capacity of HIV macrophages to produce NO was apparently not a result of the failure of these cells to respond to activation by inducing iNOS expression.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. iNOS induction in macrophages from HIV transgenic macrophages. Inflammatory peritoneal-exudate cells were harvested 16 h following plating with or without stimulation with LPS and IFN-{gamma} (1 µg/ml and 10 U/ml, respectively). Cell lysates were assayed for protein content, and 1 µg protein was fractionated in 10% acrylamide and subjected to Western analysis. The primary antibody was an iNOS mAb that detected purified iNOS monomer at 130 kDa (Std). HIV macrophages are compared with nontransgenic, sibling mice (FVB/N). The autoradiogram represents one of three experiments using macrophages pooled from at least three mice. Cumulative data from the three experiments are presented in the box.

 
Previous studies indicated that although numbers of peritoneal inflammatory macrophages harvested from control and HIV lines were similar, they may have differed phenotypically [38 ] and adhered differently to culture dishes. To control for differences in plating efficacy, experiments were conducted in which NO production was normalized to the viability (by MTT assay) of elicited macrophages (Fig. 5 ). The NO deficiency of wild-type HIV macrophages was confirmed; furthermore, the defect was observed in macrophages activated with TNF-{alpha} and IFN-{gamma}. This confirmation suggested that wild-type HIV macrophages responded differently to stimulation, not necessarily in the production of iNOS but in the regulation of iNOS activity. One manner of regulating iNOS activity is through substrate availability, controlled in part by the activity of arginase, an enzyme that converts arginine to ornithine and urea [46 , 47 ]. Supplementing elicited macrophage cultures with arginine (1 mM) failed to correct the deficit in NO production (unpublished results). Alternatively, total arginase activity in lysates of macrophage monolayers was measured. Constitutive levels of arginase activity were comparable amongst control and HIV-positive cells. The low-level induction of arginase activity that typically follows endotoxin activation [48 ] was observed in control FVB/N macrophages (Fig. 5) . However, arginase activity was uniformly reduced, not induced, in activated HIV cultures. The reduction was statistically significant in all induced cultures (P<0.01 for all with the exception of P<0.025 for X5 cultures treated with IFN-{gamma}/LPS). Thus, the unique deficiency of wild-type HIV macrophages to produce normal levels of NO could not be attributed to elevations in arginase activity.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. NO production relative to arginase activity in plated macrophages. NO production in adherent, peritoneal macrophages was measured as before (see Fig. 2 legend) but normalized to cell viability as measured by MTT assay (left panel). Cultures were also induced with a combination of TNF-{alpha} (25 µg/ml) and IFN-{gamma} (10 U/ml). In addition, parallel cultures (also plated at 2x105 exudate cells/well) were harvested after 48 h incubation, and cell lysates were analyzed for arginase activity (right panel). Presented is one of duplicate experiments using macrophages pooled from five mice. Error bars denote one standard deviation in triplicate measurements of one representative experiment.

 
NO modulation of HIV gene expression in peritoneal macrophages
Reduced generation of NO by chronically infected cells may benefit HIV persistence in vivo. In cells of HIV-1 transgenic mice, it was possible to directly explore the influence of NO on the expression of HIV-1 gene products. Transgenic, elicited macrophage monolayers were treated with inhibitors of NOS coincident with IFN-{gamma} and LPS activation. Inhibitors with different specificities for the constitutive and inducible forms of NOS (cNOS and iNOS, respectively) were applied. AG, having a specificity for iNOS [49 ], blocked NO production in response to inflammatory activation (Fig. 6A ). In contrast, use of the cNOS inhibitor L-NAME [50 ] permitted a NO burst following macrophage activation (Fig. 6A) . The induction of HIV transcripts (by Northern analysis) in adherent-elicited macrophages was measured through time pursuant to stimulation (Fig. 6B) . In cell populations from the three HIV transgenic lines, levels of HIV transcription increased with time following plating and activation. Administration of either NOS inhibitor failed to block an early induction of viral transcription but was effective at interfering with transcription after the 8-h time point. The inhibition achieved with L-NAME (to 29±9% at 24 h) exceeded that achieved with AG (to 55±10% at 24 h) with marginal significance (P<0.05). This confirmed that the later, prolonged stage of HIV gene induction was dependent on NOS function. However, NO production remained high in L-NAME cultures (22.5–35 nmoles NO/million cells as opposed to 2.4–3.9 nmoles NO/million cells in the presence of AG), implying that cNOS-derived NO, not antimicrobial NO bursts from iNOS activation, was important in viral gene regulation.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. Inhibition of viral gene transcription with inhibitors of NOS. Two metabolic inhibitors of NOS, L-NAME and AG, were applied at 1 mM to differentially affect the production of NO in control (FVB/N), inflammatory peritoneal macrophages activated with LPS (1 µg/ml) and IFN-{gamma} (10 U/ml) (A). AG blocked the production of exogenous NO as detected in culture fluids measured 24 h after stimulation, whereas L-NAME had little effect on exogenous NO production. (B) The effect of NOS inhibitors on the induction of HIV gene transcription in inflammatory peritoneal macrophages is shown. Plated cells (2x106 exudate cells/well) were activated in the presence of inhibitors, and total RNA was extracted at the designated times post-stimulation. RNA yields from each well were subjected to Northern analysis, and HIV transcription was measured digitally from scanned autoradiograms and normalized to actin mRNA levels. One of two experiments with similar results, in which macrophages were pooled from five mice/group, is shown.

 
The partial inhibition of viral gene transcription with AG and the persistence of HIV gene expression despite high levels of NO generation suggested that iNOS activity did not suppress HIV transcription. To substantiate this supposition and to address further the possibility that a high level of NO production may potentiate viral gene expression, the effect of an exogenous NO donor SNAP was tested on elicited macrophage monolayers in the absence of endotoxin/cytokine activation. SNAP treatment led to a rapid accumulation of NO metabolites in the medium, the levels of which were comparable in control and HIV transgenic macrophages after 8 h incubation (83±8 nmoles/106 cells for control macrophages; 74±6, 79±3, and 74±3 for T26, X5, and V13 cells, respectively). Viral RNA expression in HIV macrophage populations was measured after 14 h culture in the presence of SNAP (Fig. 7 ). The high release of NO was cytotoxic, and in a 24-h incubation period, viability as measured by mitochondrial activity (MTT assay) was reduced on average to 67±4% in the four populations of cells. On a per-cell basis, there appeared to be little difference between the expression of HIV RNA in macrophage monolayers treated with SNAP and those left untreated (Fig. 7A) . However, normalization of HIV transcription to actin RNA levels produced data suggesting that exogenous NO did enhance viral gene transcription (Fig. 7B) . Alternatively, when HIV RNA was normalized to cell viability, only a modest increase in viral gene expression, of marginal statistical significance (P<0.05), was computed. The discordance between actin levels and cell viability was because of a time-dependent reduction in actin message (34±18%) that exceeded the drop in apparent viability of cultures at 14 h (77%). Consistent with earlier results, exogenous NO did not inhibit HIV transcription, and its net inductive effect, if present, was modest.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 7. Effect of exogenous NO on HIV transcription in plated macrophages. (A) Inflammatory peritoneal macrophages were treated with the NO-releasing compound SNAP, activated with LPS and IFN-{gamma} (1 µg/ml and 10 U/ml, respectively), or left untreated. RNA was extracted 14 h after treatment, and total well yields were subjected to Northern analysis. A representative autoradiogram used in quantitating HIV and ß-actin expression is shown. (B) The cumulative data measuring HIV RNA expression from two such experiments (each with duplicate samples) are shown normalized to ß-actin RNA levels (left panel) or to cell viability as determined by MTT assay (right panel). All values are normalized to untreated X5 samples (arbitrarily set at 1).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HIV transgenic mice have been developed in order to model chronic viral gene expression in macrophages. They can be useful for determining factors that influence viral gene expression in vivo and for defining mechanisms of HIV-induced dysfunction in macrophage populations. The effect to which the antimicrobial and cytotoxic factor NO influences HIV expression and conversely the effect the virus has on NO production were explored in this study. A dysfunctional population of inflammatory macrophages from HIV transgenic mice was apparent from the in vitro and in vivo demonstration of subnormal NO responses to inflammatory activation. Furthermore, we found little evidence that inflammatory bursts of iNOS-generated NO influenced proviral gene expression dramatically, whereas cNOS-derived NO may have had a significant, positive influence on proviral activation.

In contrast to our observations with HIV transgenic mice, systemic NO production is often elevated in HIV-positive individuals [9 , 10 ]. Acute infection by HIV [12 , 13 ] and SIV [11 ] in vitro resulted in an induction of NO release in what might be considered a normal, antiviral response [51 ]. In particular, AIDS dementia has been associated with elevated NO production in astrocytes, induced by factors released from adjacent HIV-infected cells [18 , 19 , 26 ]. However, there are studies associating HIV infection with a reduced capacity to produce NO. In a cohort of infected hemophiliacs [52 ], higher-than-normal levels of serum nitrates characterized asymptomatic patients, but lower-than-normal levels characterized advanced patients with HIV-related disease. Considered in conjunction with studies demonstrating reduced NO in exhaled air of HIV-positive subjects [30 ] and reduced NO production associated with toxoplasmic encephalitis [28 ] and pulmonary tuberculosis [29 ] in HIV-positive patients, there is cause to consider the contributions of ineffective NO production in certain aspects of AIDS. These contrasting observations in human patients could reflect situational differences between acute HIV infection (productive infection), where NO production is increased in a normal, antiviral response, and chronic HIV infection (or nonproductive infection), where a viral-induced impairment in NO production may predominate. The latter scenario may be modeled in HIV transgenic mice. In a broader interpretation, demonstration of abnormal NO production in HIV transgenic macrophages reflects the capacity of HIV gene products to perturb macrophage function. For example, given the propensity of human alveolar macrophages to be infected [53 , 54 ], this perturbation may have significant ramifications about the development of pulmonary disease and HIV persistence in this compartment.

Mechanistically, it remains unclear how HIV down-modulates NO production in transgenic, murine macrophages. The capacity of HIV proteins Env (gp120) and Tat to induce iNOS expression in human cell-culture systems [17 , 24 ] had no apparent effect on the murine system, perhaps because of inefficient viral protein expression, inappropriate host cofactors, or its counteraction by yet-undefined mechanisms. It should be noted that in rodent macrophages, Tat did exert a negative effect on iNOS synthesis, but this was in response to IFN-{gamma} stimulation, and a negative effect was not observed with LPS stimulation [55 ]. We found no apparent abnormality in iNOS levels in HIV macrophage cultures that would implicate a defect in the induction of iNOS synthesis and explain the down-regulation of NO production in wild-type HIV mice. Thus, it seems likely that Nef- and Vpr-expressing cells regulate iNOS activity differentially. This may be unusual because iNOS is principally regulated at the level of transcription [1 ]. However, NOS monomers require four allosteric regulators for functional dimerization: heme, tetrahydropterin, calmodulin, and arginine. One alternative mechanism of iNOS regulation, the depletion of the substrate arginine by the enzyme arginase [46 , 47 ], was explored but dismissed on the basis that, if anything, lower- and not higher-than-normal levels of arginase activity were detected in wild-type HIV macrophage cultures. In fact, uniformly lower levels of arginase activity were observed in HIV-positive macrophage cultures despite their different abilities to generate NO. This arginase response may reflect an abnormal cell condition dependent on a viral gene other than nef or vpr. A dissociation of the reciprocal relationship between arginase and iNOS activities has been observed elsewhere [56 ]. Another potential regulatory mechanism, as yet unexplored, involves the availability of tetrahydrobiopterin (BH4), which is normally induced in response to TNF-{alpha} and IFN-{gamma} [57 ]. Experimentally, at least, a dissociation of BH4 and iNOS biosynthesis has been demonstrated [58 , 59 ].

The involvement of Nef and Vpr in inducing this altered behavior in murine macrophages suggests that their biological functions overlap. This also may be the case in the development of HIV-associated nephropathy in HIVd1443-transgenic murine models [38 ]. Although their viral-related functions are far from defined, Nef interferes with activation-signal pathways involving protein tyrosine kinases, and Vpr alters cellular differentiation states [60 ]. Hence, the effect of these viral proteins may be to determine a cellular state in which iNOS is differentially regulated. It is reasonable to surmise that reduced NO production by chronic HIV-expressing cells, apart from reflecting an altered differentiation/activation state, may contribute to the survival of that cell under inflammatory conditions. The anti-HIV effects of NO may be a significant component of the host’s response to acute infection. A reduced capacity to produce NO in chronically infected cells may also sustain chronic infections by prohibiting NO-induced apoptosis [47 , 61 , 62 ], by limiting the reactivation of quiescent virus, and by promoting virus transmission to sensitive T cells.

Defective NO production by chronically infected cells could be a basis for a host innate-immune defect. Although NO has proven antimicrobial effects in mice, it is less clear in humans where much lower levels of NO are produced by monocytes/macrophages in response to bacterial activation [1 , 63 ]. We have preliminary evidence demonstrating that HIVd1443 wild-type, transgenic mice, but not their mutant counterparts, are more susceptible to infection with the bacterium Listeria monocytogenes (unpublished results). However, murine host defense against Listeria is not predicated on a NO response alone [64 ]; thus, additional macrophage defects may be pertinent to innate resistance in mice. Definition of the full extent of macrophage dysfunction in murine cells expressing HIV may be pertinent to human immunology, particularly if the observed NO effects reflect a broader perturbation in the expression of immune-modulating cytokines or chemokines.

NO, in turn, was shown to have a role in the induction of HIV gene transcription. Our results suggested that cNOS-derived NO in particular was required for the induction of HIV gene expression observed in our system. Exogenous NO or iNOS-derived NO had a much lesser impact on viral gene expression. In support of this conclusion, (i) viral gene transcription increased at a time when NO output was high in activated macrophages; (ii) the NO donor, SNAP, did not dramatically affect viral gene expression; and (iii) the selective cNOS inhibitor L-NAME effectively blocked the time-dependent increase in HIV transcription without markedly affecting levels of NO production in macrophage cultures. NO has dual effects on the activation of HIV transcription in human cells. Two known studies implicate NO in the activation of HIV in chronically infected human U1 macrophages [31 , 37 ], whereas NO inhibited HIV transcription in transiently infected cell-culture systems [35 , 36 ]. It is conceivable that NO effects are a dose-dependent phenomenon. Low levels of NO production may activate the virus, perhaps through an effect on NF-{kappa}B activation [65 ] or the capacity of NO to modulate cellular differentiation [66 , 67 ], processes that favor HIV induction [68 ]. At high concentrations of NO, the effect may be negative through a direct inhibitory effect on NF-{kappa}B. Alternatively, we suspect that endogenous NO determined levels of HIV transcription indirectly through controlling effects on the differentiation/activation of the host cell. We reiterate that exogenous NO may have had dual effects: negative effects on transcription balanced by a positive effect gained through the induction of cellular activation or differentiation. Importantly, however, we can conclude that high levels of NO were not inhibitory in this model of chronic HIV expression.

In conclusion, HIV transgenic macrophages have a reduced capacity to produce NO in response to inflammatory stimuli and tolerate high doses of exogenous NO to sustain HIV transcription. Apart from any advantage that a reduced NO response has on virus survival, this macrophage-related perturbation may identify a more-general, functional condition resulting in immunologic complications. By inference, chronically infected, human macrophage-like cells may adopt a functionally compromised state that promotes cell survival and virus transmission. Modulating NO production to activate quiescent virus in reservoir cells as suggested [31 ] may be a feasible means of exposing these cells to normal, immune mechanisms and drug therapy. A more effective means may be revealed with an improved understanding of viral mechanisms that promote HIV persistence. An alternative approach may be to target viral genes such as nef and/or vpr, which appear to determine the functional suppression of chronically infected cells. Thus, the HIV transgenic mouse system, in its modeling of the chronic infection of macrophages, may be useful for understanding functional deficiencies in this unique cell population and for developing novel anti-HIV approaches.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grant MT-14135 from the Medical Research Council of Canada. The authors are indebted to the University of Alberta’s Department of Health Sciences Laboratory Animal Services for the continual care of animals and members of the Department of Medical Microbiology for their critical comments during the execution of the experimental work. We also thank Ricardo Gazzinelli who first observed anomalies in NO production in HIV provirus-transgenic mice.

Received January 13, 2001; revised April 23, 2001; accepted April 24, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nathan, C., Xie, Q-W. (1994) Nitric oxide synthases: roles, tolls and controls Cell 78,915-918[Medline]
  2. Schmidt, H. H. H. W., Walter, U. (1994) NO at work Cell 78,919-925[Medline]
  3. Green, S. J., Scheller, L. F., Marletta, M. A., Seguin, M. C., Klotz, F. W., Slater, M., Nelson, B. J., Nacy, C. A. (1994) Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens Immunol. Lett. 43,87-94[Medline]
  4. Liew, F. Y. (1994) Regulation of nitric oxide synthesis in infectious and autoimmune diseases Immunol. Lett. 43,95-98[Medline]
  5. Stamler, J. S. (1994) Redox signaling: nitrosylation and related target interactions of nitric oxide Cell 78,931-936[Medline]
  6. Weinberg, J. B., Granger, D. L., Pisetsky, D. S., Seldin, M. F., Misukonis, M. A., Mason, S. N., Pippen, A. M., Ruiz, P., Wood, E. R., Gilkeson, E. R. (1994) The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine J. Exp. Med. 179,651-660[Abstract/Free Full Text]
  7. Brenner, T., Brocke, S., Szafer, F., Sobel, R. A., Parkinson, J. F., Perez, J. F., Steinman, L. (1997) Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalitis J. Immunol. 158,2940-2946[Abstract]
  8. Modolell, M., Corraliza, I. M., Link, F., Soler, G., Eichmann, K. (1995) Reciprocal regulation of nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by Th1 and Th2 cytokines Eur. J. Immunol. 25,1101-1104[Medline]
  9. Groeneveld, P. H., Kroon, F. P., Nibbering, P. H., Bruisten, S. M., van Swieten, P., van Furth, R. (1996) Increased production of nitric oxide correlates with viral load and activation of mononuclear phagocytes in HIV-infected patients Scand. J. Infect. Dis. 28,341-345[Medline]
  10. Zangerle, R., Fuchs, D., Reibnegger, G., Werner-Felmayer, G., Gallati, H., Wachter, H., Warner, E. R. (1995) Serum nitrite and nitrate in infection with human immunodeficiency virus type-1 Immunobiology 193,59-70[Medline]
  11. Blond, D., Cheret, A., Raoul, H., Le Grand, R., Caufour, P., Theodoro, F., Dormont, D. (1998) Nitric oxide synthesis during acute SIV mac251 infection of macaques Res. Virol. 149,75-86[Medline]
  12. Bukrinsky, M. I., Nottet, H. S. L. M., Schmidtmayerova, H., Dubrovsky, L., Flanagan, C. R., Mullins, M. E., Lipton, S. A., Gendelman, H. E. (1995) Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease J. Exp. Med. 181,735-745[Abstract/Free Full Text]
  13. Obregon, E., Punzon, C., Fernandez-Cruz, E., Fresno, M., Munoz-Fernandez, M. A. (1999) HIV-1 infection induces differentiation of immature neural cells through autocrine tumor necrosis factor and nitric oxide production Virology 261,193-204[Medline]
  14. Fehsel, K., Kroncke, K-D., Meyer, K. L., Huber, H., Wahn, V., Kolb-Bachofen, V. (1995) Nitric oxide induces apoptosis in mouse thymocytes J. Immunol. 155,2858-2865[Abstract]
  15. Torre, D., Ferrario, G. (1996) Immunological aspects of nitric oxide in HIV-1 infection Med. Hypotheses 47,405-407[Medline]
  16. Taylor-Robinson, A. W. (1997) Inhibition of IL-2 production by nitric oxide: a novel self-regulatory mechanism for Th1 proliferation Immunol. Cell Biol. 75,167-175[Medline]
  17. Pietraforte, D., Tritarelli, E., Testa, U., Minetti, M. (1994) gp120 HIV envelope glycoprotein increases the production of nitric oxide in human monocytes-derived macrophages J. Leukoc. Biol. 55,175-182[Abstract]
  18. Mollace, V., Colasanti, M., Persichini, T., Bagetta, G., Lauro, G. M., Nistico, G. (1993) HIV gp120 glycoprotein stimulates the inducible isoform of NO synthase in human cultured astrocytoma cells Biochem. Biophys. Res. Commun. 194,439-445[Medline]
  19. Koka, P., He, K., Zack, J. A., Kitchen, S., Peacock, W., Fried, I., Tran, T., Yashar, S. S., Merrill, J. (1995) Human immunodeficiency virus 1 envelope proteins induce interleukin 1, tumor necrosis factor {alpha}, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain J. Exp. Med. 182,941-952[Abstract/Free Full Text]
  20. Adamson, D. C., McAurthur, J. C., Dawson, T. M., Dawson, T. M. (1999) Rate and severity of HIV-associated dementia (HAD): correlations with gp41 and iNOS Mol. Med. 5,98-109[Medline]
  21. Rostasy, K., Monti, L., Yiannoutsos, C., Kneissl, M., Bell, J., Kemper, T. L., Hedreen, J. C., Navia, B. A. (1999) Human immunodeficiency virus infection, inducible nitric oxide synthase expression, and microglial activation: pathogenetic relationship to the acquired immunodeficiency syndrome dementia complex Ann. Neurol. 46,207-216[Medline]
  22. Lipton, S. A., Sucher, N. J., Kaiser, P. K., Dreyer, E. B. (1991) Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity Neuron 7,111-118[Medline]
  23. Corasaniti, M. T., Melino, G., Navarra, M., Garaci, E., Finazzi-Agro, A., Nistico, G. (1995) Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase Neurodegeneration 4,315-321[Medline]
  24. Polazzi, E., Levi, G., Minghetti, L. (1999) Human immunodeficiency virus type 1 Tat protein stimulates inducible nitric oxide synthase expression and nitric oxide production in microglial cultures J. Neuropathol. Exp. Neurol. 58,825-831[Medline]
  25. Hermann, E., Idziorek, T., Kusnierz, J. P., Mouton, Y., Capron, A., Bahr, G. M. (1997) Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication Int. J. Immunopharmacol. 19,387-397[Medline]
  26. Hori, K., Burd, P. R., Furuke, K., Kutza, J., Weih, K. A., Clouse, K. A. (1999) Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome Blood 93,1843-1850[Abstract/Free Full Text]
  27. Vullo, V., Mastroianni, C. M., Lichtner, M., Mengoni, F., D’Agostino, C., Forcina, G., Corpolongo, A., Delia, S. (1998) Rhodococcus equi infection of monocytes/macrophages from human immunodeficiency virus (HIV)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production FEMS Immunol. Med. Microbiol. 21,11-17[Medline]
  28. Torre, D., Zeroli, C., Ferrario, G., Pugliese, A., Speranza, F., Orani, A., Casari, S., Bassi, P., Poggio, A., Carosi, G. P., Fiori, G. P. (1999) Levels of nitric oxide, gamma interferon and interleukin-12 in AIDS patients with toxoplasmic encephalitis Infection 27,218-220[Medline]
  29. Schon, T., Gebre, N., Sundqvist, T., Aderaye, G., Britton, S. (1999) Effects of HIV co-infection and chemotherapy on the urinary levels of nitric oxide metabolites in patients with pulmonary tuberculosis Scand. J. Infect. Dis. 31,123-126[Medline]
  30. Loveless, M. O., Phillips, C. R., Giraud, G. D., Holden, W. E. (1997) Decreased exhaled nitric oxide in subjects with HIV infection Thorax 52,185-186[Abstract]
  31. Mannick, J. B., Stamler, J. S., Teng, E., Simpson, N., Lawrence, J., Jordan, J., Finberg, R. W. (1999) Nitric oxide modulates HIV-1 replication J. Acquir. Immune Defic. Syndr. 22,1-9
  32. Persichini, T., Colasanti, M., Fraziano, M., Colizzi, V., Ascenzi, P., Lauro, G. M. (1999) Nitric oxide inhibits HIV-1 replication in human astrocytoma cells Biochem. Biophys. Res. Commun. 254,200-202[Medline]
  33. Persichini, T., Colasanti, M., Lauro, G. M., Ascenzi, P. (1998) Cysteine nitrosylation inactivates the HIV-1 protease Biochem. Biophys. Res. Commun. 250,575-576[Medline]
  34. Persichini, T., Colasanti, M., Fraziano, M., Colizzi, V., Medana, C., Polticelli, F., Venturini, G., Ascenzi, P. (1999) Nitric oxide inhibits the HIV-1 reverse transcriptase activity Biochem. Biophys. Res. Commun. 258,624-627[Medline]
  35. Sekkai, D., Aillet, F., Isreal, N., Lepoivre, M. (1998) Inhibition of NF-{kappa}B and HIV-1 long terminal repeat transcriptional activation by inducible nitric oxide synthase 2 activity J. Biol. Chem. 273,3895-3900[Abstract/Free Full Text]
  36. Chen, F., Lu, Y., Castranova, V., Rojanasakul, Y., Miyahara, K., Shizuta, Y., Vallyathan, V., Shi, X., Demers, L. M. (1999) Nitric oxide inhibits HIV tat-induced NF-{kappa}B activation Am. J. Pathol. 155,275-284[Abstract/Free Full Text]
  37. Ouaaz, F., Ruscetti, F. W., Dugas, B., Mikovits, J., Agut, H., Debre, P., Mossalayi, M. D. (1996) Role of IgE immune complexes in the regulation of HIV-1 replication and increased cell death of infected U1 monocytes: involvement of CD23/Fc{varepsilon} RII-mediated nitric oxide and cyclic AMP pathways Mol. Med. 2,38-49[Medline]
  38. Dickie, P. (2000) Nef modulation of HIV type 1 gene expression and cytopathicity in tissues of HIV transgenic mice AIDS Res. Hum. Retrovir. 16,777-790[Medline]
  39. Gazzinelli, R. T., Sher, A., Cheever, A., Gerstberger, S., Martin, M. A., Dickie, P. (1996) Infection of human immunodeficiency virus 1 transgenic mice with Toxoplasma gondii stimulates proviral transcription in macrophages in vivo J. Exp. Med. 183,1645-1655[Abstract/Free Full Text]
  40. Dickie, P., Felser, J., Eckhaus, M., Bryant, J., Silver, J., Marinos, N., Notkins, A. L. (1991) HIV-associated nephropathy in transgenic mice expressing HIV-1 genes Virology 185,109-119[Medline]
  41. Dickie, P. (1996) Nef perturbs eye lens development in transgenic mice AIDS Res. Hum. Retrovir. 12,177-189[Medline]
  42. Granger, D. L., Hibbs, J. B., Broudnax, L. M. (1991) Urinary nitrate excretion in relation to murine macrophage activation: influence of dietary L-arginine and oral NG-monomethyl-L-arginine J. Immunol. 146,1295-1302
  43. Stuehr, D. J., Nathan, C. F. (1989) Nitric oxide A macrophage product responsible for cytostasis and respiratory inhibition in tumor targets. J. Exp. Med. 169,1543-1555
  44. Corraliza, I. M., Campo, M. L., Soler, G., Modolell, M. (1994) Determination of arginase activity in macrophages: a micromethod J. Immunol. Methods 174,231-235[Medline]
  45. Chomczynisky, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal. Biochem. 162,156-159[Medline]
  46. Chang, C. I., Liao, J. C., Kuo, L. (1998) Arginase modulates nitric oxide production in activated macrophages Am. J. Physiol. 274,H342-H348[Abstract/Free Full Text]
  47. Gotoh, T., Mori, T. (1999) Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells J. Cell Biol. 144,427-434[Abstract/Free Full Text]
  48. Munder, M., Eichmann, K., Moran, J. M., Centeno, F., Soler, G., Modolell, M. (1999) Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells J. Immunol. 163,3771-3777[Abstract/Free Full Text]
  49. Corbett, J. A., Tilton, R. G., Chang, K., Hason, K. S., Ido, Y., Wang, J. L., Sweetland, M. A., Lancaster, J. R., Jr, Williamson, J. R., McDaniel, M. L. (1992) Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction Diabetes 41,552-556[Abstract]
  50. Gross, S. S., Stuehr, D. J., Aisaka, K., Jaffe, E. A., Levi, R., Griffith, O. W. (1990) Macrophage and endothelial cell nitric oxide synthesis: cell-type specific selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine Biochem. Biophys. Res. Commun. 170,96-103[Medline]
  51. de Bracco, M. M. E., Felippo, M., Vernava, D., Perez-Bianco, R., de Tezanos Pinto, M. (1997) Nitric oxide production and disease evolution in HIV-infected hemophilic patients AIDS 11,822-824[Medline]
  52. Persichini, T., Ascenzi, P., Colizzi, V., Fraziano, M., Venturini, G., Colasanti, M. (1999) Molecular bases for the anti-HIV-1 effect of NO Int. J. Mol. Med. 4,365-368[Medline]
  53. Lewin, S. R., Kirihara, J., Sonza, S., Irving, L., Mills, J., Crowe, S. M. (1998) HIV-1 DNA and mRNA concentrations are similar in peripheral blood monocytes and alveolar macrophages in HIV-infected individuals AIDS 12,719-727[Medline]
  54. Rich, E. A. (1998) Activation-inactivation of HIV-1 in the lung J. Biomed. Sci. 5,1-10[Medline]
  55. Barton, C. H., Biggs, T. E., Mee, T. R., Mann, D. A. (1996) The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNOS activity in a murine macrophage cell line J. Gen. Virol. 77,1643-1647[Abstract/Free Full Text]
  56. Fligger, J., Blum, J., Jungi, T. W. (1999) Induction of intracellular arginase activity does not diminish the capacity of macrophages to produce nitric oxide in vitro Immunobiology 200,169-186[Medline]
  57. Werner, E. R., Werner-Felmayer, G., Wachter, H. (1993) Tetrahydrobiopterin and cytokines Proc. Soc. Exp. Biol. Med. 203,1-12[Abstract]
  58. Bune, A. J., Brand, M. P., Heales, S. J. R., Shergill, J. K., Cammack, R., Cook, H. T. (1996) Inhibition of tretrahydrobiopterin synthesis reduces in vivo nitric oxide production in experimental endotoxic shock Biochem. Biophys. Res. Commun. 220,13-19[Medline]
  59. Vann, L. R., Twitty, S., Spiegel, S., Milstien, S. (2000) Divergence in regulation of nitric oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-alpha Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity. J. Biol. Chem. 275,13275-13281
  60. Trono, D. (1995) HIV accessory proteins: leading roles for the supporting cast Cell 82,189-192[Medline]
  61. Albina, J. E., Cui, S., Mateo, R. B., Reichner, J. S. (1993) Nitric oxide-mediated apoptosis in murine peritoneal macrophages J. Immunol. 150,5080-5085[Abstract]
  62. von Knethen, A., Brockhaus, F., Kleiter, I., Brune, B. (1999) NO-evoked macrophage apoptosis is attenuated by camp-induced gene expression Mol. Med. 5,672-684[Medline]
  63. Denis, M. (1994) Human monocytes/macrophages: NO or no NO? J. Leukoc. Biol. 55,682-684[Abstract]
  64. Shiloh, M. U., MacMicking, J. D., Nicholson, S., Brause, J. E., Potter, S., Marino, M., Fang, F., Dinauer, M., Nathan, C. (1999) Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase Immunity 10,29-38[Medline]
  65. Lander, H. M., Sehajpal, P., Levine, D. M., Novogrodsky, A. (1993) Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds J. Immunol. 150,1509-1516[Abstract]
  66. Kim, S. J., Bang, O. S., Lee, Y. S., Kang, S. S. (1998) Production of inducible nitric oxide is required for monocytic differentiation of U937 cells induced by vitamin E-succinate J. Cell Sci. 111,435-441[Abstract]
  67. Nisoli, E., Clementi, E., Tonelli, C., Sciorati, C., Briscini, L., Carruba, M. O. (1998) Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures Br. J. Pharmacol. 125,888-894[Medline]
  68. Rich, E. A., Chen, I. S., Zack, J. A., Leonard, M. L., O’Brien, W. A. (1992) Increased susceptibility of differentiated mononuclear phagocyte to productive infection with human immunodeficiency virus-1 (HIV-1) J. Clin. Investig. 89,176-183



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. R. Kline, D. J. Kleinhenz, B. Liang, S. Dikalov, D. M. Guidot, C. M. Hart, D. P. Jones, and R. L. Sutliff
Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2792 - H2804.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dickie, P.
Right arrow Articles by Lee, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dickie, P.
Right arrow Articles by Lee, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS