Inducible Nitric-oxide Synthase and Nitric Oxide Production in Human Fetal Astrocytes and Microglia
A KINETIC ANALYSIS*

(Received for publication, November 20, 1996, and in revised form, February 13, 1997)

Minzhen Ding Dagger §, Barbara A. St. Pierre Dagger , John F. Parkinson , Poonam Medberry , Joyce L. Wong Dagger , Norma E. Rogers par , Louis J. Ignarro par and Jean E. Merrill

From the Department of Dagger  Neurology and par  Medical and Molecular Pharmacology, UCLA School of Medicine, Los Angeles, California 90095 and the  Department of Immunology, Berlex Biosciences, Richmond, California 94804

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The understanding of the induction and regulation of inducible nitric-oxide synthase (iNOS) in human cells may be important in developing therapeutic interventions for inflammatory diseases. In the present study, we not only demonstrated that human fetal mixed glial cultures, as well as enriched microglial cultures, synthesize iNOS and nitric oxide (NO) in response to cytokine stimulation, but also assessed the kinetics of iNOS and NO synthesis in human fetal mixed glial cultures. The iNOS mRNA was expressed within 2 h after stimulation and decreased to base line by 2 days. Significant levels of iNOS protein appeared within 24 h after stimulation and remained elevated during the culture period. A dramatic increase in NO production and NO-mediated events, such as the induction of cyclic guanosine monophosphate (cGMP), NADPH diaphorase activity, and nitrotyrosine occurred 3 days after stimulation, a delay of 48 h from the time of the first expression of iNOS enzyme. This delay of NO production was altered by the addition of tetrahydrobiopterin, but not by the addition of L-arginine, heme, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), or NADPH. These findings suggest that a post-translational regulatory event might be involved in iNOS-mediated NO production in human glia.


INTRODUCTION

Nitric oxide (NO) mediates functions as diverse as vasodilation (1-3), neurotransmission (4, 5), and immune-mediated cytotoxicity (6-8). NO production is catalyzed by NO synthases of which there are three isoforms (9-11). Multiple sclerosis (MS)1 is a central nervous system disorder with immune-mediated destruction of myelin and the myelin-producing cells, oligodendrocytes. The presence of inducible nitric-oxide synthase (iNOS or Type II NOS) and the "footprints" of NO in tissues of patients with MS and animals with experimental allergic encephalomyelitis, a model for MS, suggests that NO may play a role in this central nervous system autoimmune disease (12-15).

Glial cells including astrocytes, microglia, and oligodendrocytes are all involved in the lesion and plaque formation in MS and experimental allergic encephalomyelitis. Rodent astrocytes and microglia express high levels of Type II/iNOS and release significant NO within hours after lipopolysaccharide stimulation in vitro (8, 16, 17). In culture NO produces mitochondrial dysfunction, DNA damage, morphological changes, and necrotic cell death in rat oligodendrocytes (8, 18), while rat astrocytes and microglia are more resistant to NO-mediated damage (19, 20). If such glial cell-mediated NO-dependent damages were to occur in vivo in MS, it could contribute to the plaque formation and a loss of myelin.

Since MS is a human central nervous system disorder, insights into the role of NO in the immnopathology of MS require a better understanding of NO induction in human glial cells. However, the induction and the regulation of iNOS in human glial cells is still unclear and seems substantially different from that in rodents. First of all, the nature of inducing signals is different. Lipopolysaccharide, a potent NO inducer in rodent cells, does not induce NO in human glial cells when used alone or in combination with cytokines (21, 22). Second, the issue of whether or not human microglia synthesize iNOS and release NO is still unresolved (22-25). In contrast to what has been observed in rodent cultures (8, 16, 17), cultures of human astrocytes produce low amounts of NO, and its detection in culture supernatants is delayed from the time of stimulation (21, 22, 26, 27).

To understand the mechanism of iNOS production in human glial cells, we performed a kinetic analysis of iNOS mRNA expression, protein synthesis, enzyme activity, and NO production as well as an evaluation of NO-mediated events or "footprints" in enriched microglial and also in mixed cultures of astrocytes and microglia from human fetal brain tissue. We found that there is a temporal lag between iNOS enzyme synthesis and NO production. Insufficient intracellular levels of the iNOS cofactor tetrahydrobiopterin (BH4) may explain the deficiency of enzyme activity leading to delayed onset of NO production. These findings suggest a post-translational regulation of iNOS enzyme activity in human glial cells.


EXPERIMENTAL PROCEDURES

Primary Human Glial Cells: Establishment of Cultures and Glial Cell Type Identification

The human glial primary cultures were established from fetal brain tissues of fetuses 19.8 ± 1.8 weeks old (range 17.5-24.0 weeks). Entire brains of aborted fetuses were obtained from Advanced Bioscience Resources, Inc. (Alameda, CA). Cultures from a single brain were used for each experiment. A total of eight different brains were used to establish primary cultures throughout the course of these experiments.

Human brain tissue was prepared as described previously (27) and cultured in Iscove's modified Dulbecco's medium (Irvine Scientific, Santa Ana, CA) containing 10% non-heat-inactivated fetal calf serum (Gemini Bioproducts, Inc., Calabasas, CA) as well as L-glutamine (2 mM) and gentamicin sulfate (50 µg/ml), both purchased from Irvine Scientific. Once cultures were established (1-4 weeks), they were split at passage 1 and maintained in Iscove's medium containing microglial growth factors: macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) (each used at 2.8 ng/ml, Immunex Corp., Seattle, WA). Enriched microglial cells were harvested from attached mixed cultures by gentle shaking.

For phenotyping, viable or fixed glial cells were stained as described previously (27). Astrocytes were detected by a polyclonal anti-glial fibrillary acidic protein antibody (1:100; Boehringer Mannheim). Microglia were labeled by monoclonal EBM-11 (anti-CD68, 1:50; DAKO Corp., Carpinteria, CA), fluorescein-conjugated Ricinus communis agglutinin 1 (RCA-1) (1:50; Vector Laboratories, Inc., Burlingame, CA), and, for receptors for acetylated low density lipoprotein, 1 µg/ml 1,1'-dioctade cyl-1-3,3,3'3'-tetramethyl indocarbocyanine perchlorate-conjugated LDL (Dil-alpha -LDL) (Biomedical Technologies, Inc., Stoughton, MA). Presence of neurons was examined by staining with anti-neuronal specific enolase (1:50; DAKO), and oligodendrocytes was detected by staining with a polyclonal antibody to galactocerebroside (GalC 1:20, Boehringer Mannheim). Polyclonal Factor VIII antibody (1:50; DAKO) was used for determining whether there were endothelial cells in the cultures.

Northern Blot Analysis for iNOS mRNA

The cultures of mixed human microglia and astrocytes (50:50) were either left unstimulated or they were stimulated with 70 ng/ml human recombinant gamma  interferon (IFN-gamma ; Genentech, San Francisco, CA) and 5 ng/ml human recombinant interleukin-1beta (IL-1beta ; Immunex Corp, Seattle, WA) for various time periods from 2 h to 7 days. The cell pellets were harvested, and total RNA was isolated by guanidinium isothiocyanate according to published methods (28). RNA concentration was measured in a Beckman DU-65 spectrophotometer equipped with a 5-carat microcell. RNA (50 µg/lane) was mixed with ethidium bromide (1-2 µg) and loaded into a 1.2% agarose gel containing formaldehyde. After separation by electrophoresis, RNA was transferred to a Nytran membrane (Schleicher & Schuell) and cross-linked with ultraviolet irradiation. The human hepatocyte iNOS cDNA fragment of 2.1 kb was digested from pBluescript SK with EcoRI and BamHI (obtained from Dr. D. Geller, University of Pittsburgh, Pittsburgh, PA), labeled with [alpha -32P]dATP (10.0 mCi/ml, DuPont), and hybridized with the membrane. A 0.7-kb BamHI cDNA fragment of rat cyclophilin gene, isolated from pCD vector (obtained from Dr. J. G. Sutcliffe, The Scripps Research Institute, La Jolla, CA) was used as a control for the amount of total RNA loaded in each lane. After 1 h of prehybridization, hybridization was performed for 2 h at 65 °C using QuikHYB Rapid Hybridization solution (Stratagene, La Jolla, CA). The blot was washed to 65 °C, and exposed to Kodak XAR-5 film at -70 °C. For iNOS, the exposure was 6-9 days, while, for cyclophilin, exposure was overnight. For reprobing of the same membrane with another cDNA, the membrane was stripped of the hybridized probe with 0.1 × SSC, 0.1% SDS at boiling temperature for 20 min.

In Situ Hybridization for iNOS mRNA

Human glial cells were plated on four-well glass Lab-Tek chamber slides at 2 × 105 cell/ml. After an incubation period similar to that described above, with or without cytokine stimulation, cultures were rinsed with diethylpyrocarbonate-treated PBS and fixed for 10 min in 4% paraformaldehyde. After rinsing twice with diethylpyrocarbonate-treated PBS, cells were incubated with 0.1 M triethanolamine and then 0.1 M triethanolamine,0.25% acetic anhydride for 10 min each. After two 5-min rinses in 2 × SSC, cells were dehydrated through graded alcohol up to 100%. After two 5-min incubations with chloroform, cells went through 100% and 95% alcohol and were dried in air. All of the above steps were performed at room temperature. 33P-Labeled antisense and sense riboprobes of the human hepatocyte-iNOS were synthesized by in vitro transcription. The pBluescript plasmid containing 2.1-kb fragment of the human hepatocyte-iNOS (inserted into EcoRI/BamHI site) was linearized at NotI and HindIII sites for antisense and sense probe, respectively. [alpha -33P]UTP (10.0 mCi/ml, DuPont) was incubated with cold nucleotides, DTT, RNasin, linearized template plasmids, and RNA polymerase at 37 °C for 1 h. The template DNA was then degraded by RNase-free DNase I at 37 °C for 15 min. Finally, labeled probes were precipitated at -20 °C overnight. For hybridization, probes were heat-denatured at 65 °C for 5 min and quenched on ice, and then added into the hybridization buffer containing 0.6 M NaCl, 0.04 M EDTA, 0.1% sodium pyrophosphate, 10% dextran, 0.2% SDS, 0.02% heparin, and 50% formamide. The hybridization buffer was added to the cells on slides which were then covered with cover slides and incubated at 53 °C overnight. After a brief wash in 2 × SSC, the slides were transferred to RNase A digestion buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 300 mM NaCl, 10 mM DTT, 30 mg/ml RNase A) for 30 min at room temperature. Thereafter, slides were washed with gradually increased stringency from 2 × SSC at room temperature to 0.2 × SSC at 60 °C. The slides were allowed to cool to room temperature, dehydrated, coated with NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), and exposed for 5 days. Slides were developed and counterstained with methylene blue and examined for silver grains on a Zeiss Axioskop microscope.

The Preparation and Specificity of the Polyclonal iNOS Antibody

The iNOS antibody used for iNOS immunocytochemical staining of cells and on Western blots was a polyclonal antibody raised in rabbits against a specific human iNOS peptide sequence (NESPQPLVETGK, encoding residues 54-65 of human iNOS) synthesized at Berlex Biosciences, Richmond, CA. This sequence is not found in Type I or III NOS. The specificity of the antibody was demonstrated by Western blot analysis where a single band at 130 kDa was detected in the astrocytoma line A-172 when stimulated with human recombinant IL-1beta and IFN-gamma (Fig. 1). The band could be blocked by preincubation of the antibody with the inducing peptide overnight at 4 °C (data not shown). The antibody did not stain neurons (containing Type I NOS) or endothelial cells (containing type III NOS) (data not shown).


Fig. 1. Western blot of iNOS protein expression in human glioblastoma cell line A-172. A-172 cells were incubated with or without 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma . Loading of protein at 100 µg/lane was performed. The blot was probed with the polyclonal Ab specific for human iNOS peptide (1:500) overnight at 4 °C. The iNOS protein was visualized by chemiluminescence.
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Western Blot Analysis for iNOS Protein

Mixed cultures of human fetal glia (both microglial and astrocytes 50:50) were stimulated with IL-1beta /IFN-gamma or left unstimulated over the same time course as above. Cell pellets were harvested and homogenized with 50 mM Tris buffer, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, and 0.5 mM dithiothreitol at 0-4 °C. Homogenates were centrifuged at 20,000 × g for 60 min at 4 °C. The protein concentration in the supernatant of the cellular homogenates was determined by the Bradford Coomassie Brilliant Blue method (Bio-Rad). Bovine serum albumin was used as the standard. The cell lysates were diluted, electrophoresed on a 10% polyacrylamide gel, and transferred overnight to a nitrocellulose blot. The nitrocellulose blot was blocked with a solution containing 2% bovine serum albumin and 5% nonfat milk for 1 h at room temperature and washed with 0.2% Tween 20 in PBS three times for 5 min. The blot was probed with the polyclonal antibodies specific for human iNOS peptide (1:500) overnight at 4 °C. The blot was washed three times and then incubated with the secondary antibody, a goat anti-rabbit horseradish peroxidase-conjugated IgG, for 1 h at room temperature. The iNOS protein was visualized by chemiluminescence (Amersham).

Immunocytochemistry for iNOS and Nitrotyrosine

Mixed glia or enriched cultures of microglia or astrocytes were cultured in Lab-Tek glass chamber slides with or without cytokines as above for times ranging from 6 h to 7 days, after which cells were fixed in 4% paraformaldehyde at room temperature for 5 min. They were then rinsed in PBS, and permeabilized with 0.1% Triton in PBS. After three 5-min washes with PBS, cells were incubated with 1% normal goat serum in PBS for 30 min to block nonspecific binding. They were then incubated with the primary iNOS specific polyclonal antibody (see above) overnight in PBS at 4 °C. To look for one of the footprints of NO production, the nitration of tyrosines, cells were also incubated with polyclonal rabbit NT antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) at 1:100. After PBS wash, cells were incubated at 4 °C with a biotinylated swine anti-rabbit IgG antibody (1:200 dilution) in PBS for 30 min, washed in PBS, and then incubated with an alkaline phosphatase-linked avidin (Vector) for 30 min at room temperature. Finally, cells were exposed to alkaline phosphatase substrate p-nitrophenyl phosphate (Vector) for 5 min. The slides were then washed in water and mounted with a glycerol/gelatin mounting medium (Sigma). As a control for nonspecific staining, either the primary antibody was omitted or normal rabbit IgG was used as the primary antibody.

Semi-quantitation of Histochemical Results

To semi-quantitate the histochemical staining results, we scored cells in a double-blind experiment after staining for iNOS and NT. According to the intensity, a 0-5 scale was set to describe the staining level and the number of positive cells. More than 200 cells in each group were examined, and the weighted average of the score for each group was derived as a measure of the presence of positive cells. For in situ hybridization results, cell images were captured into a Macintosh computer and scanned by the NIH Image Program. The mean optical density of each cell was recorded, and the average of 200 cells in each group was used as a semi-quantitative measurement.

Determination of iNOS Enzyme Activity by L-Citrulline Levels

Human fetal glial iNOS activity was measured by determining the conversion of L-[3H]arginine to L-[3H]citrulline as described previously (29). Briefly, cell pellets were harvested and homogenized with 50 mM TEA buffer, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 1 µM pepstatin A, and 2 µM leupeptin at 0-4 °C. Homogenates were centrifuged at 20,000 × g for 60 min at 4 °C, and the supernatant was used as the source of NO synthase. Enzymatic reactions were conducted at 37 °C in 50 mM TEA, pH 7.4., containing 50 µM L-arginine, 100 µM NADPH, 10 µM BH4, 10 µM FMN, 10 µM FAD, 0.2-0.4 mg of supernatant protein, and approximately 200,000 dpm of purified L-2,3,4,5-[3H]arginine HCl (77 Ci/mmol; Amersham). The reactions were terminated by addition of ice-cold 20 mM sodium acetate buffer (pH 5.5) containing 2 mM EDTA, 0.2 mM EGTA, and 1 mM citrulline. The samples were chromatographed on columns of Dowex AG 1-X8, OH- form (prepared from the acetate form). The eluate was collected, and counted in Beckman LS 3801 liquid scintillation spectrometer.

The protein concentration in cellular homogenates was determined by the Bradford Coomassie Brilliant Blue method (Bio-Rad, Richmond, CA). Bovine serum albumin was used as the standard.

Determination of Total Nitric Oxide (NOx-) by Griess Reagent Reaction and Nitrate Reductase

NOx- was determined by measuring the formation of both the stable oxidation products of NO, namely nitrite (NO2-) and nitrate (NO3-). NO in oxygen-containing solutions is chemically unstable and undergoes rapid oxidation to NO2-. The presence of various biological tissue components catalyzes this oxidation and promotes further oxidation of NO2- to NO3- (10, 31). Therefore, it is necessary to measure both NO2- and NO3- to accurately determine the level of total NO. The concentrations of NO2- can be determined by Griess reagent reaction as described by Tracey (30). Nitrate in cell culture supernatants was first reduced to nitrite by incubation the samples for 30 min with nitrate reductase (0.1 units/ml, Boehringer Mannheim) in the presence of 100 µM NADPH and 10 µM FAD. Any remaining NADPH was oxidized with lactate dehydrogenase (10 units/ml) in the presence of 10 mM sodium pyruvate. The total nitrite concentration was then determined by using the procedure based on the Griess reagent reaction. Background nitrite and nitrate levels in the medium (about 8 µM) were subtracted from the experimental values.

NADPH Diaphorase Staining

For NADPH diaphorase staining, fixed cells were incubated in 0.1 M phosphate buffer, pH 7.4, containing 1 mg/ml beta NADPH, 0.1 mg/ml nitro blue tetrazolium, and 0.3% Triton X-100 at 37 °C for 1 h and rinsed twice with 1 × PBS. The positive staining was also assessed in a semi-quantitative way as for iNOS and NT staining.

Determination of Cyclic Guanosine Monophosphate (cGMP) by Radioimmunoassay

The cGMP radioimmunoassay as a bioassay of NO production was adapted from the procedure of Ishii et al. (31) and modified by Simmons and Murphy (32). All the reagents for this assay were purchased from Sigma. Briefly, cell cultures in 12-well plates were washed twice with HBSS and then equilibrated in HBSS for 20 min. Buffer was then replaced with HBSS containing 0.6 mM isobutylmethylxanthine, 20 units/ml superoxide dismutase, and L-arginine (10-4 M) and/or arginine analogues Nomega -nitro-L-arginine methyl ester or NG-monomethyl-L-arginine. Incubation was for 15 min at 37 °C, after which buffer was replaced by 150 µl of cold assay buffer (50 mM sodium acetate with 0.1% sodium azide, pH 6.2), and cells were scraped out with 300 µl of assay buffer and frozen at -20 °C until cGMP radioimmunoassay.

Cell pellets were generated by centrifugation at 14,000 × g for 10 min at 4 °C, and 100 µl of supernatants was then assayed in duplicate using radioimmunoassay procedures adapted from those of Steiner et al. (33). Rabbit antiserum against cGMP was provided by Dr. S. Murphy (University of Iowa, Iowa City, IA). It was prepared using limpet hemocyanin-conjugated cGMP as immunogen. The samples were acetylated with 5 µl of triethylamine/acetic anhydride (2:1 v/v) and then incubated with equal volumes of antiserum (diluted 1:10,000) and 125I-cyclic GMP-tyrosine methyl ester (20,000 cpm/reaction, DuPont) in assay buffer for 16-20 h at 4 °C. They were then incubated with 200 µl/reaction magnetic goat anti-rabbit IgG (BioMag, Cambridge, MA) for 20 min at room temperature and placed on a magnetic plate (BioMag) for 10 min to achieve separation. The supernatant was decanted, and the pellets were counted for radioactivity in a Beckman 4007 gamma  counter.

Administration of Exogenous Cofactors and Substrate

The iNOS substrate L-arginine or NOS cofactors, including BH4, FAD, FMN, and NADPH, were added separately or together to the mixed microglia/astrocyte cultures. The total NOx- in the culture supernatants was determined as above. The above substrates were used in a range of concentrations from 10 µM to 500 µM.

Statistical Analysis

The levels of NOx-, cGMP, and citrulline produced by stimulation of glial cells were analyzed for significance by means of a multivariate analysis of variance (ANOVA). Statistical significance was established at p < 0.05.


RESULTS

Cell Composition of Human Fetal Enriched Microglia and Mixed Glial Cultures

There were about 50% microglia and 50% astrocytes in the mixed glial cultures, as determined by Dil-alpha -LDL and specific microglial antibody staining and glial fibrillary acidic protein staining, respectively. More than 98% of the cells in the enriched microglial cultures were Dil-alpha -LDL positive. These cultures contained CD4-negative parenchymal microglia, and not the perivascular microglia (27). When cultures were stained with antibodies to neuronal specific enolase, galactocerebroside, or Factor VIII, no significant numbers of neurons, oligodendrocytes, or endothelial cells, respectively, were found in either the mixed human fetal glial cultures or the enriched microglial cultures (data not shown).

The Kinetics of iNOS mRNA Expression in Human Fetal Glial Cultures

Northern blot analysis was used to determine iNOS mRNA expression in human fetal mixed glial cultures. As indicated in Fig. 2A, the iNOS probe identified a single 4.1-kb mRNA in cultures treated by IL-1beta and IFN-gamma . The iNOS mRNA signal was detected within 2 h after cytokine stimulation, and a significant level was maintained for at least 2 days. It gradually decreased after day 3, and was undetectable at day 5 and day 7. By a similar method, iNOS mRNA was also detected in enriched human microglial cultures after cytokine stimulation as shown in Fig. 2B.


Fig. 2. Northern blot analysis of iNOS mRNA induction in human fetal glial cultures. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma for 6 h. The positive control was the cytokine-stimulated A-172 cell line. As a negative control, unstimulated glial cell cultures were used. Electrophoresis of total RNA at 30 µg/lane was performed. Cyclophilin mRNA signal was used as a loading control. A, mixed glial cultures; B, enriched microglial cultures.
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To confirm the Northern blot results, in situ hybridization was also used to identify iNOS mRNA in human mixed glial cells in vitro. Cells with both astrocytic and microglial cell morphology demonstrated a more intense signal of silver grain deposition with the antisense probe (Fig. 3A, d) compared with the sense probe (Fig. 3A, c). Unstimulated cells had a very low background of silver grain deposition with either antisense (Fig. 3A, b) or sense probe (Fig. 3A, a). The silver grain deposition in the cells was semi-quantitatively analyzed using a Macintosh computer and NIH Image Program. The results are shown in Fig. 3B. As with the Northern blot analysis, the iNOS mRNA was induced within 2 h after cytokine stimulation and back to base line by 48 h.


Fig. 3. In situ hybridization of iNOS mRNA expression in human fetal mixed glial cultures. Glial cells were incubated for 6 h with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma . A: a, unstimulated control cultures hybridized with sense probe; b, unstimulated cultures hybridized with antisense probe; c, stimulated control cultures hybridized with sense probe; d, stimulated cultures hybridized with antisense probe. B, semi-quantitative measurement of the silver grain deposition using a Macintosh computer and NIH image program. The bar represents 50 µm.
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The Kinetics of iNOS Protein Synthesis in Human Fetal Mixed Glial Cultures

Western blot analysis was used to detect iNOS protein synthesis in mixed glial cultures. Using a polyclonal antibody directed against an iNOS-specific peptide, we detected a band of iNOS protein at 130 kDa in all lanes. The band was significantly increased in IL-1beta and IFN-gamma -treated cultures (Fig. 4). A significant level of iNOS protein was detected at day 1, reaching a maximal level at day 3 and decreasing by day 5. The faint band found in unstimulated control cultures suggested a very low level of iNOS protein synthesis in these unstimulated cells (Fig. 4).


Fig. 4. Western blot of iNOS protein expression in human fetal mixed glial cultures. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma for 1, 3, 5, and 7 days. One hundred µg of total protein was loaded in each lane. The blot was probed with the polyclonal Ab specific for human iNOS peptide (1:500) overnight at 4 °C. The iNOS protein was visualized by chemiluminescence.
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The induction of iNOS protein was further assessed by immunohistochemical staining. The unstimulated and IL-1beta /IFN-gamma -stimulated cultures were stained with the polyclonal antibody to the synthetic human iNOS peptide to assess the presence of iNOS from day 1 to 7. iNOS staining of cultures was seen at 24 h and was maintained through day 7 (Fig. 5A, a and b; day 3 as an example). Some punctate staining was also found in a few unstimulated astrocytes or microglia (Fig. 5A, a and c). Normal rabbit IgG or no primary antibody gave no staining (data not shown). iNOS protein staining was also found in the enriched microglial cultures stimulated with cytokines (Fig. 5A, c and d). The staining of iNOS in the mixed glial cell cultures was semi-quantitated, and the results are shown in Fig. 5B.


Fig. 5. Immunohistochemical staining of iNOS protein in human fetal mixed glial and enriched microglial cultures. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma . A, photographs of day 3 are shown as examples. A: a, mixed glial cultures without cytokines; b, mixed glial cultures with cytokines; c, enriched microglial cultures without cytokines; d, enriched microglial cultures with cytokines. B, semi-quantitative measurement of iNOS staining intensity in mixed glial cultures. The bar represents 50 µm.
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The Kinetics of iNOS Enzyme Activity and NO Production

As determined by Griess reagent reaction, no significant increase of NO was detected in supernatants of IL-1beta /IFN-gamma -stimulated cultures until day 3. NO levels continued to accumulate over the remainder of the 7 days, reaching the highest level of more than 40 µM (Fig. 6A). The NO accumulation was diminished 52% by an arginine analogue Nomega -nitro-L-arginine methyl ester treatment (data not shown). Even though unstimulated cultures expressed low levels of iNOS protein in the first 48 h, no NO above these base-line levels in the medium (8 µM) was apparently released into extracellular medium (Fig. 6A). Similar NO production was also detected in the enriched microglial cultures (Fig. 6B). These results imply that NOx- production occurs 48-72 h after iNOS enzyme synthesis.


Fig. 6. Nitric oxide induction in human fetal glial cultures. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma from day 1 to day 7 in mixed glial cultures (A) and for 1 day and 3 days in enriched microglial cultures (B). NOx- was measured in supernatants by Griess reagent reaction with nitrate reductase. Means ± S.D. are shown for eight different cultured fetal brains (*, p < 0.01, ANOVA).
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We extracted the cytosolic iNOS from cytokine-treated or untreated cells and analyzed its efficiency in converting L-arginine to L-citrulline in the presence of substrate L-arginine and the cofactors NADPH, BH4, FMN, and FAD at non-limiting concentrations. With the addition of substrate and iNOS cofactors, cytokine-stimulated cells showed a small to insignificant iNOS enzyme activity by day 1, which was not detectable in controls. The citrulline level peaked from day 3 to day 5, and returned to base line at day 7. This suggested that the enzyme produced could be functional if cofactors or substrate were not limiting (Fig. 7).


Fig. 7. Enzyme activity of iNOS in human fetal mixed glial cultures as determined by citrulline assay. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma from day 1 to day 7 in mixed glial cultures. Cytosolic iNOS was extracted and analyzed for its efficiency in converting L-arginine to L-citrulline. Means ± S.D. are shown for four different cultured fetal brains (*, p < 0.05, ANOVA).
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The Kinetics of NO-mediated Events

The 2-3-day lag in NO production compared with iNOS production prompted us to assess iNOS function in a separate assay. Since NADPH-d staining has been routinely taken as a nonspecific histochemical indicator of NOS catalytic activity, especially after formaldehyde fixation of cells or tissue, we stained for NADPH-d in cytokine-stimulated and unstimulated mixed glial cultures. There was no significant amount of NADPH-d staining over base line detected between day 1 and 3. NADPH-d activity was eventually seen 3-4 days after stimulation (Fig. 8A, a and b; day 3 is shown as an example). The kinetics of NADPH-d were similar to NO and NO-mediated events, again suggesting a delayed onset of iNOS catalytic activity (Fig. 8B, a).


Fig. 8.

The kinetics of NO-mediated events in human fetal mixed glial cultures. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma from day 1 to day 7 in mixed glial cultures. The photographs of day 3 are shown as example. A: a and b, NADPH-diaphorase staining; c and d, nitrotyrosine staining; a and c, unstimulated cultures; b and d, stimulated cultures. B, semi-quantitative measurement of the stainings. a, NADPH-diaphorase; b, nitrotyrosine. C, radioimmunoassay of cGMP levels. Means ± S.D. are shown for eight different cultured fetal brains (*, p < 0.01, ANOVA). The bar represents 50 µm.


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NT residues in proteins are considered to be specific "footprints" of peroxynitrite (ONOO-), a powerful oxidant and cytotoxic species formed by the rapid reaction between NO and superoxide anion (O2-) (34). ONOO- and other reactive nitrogen species would act as nitrosating agents to modify intracellular tyrosine to form 3-NT and dityrosine (35-37). Thus, NT has also been often used as a "footprint" for NO. We used a polyclonal antibody against NT to detect NT immunoreactivity, and found no significant increase of NT staining over the base line until 3 days after cytokine stimulation. The staining increased reaching the highest level at 7 days post-stimulation (Fig. 8A, c and d; day 3 is shown as an example). No such staining was seen in unstimulated cultures. The staining intensity was semi-quantitated by double-blind examiners (Fig. 8B, b).

Since NO has been reported to stimulate activity of soluble guanylate cyclase, and hence increase the production of cGMP (38, 39), we measured cGMP levels by radioimmunoassay. As with NO production and NADPH diaphorase and NT staining, significant levels of cGMP were not detected until 3 days after cytokine stimulation, reaching a day 7 peak (Fig. 8C).

Tetrahydrobiopterin Increases NO Production in Human Fetal Mixed Glial Cultures

To test the hypothesis that the delay of NO production in human glia might be due to insufficient endogenous iNOS substrate or cofactors, we added a range of concentrations of L-arginine and FAD, FMN, NADPH, heme, and BH4 into the cultures alone or in combinations. The exogenous BH4 increased NO levels in a dose-dependent (Fig. 9A) and time-dependent (Fig. 9B) manner. As indicated by Fig. 9B, after BH4 was added at an optimal concentration of 150 µM, significant NO levels were detected at 24 h and elevated levels of NO were seen at all subsequent time points compared with cultures not receiving BH4. Addition of substrate or any other cofactors, either alone or in various combinations, had no effect upon NO production in these cultures (data not shown). These results demonstrate that BH4 levels may be limited in human glial cells.


Fig. 9. Dose response and time response of the effect of BH4 upon NO production in human fetal mixed glial cultures. A, dose response. Glial cells were incubated with 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma for 3 days. A range of concentrations of BH4 was added into the cultures. B, time response. Glial cells were incubated with or without 5 ng/ml human recombinant IL-1beta plus 70 ng/ml human recombinant IFN-gamma from day 1 to day 7. An concentration of 150 µM of BH4 was added into the culture. NOx- was measured in supernatants by Griess reagent reaction plus nitrate reductase. Means ± S.D. are shown for four different experiments (*, p < 0.01, ANOVA).
[View Larger Version of this Image (22K GIF file)]



DISCUSSION

This is the first complete kinetic study comparing iNOS transcription, translation, enzyme activity, NO production, and NO-mediated events in both human astrocytes and microglia in response to cytokine stimulation as well as being the first study providing in situ hybridization and immunocytochemical staining evidence of iNOS expression in both cell types. By comparing the kinetics of these events, we found that iNOS was transcribed within 2 h after cytokine stimulation, followed by the translation of iNOS protein within 24 h. However, the iNOS catalytic activity, NO production, and NO-mediated events were not detected until 3 days after stimulation. Addition of iNOS cofactor BH4 significantly increased NO level in a dose- and time-dependent manner, suggesting that this cofactor works as a post-translational regulator of iNOS function and may be limiting in human glial cells at least in vitro. Moreover, we verified that, in addition to astrocytes, human microglia are capable of synthesizing iNOS and producing NO upon cytokine stimulation.

Many human cells such as hepatocytes, chondrocytes, endothelial cells, and glioblastoma cells have been shown to express iNOS and release large amounts of NO after stimulation with bacterial products and/or cytokines, showing kinetic patterns similar to those of rodent glial cells (40-43). The present study has demonstrated that normal human glial cells display similar transcription and translation of iNOS as other human cell types or rodent glia, but human glial iNOS is not immediately functional in these cells; therefore, NO production has a delayed kinetics. As with other human cells, iNOS mRNA in human glial cells reaches a maximum level at 3-6 h after stimulation, followed shortly thereafter by the translation into protein. As in rodents, iNOS protein is stable in glia in vitro and can be maintained in cells in culture for several days. This may be the result of a lack of degradation of iNOS protein in vitro, an event that can be mediated by cytokines like TGFbeta (44). Nevertheless, while NO synthesis occurs in rodent glial cells within 24 h post-stimulation (8, 16, 45), significant levels of NO and NO-related events in human glial cells were not detected until 3 days after stimulation, which was at least 48 h after the appearance of iNOS protein. In other human cells that have been studied in vitro (40-43), levels of iNOS mRNA begin declining by 8 h. However, the steady state iNOS mRNA levels was prolonged out to 48 h in this study of human glial cells. Because NO (46) and other factors like TGFbeta (44) suppress iNOS mRNA expression or inhibit iNOS mRNA stability, and these cultures contained neither in the first 2-3 days, the iNOS mRNA levels may have been maintained.

The lag in NO production following the appearance of iNOS suggested a block in functional iNOS enzyme activity. While NO release into culture supernatants became significant between day 3 and 4, the iNOS protein in homogenized cytosol displayed significantly more catalytic activity at earlier time points (days 2-3) in the presence of added substrate and cofactors. This suggested that a deficiency of either substrate or cofactors in glial cells might have led to the delay in functional enzyme. The addition of the substrate L-arginine into the cultures had no significant effect on NO levels. Recent studies have suggested that NO synthase monomers require dimerization for the enzyme to be functional (47, 48). The binding of heme and BH4 play a significant role in forming and stabilizing active dimeric NOS (48-51). Interestingly, other studies have also demonstrated that de novo synthesis of BH4 can be strongly stimulated by cytokines in human macrophages (52). BH4 synthesis was found to be required for iNOS-derived NO production in human endothelial cells (53). Consistent with these studies, we demonstrated here that addition of BH4, but not other cofactors, induced NO at time points when none was previously detected above background (days 1 and 2) and increased NO levels at other time points, shifting the kinetics of NO induction to time points more consistent with iNOS presence in cells. We are currently measuring BH4 levels and iNOS monomer/dimer ratios in these cells to determine if post-translational modification is the limiting step in NO production.

Unlike murine macrophages, which produce high levels of NO (54, 55), human mononuclear phagocytes produce low levels of NO in vitro after cytokine stimulation (56-58), although significant amounts of NO were found in patients with inflammatory diseases (59). Post-translational regulation of iNOS by cofactors has been suggested to be important in the human macrophage. BH4 production is low in macrophages that lack 6-pyruvoyl-tetrahydropterin synthase; nevertheless, addition of exogenous BH4 does not enable macrophages to produce an increased amount of NO, suggesting additional regulatory events in these cells, at least in vitro (57). A second issue critical in the induction of NO in human macrophages is the stimulation used to induce iNOS. Cross-linking of CD23 or CD69 as well as combinations of cytokines like GM-CSF, IL-4, IFN-gamma , and IL-1beta may be optimal inducers in macrophages, thereby distinguishing them from glia.

There is accumulating in vitro and in vivo evidence that, as we have shown here, human microglia are capable of synthesizing iNOS and produce 10-20 µM NO in response to various stimuli (23, 24, 46). However, recent studies by Liu et al. (60), as well as previous reports from the same laboratory (21, 22), failed to detect iNOS in human microglia. Moreover, in contrast to the observations reported here, the Liu study using purified astrocytes showed a delay in both iNOS mRNA and protein expression, but no lag in nitrite production. IL-4 and TGFbeta failed to inhibit both iNOS expression and nitrite production by human astrocytes in the Liu study. However, we have shown that IL-4 and TGFbeta inhibit NO production by human glial cells (61). Both the culture conditions (we used IL-3, M-CSF, and GM-CSF to expand microglia) and the microglial cell phenotype were different in the two studies (27, 60). Furthermore, in our studies, microglia were cultured with astrocytes for up to 2 weeks before harvest, whereas in the Liu study, microglia and astrocytes were separated from the beginning of the culture period (60). Finally, different cytokine doses were used in the two studies. Since the antibody reagents utilized in the two studies were also different, it is difficult to assess the nature of the cell localization of iNOS in their study compared with ours. Perivascular and parenchymal microglia do not have completely identical phenotype. Perivascular microglia are CD4-positive and are derived from blood-borne macrophages, while the parenchymal microglia which we have studied here are CD4-negative (27). The perivascular, CD4-positive, microglia used in the Liu study may thus explain their failure to show NO production (21, 22, 60).

We have consistently seen very low basal levels of iNOS mRNA and protein in unstimulated cultures. This may be the consequence of 1) activation by culture conditions, 2) stimulation of cells during the process of mechanical disruption into a single cell suspension which simulates injury, or 3) the fact that these cells came from an actively developing fetal brain, where iNOS may play a role. Nevertheless, there is also evidence suggesting that iNOS might be present in some normal adult and fetal tissues (62, 63).

In summary, we have demonstrated that human astrocytes and microglia are capable of synthesizing iNOS and NO in response to cytokine stimulation. The potential deficiency of the iNOS cofactor tetrahydrobiopterin in these human glial cells may have resulted in a block in the post-translational dimerization necessary for iNOS catalytic activity and thus a delay of NO production. Further studies are necessary to understand the role of this cofactor in regulating NO production in human glial cells. Such studies will be valuable in understanding the physiological and pathological role of NO in human neurological disorders.


FOOTNOTES

*   This work was supported in part by grants from the Conrad N. Hilton Foundation (to J. E. M.) and the Nancy Davis Foundation for Multiple Sclerosis (to J. E. M. and M. D.), and by National Institutes of Health-National Institute of Mental Health Postdoctoral Fellowship 5T32MH17140-11 and an Oncology Nursing Foundation/Bristol Myers research grant (to B. A. St. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Neurology, School of Medicine, UCLA, Reed Neurology Research Bldg., Rm. A-134, 710 Westwood Plaza, Los Angeles, CA 90095. Tel.: 310-206-8999; Fax: 310-206-9801; E-mail: mding{at}ucla.edu.
1   The abbreviations used are: MS, multiple sclerosis; NOS, nitric- oxide synthase; iNOS, inducible nitric-oxide synthase; LDL, low density lipoprotein; Dil-alpha -LDL, 1,1'-dioctade cyl-1-3,3,3'3'-tetramethyl indocarbocyanine perchlorate-conjugated LDL; kb, kilobase(s); PBS, phosphate-buffered saline; BH4, tetrahydrobiopterin; IL, interleukin; IFN, interferon; TGF, transforming growth factor; HBSS, Hepes-buffered saline solution; NADPH-d, NADPH-diaphorase; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; NT, nitrotyrosine.

ACKNOWLEDGEMENTS

We thank Genentech Inc., South San Francisco, CA for the supply of human IFN-gamma] and Immunex Corp., Seattle, WA for human IL-1beta .


REFERENCES

  1. Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S. (1987) Circ. Res. 61, 866-879 [Abstract]
  2. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 9265-9269 [Abstract]
  3. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987) Nature 327, 524-526 [CrossRef][Medline] [Order article via Infotrieve]
  4. Garthwaite, J. (1995) Trends Neurosci. 18, 51-52 [CrossRef][Medline] [Order article via Infotrieve]
  5. Garthwaite, J., Charles, S. J., and Chess-William, R. (1988) Nature 336, 385-388 [CrossRef][Medline] [Order article via Infotrieve]
  6. Hibbs, J. B., Jr., Taintor, R. R., Vavrin, Z., and Rachlin, E. M. (1988) Biochem. Biophys. Res. Commun. 157, 87-94 [Medline] [Order article via Infotrieve]
  7. Chao, C., Hu, S., Molitor, T. W., Shaskan, E. G., and Peterson, P. K. (1992) J. Immunol. 149, 2736-2741 [Abstract/Free Full Text]
  8. Merrill, J. E., Ignarro, L. J., Sherman, R. P., Melinek, J., and Lane, T. E. (1993) J. Immunol. 151, 2132-2141 [Abstract/Free Full Text]
  9. Lowenstein, C. J., Glatt, C. S., Bredt, D. S., and Snyder, S. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6711-6715 [Abstract]
  10. Xie, Q. W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225-228 [Medline] [Order article via Infotrieve]
  11. Lamas, S., Marsden, P. A., Li, G. K., Tempst, P., and Michel, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6348-6352 [Abstract]
  12. Lin, R. F., Lin, T. S., Tilton, R. G., and Cross, A. H. (1993) J. Exp. Med. 178, 643-648 [Abstract]
  13. Bö, L., Dawson, T. M., Wesselingh, S., Mork, S., Choi, S., Kong, P. A., Hanley, D., and Trapp, B. D. (1994) Ann. Neurol. 36, 778-786 [Medline] [Order article via Infotrieve]
  14. Cross, A. H., Misko, T. P., Lin, R. F., Hickey, W. F., Trotter, J. L., and Tilton, R. G. (1994) J. Clin. Invest. 93, 2684-2690 [Medline] [Order article via Infotrieve]
  15. Bagasra, O., Michaels, F. H., Zheng, Y. M., Bobroski, L. E., Spitsin, S. V., Fu, Z. F., Tawadros, R., and Koprowski, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12041-12045 [Abstract]
  16. Zielasek, J., Tausch, M., Toyka, K. V., and Hartung, H. P. (1992) Cell. Immunol. 141, 111-120 [Medline] [Order article via Infotrieve]
  17. Murphy, S., Simmons, M. L., Agullo, L., Garcia, A., Feistein, D. L., Gallea, E., Reis, D. J., Minc-Golomb, D., and Schwartz, J. P. (1993) Trends Neurosci. 16, 323-328 [CrossRef][Medline] [Order article via Infotrieve]
  18. Mitrovic, B., Ignarro, L. J., Vinters, H. V., Akers, M., Schmid, I., Uittenbogaart, C., and Merrill, J. E. (1995) Neuroscience 65, 531-538 [CrossRef][Medline] [Order article via Infotrieve]
  19. Bolaños, J. P., Peuchen, S., Heales, S. J. R., Land, J. M., and Clark, J. B. (1994) J. Neurochem. 63, 910-916 [Medline] [Order article via Infotrieve]
  20. Brown, G. C., Bolanos, J. P., Heales, S. J. R., and Clark, J. B. (1995) Neurosci. Lett. 193, 201-204 [CrossRef][Medline] [Order article via Infotrieve]
  21. Lee, S. C., Dickson, D. W., Liu, W., and Brosnan, C. F. (1993) J. Neuroimmunol. 46, 19-24 [CrossRef][Medline] [Order article via Infotrieve]
  22. Brosnan, C. F., Battistini, L., Raine, C. S., Dickson, D. W., Casadevall, A., and Lee, S. C. (1994) Dev. Neurosci. 16, 152-161 [Medline] [Order article via Infotrieve]
  23. Colasanti, M., Pucchio, T. D., Persichini, T., Sogos, V., Presta, M., and Lauro, G. (1995) Neurosci. Lett. 195, 45-48 [CrossRef][Medline] [Order article via Infotrieve]
  24. Peterson, P. K., Hu, S., Anderson, W. R., and Chao, C. C. (1994) J. Infect. Dis. 170, 457-460 [Medline] [Order article via Infotrieve]
  25. Walker, D. G., Kim, S. U., and McGeer, P. L. (1995) J. Neurosci. Res. 40, 478-493 [Medline] [Order article via Infotrieve]
  26. Mitrovic, B., Ignarro, L. J., Montestruque, S., Smoll, A., and Merrill, J. E. (1994) Neuroscience 61, 575-585 [CrossRef][Medline] [Order article via Infotrieve]
  27. Koka, P., He, K., Zack, J. A., Kitchen, S., Peacock, W., Fried, I., Tran, T., Yashar, S. S., and Merrill, J. E. (1995) J. Exp. Med. 182, 941-951 [Abstract]
  28. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  29. Bush, P. A., Gonzalez, N. E., Griscavage, J. M., and Ignarro, L. J. (1992) Biochem. Biophys. Res. Commun. 185, 960-966 [Medline] [Order article via Infotrieve]
  30. Tracey, W. R. (1992) Neuroprotocol: Companion Methods Neurosci. 1, 125-131
  31. Ishii, K., Sheng, H., Warner, T. D., Forstermann, U., and Murad, F. (1991) Am. J. Physiol. 261, H598-H603 [Abstract/Free Full Text]
  32. Simmons, M. L., and Murphy, S. (1992) J. Neurochem. 59, 897-905 [Medline] [Order article via Infotrieve]
  33. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972) J. Biol. Chem. 247, 1106-1113 [Abstract/Free Full Text]
  34. Huie, R. E., and Padmaja, S. (1993) Free Radical Res. Commun. 18, 195-199 [Medline] [Order article via Infotrieve]
  35. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431-437 [Medline] [Order article via Infotrieve]
  36. Ischiropoulos, H., and al-Mehdi, A. B. (1995) FEBS Lett. 364, 279-282 [CrossRef][Medline] [Order article via Infotrieve]
  37. van der Vliet, A., Eiserich, J. P., O'Neill, C. A., Halliwell, B., and Cross, C. E. (1995) Arch. Biochem. Biophys. 319, 341-349 [CrossRef][Medline] [Order article via Infotrieve]
  38. Ignarro, L. J. (1992) Biochem. Society Trans. 20, 465-469 [Medline] [Order article via Infotrieve]
  39. Marsault, R., and Frelin, C. (1992) J. Neurochem. 59, 942-945 [Medline] [Order article via Infotrieve]
  40. Chakravarthy, U., Stitt, A. W., McNally, J., Bailie, J. R., Hoey, E. M., and Duprex, P. (1995) Curr. Eye Res. 14, 285-294 [Medline] [Order article via Infotrieve]
  41. Fujisawa, H., Ogura, T., Hokari, A., Weisz, A., Yamashita, J., and Esumi, H. (1995) J. Neurochem. 64, 85-91 [Medline] [Order article via Infotrieve]
  42. Grabowski, P. S., Macpherson, H., and Ralston, S. H. (1996) Br. J. Rheumatol. 35, 207-212 [Medline] [Order article via Infotrieve]
  43. Blanco, F. J., Geng, Y., and Lotz, M. (1995) J. Immunol. 154, 4018-4026 [Abstract/Free Full Text]
  44. Vodovotz, Y., Bogdan, C., Paik, J., Xie, Q. W., and Nathan, C. (1993) J. Exp. Med. 178, 605-613 [Abstract]
  45. Park, S. K., and Murphy, S. (1994) J. Neurosci. Res. 39, 405-411 [Medline] [Order article via Infotrieve]
  46. Colasanti, M., Persichini, T., Menegazzi, M., Mariotto, S., Giordano, E., Caldarera, C. M., Sogos, V., Lauro, G. M., and Suzuki, H. (1995) J. Biol. Chem. 270, 26731-26733 [Abstract/Free Full Text]
  47. Hevel, J. M., and Marletta, M. A. (1992) Biochemistry 31, 7160-7165 [Medline] [Order article via Infotrieve]
  48. Baek, K. J., Thiel, B. A., Lucas, S., and Stuehr, D. J. (1993) J. Biol. Chem. 268, 21120-21129 [Abstract/Free Full Text]
  49. Wang, J., Rousseau, D. L., Abu-Soud, H. M., and Stuehr, D. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10512-10516 [Abstract/Free Full Text]
  50. Wang, J., Stuehr, D., J., and Rousseau, D. L. (1995) Biochemistry 34, 7080-7087 [Medline] [Order article via Infotrieve]
  51. Tzeng, E., Billiar, T. R., Robbins, P. D., Loftus, M., and Stuehr, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11771-11775 [Abstract]
  52. Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., Yim, J. J., Pfleiderer, W., and Wachter, H. (1990) J. Biol. Chem. 265, 3189-3192 [Abstract/Free Full Text]
  53. Werner, E. R., Werner-Felmayer, G., Weiss, G., and Wachter, H. (1993) Adv. Exp. Med. Biol. 338, 203-209 [Medline] [Order article via Infotrieve]
  54. Assreuy, J., cunha, F. Q., Epperlein, M., Noronha-Dutra, A., O'Donnell, C. A., Liew, F. Y., and Moncada, S. (1994) Eur. J. Immunol. 24, 672-676 [Medline] [Order article via Infotrieve]
  55. Evans, T., Carpenter, A., and Cohen, J. (1994) Eur. J. Biochem. 219, 563-569 [Abstract]
  56. Albina, J. E. (1995) J. Leukocyte Biol. 58, 643-649 [Abstract]
  57. Weinberg, J. B., Misukonis, M. A., Shami, P. J., Mason, S. N., Sauls, D. L., Dittman, W. A., Wood, E. R., Smith, G. K., McDonald, B., and Bachus, K. E. (1995) Blood 86, 1184-1195 [Abstract/Free Full Text]
  58. Padgett, E. L., and Pruett, S. B. (1992) Biochem. Biophys. Res. Commun. 186, 775-781 [CrossRef][Medline] [Order article via Infotrieve]
  59. Nicholson, S., Bonecini-Almeida, M. d., Lapa e Silva, J. R., Nathan, C., Xie, D. W., Mumford, R., Weidner, J. R., Calaycay, J., Geng, J., Boechat, N., Linhares, C., Rom, W., and Ho, J. L. (1996) J. Exp. Med. 183, 2293-2302 [Abstract]
  60. Liu, J., Zhao, M.-L., Brosnan, C. F., and Lee, S. C. (1996) J. Immunol. 157, 3569-3576 [Abstract]
  61. St. Pierre, B. A., Wong, J. L., and Merrill, J. E. (1997) in Cell Bioloby and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches (Devon, R. M., Doucette, R., Juunink, B. H. J., Nazarali, A. J., Schreyer, D. J., and Verge, N. K., eds), Plenum Publishing Corp., New York in press
  62. Kobzik, L., Bredt, D. S., Lowenstein, C. J., Drazen, J., Gaston, B., Sugarbaker, D., and Stamler, J. S. (1994) Am. J. Res. Cell Mol. Biol. 9, 371-377
  63. Nathan, C., and Xie, Q. (1994) J. Biol. Chem. 269, 13725-13728 [Free Full Text]

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