* Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; and
Mucosal Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
Correspondence: Mark Gilchrist, Room 574 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail: Mark.Gilchrist{at}ualberta.ca
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(IFN-), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-, or anti-CD8, nitrite production was increased significantly (8.4±0.6, 7.6±0.9, and 6.6±0.9 µM/2x105 cells/48 h NO2-, respectively; P<0.01), whereas unstimulated PMC released 2.1 ± 0.3 µM/2 x 105 cells/48 h NO2-. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.
Key Words: interferon- • RT-PCR • L-NMMA
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mast cells (MC) are strategically located throughout the body at neural, epidermal, mucosal, and vascular sites. They play a critical role in allergy and other inflammatory states through the release of multifunctional mediators, such as histamine, cytokines, arachidonic acid metabolites, and proteinases [9
]. Production of NOS and NO by rat MC has been investigated previously by a variety of means, in many MC types, and using a variety of time courses. Direct localization of iNOS protein to MC has been described using immunohistochemistry in rat intestinal biopsies, mouse-cultured MC, and rat brain tissue [10
11
12
]. Bioassays, including platelet aggregation [13
, 14
] and epithelial permeability [15
], have been used to detect the production of NO indirectly by MC after short-term activation in mouse and rat peritoneal MC (PMC). Detection of iNOS mRNA was accomplished in MC cultured from mouse bone marrow [11
] and subsequently stimulated with immunoglobulin E (IgE). The NO produced by mouse MC has been detected and quantified as its stable breakdown product nitrite (NO2-) using the Griess assay following long-term stimulation with IgE [11
]. However, the amounts of NO obtained were low, and there was little information concerning events at the cellular level. Furthermore, there is debate about whether NO is made by MC or by contaminating cells in isolated cell populations [14
] or if indeed MC have the cellular capacity to react to cytokines, such as interferon- (IFN-), to produce NO [16
]. Therefore, despite recent studies giving further indication that rodent MC produce NO [11
, 12
], the NOS isoform involved, the balance between constitutive and inducible NO production, and the precise stimulatory events and time-course that initiate the NO production remain unresolved.
To investigate the regulation of NO in PMC, we studied the expression of NOS isoforms constitutively and following induction by cytokines and antigens (Ag). In this study, we demonstrate the production of constitutive eNOS mRNA and up-regulated iNOS mRNA expression by PMC after treatment with IFN-, stimulating Ag or CD8 ligation with antibody. Secondly, we localize the iNOS mRNA directly to MC by an in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Third, we demonstrate that PMC synthesize iNOS protein as detected by immunohistochemistry. Last, this protein is active and stimulates the production of NO.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ag
Ag used to activate in vivo-sensitized PMC were a collection of soluble excretory and secretory products of adult N. brasiliensis, prepared by incubating worms in phosphate-buffered solution (PBS; pH 7.2) at 37°C for 4 h [18
]. The Ag concentration was described as worm equivalents (WE) per mL, and the final protein concentration of Ag (5 WE/mL) was 7.1 ± 0.8 µg/mL. Lipopolysaccharide levels were negative as tested by E-toxate reagent (Sigma Chemical Co., St. Louis, MO).
MC isolation and stimulation
Total peritoneal cells were obtained by peritoneal lavage with 15 mL cold HEPES (10 mM)-buffered Tyrode’s solution containing 0.1% bovine serum albumin (BSA). PMC were purified by centrifuging the total peritoneal cells through a two-step, discontinuous gradient of Percoll (Pharmacia Ltd., Upsala, Sweden), as previously described [18
]. The number of PMC recovered was 1.1 x 106 cells/rat, and PMC purity was >98%, as determined by staining with toluidine blue. Contaminating cells (<2%) included eosinophils, pycnotic neutrophils, small dense lymphocytes, and erythrocytes. Cell viability, as determined by trypan blue staining was >99%. PMC were resuspended in RPMI-1640 medium (Gibco-BRL, Grand Island, NY) containing L-glutamine, pH 7.2, supplemented with 5% heat inactivated fetal bovine serum and 40 U/mL penicillin/streptomycin (Gibco-BRL). For RT-PCR experiments, PMC from unsensitized rats were treated with medium containing IFN- (800 U/mL; Gibco-BRL) or anti-CD8 antibody (OX-8, 5 µg/mL; Serotec, Toronto, Canada) for 6 h. PMC from sensitized rats were treated with sensitizing Ag (5 WE/mL) for 16 h. As controls for all experiments, PMC from unstimulated rats were incubated without treatment for the described period of time. For immunohistochemistry experiments, PMC were stimulated with IFN- (800 U/mL), anti-CD8 (5 µg/mL), or Ag (5 WE/mL) for 16 h. As a positive control, isolated macrophages were also incubated with IFN- (800 U/mL; Gibco-BRL) for 16 h. For NO2- determinations, PMC were incubated with IFN- (800 U/mL), anti-CD8 (5 µg/mL), or Ag (5 WE/mL) for 48 h.
RT-PCR
Total RNA was isolated from PMC using a modification of the Chomczynski/Sacchi method as described previously. The isolated RNA was then pretreated with heparinase I (Sigma Chemical Co.) to remove contaminating heparin [19
]. RNA (1 µg) was converted to cDNA by the reverse-transcription reaction (MMLV-RT; Gibco-BRL) in a total volume of 20 µL. PCR amplification was performed in a final volume of 20 µL to which 2 µL cDNA had been added. The reaction was performed on a PTC-100 thermal cycler (MJ Research, Boston, MA). The primers were designed to be intron-spanning, and the expected product sizes are listed in Table 1
. The numbers of cycles used for the NOS primer sets were 25–30 (depending on the linear range) and 20 cycles for ß-actin. The conditions for PCR amplification are as follows: denaturing at 95°C for 45 s, annealing for 45 s, and extension at 72°C for 2 min. The annealing temperature was 56°C for ß-actin and iNOS, 54°C for eNOS, and 50°C for bNOS. For semiquantitative PCR analysis of iNOS induction during amplification, aliquots were withdrawn at 26, 29, 32, and 35 cycles to ensure that amplification was within the exponential range. Products were run on a 1.2% agarose gel and stained with ethidium bromide (Sigma Chemical Co.).
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View this table: [in a new window] |
Table 1. Primer Sets Used for RT-PCR Experiments
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Immunohistochemistry for iNOS
Immunohistochemistry was performed on preparations of PMC to localize iNOS protein further within the cells and to confirm that the iNOS detected was derived from PMC and not from contaminating cells. Briefly, IFN-, anti-CD8, or Ag-treated PMC along with untreated PMC were washed in PBS and then cytocentrifuged onto slides and air-dried overnight. After treatment with 3% hydrogen peroxide to block endogenous peroxidase, slides were incubated overnight at 4°C with a 1/100 (1 µg/mL) dilution of monoclonal iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Specific antibody-binding was detected with a peroxidase-labeled avidin-biotin complex (Vector Laboratories, Burlingame, CA), and diaminobenzidine was used as the chromagen. Negative-control cytospins labeled with isotype (IgG1)-antibody control at a concentration of 1 µg/mL were run concurrently. To confirm the specificity of staining, iNOS antibody was absorbed with immunizing peptide according to the manufacturer’s protocol (Santa Cruz Biotechnology), and this absorbed antibody was also used concurrently to stain PMC.
Measurement of NO2- production
NO2- in culture (phenol red-free) supernatants was measured by the Griess reaction [22
]. Results were expressed as µM/2 x 105 cells/48 h following incubation of 2 x 105 cells in 200 µL volume. Equal volumes of cell-free supernatant and Griess reagent (1% sulfanilamine, 0.1% N-(1-naphyl)-ethylene-diamine dihydrochloride, 2.5% H3PO4) were mixed. NaNO2 was used as a standard. Plates were read on a Vmax kinetic microplate reader (Molecular Devices Co., Menlo Park, CA) at 540 nm.
Assay of NOS activity
To further categorize NO production in PMC, NOS activity was measured by the conversion of L-[14C] arginine to L-[14C] citrulline, as described previously [23
]. PMC (5x106) were incubated in the presence of 800 U/mL IFN- for 18 h, and unstimulated PMC (control) were isolated and used immediately (0 h). The cells were centrifuged at 900 g for 5 min, and the resulting pellet was retained. The pellet was homogenized in buffer containing 50 mM Tris HCl, 320 mM sucrose, 1 mM dithiothreitol, 10 µg/mL leupeptin, 10 µg/mL soybean trypsin inhibitor, and 2 µg/mL aprotinin (Sigma Chemical Co.). To start the assay, 40 µL cell homogenate was added to tubes containing 100 µL reaction buffer consisting of 50 mM KH2PO4 (pH 7.2), 50 mM valine, 100 µM reduced nicotinamide adenine dinucleotide phosphate, 1 mM L-citrulline, 0.2 mM CaCl2, 5 µM tetrahydrobiopterin, 20 µM L-arginine, and L-[14C] arginine (ICN, Costa Mesa, CA). Duplicate incubations at 37°C for each sample were run for 30 min in the presence of ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (2 mM) to determine the levels of calcium-dependent (constitutive) NOS activity. Calcium-independent (inducible) NOS activity was determined by subtracting the constitutive activity from the total NOS activity in the sample. The reaction was terminated by the addition of 1.0 mL Dowex resin AF 50W-X8 (pH 7.5; Sigma Chemical Co.). The resin was allowed to settle for 45 min, and then 500 µL of the supernatant was removed and analyzed by liquid-scintillation counting. The level of citrulline produced was expressed as picomoles/min/mg protein. The protein content was determined by the Bradford technique (BioRad, Hercules, CA) using BSA as a standard. The citrulline assay was validated by determining the retention of radioactive arginine by the Dowex resin. The reaction cocktail (100 µL) was applied to 1.0 mL Dowex resin, and counts were determined as above. Greater than 99% of the applied radioactivity was retained by the Dowex resin. Blank values obtained from sample tubes containing boiled cell extracts were subtracted from all test samples. Specificity of the assay was validated further by adding the pan-NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA; 100 µM) to ensure that the citrulline detected arose from the activity of NOS.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated macrophages (iNOS), and rat kidney (eNOS), respectively. All PCR reactions were negative when the RT step was eliminated, indicating that there was no contamination from genomic DNA.
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Figure 1. Expression of NOS mRNA in unstimulated PMC. Representative RT-PCR analysis of total RNA isolated from untreated PMC using eNOS (lanes 2–5)-, bNOS (lanes 7–10)-, and iNOS (lanes 12–15)-specific primers. RT-negative controls are as indicated. Rat kidney (lanes 2 and 3), rat brain (lanes 7 and 8), and IFN--stimulated rat macrophages (lanes 12 and 13) were used as the appropriate controls. Molecular weight markers are in lanes 1, 6, and 11. Results are representative of four different RNA isolations.
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, CD8 ligation, IgE cross-linking with stimulating Ag). iNOS mRNA production was then assessed by semiquantitative RT-PCR (Fig. 2
). iNOS signal (26 cycles) in PMC increased following treatment with all three stimulants. IFN- [0.71±0.05 arbitrary units (a.u.)] or anti-CD8 (0.70±0.08 a.u.) treatment produced approximately twofold greater iNOS mRNA induction compared with stimulating Ag (0.37±0.12 a.u.) as measured by band densitometry versus a ß-actin control. eNOS mRNA remained unaltered when PCR was run on the same samples. As seen previously, no bNOS mRNA was detected under any of the treatment conditions studied. Consistent with our initial studies on unstimulated PMC, iNOS expression was not detected in untreated PMC. Results are from PMC RNA obtained from three independent batches of cells.
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Figure 2. Measurement of mRNA levels for iNOS and ß-actin by RT-PCR in PMC cultured in media alone (lane 1), with IFN- (800/U/mL; lane 2), with anti-CD8 (5 µg/mL; lane 3), and with Ag (5 WE/mL; lane 4) for 6 h. Densitometric analyses are shown as the iNOS/ß-actin ratio, expressed in a.u. eNOS mRNA levels are stable under similar treatment conditions. No bNOS mRNA was detected. The PCR gels shown are representative of two others performed using PMC from different cell isolations. Error bars represent SE from three separate experiments.
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Figure 3. In situ RT-PCR localization of iNOS mRNA in formalin-fixed smears of untreated PMC (a–c) and anti-CD8 (5 µg/mL)-stimulated PMC (d–f). (a) Only a few untreated cells show positivity for iNOS mRNA (13.7±1.5%); (d) a significant (P<0.001) percentage (70.5±2.8%) of anti-CD8-stimulated cells are positive (arrow). Note the intense perinuclear signal (arrowhead). (b and e) All cells showed intense nuclear staining (arrows) in the positive control, indicating nuclear DNA priming and that the pepsin treatment was optimal, thereby allowing PCR reagents to enter the cell. The cytoplasm remains negative. (c and f) The cells were treated with DNase, the RT step was eliminated, and no staining was shown in the negative control, indicating that the product detected was indeed cDNA and not from contaminating genomic DNA. Original magnification: x1000; bar = 10 µm. Results are representative of three separate experiments, counting 200 cells per slide.
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shown; Fig. 4b
). Cell counts revealed that upon IFN- treatment, 97.5 ± 0.4% of cells were positive. iNOS immunostaining was also increased in anti-CD8 and Ag-stimulated cells, 96.2 ± 0.6% and 94.1 ± 0.3%, respectively. Staining was predominantly in the cytoplasm (arrow), and some protein was also localized to the granules (arrowhead). Very little iNOS staining (4.3±0.7%) was detected in untreated PMC (Fig. 4a)
. These data are summarized in Table 2
. Staining was blocked when the iNOS antibody was preincubated with the iNOS-immunizing peptide (Fig. 4c)
, and staining was also absent in slides incubated with IgG1 control antisera (Fig. 4d)
.
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Figure 4. Immunohistochemical identification of iNOS protein in PMC. Staining for iNOS protein in IFN--stimulated PMC was detected (b) predominantly in the cytoplasm (arrow), with some protein also being localized to the granules (arrowhead). Few iNOS-staining cells were detected in untreated PMC (a). Staining was blocked when the iNOS antibody was preincubated with the iNOS-immunizing peptide (c); staining was also absent in slides incubated with IgG1 control antisera (d). Original magnification: x1000; bar = 10 µm.
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Table 2. Immunohistochemical Analysis of iNOS Expression in PMC
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, anti-CD8, or Ag were compared with untreated PMC for their NO production capacity. Upon activation with IFN-, anti-CD8, or Ag (all present for 48 h), PMC released significantly greater (P<0.01) amounts of NO2- (7.6±0.9, 6.6±0.9, and 8.4±0.6 µM/2x105/48 h, respectively). Unstimulated PMC released low levels of NO2- spontaneously, after 48 h of incubation (2.1±0.3 µM/2x105/48 h; Fig. 5
). To determine if the NO produced arises from the conversion of L-arginine by NOS, the pan-NOS inhibitor L-NMMA (100 µM) was added concomitantly to IFN--stimulated cells. NO production was reduced back to below baseline levels in the presence of L-NMMA.
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Figure 5. The release of NO (measured by nitrite) by the Griess assay from PMC stimulated with anti-CD8, Ag, and IFN-. Cells treated concomitantly with IFN- and the NOS inhibitor L-NMMA (100 µM) were used to determine the specificity of NO production. Cells from unsensitized rats were used as the unstimulated control. Data shown as mean ± SE of five experiments, 2 x 105 cells/sample, were analyzed. *, P < 0.05 by comparison with untreated cells.
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treatment was a result of the induction of iNOS or eNOS, the citrulline assay was performed. This assay is based on the conversion of L-arginine to L-citrulline and allows detection and differentiation of constitutive and inducible isoenzyme activity in PMC homogenates (Fig. 6
). Blank values obtained from concordant incubations with heat-treated samples (to inactivate NOS activity) were subtracted from all test values. NOS activity in unstimulated PMC (0 h) was produced exclusively by Ca2+-dependent NOS activity (43.7±5.1 pmol/min/mg protein) with no detectable iNOS activity. Treatment with IFN- (18 h) increased citrulline production significantly (P<0.01) in PMC by iNOS activity (52.8±18.4 pmol/min/mg protein) but with no significant increase in cNOS (eNOS/bNOS) activity (31.8±22.5 pmol/min/mg protein). This correlates with mRNA data showing induction of an iNOS message following stimulation with IFN-. NOS activity was inhibited by the addition of L-NMMA, indicating that the citrulline conversion detected was a result of NOS (unpublished results).
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Figure 6. NOS activity in untreated (0 h) and IFN- (18 h)-stimulated PMC, as measured by the citrulline assay. The results are expressed as picomoles L-citrulline formed per minute per mg protein. Data shown as mean ± SE for three independent experiments. *, P < 0.05 by comparison with levels of inducible NOS activity in untreated cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have demonstrated that rat PMC are a source of eNOS and iNOS mRNA as well as iNOS protein. We show that unstimulated PMC express no detectable iNOS or bNOS mRNA. However, low baseline levels of eNOS mRNA are expressed. It is interesting that unstimulated PMC have Ca2+-dependent NOS activity as measured by citrulline production. To our knowledge, this is the first demonstration of Ca2+-dependent NOS activity in PMC. Furthermore, iNOS mRNA is induced rapidly (6 h) upon stimulation with Ag, anti-CD8 antibody, or IFN-. This mRNA is also translated, and iNOS protein was detected in stimulated PMC using immunohistochemistry. Furthermore, the translated iNOS protein is biologically active, as quantified by the citrulline assay, and produces increased levels of NO, measured as nitrite, by the Griess assay.
Because PMC were isolated from mixed-peritoneal cells, the possibility exists that the NOS/NO expression observed may be a result of the presence of contaminating cells such as macrophages. Furthermore, several early studies of MC-NO production were derived from populations that contained up to 15% contaminating cells [14 ], and recent studies have confirmed that in peritoneal cell populations, these contaminating cells contributed significantly to the NO produced [16 ]. To confirm the cellular source of iNOS, we used in situ RT-PCR to localize the mRNA directly to PMC. PMC treated with anti-CD8 showed a significant increase in the cytoplasmic accumulation of iNOS mRNA after 18 h. As further verification, iNOS protein was confirmed to arise from PMC by the immunocytochemistry results. It is interesting that the pattern of iNOS staining obtained showed similar cytoplasmic and granular localization as that described recently by Messina et al. [12 ], who showed iNOS immunoreactivity in brain MC after ischemia, providing further evidence that MC produce iNOS protein.
These results present an important point because they differ from the observations of other groups that MC isolated from mixed peritoneal populations do not produce detectable amounts of NO, particularly following IFN- stimulation [16
, 26
]. This result may be because of methodological differences in the Griess assay, including such variables as the ratio of sample:Griess reagent added (3:1 vs. 1:1) or time of incubation before supernatant removal (24 vs. 48 h). Our results obtained using the more sensitive citrulline assay, which allows detection of the low amounts of NOS activity found in some cell systems [23
], confirm that PMC have low-level NOS activity. Unfortunately, neither assay used allows for the direct cellular detection of NO; thus, we are unable to equivocally determine the cellular source.
The fact that PMC produce so little NO in relation to other peritoneal cells, especially macrophages, leads to the question of physiological relevance of PMC-derived NO. Indeed, Eastmond et al. [26 ] have shown recently that in peritoneal populations, PMC function is regulated substantially by macrophage-derived NO. However, NO produced by MC in other tissue environments, such as in the lung or intestine, may make a significant contribution to tissue function. Studies by Gaboury et al. [15 ] show that MC-derived NO is important in down-regulating neutrophil adhesion to the vascular endothelium. Furthermore, studies by Bidri et al. [11 ] on pure MC populations implicated MC in NO production, and investigations using MC lines show further that MC-derived NO is important in endogenous regulation of the cell [27 ]. Thus, the contribution of the extracellular environment must be considered when determining the amount and functional significance of MC-derived NO.
Because MC have the ability to act in homeostatic and pathological roles, a precise function of MC-derived NO is difficult to delineate. MC-NO produced basally through the activity of eNOS could function in a physiological capacity, functioning in signal transduction via cGMP-mediated mechanisms. Previously, PMC have been shown to produce NO spontaneously without prior stimulation [13 , 14 , 24 ]. Because iNOS mRNA is not increased until 4–6 h following activation, these results must be a result of the presence of a basally active NOS enzyme. The finding in this study of the presence of a functioning eNOS system can now provide an explanation for the release of basal NO from rat PMC.
By virtue of their ability to produce NO through iNOS upon exposure to various immunological stimuli, MC may play an important role in innate immunity. MC are located strategically throughout the body and have been shown to bind various bacteria often in the absence of opsonization [28
]. Through the production of NO, MC may function directly or in combination with other mediators and reactive intermediates to eliminate bacteria and parasites [29
]. Furthermore, NO produced locally by MC may contribute to general vasodilation and the associated accumulation of inflammatory cells to the site of infection [15
]. It is interesting that although cross-linking IgE receptors or stimulating CD8 can be considered possible proinflammatory signals, IFN- is a well-known inhibitor of MC function [26
, 30
, 31
], and MC-NO derived from IFN activation may mediate MC down-regulation. However, recent studies by Deschoolmeester and others [16
] have raised the possibility that PMC may not produce function IFN- receptors on their cell surface. This could be a reason for the requirement for higher concentrations of IFN- to stimulate MC to produce NO compared with that needed to activate macrophages. Whether MC respond specifically to IFN- by a receptor or by a nonspecific mechanism such as charge interaction is an area that clearly requires more study and will rely on confirming the presence of IFN- receptor mRNA and protein in MC.
In summary, we have presented evidence for the differential production of NOS isoenzyme mRNA, protein, and NO production in unstimulated and treated PMC. However, further definition of the regulatory pathways for NO synthesis will help identify the inflammatory responses favorable to the production of NO by MC and the roles MC-NO may play in tissue homeostasis, inflammation, and host defense.
Received June 28, 2000; revised November 25, 2001; accepted November 26, 2001.
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