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The Journal of Immunology, 2001, 167: 5935-5939.
Copyright © 2001 by The American Association of Immunologists

Regulation of CD8 Expression in Mast Cells by Exogenous or Endogenous Nitric Oxide1

Osamu Nohara2,*, Marianna Kulka2,{dagger}, René E. Déry{dagger}, Fiona L. Wills{dagger}, Nadir S. Hirji{dagger}, Mark Gilchrist{dagger} and A. Dean Befus3,{dagger}

* Department of Otorhinolaryngology, Jikei University School of Medicine, Tokyo, Japan; and {dagger} Glaxo-Heritage Asthma Research Laboratories, Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently reported a novel CD8 molecule on rat alveolar macrophages and peritoneal mast cells (PMC). However, little is known about the regulation of CD8 expression and function on these cells. We investigated the regulation of CD8 expression on PMC by NO, because NO can regulate inflammatory responses and also because anti-CD8 Ab stimulates inducible NO synthase and NO production by PMC and alveolar macrophages. Ligation of CD8{alpha} on PMC with Ab (OX8) induced CD8{alpha} mRNA expression after 3–6 h, and flow cytometry demonstrated that OX8 treatment increased CD8{alpha} protein expression compared with PMC treated with isotype control IgG1. To test whether NO mediates the up-regulation of CD8{alpha}, we used the NO donor S-nitrosoglutathione (500 µM) and NO synthase inhibitors (NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester; 100 µM). S-nitrosoglutathione up-regulated both mRNA and protein expression of CD8{alpha} in PMC compared with that in sham-treated cells, while NO synthase inhibitors down-regulated OX8 Ab-induced CD8{alpha} expression. To investigate how NO regulates CD8 expression on PMC, we examined the cGMP-dependent pathway using 8-bromo-cGMP (2 mM) and the guanylate cyclase inhibitor, 1H-oxadiazoloquinoxalin-1-one (20 µM). 8-Bromo-cGMP up-regulated CD8 expression, whereas 1H-oxadiazoloquinoxalin-1-one down-regulated its expression. Thus, ligation of CD8 up-regulates CD8 expression on PMC, a response mediated at least in part by NO through a cGMP-dependent pathway. The significance of this up-regulation of CD8{alpha} on mast cells (MC) is unclear, but since ligation of CD8 on MC with OX8 Ab can alter gene expression and mediator secretion, up-regulation of CD8 may enhance the MC response to natural ligation of this novel form of CD8.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ligand CD8 on T cells plays a critical role as a ligand/receptor for MHC class I, as an adhesion molecule, and as a coreceptor in conjunction with TCR signaling (1, 2, 3, 4, 5). We recently identified CD8 expression on rat macrophages (6, 7, 8, 9) and mast cells (MC)4 (10) using OX8 Ab to the hinge region of CD8{alpha}. Furthermore, G28, an Ab that identifies the Ig-like region of CD8{alpha} on T lymphocytes, did not bind rat macrophages or peritoneal MC (PMC), suggesting that CD8{alpha} on these cells is distinct from that on T lymphocytes (6). Because little is known about this novel form of CD8, we investigated mechanisms that might modulate its expression, with a focus on MC.

MC are primary effector cells in allergic reactions. They play important roles in other kinds of immune and inflammatory responses, such as in innate defenses to microbial agents (11, 12, 13, 14), and in Ag presentation and initiation of immune responses (15, 16, 17). MC also produce a variety of cytokines, histamine, proteases, and mediators that are newly synthesized following MC activation, such as NO (18).

NO is a mediator synthesized from the conversion of L-arginine to L-citruline by the enzyme NO synthase (NOS). NO has important roles in a variety of biologic functions, including vasodilation, neurotransmission, host defense against pathogens, bronchodilation, and tumor cytotoxicity (19, 20). Salvemini et al. (21, 22) first suggested that MC make NO and that this NO can modulate selected MC activities. We established that purified rat PMC (>=99%) produce NO and that it potentiates their ability to induce TNF-mediated cytotoxicity (23). Subsequently, we showed that IL-1{beta} stimulated NO production by PMC, and that platelet-activating factor release by MC was inhibited by an NO-dependent mechanism (24). Eastmond et al. (25) established that NO is a principal effector of IFN-{gamma}-induced suppression of MC exocytosis, and we confirmed and extended this to effects on MC adhesion to fibronectin (26). Indeed, NO is well known to regulate cytokine gene expression in other cells and to influence immune and inflammatory responses (27).

In functional studies of CD8 on rat PMC, NO production was induced by OX8 and 341 (anti-CD8{beta}) Abs in a dose-dependent manner, but not by isotype controls (10). We established with similar studies using macrophages that CD8 activation induces TNF and IL-1{beta} production, as well as inducible NOS (iNOS) and NO (6, 7). Furthermore, TNF production involves an Src family kinase and an Syk kinase (9). In the present study we investigated the effect of NO on the regulation of CD8 expression on PMC. Ligation of CD8{alpha} using OX8 Ab up-regulated CD8 expression on PMC, and this was blocked by NOS inhibitors. The NO donor S-nitrosoglutathione (GSNO) also induced CD8{alpha} expression on PMC. This CD8{alpha}- and NO-induced up-regulation of CD8{alpha} expression involved the NO-cGMP pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents

Male Sprague Dawley rats (>400 g) were obtained from Charles River Breeding Laboratories (St. Constant, Canada) (6). Animals were maintained in an isolated room in filter-top cages to minimized unwanted infections. Most studies used normal rats, but in some experiments rats were infected with L3 larvae of Nippostrongylus brasiliensis 5–6 wk before MC isolation as previously described (28). Food and water were provided ad libitum, and animals were maintained on a 12-h dark, 12-h light (0700–1900) cycle. Experimental procedures were approved by the university animal care committee (Jikei University School of Medicine, Tokyo, Japan) and were in accordance with the guidelines of the Canadian Council on Animal Care (Ottawa, Canada).

Ab OX8 (anti-CD8{alpha} hinge region, IgG1) (29), OX8-FITC, and 341 (anti-CD8{beta}, IgG1) (30) were purchased from Serotec (Toronto, Canada). 341-FITC and IgG1 were purchased from BD PharMingen (Mississauga, Canada). IgG1-FITC was purchased from Accurate Chemical and Scientific (New York, NY). Heparinase I and NG-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 medium was purchased from Life Technologies (Grand Island, NY). GSNO, NG-monomethyl-L-arginine (L-NMMA), and 8-bromo-cGMP were purchased from Calbiochem (La Jolla, CA). 1H-(1, 2, 4)oxodiazolo(4,3-a)quinoxalin-1-one (ODQ) was purchased from Tocris Neuramin (Ballwin, MO). The viability of PMC was not affected by in vitro treatment with any of these compounds at the doses used.

MC isolation and in vitro stimulation

Isolation and purification of rat PMC were performed as previously described (31). Briefly, 20 ml of cold HEPES-buffered Tyrode’s solution (HBTS) was used to lavage the peritoneal cavity of each rat. The recovered cells were laid on a 30:80% discontinuous Percoll gradient and centrifuged at 600 x g for 20 min at 4°C. The purity of PMC from SD rat was >=98% (32). PMC were incubated for 3–20 h with OX8 (2.5–10 µg/ml) or IgG1 isotype (10 µg/ml) with or without L-NMMA (100 µM), 8-bromo-cGMP (2 mM), GSNO (500 µM), or ODQ (20 µM) in RPMI 1640 medium (Life Technologies) and used for RT-PCR. For analysis of CD8 protein expression, PMC were incubated for 20 h with OX8 (10 µg/ml), 341 (10 µg/ml), or IgG1 isotype (10 µg/ml) with or without NOS inhibitors (L-NMMA, L-NAME; 100 µM), 8-bromo-cGMP (2 mM), GSNO (500 µM), or ODQ (20 µM) in RPMI 1640 medium and used for flow cytometric analysis.

RT-PCR

Total RNA extraction from PMC was performed by the method of Chomczynski and Sacchi (33, 34) with some modifications. After isolation, a 1-µg aliquot of RNA was incubated in 15 µl of 333 U/ml heparinase I (Sigma-Aldrich) in a reaction mixture of 5 mM Tris (pH 7.5), 1 mM CaCl2, and 7.5 U RNase inhibitor (Life Technologies) for 2 h at 22°C to remove contaminating heparin (34).

To synthesize a first-strand cDNA, each RNA sample (1 µg) was mixed with 1 µl of oligo(dT)12–18 primer (500 µg/ml; Life Technologies). mRNA was reverse transcribed by Moloney murine leukemia virus RT (Life Technologies) using a PTC-100 Programmable Thermal Controller (Fisher Scientific, Nepean, Canada) according to the manufacturer’s protocols.

PCR was conducted using a hot start method by adding 2 µl of cDNA product to 18 µl of PCR buffer containing 67 mM Tris (pH 8.8), 1.5 mM MgCl2, 16.6 mM (NH4)2SO4, mixed dNTPs at 200 µM, 125 U/ml Taq polymerase (Life Technologies), and 0.3 µM sense and antisense primers. The primers used were rat {beta}-actin: sense primer 5'-ATGGATGACGATATCGCTG-3' and antisense primer 5'-GATTCCATACCCAAGAAGG-3'; and rat CD8{alpha}: sense primer 5'-CAGTTACAGTTGTCACCAAA-3' and antisense primer 5'-CACGAATTTCTCTGAAGGTC-3'. The PCR products for {beta}-actin and CD8{alpha} were 812 and 630 bp, respectively. After denaturing at 94°C for 2 min, the reaction was conducted for 30 cycles at 94°C for 1 min, at 60°C ({beta}-actin) or 55°C (CD8{alpha}) for 1 min, and at 72°C for 1 min. Products were electrophoresed on a 2% agarose gel and visualized by staining with ethidium bromide. Care was taken in optimizing conditions for semiquantitative analysis of RT-PCR results, including loading controls with {beta}-actin. Complementary studies of protein expression were also conducted as outlined below. All PCR data shown are from one representative experiment of four performed.

Flow cytometric analysis

In 96-well U-bottom plates, cells (5x105 cells/well) were preincubated in RPMI 1640 medium (5% FBS) and 10% normal mouse serum (for conjugate primary Abs only) for 1 h before incubation with Ab for 1 h at 4°C. Cells were washed three times (with HBTS) and resuspended in 400 µl of HBTS, and 10,000 cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA). The mean fluorescence intensity (MFI; logarithmic scale) of cell populations was determined, and the results presented are net values of MFI following subtraction of values for isotype-matched controls. The percentage of CD8{alpha}-positive cells is indicated for both responding and nonresponding populations for each stimulus used.

Measurement of NO production

PMC were incubated (2 x 105 cells/well) with 5–10 µg of Ab for 48 h. Cell-free supernatants were mixed with an equal volume of Griess reagent (1% sulfamide, 0.1% N-(l-naphthyl)ethylendiamine dihydrochloride, and 2.5% H3PO4) and incubated for 10 min at room temperature (35). The concentration of NO2- was determined by measuring the absorbance at 540 nm with a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). NaNO2 was used as a standard. In experiments with L-NMMA, inhibitor was added 10 min before addition of Ab.

Statistical analysis

For flow cytometric data, statistical analysis was performed using two-tailed Student’s t test. For PCR data, scanned images are one representative experiment of four experiments conducted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-CD8{alpha} Ab and NO donor up-regulate CD8{alpha} mRNA expression in PMC

RT-PCR was used to examine the regulation of CD8{alpha} mRNA expression by anti-CD8{alpha} and the NO donor, GSNO (Fig. 1Go). cDNA from PMC stimulated with OX8 (2.5–10 µg/ml) or GSNO (100–500 µM) were used in the PCR reaction to detect CD8{alpha} mRNA. In the time-course study (Fig. 1GoA) OX8 (5 µg/ml) up-regulated CD8{alpha} mRNA after 3–6 h of stimulation, and mRNA levels returned toward baseline by 12 h. By contrast, the expression of CD8{alpha} mRNA in cells stimulated with GSNO was maximal after 12–20 h of stimulation (Fig. 1GoB). CD8{alpha} mRNA expression was up-regulated by OX8 (3-h stimulation) and GSNO (12-h stimulation) in a dose-dependent manner (Fig. 1GoB). IgG1 isotype (10 µg/ml) was used as a control for OX8, and no up-regulation of CD8 mRNA expression was found (data not shown).



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  		FIGURE 1. RT-PCR analysis of the regulation of CD8{alpha} mRNA expression in PMC by OX8 and NO donor (GSNO). A, Time-course study. PMC were stimulated with OX8 (5 µg/ml) and GSNO (500 µM). B, Dose dependency. PMC were incubated at the various doses of OX8 (3 h) and GSNO (12 h). C, Role of endogenous NO in the up-regulation of CD8 mRNA by OX8. PMC were stimulated by OX8 (10 µg/ml) with or without L-NMMA (100 µM) for 3 h. Data are from one representative experiment of four independent sources of RNA (n = 4).

 
To test whether endogenous NO is involved in the up-regulation of CD8 mRNA by OX8, the NOS inhibitor L-NMMA (100 µM) was used (Fig. 1GoC). PMC were stimulated by OX8 (10 µg/ml) with or without L-NMMA for 3 h. L-NMMA ablated OX8-induced CD8 mRNA expression.

Regulation of CD8 protein expression on PMC

Flow cytometry was used to examine the regulation of CD8 protein on PMC (Figs. 2Go and 3Go). PMC were stimulated with GSNO (500 µM), IgG1 isotype (10 µg/ml), or OX8 (10 µg/ml) with or without NOS inhibitors (L-NMMA and L-NAME; 100 µM) for 20 h. The majority of PMC were CD8{alpha} positive (64.6 ± 15.3%), and some (24.5 ± 8.5%) were 341 (CD8{beta}) positive as we reported previously (10). No significant difference in the proportion of positive cells was seen between preincubation and after 20 h of incubation (data not shown).



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  		FIGURE 2. A, Flow cytometric analysis of the regulation of CD8{alpha} molecule on PMC in one representative experiment. PMC were incubated with OX8 (10 µg/ml), GSNO (500 µM), or IgG1 isotype (10 µg/ml) for 20 h. Cell viability was not affected by these treatments. These cells were stained with OX8-FITC (CD8{alpha}) or isotype control IgG1-FITC. B, Percentage of positive cells in each of the two cell populations (responding to treatment and nonresponding) as measured by flow cytometric analysis from one representative experiment (n = 5).

 


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  		FIGURE 3. NO dependency of anti-CD8{alpha} (OX8)-induced CD8{alpha} protein expression. PMC were treated with IgG1 isotype (10 µg/ml), OX8 (10 µg/ml), or OX8 and NOS inhibitor (L-NMMA, 100 µM) for 3 h. Data shown are from one representative experiment of five performed. A, Flow cytometric analysis showing the effect of 20-h incubation of NOS inhibitor (L-NMMA, 100 µM) on CD8{alpha} levels in PMC stimulated with OX8 (10 µg/ml). B, Percentage of positive cells in each of the two cell populations (responding to treatment and nonresponding) as measured by flow cytometric analysis (n = 5).

 
OX8 ligation induced a second peak of CD8{alpha}-positive cells (responding cells; MFI = 102.3) that was not seen using treatment with IgG1 isotype control Ab. For OX8 treatment, the populations expressing low (MFI = 34) and high (MFI = 102.3) levels of CD8{alpha} comprised 56.6 and 42.5% of the total population, respectively. Treatment of PMC with 500 µM of GSNO for 20 h also induced a second peak of CD8{alpha}-positive cells (Fig. 2GoA). For GSNO treatment, the two CD8{alpha}-positive cell populations were unevenly distributed, with 19.3% expressing low levels of CD8{alpha} (MFI = 28.3; nonresponding cells) and 76.7% expressing high levels of CD8{alpha} (MFI = 92.3; responding cells). Statistical analysis of MFI values showed that GSNO and OX8 significantly (p < 0.05) up-regulated CD8{alpha} protein expression compared with unstimulated PMC. One representative experiment is shown in Fig. 2GoB. By contrast, treatment of PMC with 341 (20 µg/ml, 20 h) or allergen (PMC from rats infected with N. brasiliensis) had no effect on CD8{alpha} protein expression (data not shown). Finally, neither OX8 (10 µg/ml, 20 h) nor GSNO (500 µM, 20 h) modified the expression of CD8{beta} protein on PMC (data not shown).

Inhibitory effect of NOS inhibitor on CD8 expression by PMC

To test whether endogenous NO regulated CD8{alpha} protein expression as it had with CD8{alpha} mRNA (Fig. 1GoC), NOS inhibitor (L-NMMA, 100 µM) was used (Fig. 3Go). Flow cytometry showed that OX8 increased CD8{alpha} expression in 46.4% of the cells (MFI = 115; responding cells). Only 19.8% of L-NMMA- and OX8-treated cells showed an increase in CD8{alpha} expression, suggesting that L-NMMA treatment inhibited the OX8 effect (MFI of the responding population = 41.2; Fig. 3GoA). L-NMMA and L-NAME (data not shown) treatment significantly (p < 0.01) decreased MFI for CD8{alpha} in the responding population compared with OX8 treatment alone (Fig. 3GoB). L-NMMA alone had no effect on the MFI or the number of CD8{alpha}-positive cells in either the responding or nonresponding population (data not shown).

Regulation of CD8 expression by NO involves a cGMP-dependent pathway

To determine how NO regulates CD8{alpha} expression, PMC were treated for 12 h with a membrane-permeable cGMP analog, 8-bromo-cGMP (2 mM). Time-course treatment indicated that 12 h was optimal for cGMP-induced expression of CD8{alpha} (data not shown). RT-PCR analysis showed that both 8-bromo-cGMP and GSNO (500 µM) up-regulated CD8{alpha} mRNA expression (Fig. 4Go). In addition, the mRNA expression evoked by GSNO was down-regulated by a selective inhibitor of NO-stimulated soluble guanylyl cyclase (sGC), ODQ (20 µM). Accordingly, to determine whether similar effects occurred with CD8{alpha} protein expression, flow cytometric analysis was conducted (Fig. 5Go). With 8-bromo-cGMP (20 h), 40.8% of the cells responded, and their MFI was 54.0. Similarly with GSNO, 55.9% of the cells responded, and their MFI was 85.1. By contrast, following treatment with GSNO and ODQ together, the MFI of the responding population (45.9%) was only 60.7 compared with 85.1 after treatment with GSNO alone. ODQ alone had no effect on CD8{alpha} protein expression.



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  		FIGURE 4. Effects of cGMP analog (8-bromo-cGMP) and sGC inhibitor (ODQ) on CD8{alpha} mRNA expression. PMC were incubated with 8-bromo-cGMP (2 mM), GSNO (500 µM), or GSNO plus ODQ (20 µM) for 12 h. Data are from a single representative experiment from a total of four independent RNA isolations (n = 4).

 


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  		FIGURE 5. Flow cytometric analysis (single representative experiment) of the regulation of CD8{alpha} molecule on PMC involves a cGMP-dependent pathway. PMC were stimulated by 8-bromo-cGMP (2 mM), GSNO (500 µM), or GSNO plus ODQ (20 µM) for 20 h. Cell viability was not affected by these treatments. B, Percentage of positive cells in each of the two cell populations (responding to treatment and nonresponding) as measured by flow cytometric analysis in one representative experiment (n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study confirms and extends our previous finding that freshly isolated rat PMC and rat basophilic leukemia cells (RBL 2H3) express a novel form of CD8{alpha} as well as CD8{beta} (10). A mouse MC line has also been reported to express CD8{alpha} (36). Importantly, in the current investigations we have established that ligation of this CD8{alpha} with OX8 Ab to its hinge region up-regulates CD8{alpha} mRNA and surface protein expression on MC. This is the first report on factors that regulate CD8{alpha} expression on MC, and to our knowledge it is the first report that NO regulates CD8{alpha} expression on any cell type (5). Interestingly, about half the MC up-regulate CD8{alpha} protein in response to OX8, GSNO, or 8-bromo-cGMP. Therefore, although most MC express CD8{alpha}, only half the cell population (responding cells) is capable of increasing protein expression of CD8{alpha} in response to the stimuli used.

We have also shown that OX8-mediated increases in CD8{alpha} expression are mediated by NO, since the NOS inhibitor L-NMMA abrogates the OX8 effect. The NO donor (GSNO) increases CD8{alpha} mRNA expression as early as 3 h and maximally at 12–20 h. This enhanced expression of CD8{alpha} is associated with an earlier increase in NO production, which in our previous studies of both alveolar macrophages (6, 7) and PMC (37) involves the induction of iNOS mRNA by OX8 Ab within 6 h of stimulation (7). Treatment with OX8 Ab increases the expression of CD8{alpha} mRNA within 3 h, whereas the maximal effect of GSNO on CD8{alpha} mRNA is at 12–20 h. L-NMMA, an NOS inhibitor, blocks the OX8 effect in 3 h. This is an interesting observation that most likely reflects a major difference between the effects of exogenously administered NO (GSNO) and endogenously produced NO. The latter could arise from either constitutive NOS or iNOS in MC and could probably involve compartmentalized production of NO in selected sites within the cell (37).

Moreover, flow cytometric data showed that GSNO and 8-bromo-cGMP increased CD8{alpha} expression by a pathway dependent upon sGC. NO can initiate its biological effects through activation of sGC, resulting in the production of cGMP and affecting pathways with other signaling systems, such as phosphoinositides, eicosanoids, cAMP, and Ca2+ (38). Therefore, our results suggest that OX8-mediated up-regulation of MC CD8{alpha} is mediated via the NO-sGC pathway and results in cell activation. We have previously shown that both TNF and NO production are induced by OX8 and 341 Ab in a dose-dependent manner (10).

Up-regulation of CD8 expression has been demonstrated in other cell types. Ligation of CD40 on Langerhans cells is associated with acquisition of CD8 and a dendritic cell phenotype by these cells (39). It is possible that when CD40 ligand on MC interacts with CD40 on B lymphocytes or other cells, MC expression of CD8 is modulated, in turn influencing selected functions of the MC such as Ag presentation (15, 16, 17). By contrast to this enhanced expression of CD8{alpha}, TGF-{beta}1, TGF-{beta}2, and PGE2 down-regulate CD8{alpha} expression on human peripheral blood lymphocytes (40). This could involve calcineurin, as has been shown in double-positive lymphocytes where PMA is known to inhibit CD8 expression (41).

It is attractive to postulate that through endogenously or exogenously produced NO, MC expression of CD8 is altered in a manner that influences its competence to respond to various signals and, in turn, selectively channels its functions in various microenvironments. For example, NO inhibits IgE-dependent histamine and serotonin secretion (21, 22, 25, 42, 43), platelet-activating factor production (24), and adhesion to fibronectin (26), but potentiates TNF-mediated cytotoxicity (23). How the up-regulation of CD8{alpha} expression by NO fits in with this spectrum of other effects of NO on MC is not currently clear. It is intriguing that ligation of CD8{alpha} on the MC surface with OX8 Ab induces NO and TNF production directly, but does not modulate IgE Ag-dependent mediator secretion. Taken together with the inhibitory effects of NO on MC secretion of stored mediators such as histamine and serotonin, it is tempting to speculate that NO depresses the functions of the MC associated with the immediate phase of allergic reactions, but enhances other components of MC function such as innate immunity and regulation of immune responses.

The ligand for the novel form of CD8 on MC is currently unknown. It might be classical (1, 5) or nonclassical (44) MHC class I. Alternatively, given that the novelty of MC and macrophage CD8 lies in the N-terminal Ig-like domain that on T cell CD8 binds MHC class I, it is possible that MC CD8{alpha} binds another ligand, such as gp180 (45) from epithelial or other cells, or a ligand that has yet to be identified. The identity of the ligand(s) for CD8 on MC is an important challenge that must be addressed if we are to fully understand the functional significance of the expression of CD8 on MC and other cells and the value of knowledge about the controls on its expression by NO and other factors.


   Acknowledgments
 
We thank Drs. Grant R. Stenton and Harissios Vliagoftis for ongoing advice and Lynelle Haug for expert secretarial assistance.


   Footnotes
 
1 This work was supported by the Canadian Institutes for Health Research (previously the Medical Research Council of Canada), Alberta Heritage Foundation for Medical Research, and Alberta Lung Association. Back

2 O.N. and M.K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. A. Dean Befus, Glaxo-Heritage Asthma Research Laboratories, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail address: dean.befus{at}ualberta.ca Back

4 Abbreviations used in this paper: MC, mast cell; PMC, peritoneal MC; GSNO, S-nitrosoglutathione; HBTS, HEPES-buffered Tyrode’s solution; NOS, NO synthase; iNOS, inducible NOS; L-NMMA, NG-monomethyl-L-arginine; L-NAME, NG-nitro-L-arginine methyl ester; MFI, mean fluorescence intensity; ODQ, 1H-(1,2,4)oxodiazolo(4,3-a)quinoxalin-1-one; sGC, soluble guanylyl cyclase. Back

Received for publication December 26, 2000. Accepted for publication August 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. Forsythe and A. D. Befus
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