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Originally published In Press as doi:10.1074/jbc.M409738200 on September 10, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48751-48759, November 19, 2004
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Rodent and Human Mast Cells Produce Functionally Significant Intracellular Reactive Oxygen Species but Not Nitric Oxide*

Emily J. Swindle{ddagger}§, Dean D. Metcalfe§, and John W. Coleman{ddagger}§

From the {ddagger}Department of Pharmacology, University of Liverpool, Liverpool L69 3GE, United Kingdom and the §Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 24, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In immunity, reactive oxygen species (ROS) and nitric oxide (NO) are important antimicrobial agents and regulators of cell signaling and activation pathways. However, the cellular sources of ROS and NO are much debated. Particularly, there is contention over whether mast cells, key secretory cells in allergy and immunity, can generate these chemical species, and if so, whether they are of functional significance. We therefore examined directly by flow cytometry the capacity of mast cells to generate intracellular ROS and NO using the respective cell-permeable fluorescent probes dichlorodihydrofluorescein and diaminofluorescein and evaluated the effects of inhibitors of ROS and NO synthesis on cell degranulation. For each of three mast cell types (rat peritoneal mast cells, mouse bone marrow-derived mast cells, and human blood-derived mast cells), degranulation stimulated by IgE/antigen was accompanied by production of intracellular ROS but not NO. Inhibition of ROS production led to reduced degranulation, indicating a facilitatory role for ROS, whereas NO synthase inhibitors were without effect. Likewise, bacterial lipopolysaccharide and interferon-{gamma} over a wide range of conditions failed to generate intracellular NO in mast cells, whereas these agents readily induced intracellular NO in macrophages. NO synthase protein, as assessed by Western blotting, was readily induced in macrophages but not mast cells. We conclude that rodent and human mast cells generate intracellular ROS but not NO and that intracellular ROS but not intracellular NO are functionally linked to mast cell degranulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are secretory cells central to specific and innate immunity, allergy, and inflammation (1–5). In specific IgE-mediated responses, they are activated by antigen to release chemical mediators such as histamine, proteases, prostaglandins, and cytokines (1), whereas in innate responses to bacteria, they promote neutrophil phagocytosis (3, 4) and lymph node hyperplasia (5) via the production of tumor necrosis factor. In keeping with a role in innate defense, mast cells can directly phagocytose and kill bacteria (6). Essential to bacterial killing by "professional" phagocytic cells such as neutrophils and macrophages is the production of reactive oxygen species (ROS)1 such as superoxide and hydrogen peroxide, nitrogen oxygen species such as nitric oxide (NO), and their combined product peroxynitrite (7–10). However, in mast cells, these pathways are less well characterized, and there is debate over whether mast cells generate biologically significant amounts of reactive oxygen and nitrogen species.

Several studies (11–13) have reported that rat peritoneal tissue type mast cells (RPMC) or mast cells of the rat RBL-2H3 cell line (14, 15) release ROS into the extracellular milieu in response to a range of stimuli. These studies measured ROS-induced light emission by scintillation counting (11) or using luminol or the luciferin-related compound 2-methyl-6-(pmethoxyphenyl)-3-7-dihydroamidazo[1,2{alpha}]pyrazine-3-one) (12–15), and all of these techniques suffer from interference and lack of specificity (16). Using the highly sensitive real-time chemiluminescent probe Pholasin, we could not detect the release of ROS by purified rat tissue mast cells activated with IgE/antigen, phorbol myristate acetate, or ionomycin, whereas macrophages from the same starting populations of peritoneal cells readily released ROS at high levels (16). Because Pholasin is not cell-permeable, it detects released rather than intracellular ROS. Therefore, ROS may be generated but not exported. Indeed, intracellular ROS generation has been reported for RPMC (17–19), a mouse mast cell line (20), RBL-2H3 cells, and mouse bone marrow-derived mast cells (21). To date, no studies have been performed with human mast cells.

Reports from several groups claim that mast cells produce NO (22–26), although again, studies from our own laboratory (27–29) have provided contradictory evidence. Salvemini et al. (22) reported the release of a NO-like activity from rat peritoneal mast cells, although their preparations contained up to 15% macrophages, a known source of NO. Bidri et al. (23) reported expression of the inducible form of NO synthase (NOS2) and nitrite production by mouse bone marrow-derived mast cells, whereas Gilchrist et al. (24–26) have demonstrated inducible or constitutive NOS expression, nitrite production, and intracellular NO-induced DAF fluorescence in activated RPMC and the human tumor-derived HMC-1 and LAD2 mast cell-like lines. In contrast, we found that interferon (IFN)-{gamma} stimulated or spontaneous NO production by rat or mouse peritoneal mast cells diminished as mast cells were progressively purified, such that even in preparations that contained 98–99% mast cells, the NO production could be fully accounted for by the residual 1–2% non-mast cells (27, 28). Furthermore, mixed populations of macrophages and mast cells produced NO in response to IFN-{gamma} only when the macrophages expressed the IFN-{gamma} receptor (29). Therefore, at least in IFN-{gamma}-driven responses, a small minority of contaminating cells can contribute substantially to nitrite production. In addition, we have consistently been unable to detect nitrite production by IgE/antigen-activated mast cells of various rodent phenotypes.2

In view of our demonstration that mast cells do not release ROS to the outside of the cell (16) and the apparent differences between our own and other groups relating to whether or not mast cells produce NO (22–29), the aims of the present study were as follows: 1) to examine directly, by flow cytometry employing specific and sensitive fluorescent intracellular probes, the capacity of rodent and human mast cells to generate intracellular ROS and NO; 2) to ascertain, using the appropriate pharmacological agents, the potential contribution of intracellular ROS and NO to mast cell secretory responses; 3) to investigate whether the capacity or inability of mast cells to generate ROS or NO relates to their species. We used exclusively tissue-derived or primary culture-derived mast cells rather than mast cells of tumor phenotype as used in many previous studies.

Our results show that activated tissue type or primary cultured rodent and human mast cells generate intracellular ROS. Inhibition of ROS production led to suppression of IgE/antigen-mediated degranulation in all mast cell types, indicating a functional link between these responses. However, activated rodent and human mast cells did not generate intracellular NO or express NOS2 protein, and inhibitors of NO synthesis were without effect on cell activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Reagents—Female Brown Norway rats (150–200 g) were obtained from Harlan Olac (Bicester, UK) and maintained in the university animal housing unit where food and water were provided ad libitum. Rats were sensitized by subcutaneous injection of ovalbumin (OVA)/alum (2 x 0.2 ml, total dose of 80 µg of OVA, 8 mg of aluminum hydroxide) on days 0 and 7, and the rats were sacrificed humanely on day 14 by asphyxiation in CO2. Experimental procedures were approved by the University of Liverpool and were in accordance with guidelines set by the Home Office (London, UK).

Aluminum hydroxide, Dulbecco's modified Eagle's medium, diphenyleneiodonium (DPI), L-glutamine, bacterial lipopolysaccharide (LPS), OVA, mouse monoclonal anti-{beta}-actin antibody, toluidine blue, trypan blue, sulfanilamide and N-(1-napthyl)ethylenediamine dihydrochloride were purchased from Sigma. Fetal calf serum, gentamicin, and Hanks' balanced saline solution were from Invitrogen. 4,5-Diaminofluorescein (DAF-2) diacetate, dichlorodihydrofluorescein (DCF) diacetate, aminoguanidine (AG), and NG-monomethyl-L-arginine (L-NMMA) were obtained from CN Biosciences (Nottingham, UK). Cytokines were obtained from Peprotech (Rocky Hill, NJ). Lysis buffer and SDS-polyacrylamide gels were from Invitrogen. Mouse monoclonal anti-NOS2 and rabbit anti-Lyn antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit polyclonal anti-NOS2 was from Calbiochem. Cell culture media and supplements were from Invitrogen or BIOSOURCE International (Camarillo, CA).

Cell Isolation and Cultures—Rat mast cells and macrophages were obtained by peritoneal lavage and purified by density gradient fractionation as described previously (16). Purified mast cell preparations contained >98% mast cells, whereas the macrophage preparations contained >95% monocyte/macrophages and <1% mast cells by metachromatic staining in 0.05% toluidine blue and by Giemsa staining of cytospin preparations. Mouse bone marrow-derived mast cells (BMMC) were grown from femoral marrow cells of BALB/c mice and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 25 mM HEPES, 1.0 mM sodium pyruvate, non-essential amino acids, 0.0035% 2-mercaptoethanol, and 30 ng/ml mouse interleukin-3. Cells were used after 4–6 weeks of culture. Human mast cells (HUMC) were grown from CD34-positive peripheral blood mononuclear cells, obtained following informed consent. CD34-positive cells were cultured in interleukin-3 (week 1 only), stem cell factor, and interleukin-6 as described (30). HUMC were >95% pure by toluidine blue staining of cytospin preparations and were used after 8–10 weeks of culture.

Cell Degranulation and Nitrite Assays—Mast cells from OVA-sensitized rats, as well as BMMC or HUMC optimally sensitized with mouse IgE anti-DNP or biotinylated human IgE, respectively (both at 100 ng/ml for 24 h), were seeded at 10–50,000/well in 80 µl in a 96-well cell culture plate. Enzyme inhibitors (10 µl) were added for 1 h, the cells were challenged with 10 µl of the appropriate antigen (OVA (1 µg/ml), DNP30-HSA (100 ng/ml), or streptavidin (SA, 100 ng/ml), respectively), and degranulation was measured as the release of serotonin or {beta}-hexosaminidase as described (16, 31). Mediator release was expressed as the percentage of total cell content after subtracting background release from unstimulated cells. NO production was monitored as accumulated supernatant nitrite (the stable product of NO) by Griess assay as described (27).

Fluorescent Detection of Intracellular ROS and NO—Intracellular ROS and NO were measured by flow cytometry employing the fluorescent probes DCF (the intracellular product of DCF diacetate that fluoresces in the presence of ROS) and DAF-2 (the intracellular product of DAF-2 diacetate that fluoresces in the presence of NO). Macrophages and purified RPMC, BMMC, or HUMC (at 105-106/ml) were incubated for 1 or 24 h at 37 °C in 5% CO2 with or without L-NMMA (100–500 µM), AG (100 µg/ml), or DPI (1–5 µM). Then DAF-2 diacetate or DCF diacetate was added (2–20 µM) for 0.5–1 h before stimulation. Forward light scatter (FS), side scatter (SS), and cell fluorescence were analyzed (10,000 events) at various times from 1 min to 48 h after stimulation (FACscan flow cytometer and associated Lysis II software, BD Biosciences). Macrophage fluorescence was monitored over the entire log FS versus log SS plot, whereas BMMC and HUMC were gated according to FS and SS parameters, and RPMC were gated in areas defined as mast cells by fluorescent staining with phycoerythrin-rabbit anti-rat IgE serum (Liverpool University in-house preparation). After flow cytometric analysis, samples were centrifuged (400 x g, 5 min), and supernatants were removed (50 µl) in duplicate for a nitrite assay. Viability of the pelleted cells was determined by trypan blue exclusion.

Immunoblots for NOS2 Protein—IgE-loaded BMMC and HUMC were incubated in complete medium with or without the appropriate antigen or LPS and IFN-{gamma} under conditions that optimally induced cell activation measured as degranulation and ROS production. At various times (2–24 h), the cells were washed twice in serum-free medium, suspended at 2 x 106/ml, and then mixed with an equal volume of boiling lysis buffer supplemented with 100 mM dithiothreitol, protease inhibitor mixture, and 2-mercaptoethanol as described (32). The cell extracts were boiled for 3 min and electrophoresed on 4–12% SDS-polyacrylamide gels. The separated proteins were then electrophoretically transferred to nitrocellulose membranes and probed with mouse monoclonal anti-NOS2 or rabbit polyclonal anti-NOS2. To monitor protein loading, blots were stripped and reprobed with monoclonal mouse anti-{beta}-actin or rabbit anti-Lyn. Blots were developed with horseradish peroxidase-linked secondary antibody followed by chemiluminescent reagent and then exposed to x-ray film.

Data Presentation and Statistical Analysis—Data were analyzed by ANOVA followed by two-tailed Student's t test. Differences were considered significant when p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Intracellular ROS Production and Relationship to Degranulation in Antigen-stimulated Mast Cells—Intracellular ROS production was monitored using the ROS-sensitive fluorescent probe DCF, and the effects of the flavoenzyme inhibitor DPI and the NOS inhibitor L-NMMA on both ROS production and cell degranulation were evaluated. In the case of RPMC, OVA produced a dramatic shift to the left of mast cell SS after 30 min (Fig. 1A), reflecting the loss of granularity associated with degranulation. As shown in scatter plots of FL1 versus SS (Fig. 1, B–E), after 30 min, OVA increased the number of ROS-positive cells (FL1, upper left and upper right quadrants) from 13% to 54% and increased the number of fully degranulated cells (lower left and upper left quadrants) from 10% to 31% (compare Fig. 1, B and C). The number of cells that were both fully degranulated and ROS-positive (upper left quadrant) increased from 1% to 19% (Fig. 1, B and C). Thus, a substantial proportion of antigen-activated RPMC degranulated and simultaneously produced intracellular ROS. Pre-exposure to DPI returned OVA-induced numbers of ROS-positive cells to control levels (from 54% back to 12%) and reduced the number of fully degranulated mast cells from 30% to 15% (Fig. 1D). L-NMMA was without effect on OVA-induced degranulation and ROS production (Fig. 1E). Histograms of cell number versus FL1 for IgE-positive RPMC populations confirmed antigen-induced DCF fluorescence (Fig. 1F) and inhibition by DPI (Fig. 1G) but not L-NMMA (Fig. 1H).



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  		FIG. 1.
Effect of antigen on mast cell degranulation and intracellular ROS production. IgE-sensitized RPMC, BMMC, or HUMC were incubated with or without DPI (1–5 µM) or L-NMMA (100–500 µM) for 1–24 h, and DCF diacetate (2–20 µM) was added for a further 0.5–1 h. The cells were then stimulated with relevant antigen: OVA (1 µg/ml) for RPMC for 30 min or DNP-HSA (100 ng/ml) for BMMC and SA (100 ng/ml) for HUMC, both for 2 min. FL1 fluorescence and SS were monitored by flow cytometry of 10,000 events within a gated area defined previously as IgE-positive mast cells. A–E show results for RPMC. A, an SS histogram for OVA-stimulated versus unstimulated cells. B–E, scatter plots of SS versus FL1 for unstimulated cells (B), OVA-stimulated cells (C), OVA-stimulated cells pretreated with DPI (D), or L-NMMA (E). Quadrants (B–E) were set to give <1% of cells both degranulated and ROS-positive (upper left quadrant) in unstimulated populations. Representative histograms of ROS generation in RPMC (F–H), BMMC (I–K), and HUMC (L–N) following antigen stimulation alone (F, I, and L) or pretreated with DPI (G, J, and M) or L-NMMA (H, K, and N) are shown.

 
Antigen stimulation of BMMC and HUMC led to ROS production that peaked at 2 min (Fig. 1, I and L). The BMMC ROS response to antigen was considerably greater than that of RPMC (compare Fig. 1, F–I), whereas the HUMC ROS response was less than that of BMMC but comparable with that of RPMC (compare Fig. 1L with I and F). In both BMMC and HUMC, the ROS response was inhibited by DPI (Fig. 1, J and M) but not L-NMMA (Fig. 1, K and N). Analysis of data pooled from replicated experiments confirmed that for each mast cell type, antigen elicited significant ROS generation, and this was blocked by DPI but not L-NMMA (Fig. 2, A–C).



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  		FIG. 2.
Effect of DPI and L-NMMA on intracellular ROS production and degranulation. Experiments were conducted as in Fig. 1, and results were averaged over 3–5 experiments. A–C show ROS generation, expressed as the percentage of antigen response, and D–F show degranulation for RPMC, HUMC, and BMMC, respectively. For degranulation experiments, RPMC (D), HUMC (E), or BMMC (F) were incubated for 1 h with L-NMMA or DPI prior to challenge with the appropriate antigen for 30 min. Degranulation was measured as net serotonin release for RPMC or {beta}-hexosaminidase for HUMC or BMMC. Results are means ± S.E. from 3 to 5 separate experiments performed in duplicate. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 for comparison with antigen alone by ANOVA followed by paired Student's t test.

 
To investigate the possible functional roles of ROS or NO in mast cell degranulation, we studied the effects of DPI and L-NMMA on antigen-induced mediator release. DPI almost completely blocked antigen-induced degranulation of RPMC and HUMC (Fig. 2, D and E) but produced only a small inhibitory effect on BMMC responses (Fig. 2F). L-NMMA, at concentrations that routinely inhibited NO production by macrophages, was without effect on antigen-induced degranulation of all three mast cell types (Fig. 2, D–F).

The relationship between intracellular ROS production and mast cell degranulation was further explored in RPMC as in this mast cell type, both responses can be measured concurrently by flow cytometry. As shown in Fig. 3A, the OVA-induced shift to the left of SS, reflecting cell degranulation, was blocked by DPI. Over five separate experiments, OVA stimulation increased significantly the number of cells that were simultaneously positive for degranulation (SS shift) and ROS production (from ~12 to 60%), and this response was blocked by DPI but not L-NMMA (Fig. 3B). These data are consistent with Figs. 1D and 2A showing that DPI blocked ROS production and Fig. 2D showing that DPI blocked antigen-induced serotonin release by RPMC. It is evident from these data that inhibition of ROS production is associated with inhibition of cell degranulation.



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  		FIG. 3.
Effect of OVA on concurrent mast cell degranulation and intracellular ROS generation. Experiments were conducted as in Fig. 1 with SS and DCF fluorescence measured 0.5 h after OVA challenge in RPMC. A representative histogram of SS for unstimulated cells, OVA-stimulated cells, and OVA-stimulated cells pretreated with DPI is shown (A). Data averaged across 5 experiments for the percentage of cells positive for concurrent degranulation and ROS generation (upper left (UL) and upper right (UR) quadrants of SS versus FL1 scatter plots from Fig. 1, B–E) are presented (B). Results are means ± S.E. from 5 experiments. ***, p < 0.001 for comparison with OVA alone by ANOVA followed by paired Student's t test with Bonferroni correction.

 
Antigen-activated Mast Cells Do Not Produce Intracellular NO—Intracellular NO was measured by DAF-2 fluorescence in OVA- and LPS- and IFN-{gamma}-stimulated RPMC, DNP-HSA-stimulated BMMC, and SA-stimulated HUMC, and the effects of NOS inhibitors on FL1 fluorescence were examined. In the first series of experiments, RPMC were incubated overnight with the NOS inhibitors L-NMMA or AG before incorporation of DAF-2 for 1 h. Cells were then stimulated with OVA under optimized conditions, and DAF-2 fluorescence and degranulation were monitored as described above. Following OVA stimulation for 0.5 h, the percentage of mast cells fully degranulated (upper left and lower left quadrants) increased from 3 to 18%, but there was only a negligible change in DAF-2 fluorescence (upper left and upper right quadrants) (Fig. 4, A and B). Furthermore, in contrast to ROS detection (Fig. 1C), OVA did not lead to the appearance of cells in the upper left quadrant, i.e. FL1-positive degranulating cells (Fig. 4B). In addition, the FL1 signal was not reversed by either L-NMMA (Fig. 4C) or AG (Fig. 4D). Histograms of DAF-2-dependent fluorescence confirmed that OVA produced no DAF-2 signal (Fig. 4E) and that L-NMMA (Fig. 4F) and AG (Fig. 4G) were without effect.



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  		FIG. 4.
Absence of NO production in mast cells activated by antigen. Experiments were conducted as in Fig. 1 except that cells were cultured with or without L-NMMA (500 µM) or AG (100 µg/ml) for 1–24 h and loaded with DAF-2 diacetate (2–20 µM) for 0.5–1 h. Results for RPMC are shown in A–D. FL1 and SS were measured in unstimulated cells (A) or in OVA-stimulated cells without pretreatment (B) or pretreated with L-NMMA (C) or AG (D). Representative histograms of DAF-2 fluorescence in RPMC (E–G), BMMC (H–J), and HUMC (K–M) following antigen stimulation alone (E, H, and K) or pretreated with L-NMMA (F, I, and L) or AG (G, J, and M) are shown. Rat macrophages were used as a positive control. Cells (500,000/tube) were treated as above except that FL1 was measured 24 h after stimulation with LPS (10 µg/ml) and IFN-{gamma} (100 ng/ml). Results are representative histograms of rat macrophages stimulated with LPS and IFN-{gamma} (N) or pretreated with L-NMMA (O) or AG (P).

 
Similar responses were observed for BMMC or HUMC. Stimulation with the appropriate antigen produced no DAF-2 signal (Fig. 4, H and K, respectively), and L-NMMA or AG had no effect (Fig. 4, I–J and L–M). The unresponsiveness of all types of antigen-stimulated mast cells with respect to DAF-2 fluorescence was in sharp contrast to rat macrophages. When these cells were stimulated with LPS and IFN-{gamma}, a dramatic shift in DAF-2 fluorescence was observed that was fully inhibited by both L-NMMA and AG (Fig. 4, N–P).

Averaged data across experiments confirmed that OVA, SA, or DNP-HSA did not induce NO production (elevated FL1) within 30 min of stimulation of RPMC, HUMC, or BMMC, respectively, and accordingly, L-NMMA and AG were without effect (Fig. 5, A–C). In RPMC, later time points of 1, 7, and 24 h after OVA stimulation were also investigated and produced similar results showing a lack of NO production over 24 h. In the same experiments, OVA did not induce the release of NO detectable as nitrite (Table I). The unresponsiveness of mast cells to antigen was in sharp contrast to rat macrophages, which showed a marked increase in DAF-2 fluorescence following stimulation with LPS and IFN-{gamma}, which was fully inhibited by both L-NMMA and AG (Fig. 5D).



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  		FIG. 5.
Effect of L-NMMA and AG on intracellular DAF-2 fluorescence. DAF-2 fluorescence by antigen-stimulated RPMC (A), HUMC (B), and BMMC (C) and by LPS and IFN-{gamma}-stimulated rat macrophages (D) was measured as in Fig. 4, and results were presented as means ± S.E. for 3 experiments performed in duplicate. **, p < 0.01 and ***, p < 0.001 for comparison with LPS and IFN-{gamma} alone by ANOVA followed by paired Student's t test.

 


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  		TABLE I
Nitrite levels (µM) in supernatants from mast cells and macrophages

Mast cells or macrophages were cultured as described with or without either L-NMMA (500 µM) or AG (100 µg/ml) before stimulation with either OVA (1 µg/ml, mast cells only) or IFN-{gamma} (100 ng/ml) + LPS (10 µg/ml) and nitrite determined in cell supernatants after 24 or 48 h. Results are means ± S.E. from three separate experiments. *, p < 0.001 for comparison with stimulated cells by ANOVA followed by paired Student's t test with Bonferroni correction. ND = not detected, assay limit was 1 µM.

 
Intracellular NO Generation by Macrophages but Not Mast Cells Stimulated with LPS and IFN-{gamma}—Since RPMC did not produce intracellular NO in response to OVA, we next examined whether they might respond to the classical NOS2 activating agents LPS and IFN-{gamma}, although these agents do not induce mast cell degranulation. As for antigen, LPS and IFN-{gamma} induced no DAF-2 signal in RPMC (Fig. 6A) but induced a strong signal in macrophages (Fig. 6B). Accordingly, L-NMMA and AG blocked the macrophage DAF-2 signal (Fig. 6B) but were without effect on RPMC fluorescence (Fig. 6A). Data averaged across experiments confirmed these results (Fig. 6, C and D). Additionally, extracellular nitrite was measured in LPS- and IFN-{gamma}-stimulated macrophages and LPS and IFN-{gamma}- or OVA-stimulated RPMC and gave comparable results to those obtained in the DAF-2 fluorescence experiments (Table I).



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  		FIG. 6.
Production of intracellular NO by macrophages but not mast cells stimulated with LPS and IFN-{gamma}. Mast cells (A and C) or macrophages (B and D) were incubated for 1 h with or without L-NMMA (500 µM) or AG (100 µg/ml) prior to incorporation of DAF-2 diacetate (20 µM) for a further 1 h. Cells were then stimulated with IFN-{gamma} (100 ng/ml) and LPS (10 µg/ml), and the median FL1 of 10,000 events was monitored at 7 and 48 h. Results are means ± S.E. from 3 separate experiments. **, p < 0.01 for comparison with LPS and IFN-{gamma} alone by ANOVA followed by paired Student's t test.

 
Expression of NOS2 Protein by Macrophages but Not Mast Cells—Incubation of IgE-sensitized BMMC or HUMC with antigen or LPS and IFN-{gamma} for 2–24 h, under conditions that optimally induced degranulation and/or ROS production, failed to induce cellular NOS2 protein detectable with either the monoclonal (Fig. 7) or the polyclonal antibody, whereas on each gel, NOS2 protein was readily detected in a positive control extract from rat macrophages stimulated with LPS and IFN-{gamma}.



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  		FIG. 7.
Immunoblots of NOS2 protein. IgE-sensitized BMMC or HUMC were challenged with antigen (DNP-HSA or SA, respectively) or LPS and IFN-{gamma} as indicated, and cell proteins were extracted. On each gel, a positive control extract from LPS- and IFN-{gamma}-activated rat peritoneal macrophages was run in lane 1. Mast cell extracts were loaded at 5–10-fold protein excess over the positive control. Results are shown for blots developed with monoclonal anti-NOS2; identical results were obtained with rabbit polyclonal anti-NOS2 and in further experiments in which both types of mast cell were incubated with the stimulating agents for 2, 4, 8, or 24 h. M, macrophage; U, unstimulated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that primary cultured mast cells of three different species (rat peritoneal tissue type, mouse bone marrow-derived, and human CD34+ blood cell-derived) generate intracellular ROS when activated by IgE/antigen but do not produce intracellular NO when activated by either IgE/antigen or LPS and IFN-{gamma}. In each case, cells were activated by antigen under conditions that gave the optimal release of granule mediators. LPS and IFN-{gamma} readily induced NO in control cells (rat macrophages). Furthermore, antigen or LPS and IFN-{gamma} failed to elicit expression of the inducible form of NO synthase (NOS2) in mouse and human mast cells. Intracellular ROS production by mast cells was inhibited by the flavoenzyme inhibitor DPI, whereas macrophage NO production was inhibited by the NOS inhibitors L-NMMA or AG, confirming the identity of these products.

We reported recently that activated rat peritoneal tissue type mast cells do not release ROS detectable by the sensitive real-time chemiluminescent probe Pholasin (16). In the present study, these same cells are shown to generate ROS intracellularly. Thus, ROS generated intracellularly by mast cells are not exported to the outside of the cell. In this respect, mast cells differ from activated macrophages that readily export high levels of ROS (16). Mast cells contain intracellular mechanisms for neutralization or chemical conversion of ROS including intracellular superoxide dismutase and glutathione, levels of which are enhanced following mast cell activation (17), and presumably, these are sufficient to breakdown ROS before they can be exported.

In our experiments, the IgE/antigen-induced production of intracellular ROS was accompanied by 20–50% release of granule-associated mediators in all three types of mast cell studied. To examine the potential regulatory role of intracellular ROS in mast cell activation, we blocked ROS production using the flavoenzyme inhibitor DPI. This agent blocked completely antigen-induced ROS production by all mast cell types. Concurrently, DPI inhibited granule-associated mediator release by >80% for RPMC and HUMC but by <15% for BMMC. We conclude that the production of ROS in mast cells is functionally linked to degranulation in RPMC and HUMC but less so in BMMC. However, these processes may not necessarily be interdependent since under certain conditions, for example combinations of diethyldithiocarbamate with calcium ionophore or compound 48/80 (33) or at subthreshold concentrations of antigen or substance P (19), the mast cell production of ROS can be dissociated from amine release. However, studies with the rat RBL-2H3 mast cell line, as in the present study, reveal a positive correlation between inhibition of ROS production and inhibition of mast cell degranulation (14, 15, 21). Inhibition of RBL-2H3 cell ROS production and degranulation by DPI is accompanied by decreased elevations of cellular calcium and reduced activation of phospholipase C{gamma} and the linker for activation of T cells, implicating signaling events prior to calcium mobilization as ROS targets (21). DPI inhibits flavoenzymes other than NADPH oxidase including mitochondrial complex I (34–36), which leads in turn to inhibition of mitochondrial-associated ROS production (37). There is an increasing body of evidence that mitochondrial-derived ROS are not simply by-products of respiration but may act as signaling molecules in normal cell function (38). Therefore, the DPI-dependent inhibition of either ROS production and/or degranulation observed within mast cells may occur at the mitochondrial level.

Our previous study (16) and others (39, 40) reported that cell- or chemical-derived hydrogen peroxide (a ROS generated from superoxide) inhibits antigen-driven mast cell mediator release. Thus, there is a potential dual role for ROS in mast cell activation; exogenous hydrogen peroxide, generated for example by macrophages at high levels, inhibits mast cell activation, whereas low level endogenously generated ROS may be facilitatory for exocytotic degranulation.

There is debate over whether mast cells are capable of generating NO, and if so, whether the radical exerts autoregulatory effects on mast cell activation (22–29). Our own studies (27–29) failed to confirm reports from others (22–26) that mast cells generate NO. We found that IFN-{gamma}-induced nitrite production by mixed mast cell/macrophage populations was almost entirely abolished as mast cells were purified, such that even in preparations that contained 98–99% mast cells, the NO production could be fully accounted for by the residual 1–2% macrophages (27, 28). Furthermore, mixed populations of macrophages and mast cells produced NO only when the macrophages expressed the IFN-{gamma} receptor (29). Therefore, nitrite production and NOS2 mRNA detected by reverse transcriptase-PCR (24) could be of non-mast cell origin even in highly purified mast cell populations. In the current study, we show that mast cells of three species, rat peritoneal tissue type, mouse bone marrow-derived, and human CD34+ blood cell-derived, do not generate detectable intracellular NO following activation with IgE/antigen or LPS and IFN-{gamma}, whereas in the same assay, rat macrophages readily generated high levels of intracellular NO. Furthermore, activated macrophages but not mast cells expressed protein for NOS2. Consistent with the lack of NO production by mast cells, inhibitors of NOS were without effect on mast cell degranulation. Thus, although we cannot rule out entirely the production of low level NO by mast cells, we conclude that relative to macrophages, mast cells produce negligible NO and evidently insufficient NO to appreciably influence mediator release.

Our results and conclusions differ markedly from those of Gilchrist et al. (24–26) who, also using the intracellular NO probe DAF, reported NO production by RPMC (25) and human HMC-1 and LAD2 mast cells (26). In the case of RPMC, activation by IgE/antigen led to NO production in a minority subset of cells that showed no visible signs of degranulation, whereas degranulating cells failed to generate NO. Interestingly, the NO-producing non-degranulating cells eventually did show degranulation, and this was interpreted as NO delaying mediator release (25). Our current results, showing a complete absence of NO production in IgE/antigen-activated degranulating and non-degranulating RPMC over a wide range of time periods, casts doubt over the conclusions of Gilchrist et al. (24–26), or at least suggests that the responses of outlying cells does not reflect larger populations as a whole. In their study, cell fluorescence was measured on limited numbers of cells (8–15/experiment), whereas in our experiments, we monitored NO production simultaneously with degranulation and found no differences in distribution of DAF signal between activated and non-activated mast cells in large populations of 10,000 cells. In addition, we detected no outlying high DAF-2 fluorescing cells, as would be predicted from the studies of Gilchrist et al. (25). Furthermore, although Gilchrist et al. (25) showed that inhibition of synthesis of the NOS co-factor biopterin led to enhanced RPMC degranulation, our work (Refs. 27 and 28 and the present study) shows that direct inhibition of NO synthesis does not influence mediator release by rat, mouse, or human mast cells. Convincingly, Gilchrist et al. (26) demonstrated constitutive NOS protein expression and calcium-driven NO production in human HMC-1 and LAD2 mast cells and found that NO donors and inhibitors, respectively, inhibited and enhanced leukotriene production. Thus, in these mast cell lines, a calcium-driven, regulatory constitutive NOS-dependent NO response occurs. However, it should be stressed that the primary phenotype of HMC-1 and LAD2 cells is that of tumor cells. In the present study, using tissue type, bone marrow-, or blood-derived mast cells of rodent and human origin, we were unable to detect NO production under a range of conditions.

In conclusion, our results show that activated rat, mouse, and human mast cells generate intracellular ROS but not NO. In each species of mast cell studied, endogenous ROS were functionally linked to mast cell activation and may act to facilitate mediator release. With regard to NO, exogenous sources such as macrophages or epithelial cells are more likely than endogenous NO to be involved in regulation of mast cell activation and mast cell-dependent immune and allergic reactions.


   FOOTNOTES
 
* This work was supported by a grant (to J. W. C.) from The Wellcome Trust and by National Institutes of Health intramural funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Bldg. 10, Rm. 11C 209, Bethesda, MD 20892-1881. Tel.: 301-594-1276; Fax: 301-480-8384; E-mail: ejswindle{at}niaid.nih.gov.

1 The abbreviations used are: ROS, reactive oxygen species; NO, nitric oxide; NOS, NO synthase; AG, aminoguanidine; BMMC, mouse bone marrow-derived mast cells; HUMC, human mast cells; RPMC, rat peritoneal mast cells; DNP-HSA, dinitrophenylated human serum albumin; DPI, diphenyleneiodonium; DCF, dichlorodihydrofluoroscein; FL1, fluorescence channel 1; FS, light forward scatter; SS, light side scatter; IFN, interferon; L-NMMA, NG-monomethyl-L-arginine; LPS, bacterial lipopolysaccharide; OVA, ovalbumin; SA, streptavidin; DAF, diaminofluorescein; DAF-2, 4,5-diaminofluorescein; ANOVA, analysis of variance. Back

2 E. J. Swindle and J. W. Coleman, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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