Rodent and Human Mast Cells Produce Functionally SignificantIntracellular Reactive Oxygen Species but Not Nitric Oxide*

Received for publication, August 24, 2004Published, JBC Papers in Press, September 10, 2004, DOI 10.1074/jbc.M409738200

Emily J. Swindle‡§¶, Dean D. Metcalfe§, and John W. Coleman‡§

From the ‡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

In immunity, reactive oxygen species (ROS) and nitricoxide (NO) are important antimicrobial agents and reg-ulators of cell signaling and activation pathways. How-ever, the cellular sources of ROS and NO are much de-bated. Particularly, there is contention over whethermast cells, key secretory cells in allergy and immunity,can generate these chemical species, and if so, whetherthey are of functional significance. We therefore exam-ined directly by flow cytometry the capacity of mastcells to generate intracellular ROS and NO using therespective cell-permeable fluorescent probes dichlo-rodihydrofluorescein and diaminofluorescein and eval-uated the effects of inhibitors of ROS and NO synthesison cell degranulation. For each of three mast cell types(rat peritoneal mast cells, mouse bone marrow-derivedmast cells, and human blood-derived mast cells), degran-ulation stimulated by IgE/antigen was accompanied byproduction of intracellular ROS but not NO. Inhibitionof ROS production led to reduced degranulation, indi-cating a facilitatory role for ROS, whereas NO synthaseinhibitors were without effect. Likewise, bacterial li-popolysaccharide and interferon-� over a wide rangeof conditions failed to generate intracellular NO inmast cells, whereas these agents readily induced intra-cellular NO in macrophages. NO synthase protein, asassessed by Western blotting, was readily induced inmacrophages but not mast cells. We conclude that ro-dent and human mast cells generate intracellular ROSbut not NO and that intracellular ROS but not intra-cellular NO are functionally linked to mast celldegranulation.

Mast cells are secretory cells central to specific and innateimmunity, allergy, and inflammation (1–5). In specific IgE-mediated responses, they are activated by antigen to releasechemical mediators such as histamine, proteases, prostaglan-dins, and cytokines (1), whereas in innate responses to bacte-ria, they promote neutrophil phagocytosis (3, 4) and lymphnode hyperplasia (5) via the production of tumor necrosis fac-tor. In keeping with a role in innate defense, mast cells candirectly phagocytose and kill bacteria (6). Essential to bacterialkilling by “professional” phagocytic cells such as neutrophilsand macrophages is the production of reactive oxygen species

(ROS)1 such as superoxide and hydrogen peroxide, nitrogenoxygen species such as nitric oxide (NO), and their combinedproduct peroxynitrite (7–10). However, in mast cells, thesepathways are less well characterized, and there is debate overwhether mast cells generate biologically significant amounts ofreactive oxygen and nitrogen species.

Several studies (11–13) have reported that rat peritonealtissue type mast cells (RPMC) or mast cells of the rat RBL-2H3cell line (14, 15) release ROS into the extracellular milieu inresponse to a range of stimuli. These studies measured ROS-induced light emission by scintillation counting (11) or usingluminol or the luciferin-related compound 2-methyl-6-(p-methoxyphenyl)-3-7-dihydroamidazo[1,2�]pyrazine-3-one)(12–15), and all of these techniques suffer from interferenceand lack of specificity (16). Using the highly sensitive real-timechemiluminescent probe Pholasin, we could not detect the re-lease of ROS by purified rat tissue mast cells activated withIgE/antigen, phorbol myristate acetate, or ionomycin, whereasmacrophages from the same starting populations of peritonealcells readily released ROS at high levels (16). Because Pholasinis not cell-permeable, it detects released rather than intracel-lular ROS. Therefore, ROS may be generated but not exported.Indeed, intracellular ROS generation has been reported forRPMC (17–19), a mouse mast cell line (20), RBL-2H3 cells, andmouse bone marrow-derived mast cells (21). To date, no studieshave been performed with human mast cells.

Reports from several groups claim that mast cells produceNO (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 perito-neal mast cells, although their preparations contained up to15% 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-derivedmast cells, whereas Gilchrist et al. (24–26) have demonstratedinducible or constitutive NOS expression, nitrite production,and intracellular NO-induced DAF fluorescence in activatedRPMC and the human tumor-derived HMC-1 and LAD2 mastcell-like lines. In contrast, we found that interferon (IFN)-�stimulated or spontaneous NO production by rat or mouseperitoneal mast cells diminished as mast cells were progres-sively purified, such that even in preparations that contained

* This work was supported by a grant (to J. W. C.) from The WellcomeTrust and by National Institutes of Health intramural funds. The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

¶ To whom correspondence should be addressed: Laboratory of Aller-gic Diseases, NIAID, National Institutes of Health, Bldg. 10, Rm. 11C209, Bethesda, MD 20892-1881. Tel.: 301-594-1276; Fax: 301-480-8384;E-mail: ejswindle@niaid.nih.gov.

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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 47, Issue of November 19, pp. 48751–48759, 2004Printed in U.S.A.

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98–99% mast cells, the NO production could be fully accountedfor by the residual 1–2% non-mast cells (27, 28). Furthermore,mixed populations of macrophages and mast cells produced NOin response to IFN-� only when the macrophages expressed theIFN-� receptor (29). Therefore, at least in IFN-�-driven re-sponses, a small minority of contaminating cells can contributesubstantially to nitrite production. In addition, we have con-sistently been unable to detect nitrite production by IgE/anti-gen-activated mast cells of various rodent phenotypes.2

In view of our demonstration that mast cells do not releaseROS to the outside of the cell (16) and the apparent differencesbetween our own and other groups relating to whether or notmast cells produce NO (22–29), the aims of the present studywere as follows: 1) to examine directly, by flow cytometry em-ploying specific and sensitive fluorescent intracellular probes,the capacity of rodent and human mast cells to generate intra-cellular ROS and NO; 2) to ascertain, using the appropriatepharmacological agents, the potential contribution of intracel-lular ROS and NO to mast cell secretory responses; 3) to in-vestigate whether the capacity or inability of mast cells togenerate ROS or NO relates to their species. We used exclu-sively tissue-derived or primary culture-derived mast cellsrather than mast cells of tumor phenotype as used in manyprevious studies.

Our results show that activated tissue type or primary cul-tured 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, activatedrodent and human mast cells did not generate intracellular NOor express NOS2 protein, and inhibitors of NO synthesis werewithout effect on cell activation.

EXPERIMENTAL PROCEDURES

Animals and Reagents—Female Brown Norway rats (150–200 g)were obtained from Harlan Olac (Bicester, UK) and maintained in theuniversity animal housing unit where food and water were provided adlibitum. Rats were sensitized by subcutaneous injection of ovalbumin(OVA)/alum (2 � 0.2 ml, total dose of 80 �g of OVA, 8 mg of aluminumhydroxide) on days 0 and 7, and the rats were sacrificed humanely onday 14 by asphyxiation in CO2. Experimental procedures were ap-proved by the University of Liverpool and were in accordance withguidelines set by the Home Office (London, UK).

Aluminum hydroxide, Dulbecco’s modified Eagle’s medium, diphen-yleneiodonium (DPI), L-glutamine, bacterial lipopolysaccharide (LPS),OVA, mouse monoclonal anti-�-actin antibody, toluidine blue, trypanblue, sulfanilamide and N-(1-napthyl)ethylenediamine dihydrochloridewere purchased from Sigma. Fetal calf serum, gentamicin, and Hanks’balanced saline solution were from Invitrogen. 4,5-Diaminofluorescein(DAF-2) diacetate, dichlorodihydrofluorescein (DCF) diacetate, amino-guanidine (AG), and NG-monomethyl-L-arginine (L-NMMA) were ob-tained from CN Biosciences (Nottingham, UK). Cytokines were obtainedfrom Peprotech (Rocky Hill, NJ). Lysis buffer and SDS-polyacrylamidegels were from Invitrogen. Mouse monoclonal anti-NOS2 and rabbitanti-Lyn antibodies were from Santa Cruz Biotechnology (Santa Cruz,CA), and rabbit polyclonal anti-NOS2 was from Calbiochem. Cell cul-ture media and supplements were from Invitrogen or BIOSOURCEInternational (Camarillo, CA).

Cell Isolation and Cultures—Rat mast cells and macrophages wereobtained by peritoneal lavage and purified by density gradient fraction-ation as described previously (16). Purified mast cell preparations con-tained �98% mast cells, whereas the macrophage preparations con-tained �95% monocyte/macrophages and �1% mast cells bymetachromatic staining in 0.05% toluidine blue and by Giemsa staining2 E. J. Swindle and J. W. Coleman, unpublished observations.

FIG. 1. Effect of antigen on mast celldegranulation and intracellular ROSproduction. IgE-sensitized RPMC,BMMC, or HUMC were incubated with orwithout DPI (1–5 �M) or L-NMMA (100–500 �M) for 1–24 h, and DCF diacetate(2–20 �M) was added for a further 0.5–1 h.The cells were then stimulated with rele-vant antigen: OVA (1 �g/ml) for RPMC for30 min or DNP-HSA (100 ng/ml) forBMMC and SA (100 ng/ml) for HUMC,both for 2 min. FL1 fluorescence and SSwere monitored by flow cytometry of10,000 events within a gated area definedpreviously as IgE-positive mast cells. A–Eshow results for RPMC. A, an SS histo-gram for OVA-stimulated versus un-stimulated cells. B–E, scatter plots of SSversus FL1 for unstimulated cells (B),OVA-stimulated cells (C), OVA-stimu-lated cells pretreated with DPI (D), orL-NMMA (E). Quadrants (B–E) were setto give �1% of cells both degranulatedand ROS-positive (upper left quadrant) inunstimulated populations. Representa-tive histograms of ROS generation inRPMC (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) areshown.

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of cytospin preparations. Mouse bone marrow-derived mast cells(BMMC) were grown from femoral marrow cells of BALB/c mice andcultured in RPMI 1640 medium supplemented with 10% fetal bovineserum, 100 units/ml penicillin, 100 �g/ml streptomycin, 25 mM HEPES,1.0 mM sodium pyruvate, non-essential amino acids, 0.0035% 2-mer-captoethanol, and 30 ng/ml mouse interleukin-3. Cells were used after4–6 weeks of culture. Human mast cells (HUMC) were grown fromCD34-positive peripheral blood mononuclear cells, obtained followinginformed 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 prepa-rations and were used after 8–10 weeks of culture.

Cell Degranulation and Nitrite Assays—Mast cells from OVA-sensi-tized rats, as well as BMMC or HUMC optimally sensitized with mouseIgE anti-DNP or biotinylated human IgE, respectively (both at 100ng/ml for 24 h), were seeded at 10–50,000/well in 80 �l in a 96-well cellculture plate. Enzyme inhibitors (10 �l) were added for 1 h, the cellswere challenged with 10 �l of the appropriate antigen (OVA (1 �g/ml),DNP30-HSA (100 ng/ml), or streptavidin (SA, 100 ng/ml), respectively),and degranulation was measured as the release of serotonin or �-hex-osaminidase as described (16, 31). Mediator release was expressed asthe percentage of total cell content after subtracting background releasefrom unstimulated cells. NO production was monitored as accumulatedsupernatant nitrite (the stable product of NO) by Griess assay asdescribed (27).

Fluorescent Detection of Intracellular ROS and NO—IntracellularROS and NO were measured by flow cytometry employing the fluores-cent probes DCF (the intracellular product of DCF diacetate that fluo-resces in the presence of ROS) and DAF-2 (the intracellular product ofDAF-2 diacetate that fluoresces in the presence of NO). Macrophagesand purified RPMC, BMMC, or HUMC (at 105-106/ml) were incubatedfor 1 or 24 h at 37 °C in 5% CO2 with or without L-NMMA (100–500 �M),AG (100 �g/ml), or DPI (1–5 �M). Then DAF-2 diacetate or DCF diac-etate was added (2–20 �M) for 0.5–1 h before stimulation. Forward lightscatter (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 Bio-

sciences). Macrophage fluorescence was monitored over the entire logFS versus log SS plot, whereas BMMC and HUMC were gated accordingto FS and SS parameters, and RPMC were gated in areas defined asmast cells by fluorescent staining with phycoerythrin-rabbit anti-ratIgE serum (Liverpool University in-house preparation). After flow cy-tometric analysis, samples were centrifuged (400 � g, 5 min), andsupernatants were removed (50 �l) 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 HUMCwere incubated in complete medium with or without the appropriateantigen or LPS and IFN-� under conditions that optimally induced cellactivation measured as degranulation and ROS production. At varioustimes (2–24 h), the cells were washed twice in serum-free medium,suspended at 2 � 106/ml, and then mixed with an equal volume ofboiling lysis buffer supplemented with 100 mM dithiothreitol, proteaseinhibitor mixture, and 2-mercaptoethanol as described (32). The cellextracts were boiled for 3 min and electrophoresed on 4–12% SDS-polyacrylamide gels. The separated proteins were then electrophoreti-cally transferred to nitrocellulose membranes and probed with mousemonoclonal anti-NOS2 or rabbit polyclonal anti-NOS2. To monitor pro-tein loading, blots were stripped and reprobed with monoclonal mouseanti-�-actin or rabbit anti-Lyn. Blots were developed with horseradishperoxidase-linked secondary antibody followed by chemiluminescentreagent and then exposed to x-ray film.

Data Presentation and Statistical Analysis—Data were analyzed byANOVA followed by two-tailed Student’s t test. Differences were con-sidered significant when p � 0.05.

RESULTS

Intracellular ROS Production and Relationship to Degranu-lation in Antigen-stimulated Mast Cells—Intracellular ROSproduction was monitored using the ROS-sensitive fluorescentprobe DCF, and the effects of the flavoenzyme inhibitor DPIand the NOS inhibitor L-NMMA on both ROS production andcell degranulation were evaluated. In the case of RPMC, OVAproduced a dramatic shift to the left of mast cell SS after 30 min

FIG. 2. Effect of DPI and L-NMMA on intracellular ROS production and degranulation. Experiments were conducted as in Fig. 1, andresults were averaged over 3–5 experiments. A–C show ROS generation, expressed as the percentage of antigen response, and D–F showdegranulation for RPMC, HUMC, and BMMC, respectively. For degranulation experiments, RPMC (D), HUMC (E), or BMMC (F) were incubatedfor 1 h with L-NMMA or DPI prior to challenge with the appropriate antigen for 30 min. Degranulation was measured as net serotonin release forRPMC or �-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.

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(Fig. 1A), reflecting the loss of granularity associated withdegranulation. 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) from13% to 54% and increased the number of fully degranulatedcells (lower left and upper left quadrants) from 10% to 31%(compare Fig. 1, B and C). The number of cells that were bothfully degranulated and ROS-positive (upper left quadrant) in-creased from 1% to 19% (Fig. 1, B and C). Thus, a substantialproportion of antigen-activated RPMC degranulated and si-multaneously produced intracellular ROS. Pre-exposure to DPIreturned OVA-induced numbers of ROS-positive cells to controllevels (from 54% back to 12%) and reduced the number of fullydegranulated mast cells from 30% to 15% (Fig. 1D). L-NMMAwas without effect on OVA-induced degranulation and ROSproduction (Fig. 1E). Histograms of cell number versus FL1 forIgE-positive RPMC populations confirmed antigen-inducedDCF fluorescence (Fig. 1F) and inhibition by DPI (Fig. 1G) butnot L-NMMA (Fig. 1H).

Antigen stimulation of BMMC and HUMC led to ROS pro-duction that peaked at 2 min (Fig. 1, I and L). The BMMC ROSresponse to antigen was considerably greater than that ofRPMC (compare Fig. 1, F–I), whereas the HUMC ROS responsewas less than that of BMMC but comparable with that ofRPMC (compare Fig. 1L with I and F). In both BMMC andHUMC, the ROS response was inhibited by DPI (Fig. 1, J andM) but not L-NMMA (Fig. 1, K and N). Analysis of data pooledfrom replicated experiments confirmed that for each mast celltype, antigen elicited significant ROS generation, and this wasblocked by DPI but not L-NMMA (Fig. 2, A–C).

To investigate the possible functional roles of ROS or NO inmast cell degranulation, we studied the effects of DPI andL-NMMA on antigen-induced mediator release. DPI almostcompletely blocked antigen-induced degranulation of RPMCand HUMC (Fig. 2, D and E) but produced only a small inhib-itory effect on BMMC responses (Fig. 2F). L-NMMA, at concen-trations that routinely inhibited NO production by macro-phages, was without effect on antigen-induced degranulation ofall three mast cell types (Fig. 2, D–F).

The relationship between intracellular ROS production andmast cell degranulation was further explored in RPMC as inthis mast cell type, both responses can be measured concur-rently by flow cytometry. As shown in Fig. 3A, the OVA-in-duced shift to the left of SS, reflecting cell degranulation, wasblocked by DPI. Over five separate experiments, OVA stimu-lation increased significantly the number of cells that weresimultaneously positive for degranulation (SS shift) and ROSproduction (from �12 to 60%), and this response was blockedby DPI but not L-NMMA (Fig. 3B). These data are consistentwith Figs. 1D and 2A showing that DPI blocked ROS produc-tion and Fig. 2D showing that DPI blocked antigen-inducedserotonin release by RPMC. It is evident from these data thatinhibition of ROS production is associated with inhibition ofcell degranulation.

Antigen-activated Mast Cells Do Not Produce IntracellularNO—Intracellular NO was measured by DAF-2 fluorescence inOVA- and LPS- and IFN-�-stimulated RPMC, DNP-HSA-stim-ulated BMMC, and SA-stimulated HUMC, and the effects ofNOS inhibitors on FL1 fluorescence were examined. In the firstseries of experiments, RPMC were incubated overnight withthe NOS inhibitors L-NMMA or AG before incorporation ofDAF-2 for 1 h. Cells were then stimulated with OVA underoptimized conditions, and DAF-2 fluorescence and degranula-tion were monitored as described above. Following OVA stim-ulation for 0.5 h, the percentage of mast cells fully degranu-

lated (upper left and lower left quadrants) increased from 3 to18%, but there was only a negligible change in DAF-2 fluores-cence (upper left and upper right quadrants) (Fig. 4, A and B).Furthermore, in contrast to ROS detection (Fig. 1C), OVA didnot lead to the appearance of cells in the upper left quadrant,i.e. FL1-positive degranulating cells (Fig. 4B). In addition, theFL1 signal was not reversed by either L-NMMA (Fig. 4C) or AG(Fig. 4D). Histograms of DAF-2-dependent fluorescence con-firmed that OVA produced no DAF-2 signal (Fig. 4E) and thatL-NMMA (Fig. 4F) and AG (Fig. 4G) were without effect.

Similar responses were observed for BMMC or HUMC. Stim-ulation with the appropriate antigen produced no DAF-2 signal(Fig. 4, H and K, respectively), and L-NMMA or AG had noeffect (Fig. 4, I–J and L–M). The unresponsiveness of all typesof antigen-stimulated mast cells with respect to DAF-2 fluores-cence was in sharp contrast to rat macrophages. When thesecells were stimulated with LPS and IFN-�, a dramatic shift inDAF-2 fluorescence was observed that was fully inhibited byboth 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 withouteffect (Fig. 5, A–C). In RPMC, later time points of 1, 7, and 24 hafter OVA stimulation were also investigated and producedsimilar results showing a lack of NO production over 24 h. Inthe same experiments, OVA did not induce the release of NOdetectable as nitrite (Table I). The unresponsiveness of mastcells to antigen was in sharp contrast to rat macrophages,which showed a marked increase in DAF-2 fluorescence follow-ing stimulation with LPS and IFN-�, which was fully inhibitedby both L-NMMA and AG (Fig. 5D).

FIG. 3. Effect of OVA on concurrent mast cell degranulationand intracellular ROS generation. Experiments were conducted asin Fig. 1 with SS and DCF fluorescence measured 0.5 h after OVAchallenge in RPMC. A representative histogram of SS for unstimulatedcells, OVA-stimulated cells, and OVA-stimulated cells pretreated withDPI is shown (A). Data averaged across 5 experiments for the percent-age of cells positive for concurrent degranulation and ROS generation(upper left (UL) and upper right (UR) quadrants of SS versus FL1scatter plots from Fig. 1, B–E) are presented (B). Results are means �S.E. from 5 experiments. ***, p � 0.001 for comparison with OVA aloneby ANOVA followed by paired Student’s t test with Bonferronicorrection.

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Intracellular NO Generation by Macrophages but Not MastCells Stimulated with LPS and IFN-�—Since RPMC did notproduce intracellular NO in response to OVA, we next exam-ined whether they might respond to the classical NOS2 acti-vating agents LPS and IFN-�, although these agents do notinduce mast cell degranulation. As for antigen, LPS and IFN-�induced no DAF-2 signal in RPMC (Fig. 6A) but induced astrong signal in macrophages (Fig. 6B). Accordingly, L-NMMAand AG blocked the macrophage DAF-2 signal (Fig. 6B) butwere without effect on RPMC fluorescence (Fig. 6A). Data

averaged across experiments confirmed these results (Fig. 6, Cand D). Additionally, extracellular nitrite was measured inLPS- and IFN-�-stimulated macrophages and LPS and IFN-�-or OVA-stimulated RPMC and gave comparable results tothose obtained in the DAF-2 fluorescence experiments(Table I).

Expression of NOS2 Protein by Macrophages but Not MastCells—Incubation of IgE-sensitized BMMC or HUMC with an-tigen or LPS and IFN-� for 2–24 h, under conditions thatoptimally induced degranulation and/or ROS production, failed

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

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to induce cellular NOS2 protein detectable with either themonoclonal (Fig. 7) or the polyclonal antibody, whereas on eachgel, NOS2 protein was readily detected in a positive controlextract from rat macrophages stimulated with LPS and IFN-�.

DISCUSSION

In this study, we show that primary cultured mast cells ofthree different species (rat peritoneal tissue type, mouse bonemarrow-derived, and human CD34� blood cell-derived) gener-ate intracellular ROS when activated by IgE/antigen but do notproduce intracellular NO when activated by either IgE/antigenor LPS and IFN-�. In each case, cells were activated by antigenunder conditions that gave the optimal release of granule me-diators. LPS and IFN-� readily induced NO in control cells (ratmacrophages). Furthermore, antigen or LPS and IFN-� failedto 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 flavoenzymeinhibitor DPI, whereas macrophage NO production was inhib-ited by the NOS inhibitors L-NMMA or AG, confirming theidentity of these products.

We reported recently that activated rat peritoneal tissuetype mast cells do not release ROS detectable by the sensitivereal-time chemiluminescent probe Pholasin (16). In the presentstudy, these same cells are shown to generate ROS intracellu-larly. Thus, ROS generated intracellularly by mast cells are notexported to the outside of the cell. In this respect, mast cellsdiffer from activated macrophages that readily export highlevels of ROS (16). Mast cells contain intracellular mechanismsfor neutralization or chemical conversion of ROS includingintracellular superoxide dismutase and glutathione, levels ofwhich are enhanced following mast cell activation (17), andpresumably, these are sufficient to breakdown ROS before theycan be exported.

TABLE INitrite levels (�M) in supernatants from mast cells and macrophages

Mast cells or macrophages were cultured as described with or without either L-NMMA (500 �M) or AG (100 �g/ml) before stimulation with eitherOVA (1 �g/ml, mast cells only) or IFN-� (100 ng/ml) � LPS (10 �g/ml) and nitrite determined in cell supernatants after 24 or 48 h. Results aremeans � S.E. from three separate experiments. *, p � 0.001 for comparison with stimulated cells by ANOVA followed by paired Student’s t testwith Bonferroni correction. ND � not detected, assay limit was 1 �M.

Mast cells Macrophages

OVA LPS � IFN-� LPS � IFN-� LPS � IFN-� LPS � IFN-�

24 h 24 h 48 h 24 h 48 h

Unstimulated ND ND ND 2.4 � 0.8* 2.6 � 0.8*Stimulated ND ND ND 9.8 � 2.0 13.2 � 0.3*Stimulated � L-NMMA ND ND ND ND NDStimulated � AG ND ND ND 2.3 � 0.7* 7.1 � 0.7*

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-�-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-� alone by ANOVA followed by pairedStudent’s t test.

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In our experiments, the IgE/antigen-induced production ofintracellular ROS was accompanied by 20–50% release of gran-ule-associated mediators in all three types of mast cell studied.To examine the potential regulatory role of intracellular ROSin mast cell activation, we blocked ROS production using theflavoenzyme inhibitor DPI. This agent blocked completely an-tigen-induced ROS production by all mast cell types. Concur-rently, DPI inhibited granule-associated mediator release by�80% for RPMC and HUMC but by �15% for BMMC. Weconclude that the production of ROS in mast cells is function-ally linked to degranulation in RPMC and HUMC but less so inBMMC. However, these processes may not necessarily be in-terdependent since under certain conditions, for example com-binations of diethyldithiocarbamate with calcium ionophore orcompound 48/80 (33) or at subthreshold concentrations of an-tigen or substance P (19), the mast cell production of ROS canbe dissociated from amine release. However, studies with therat RBL-2H3 mast cell line, as in the present study, reveal apositive correlation between inhibition of ROS production andinhibition of mast cell degranulation (14, 15, 21). Inhibition ofRBL-2H3 cell ROS production and degranulation by DPI isaccompanied by decreased elevations of cellular calcium andreduced activation of phospholipase C� and the linker for acti-vation of T cells, implicating signaling events prior to calciummobilization as ROS targets (21). DPI inhibits flavoenzymesother 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 ofevidence that mitochondrial-derived ROS are not simply by-

products of respiration but may act as signaling molecules innormal cell function (38). Therefore, the DPI-dependent inhi-bition of either ROS production and/or degranulation observedwithin mast cells may occur at the mitochondrial level.

Our previous study (16) and others (39, 40) reported thatcell- or chemical-derived hydrogen peroxide (a ROS generatedfrom superoxide) inhibits antigen-driven mast cell mediatorrelease. Thus, there is a potential dual role for ROS in mast cellactivation; exogenous hydrogen peroxide, generated for exam-ple by macrophages at high levels, inhibits mast cell activation,whereas low level endogenously generated ROS may be facili-tatory for exocytotic degranulation.

There is debate over whether mast cells are capable of gen-erating NO, and if so, whether the radical exerts autoregula-tory effects on mast cell activation (22–29). Our own studies(27–29) failed to confirm reports from others (22–26) that mastcells generate NO. We found that IFN-�-induced nitrite pro-duction by mixed mast cell/macrophage populations was al-most entirely abolished as mast cells were purified, such thateven in preparations that contained 98–99% mast cells, the NOproduction could be fully accounted for by the residual 1–2%macrophages (27, 28). Furthermore, mixed populations of macro-phages and mast cells produced NO only when the macrophagesexpressed the IFN-� receptor (29). Therefore, nitrite productionand NOS2 mRNA detected by reverse transcriptase-PCR (24)could be of non-mast cell origin even in highly purified mast cellpopulations. In the current study, we show that mast cells ofthree species, rat peritoneal tissue type, mouse bone marrow-derived, and human CD34� blood cell-derived, do not generate

FIG. 6. Production of intracellularNO by macrophages but not mastcells stimulated with LPS and IFN-�.Mast cells (A and C) or macrophages (Band D) were incubated for 1 h with orwithout L-NMMA (500 �M) or AG (100�g/ml) prior to incorporation of DAF-2 di-acetate (20 �M) for a further 1 h. Cellswere then stimulated with IFN-� (100 ng/ml) and LPS (10 �g/ml), and the medianFL1 of 10,000 events was monitored at 7and 48 h. Results are means � S.E. from3 separate experiments. **, p � 0.01 forcomparison with LPS and IFN-� alone byANOVA followed by paired Student’s ttest.

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detectable intracellular NO following activation with IgE/anti-gen or LPS and IFN-�, whereas in the same assay, rat macro-phages readily generated high levels of intracellular NO. Fur-thermore, activated macrophages but not mast cells expressedprotein for NOS2. Consistent with the lack of NO production bymast cells, inhibitors of NOS were without effect on mast celldegranulation. Thus, although we cannot rule out entirely theproduction of low level NO by mast cells, we conclude thatrelative to macrophages, mast cells produce negligible NO andevidently insufficient NO to appreciably influence mediatorrelease.

Our results and conclusions differ markedly from those ofGilchrist et al. (24–26) who, also using the intracellular NOprobe DAF, reported NO production by RPMC (25) and humanHMC-1 and LAD2 mast cells (26). In the case of RPMC, acti-vation by IgE/antigen led to NO production in a minority sub-set of cells that showed no visible signs of degranulation,whereas degranulating cells failed to generate NO. Interest-ingly, the NO-producing non-degranulating cells eventually didshow degranulation, and this was interpreted as NO delayingmediator release (25). Our current results, showing a completeabsence of NO production in IgE/antigen-activated degranulat-ing and non-degranulating RPMC over a wide range of timeperiods, casts doubt over the conclusions of Gilchrist et al.(24–26), or at least suggests that the responses of outlying cellsdoes 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 monitoredNO production simultaneously with degranulation and foundno differences in distribution of DAF signal between activatedand non-activated mast cells in large populations of 10,000cells. In addition, we detected no outlying high DAF-2 fluoresc-ing cells, as would be predicted from the studies of Gilchrist etal. (25). Furthermore, although Gilchrist et al. (25) showed that

inhibition of synthesis of the NOS co-factor biopterin led toenhanced RPMC degranulation, our work (Refs. 27 and 28 andthe present study) shows that direct inhibition of NO synthesisdoes not influence mediator release by rat, mouse, or humanmast cells. Convincingly, Gilchrist et al. (26) demonstratedconstitutive NOS protein expression and calcium-driven NOproduction in human HMC-1 and LAD2 mast cells and foundthat NO donors and inhibitors, respectively, inhibited and en-hanced leukotriene production. Thus, in these mast cell lines, acalcium-driven, regulatory constitutive NOS-dependent NO re-sponse occurs. However, it should be stressed that the primaryphenotype of HMC-1 and LAD2 cells is that of tumor cells. Inthe present study, using tissue type, bone marrow-, or blood-derived mast cells of rodent and human origin, we were unableto 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 werefunctionally linked to mast cell activation and may act to facil-itate mediator release. With regard to NO, exogenous sourcessuch as macrophages or epithelial cells are more likely thanendogenous NO to be involved in regulation of mast cell acti-vation and mast cell-dependent immune and allergic reactions.

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Emily J. Swindle, Dean D. Metcalfe and John W. ColemanReactive Oxygen Species but Not Nitric Oxide

Rodent and Human Mast Cells Produce Functionally Significant Intracellular

doi: 10.1074/jbc.M409738200 originally published online September 10, 20042004, 279:48751-48759.J. Biol. Chem. 

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