European Journal of Pharmacology 543 (2006) 190–193www.elsevier.com/locate/ejphar

Short communication

Aspirin induces the production of the inflammatorymediator 8-epi-PGF in mast cells

Esmaeil Mortaz, Frank A. Redegeld, Frans P. Nijkamp, Ferdi Engels ⁎

Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,PO BOX 80082, 3508 TB Utrecht, The Netherlands

Received 24 January 2006; received in revised form 14 May 2006; accepted 16 May 2006Available online 2 June 2006

Abstract

Recently it has been shown that mast cells, through release of their pro-inflammatory mediators, are involved in aspirin attacks in aspirin-sensitive patients. To date, little information is available concerning 8-isoprostane (8-epi-prostaglandin F) production by mast cells. Therefore, weexamined whether exposure of mast cells to aspirin can lead to isoprostane production. In this study we show that in mast cells, IgE and antigenstimulates an intracellular oxidative burst inducing H2O2 and 8-epi-PGF production. Moreover, we show that exposure of mast cells to aspirindirectly induces the production of 8-epi-PGF. Our study suggests that production of 8-epi-PGF by mast cells could contribute to the inflammatoryresponse in e.g. aspirin-sensitive asthma patients.© 2006 Elsevier B.V. All rights reserved.

Keywords: 8-Isoprostane; Mast cell; Aspirin

1. Introduction

Aspirin-induced asthma consists of the clinical triad ofasthma, chronic rhinosinusitis with nasal polyps, and precipi-tation of asthma and rhinitis attacks in response to aspirin andother nonsteroidal anti-inflammatory drugs (NSAIDs) (Szczek-lik and Stevenson, 1999).

The prevalence of the syndrome in the adult asthmaticpopulation is approximately 4–10% (Szczeklik, 1997). Relatedstudies provide evidence for abnormal regulation of thelipoxygenase pathway, demonstrating elevated levels ofcysteinyl leukotrienes in urine, sputum and peripheral blood,before and following aspirin challenge in aspirin-inducedasthma patients. Besides cysteinyl leukotrienes, it has beenreported that levels of the isoprostane 8-epi-PGF are increasedin exhaled air of aspirin-induced asthma patients (Antczak et al.,2002). The isoprostanes are families of prostaglandin isomersformed in a free radical catalyzed manner from arachidonic acid(Morrow et al., 1990). 8-Epi-PGF is a biomarker of oxidativestress in breath condensate in inflammatory airway diseases

⁎ Corresponding author. Tel.: +31 30 2537356; fax: +31 30 2537420.E-mail address: G.M.H.Engels@pharm.uu.nl (F. Engels).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.05.044

such as asthma, chronic obstructive pulmonary disease, cysticfibrosis, and in diabetes (Montuschi et al., 1998; Gopaul et al.,1995).

While it would be hard to predict the relative amounts ofdifferent isoprostanes, it is conceivable that such a mixture ofisoprostanes in the lung could account for many symptomsfound in those diseases states. For example, isoprostanes triggerconstriction of the airways, the lymphatics, and the bronchialand pulmonary vasculature, and augment enthothelial perme-ability (Janssen, 2004). Isoprostanes would likely also lead tolung inflammation via increased production of pro-inflamma-tory cytokines by smooth muscle and endothelial cells (Janssenet al., 2000), and via direct actions on the inflammatory cellsthemselves. The inflammatory response in turn would exacer-bate bronchoconstriction and edema formation (Kawikova etal., 1996).

Moreover, over a longer time-course, the isoprostanes couldcontribute to smooth muscle hypertrophy and hyperresponsive-ness (Saunders et al., 2001). Montuschi et al. demonstrated thatthe level of oxidative stress is enhanced in patients withinterstitial lung diseases as reflected by increased concentrationsof 8-epi-PGF2α (Montuschi et al., 1999). Recently, it has beenshown that mast cells, through release of their proinflammatory

Fig. 1. Time-dependent production of 8-epi-PGF in mast cells triggered by IgE/Ag or exposed to aspirin. Cells were incubated with aspirin (ASA, 1 mM) orstimulated with IgE and DNP–HSA (IgE/Ag). At different time-points afteractivation the production of 8-epi-PGF was quantified in the medium. Resultsare mean±S.E.M. from three independent experiments. The asterisks representsignificant differences (⁎P<0.05; ⁎⁎P<0.01, ⁎⁎⁎P<0.001).

Fig. 2. Cross-linking of IgE/antigen or aspirin induce production of 8-epi-PGF.Cells were incubated with aspirin (ASA, 1 or 10 mM) or activated with IgE andDNP–HSA (IgE/Ag) for 5 h in the presence or absence of pyrrolidine dithio-carbamate (PDTC). 8-epi-PGF released into the medium was determined using acommercial EIA kit. Results are mean±S.E.M. from 3 independent experiments.The asterisks represent significant differences (⁎P<0.05; ⁎⁎P<0.01, ⁎⁎⁎P<0.001).

191E. Mortaz et al. / European Journal of Pharmacology 543 (2006) 190–193

mediators, are involved in aspirin attacks in aspirin-sensitivepatients (Mita et al., 2001). This prompted us to investigate theeffects of aspirin on the 8-isoprostane production from mastcells.

2. Materials and methods

2.1. Materials

A specific enzyme immunoassay (EIA) kit for measurementof 8-epi-PGF2α was obtained from Cayman Chemical (ITKDiagnostics, Uithoorn, The Netherlands). RPMI 1640, fetal calfserum, and nonessential amino acids were purchased fromGibCo BRL. Penicillin, streptomycin, 1-glutamine, sodiumpyruvate, 2-mercaptoethanol, dinitrophenol–human serum al-bumin (DNP–HSA), aspirin and pyrrolidine dithiocarbamate(PDTC) were obtained from Sigma (Sigma-Aldrich, Zwijn-drecht, The Netherlands).

2.2. Cell culture and activation of cells

Bone marrow-derived mast cells were generated from bonemarrow of male BALB/cBy mice as described before (Mortaz etal., 2005). After 3–4 weeks, mast cell purity of >95% wasachieved as assessed by toluidine blue staining. For activation,cells were sensitized with 1 μg/ml of anti-DNP IgE fromhybridoma clone 26.82 at 37 °C for 1 h. Then cells were washedwith medium and stimulated with 10 ng/ml of DNP–HSA. Non-sensitized cells were incubated with aspirin at 1 or 10 mM for5 h. Incubations were stopped by centrifugation at 1200×g for5 min at 4 °C. Cell-free supernatants were isolated and stored at−20 °C until further analysis.

2.3. Measurement of 8-epi-PGF2α

8-Epi-PGF2α concentrations in supernatants were measuredby a specific enzyme immunoassay (EIA) kit. The antiserumused in this assay has 100% cross-reactivity with 8-epi-PGF2α,0.2% each with prostaglandin F2α (PGF2α), PGF3α, prostaglan-din E1 (PGE1) and prostaglandin E2 (PGE2) and 0.1% with 6-keto-PGF1α. The detection limit of the assay is 4 pg/ml.

2.4. Measurement of H2O2

Hydrogen peroxide production was measured by kits obtainedfrom Stressgen (Sanbio, Uden, The Netherlands). The sensitivityof this assay was determined to be 2.39 ng/ml. Briefly, IgE-sensitized cells were washed and added in Hanks' Balanced SaltSolution (HBSS) containing 1mMNaN3 and then stimulatedwithantigen (30 ng/ml) for 2 or 10 min. In another set of experimentscells were incubated with aspirin for 5 h. In both conditions cellswere pelleted by centrifugation and the supernatants wereharvested and the pellets were lysed with lysis buffer containing1% Triton X-100, and analyzed for H2O2 release. Supernatants orcell lysates were added to the reactionmixture provided in kits in a96-well plate and incubated for 40 min. The decrease influorescence was measured using a luminometer (with excitationand emission at 335 and 460 nm, respectively) for 5 s. Theconcentration of the oxidant in the samples was calculated usingthe standard curve, which was made by adding knownconcentrations of the authentic H2O2 instead of the samples.

2.5. Survival assay

Viability of cells was determined by staining cells withAnnexin-V or 7-ADD by flow cytometry analysis (FACS) asdescribed before (Schmid et al., 1994).

2.6. Statistical analysis

For parametric data Student's unpaired t test was used to com-pare groups. All data were expressed as means±standard error ofmean (S.E.M.). Significance was defined as a P value of <0.05.

3. Results

3.1. Cross-linking of IgE/antigen and aspirin induce produc-tion of 8-epi-PGF

The production of 8-epi-PGF by mast cells upon stimulationwith IgE and antigen or treatment with aspirin was measured.

Fig. 3. Cross-linking of IgE/antigen or aspirin induce H2O2 release. Cells weresensitized with anti-DNP IgE at 37 °C for 1 h and then activated with antigen(IgE/Ag) for 2 min or treated with aspirin (ASA) for 5 h. Then aftercentrifugation and lysis of the cells the amount of H2O2 was measured in celllysates. Results are mean±S.E.M. from 3 independent experiments. Theasterisks represent significant differences compared with controls, i.e. un-activated cells alone (⁎P<0.05; ⁎⁎P<0.01).

192 E. Mortaz et al. / European Journal of Pharmacology 543 (2006) 190–193

Cross-linking of IgE by antigen significantly enhanced produc-tion of 8-epi-PGFwith amaximum at 5 h after activation (Fig. 1).Aspirin at 1 and 10 mM also resulted in a significant productionof 8-epi-PGF at the same time point (Figs. 1 and 2). Cell viabilitywas not affected by aspirin (92±2% vs. 98±1% in control).

If the reactive oxygen species produced play a role inregulating 8-isoprostane production, then decreasing their levelsby blocking them may shed more light on the mechanism ofproduction of 8-isoprostane. As shown in Fig. 2, pre-incubationof cells with the cell permeable antioxidant, PDTC decreasedproduction of 8-epi-PGF from cells stimulated with IgE andantigen (24±5 pg/ml vs. 75±2 pg/ml, Fig. 2) or when incubatedwith aspirin (1 and 10 mM).

3.2. Aspirin and IgE/antigen enhance production of H2O2

It has been reported that cross-linking IgE with antigenincreases production of H2O2 in RBL-2H3 cells (Morrow et al.,1990). To gain some insight into production of reactive oxygenspecies we studied the release of H2O2 by IgE/antigen or aspirinin mast cells. The increase in H2O2 production was observed by2 min after stimulation with IgE/antigen (Fig. 3) reaching itspeak by 10 min (data not shown). Aspirin alone concentration-dependently increased H2O2 production at the same time points(Fig. 3).

4. Discussion

We have demonstrated that bone marrow-derived mast cellsare able to produce and release the isoprostane 8-epi-PGF2α inresponse to IgE cross-linking by antigen as well as byincubation with aspirin. The release of 8-epi-PGF2α wasparalleled by the production of reactive oxygen species, notablyH2O2. Pharmacological inhibition of reactive oxygen speciesproduction also suppressed the release of 8-epi-PGF2α suggest-ing that reactive oxygen species are involved in the productionof isoprostanes by mast cells. Synthesis of isoprostanes isusually considered to be mediated by free radicals in a non-enzymatic fashion (Janssen, 2001). However, in endothelialcells the cyclooxygenase enzyme has been implicated in theformation of isoprostanes (Watkins et al., 1999). Indeed,

isoprostane formation could be inhibited by indomethacin oraspirin. We can rule out such a mode of action in mast cells,since aspirin did not inhibit the release of 8-epi-PGF2α. On thecontrary, aspirin induced the release of 8-epi-PGF2α in thesecells. It is conceivable that this effect was a consequence of theincrease of H2O2, since both responses could be inhibited by acell permeable antioxidant. Similar results were found in gastriccells (Kusuhara et al., 1999). Thus, it seems likely that also inmast cells the formation of isoprostanes is a non-enzymaticphenomenon mediated by reactive oxygen species.

It is clear from our present data and from literature thatisoprostanes can be viewed as markers of oxidative stress.However, it is also evident that many of the isoprostane speciescan function as mediators affecting many different cell types(Janssen, 2001). Amongst the many isoprostanes tested, 8-epi-PGF2α and 8-epi-PGE2α were the most potent in contractingsmall-diameter pulmonary arteries and veins (Janssen et al.,2000, 2001). Airway smooth muscle also displays contractileresponses to these isoprostanes, as observed in rat (Kang et al.,1965), guinea pig (Kawikova et al., 1996) and human (Janssen etal., 2000) tissues. Two lines of evidence suggest the specificityof these effects. First, other isoprostane species that differ verylittle in structure from 8-epi-PGF2α and 8-epi-PGE2α are lessactive or not active at all in contracting smooth muscle (Janssen,2001; Janssen et al., 2000). Second, the observed effects aremediated by specific receptors since they can be antagonized byappropriate receptor antagonists (Janssen, 2001).

What are the pathophysiological consequences of ourobservations that mast cells produce isoprostanes in responseto allergic activation or aspirin? In allergic asthma, allergenchallenge results in activation of various types of inflammatorycells, including mast cells that release a plethora of mediators inresponse. We are the first to identify 8-epi-PGF2α as one of themediators from mast cells that make up the inflammatory soupthat is responsible for many of the effects seen after allergenchallenge in allergic individuals. The exact mechanisms bywhich mast cells produce 8-epi-PGF2α in response to aspirinremains to be elucidated. Recently, we have shown thatexposure to aspirin results in the release of heat shock proteinsfrom mast cells, which in turn can activate toll-like receptors(Mortaz et al., 2006). Possibly these effects play a role in theproduction of isoprostanes.

Our present findings may shed more light on the poorlyunderstood aspirin sensitivity in aspirin-induced asthma. Asmast cells have been implicated in aspirin-induced asthma (Mitaet al., 2001) this could possibly be related to the mast cell-derived 8-epi-PGF2α. It would be interesting to investigate if adifferential sensitivity of mast cells to aspirin in aspirin-inducedasthma, for example related to an altered functional expressionof toll-like receptors, could account for the increased 8-epi-PGF2α production found in these patients.

References

Antczak, A., Montuschi, P., Kharitonov, S., Gorski, P., Barnes, P.J., 2002.Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirin-induced asthma. Am. J. Respir. Crit. Care Med. 166, 301–306.

193E. Mortaz et al. / European Journal of Pharmacology 543 (2006) 190–193

Gopaul, N.K., Anggard, E.E., Mallet, A.I., Betteridge, D.J., Wolff, S.P.,Nourooz-Zadeh, J., 1995. Plasma 8-epi-PGF2 alpha levels are elevated inindividuals with non-insulin dependent diabetes mellitus. FEBS Lett. 368,225–229.

Janssen, L.J., 2001. Isoprostanes: an overview and putative roles in pulmonarypathophysiology. Am. J. Physiol., Lung Cell Mol. Physiol. 280,L1067–L1082.

Janssen, L.J., 2004. The pulmonary biology of isoprostanes. Chem. Phys. Lipids128, 101–116.

Janssen, L.J., Premji, M., Netherton, S., Catalli, A., Cox, G., Keshavjee, S.,Crankshaw, D.J., 2000. Excitatory and inhibitory actions of isoprostanes inhuman and canine airway smooth muscle. J. Pharmacol. Exp. Ther. 295,506–511.

Janssen, L.J., Premji, M., Netherton, S., Coruzzi, J., Lu-Chao, H., Cox, P.G.,2001. Vasoconstrictor actions of isoprostanes via tyrosine kinase and Rhokinase in human and canine pulmonary vascular smooth muscles. Br. J.Pharmacol. 132, 127–134.

Kang, K.H., Morrow, J.D., Roberts, L.J., Newman, J.H., Banerjee, M., 1965.Airway and vascular effects of 8-epi-prostaglandin F2α in isolated perfusedrat lung. J. Appl. Physiol. 74, 460–465.

Kawikova, I., Barnes, P.J., Takahashi, T., Tadjkarimi, S., Yacoub, M.H., Belvisi,M.G., 1996. 8-Epi-PGF2 alpha, a novel noncyclooxygenase-derivedprostaglandin, constricts airways in vitro. Am. J. Respir. Crit. Care Med.153, 590–596.

Kusuhara, H., Komatsu, H., Sumichika, H., Sugahara, K., 1999. Reactiveoxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells. Eur. J. Pharmacol. 383,331–337.

Mita, H., Endoh, S., Kudoh, M., Kawagishi, Y., Kobayashi, M., Taniguchi, M.,Akiyama, K., 2001. Possible involvement of mast-cell activation in aspirinprovocation of aspirin-induced asthma. Allergy 56, 1061–1067.

Montuschi, P., Ciabattoni, G., Paredi, P., Pantelidis, P., du Bois, R.M.,Kharitonov, S.A., Barnes, P.J., 1998. 8-Isoprostane as a biomarker of

oxidative stress in interstitial lung diseases. Am. J. Respir. Crit. Care Med.158, 1524–1527.

Montuschi, P., Curro, D., Ragazzoni, E., Preziosi, P., Ciabattoni, G., 1999.Anaphylaxis increases, 8-iso-prostaglandin F2alpha release from guinea-piglung in vitro. Eur. J. Pharmacol. 365, 59–64.

Morrow, J.D., Hill, K.E., Burk, R.F., Nammour, T.M., Badr, K.F., Roberts II, L.J., 1990. A series of prostaglandin F2-like compounds are produced in vivoin humans by a non-cyclooxygenase, free radical-catalyzed mechanism.Proc. Natl. Acad. Sci. 87, 9383–9387.

Mortaz, E., Redegeld, F.A., Nijkamp, F.A., Engels, F., 2005. Dual effects ofacetylsalicylic acid on mast cell degranulation, expression of cyclooxygen-ase-2 and release of pro-inflammatory cytokines. Biochem. Pharmacol. 69,1049–1057.

Mortaz, E., Redegeld, F.A., Nijkamp, F.A., Engels, F., 2006. Acetylsalicylicacid-induced release of HSP70 from mast cells results in cell activationthrough TLR pathway. Exp. Hematol. 34, 8–18.

Saunders, M.A., Sansores-Garcia, L., Gilroy, D.W., Wu, K.K., 2001. Selectivesuppression of CCAAT/enhancer-binding protein beta binding and cycloox-ygenase-2 promoter activity by sodium salicylate in quiescent humanfibroblasts. J. Biol. Chem. 276, 18897–18904.

Schmid, I., Uittenbogaart, C.H., Giorgi, J.V., 1994. Sensitive method formeasuring apoptosis and cell surface phenotype in human thymocytes byflow cytometry. Cytometry 15, 12–20.

Szczeklik, A., 1997. Mechanism of aspirin-induced asthma. Allergy 52,613–619.

Szczeklik, A., Stevenson, D.D., 1999. Aspirin-induced asthma: advances inpathogenesis and management. J. Allergy Clin. Immunol. 104, 5–13.

Watkins, M.T., Patton, G.M., Soler, H.M., Albadawi, H., Humphries, D.E.,Evans, J.E., Kadowaki, H., 1999. Synthesis of 8-epi-prostaglandin F2α byhuman endothelial cells: role of prostaglandin H2 synthase. Biochem. J. 344,747–754.