Food, drug, insect sting allergy, and anaphylaxis

Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production Valerie Hox, MD, PhD, Avanti Desai, MSc, Geethani Bandara, PhD, Alasdair M. Gilfillan, PhD, Dean D. Metcalfe, MD,* and Ana Olivera, PhD* Bethesda, Md Background: Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. Objectives: We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. Methods: Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. Results: Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. Conclusion: Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide From the Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health. *These authors contributed equally to this work. Supported by the Division of Intramural Research Program within the National Institute of Allergy and Infectious Diseases/National Institutes of Health and by Research Foundation Flanders (F.W.O.). V.H. was a research fellow and the recipient of a Travel Grant from F.W.O. as well as a Gustave Bo€el–Sofina–Belgian American Educational Foundation (B.A.E.F.) fellow. Disclosure of potential conflict of interest: This work was supported by the Division of Intramural Research Program within the National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIH) and by Research Foundation Flanders (F.W.O.). V. Hox was a research fellow and the recipient of a Travel Grant from F.W.O. as well as a Gustave Bo€el–Sofina–Belgian American Educational Foundation (B.A.E.F.) fellow. The rest of the authors declare that they have no relevant conflicts of interest. Received for publication July 8, 2014; revised November 3, 2014; accepted for publication November 10, 2014. Available online December 29, 2014. Corresponding author: Valerie Hox, MD, PhD, Mast Cell Biology Section, Laboratory of Allergic Diseases, NIAID, NIH, Bldg 10, Rm 11C209, 10 Center Dr MSC-1881, Bethesda, MD 20892-1881. E-mail: [email protected]. 0091-6749 http://dx.doi.org/10.1016/j.jaci.2014.11.003

production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis. (J Allergy Clin Immunol 2015;135:729-36.) Key words: Anaphylaxis, estrogen, nitric oxide synthase, vascular permeability, mast cells

A number of clinical studies have indicated sex differences in the incidence of systemic anaphylaxis,1,2 showing that adult women have more frequent anaphylaxis induced by food,3,4 drugs,2,3,5 and radiocontrast agents,6 as well as idiopathic anaphylaxis,7 compared with adult men. The fact that this sex difference is observed during the reproductive but not the prepubertal years3 suggests that sex hormones might be involved in this sexual dimorphism. Additionally, clinical reports of catamenial (or cyclical) anaphylaxis, which is characterized by recurrent episodes of anaphylactic reactions occurring around the time of menstruation,8 suggest that female sex hormones, such as estrogens or progesterone, might be involved in susceptibility to anaphylaxis. However, other than in selected cases of suggested autoimmune progesterone sensitivity,9,10 there is little insight into how sex hormones influence anaphylaxis. Anaphylaxis is usually triggered by an antigen that recognizes and aggregates antigen-specific IgE bound to FcεRI, the high-affinity receptor of IgE, in tissue-resident mast cells of sensitized subjects. Binding of antigen to FcεRI leads to the release and generation of various mediators, including proteases, lipid-derived molecules, cytokines, and histamine.11 These mediators act on the surrounding tissues, leading to a number of biologic effects, including vasodilation, plasma exudation, and edema, causing anaphylaxis in the most severe cases. In experimental mouse models anaphylaxis can also be elicited through IgE-independent mechanisms, including activation of IgG receptors (FcgR) present on mast cells, basophils, neutrophils, and macrophages.12-14 Anaphylaxis in human subjects can also follow the administration of drugs or radiocontrast agents, the mechanisms of which are not fully understood but are believed to be independent from FcεR. Here we explore sex differences in the severity of anaphylaxis using a well-established mouse model of passive systemic anaphylaxis (PSA) and demonstrate an enhanced severity and duration of anaphylaxis in female compared with male mice. The advantage of using this anaphylaxis model in male and female mice is that the differences in severity and duration at given concentrations of IgE and antigen can be attributed to sex and not 729

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Abbreviations used BMMC: Bone marrow–derived mast cell DMSO: Dimethyl sulfoxide DNP: Dinitrophenyl E2: 17b-Estradiol eNOS: Endothelial nitric oxide synthase ER: Estrogen receptor HSA: Human serum albumin L-NAME: L-NG-nitroarginine methyl ester MCPT-1: Mast cell protease 1 MLEC: Mouse lung endothelial cell NIH: National Institutes of Health NO: Nitric oxide PAEC: Pulmonary artery endothelial cell PAF: Platelet-activating factor PSA: Passive systemic anaphylaxis

to confounding factors, including previous antigen exposure, type of antigen, antigen-specific IgE levels, underlying disease, and treatments, that might affect severity and lethality in human subjects.15 Differences in anaphylaxis severity found in this experimental model relate to incidence data only in that more severe reactions are more likely to be recognized and thus disproportionately contribute to the calculation of incidence. We find that increased severity of anaphylaxis in female mice is dependent on estradiol-mediated upregulation of endothelial nitric oxide synthase (eNOS), the enzyme responsible for production of nitric oxide (NO). The mechanisms that underlie human anaphylactic shock are not well understood in part because of the difficulties in designing appropriate clinical studies. However, we are hopeful that the insights from the mouse studies reported in this work will stimulate further research into sex differences and in the role of NO as a biologic mediator in patients with severe systemic allergic reactions.

METHODS Further details can be found in the Methods section in this article’s Online Repository at www.jacionline.org.

Mice

Wild-type C57Bl/6, BALB/c, and NOS32/2 mice, as well as ovariectomized and sham-operated mice, were obtained from the Jackson Laboratory (Bar Harbor, Me). Mice were used in accordance with National Institutes of Health (NIH) guidelines and an animal study proposal (LAD 2E) approved by the NIH/National Institute of Allergy and Infectious Diseases Institutional Animal Care and Use Committee.

Systemic anaphylaxis For IgE-induced PSA, mice were sensitized with 3 mg of dinitrophenyl (DNP)–specific IgE and challenged 24 hours later with 200 mg of antigen administered intravenously (DNP–human serum albumin [DNP-HSA]; Sigma-Aldrich, St Louis, Mo) in PBS. Anaphylactic responses were determined by measuring core body temperature. Alternatively, anaphylaxis was induced by a bolus of histamine dihydrochloride (5 mmol, Sigma Aldrich) or by 80 mg of anti-mouse CD16/CD32 2.4G2 clone in PBS administered intravenously. Mice were injected intravenously with 2.5 mg of L-NGnitroarginine methyl ester (L-NAME; Sigma-Aldrich) in 100 mL of PBS 1 hour before DNP challenge to investigate the involvement of NOS.

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In some experiments mice were killed 90 seconds after the induction of anaphylaxis, and blood was collected. Plasma histamine and mast cell protease 1 (MCPT-1) levels were measured with specific ELISA kits (Beckman Coulter, Fullerton, Calif, and eBioscience, San Diego, Calif, respectively). To determine hematocrit levels, blood was collected before and 2 hours after anaphylaxis with microhematocrit tubes (Jorvet, Loveland, Colo), and the ratio of red blood cell volume compared with total volume of blood was determined. For determination of lung fluid, left lung lobes were excised 2 hours after antigen injection. Excised lungs were weighed immediately (wet weight), as well as after drying at 558C overnight (dry weight), and the wet/dry weight ratio was calculated. The right lung lobes were excised, fixed, and embedded in paraffin. Histologic sections of the paraffin-embedded lungs were prepared and stained with hematoxylin and eosin (American Histolabs, Gaithersburg, Md).

Estrogen receptor blockage and estradiol administration in female mice Mice received 5 injections with 20 mg/kg of the estrogen receptor (ER) antagonist ICI182,780 (Tocris, Ellisville, Mo) to block estrogen binding to the ER. To restore estrogen levels in ovariectomized female mice, slow-releasing pellets (Innovative Research of America, Sarasota, Fla) containing either 17b-estradiol (E2; 0.1 mg per pellet, 21-day release) or placebo (0.1 mg per pellet, 21-day release) were implanted, and PSA was performed 2 weeks after implantation.

Statistics All data are presented as means with SEMs or as box plots with minimum/ maximum ranges. Comparisons of temperature changes between groups were performed by using 2-way ANOVA. Differences between 2 variables were compared with the 2-tailed Student t test and between multiple variables with 1-way ANOVA (GraphPad Prism 4.01; GraphPad Software, San Diego, Calif). A P value of less than .05 was considered significant.

RESULTS Sex differences in systemic anaphylaxis are estrogen dependent Resembling epidemiologic studies on systemic anaphylaxis, IgE-mediated PSAwas more severe in female C57Bl/6 mice than in male mice (Fig 1, A), as determined by a decrease in core body temperature, which correlates with increases in plasma histamine and hematocrit values, hypotension, and visible behavioral changes.16 The differential severity between age-matched male and female mice was also observed in BALB/c mice (see Fig E1, A, left panel, in this article’s Online Repository at www. jacionline.org) and was unrelated to differences in body weight because a similar difference in PSA was found in weight-matched instead of age-matched female and male mice (see Fig E1, B). We next explored the basis for the sex disparity. Ablation of the major source of sex hormones in female mice by using ovariectomy resulted in a reduced PSA response compared with that seen in sham-operated female mice and appeared similar to that seen in male mice (Fig 1, B, and see Fig E1, B, right panel). We then focused on E2, which is the predominant circulating estrogen in females the reproductive years.17 Subcutaneous implantation of estradiolreleasing pellets into ovariectomized mice restored the presence of the hormone in circulation (see Fig E1, C), as well as the severity of the anaphylactic response (Fig 1, C), demonstrating a direct link between the presence of the female hormone estradiol and the sex disparities in anaphylaxis. Furthermore, there was a linear correlation between the circulating levels of estradiol and anaphylaxis severity and duration in female mice (see Fig E1, D).

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FIG 1. Enhanced anaphylaxis in female mice is estrogen dependent. A, Temperature changes in female and male C57Bl/6 mice during anaphylaxis. Mice were sensitized with DNP-specific IgE and challenged 24 hours later with DNP-HSA. Data are from 3 independent experiments of 6 mice per group. B-D, Temperature changes during anaphylaxis in ovariectomized (OVX) or sham-operated female mice (Fig 1, B) after implantation in OVX mice of E2–releasing (OVX1E2) or placebo pellets (OVX; Fig 1, C) and in female mice after treatment with the ER antagonist ICI182,780 (ER-ant; Fig 1, D). In Fig 1, B-D, there were 6 mice per group. ***P < .001.

Estradiol is known to mediate its actions by binding to ERa and ERb.18 Thus to further confirm the involvement of estradiol in the severity of anaphylaxis, we blocked ER with the ER antagonist ICI182,780 (fulvestrant).19 Treatment of female mice with ICI182,780 for 5 days was found to reduce the severity of the anaphylaxis to essentially the response observed in male or ovariectomized female mice (Fig 1, D, compared with Fig 1, A and B). Taken together, our data point to a role for estradiol in determining the severity of anaphylactic shock in this model.

Estrogen does not affect mast cell responsiveness but promotes vascular leakage during anaphylaxis The marked differences in PSA between ovariectomized and sham-operated female mice were not accompanied by a difference in serum concentrations of the early released mast cell mediators histamine and MCPT-1 (Fig 2, A) or cytokines, such as TNF-a (see Fig E2, A and B, in this article’s Online Repository at www.jacionline.org), suggesting that the effect of estradiol on anaphylaxis is not due to an enhancement of IgE/antigenmediated mast cell responses. In agreement, degranulation (Fig 2, B) and release of TNF-a (see Fig E2, C) or IL-6 (data not shown) of mouse bone marrow–derived mast cells (BMMCs) in response to maximal or submaximal concentrations of antigen was not affected by a wide range of estradiol concentrations (from physiologic to supraphysiologic) or by pretreatment with a physiologic concentration of estradiol (35 pg/mL) hours to days before antigen stimulation (data not shown). Estradiol was also found to be ineffective in altering degranulation of human basophils,20 but it was reported to enhance IgE/antigeninduced degranulation by about 4% in transformed mast cells

(RBL-2H3 and HMC-1).21 We confirmed that ERa, the ER found in mouse mast cells,21,22 was expressed in BMMCs from male and female mice (Fig 2, B, inset), suggesting that the lack of response to estradiol in mast cells is not due to loss of ER expression. Further evidence that the effect of estradiol on anaphylaxis is dissociated from mast cell effector responses comes from the finding that anaphylaxis induced by either a bolus of histamine, a key mediator of IgE/antigen-induced systemic anaphylaxis,16 or by cross-linking FcgRIIIA with 2.4G2, an anaphylaxis model that does not require FcεRI or mast cells,23,24 was also more severe in sham-operated than in ovariectomized female mice (Fig 2, C and D). Collectively, the data suggest that the mechanism for estrogen potentiation on anaphylaxis is subsequent to mast cell mediator release. We hence investigated whether the presence of estrogens might enhance plasma exudation induced by vascular mediators. Hematocrit levels (Fig 3, A) and lung fluid accumulation, as measured by lung wet/dry ratios (Fig 3, B), which are both used to quantify vascular leakage during PSA,25 were significantly higher in female compared with male mice. Lung histology confirmed a more pronounced peribronchial edema, as well as abundance of red blood cells in the airspace in lungs of female mice (Fig 3, C). The data suggest that estrogens affect a pathway that regulates vascular permeability.

Increased severity to anaphylaxis by estrogen is mediated by eNOS eNOS and the product of its activity, NO, have been reported to regulate vascular permeability in mice induced by vascular endothelial growth factor26 and vascular reactivity in female

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FIG 2. Estrogen does not affect mast cell responses. A, Histamine and MCPT-1 released into the circulation 90 seconds after challenging ovariectomized (OVX) or sham-operated female mice with antigen (n 5 5 mice per group). B, b-Hexosaminidase release from BMMCs in the presence of different concentrations of E2. Results are the average of 4 independent cultures (3 female and 1 male donor). The inset shows ERa expression in BMMCs from male (M) and female (F) mice. C and D, Temperature changes during anaphylaxis induced by histamine (Fig 2, C) or anti-CD16/CD32 2.4G2 (Fig 2, D) in OVX and sham-operated mice. In Fig 2, C and D, there were 6 mice per group. ***P < .001.

mice undergoing anaphylaxis.27 Because eNOS expression, activity, or both are susceptible to estrogen regulation,28-30 we investigated whether levels of this enzyme might relate to the sex-related differences observed in IgE-mediated anaphylaxis in our mouse model. Both lungs (Fig 4, A and B, upper panels) and aortas (see Fig E3, A, in this article’s Online Repository at www. jacionline.org) of female mice showed higher expression of eNOS than tissues from male or ovariectomized mice at both baseline and 10 minutes after induction of PSA. Although eNOS expression in ovariectomized female mice was not statistically different from that in male mice, there was a slightly higher trend in ovariectomized female mice, which might suggest compensatory mechanisms in ovariectomized mice or a minor participation of nonestrogen factors to increased eNOS expression in female compared with male mice. The finding that treatment of cultured mouse lung endothelial cells (MLECs) with estradiol increased eNOS expression (see Fig E2, B) further supports the conclusion that the increased eNOS expression in female mice is indeed mediated through estradiol. Mirroring eNOS levels, phosphorylation of eNOS at Ser1177, which has been shown to enhance eNOS activity,31 was increased in the lungs of female mice compared with those of male or ovariectomized mice (Fig 4, A and B, lower panels). We did not find an increase in the ratio of phosphorylated Ser1177-eNOS to eNOS 10 minutes after antigen challenge, as described after administration of platelet-activating factor (PAF),27,31 a potent and usually lethal anaphylaxis mediator with a minor role in IgE/antigen-induced systemic anaphylaxis in mice,12,32 which might suggest differences in the mechanism of action or differences in the time course or robustness of eNOS phosphorylation.

We then examined whether the higher constitutive expression and phosphorylation of eNOS in tissues from female mice relates functionally to the sex bias in anaphylaxis. We first injected the NOS inhibitor L-NAME 1 hour before antigen challenge and found that inhibition of NO production obliterated the differences between female and male mice in the PSA response (Fig 5, A). Furthermore, the effect of L-NAME was specific to eNOS inhibition because anaphylactic responses in female and male mice were indistinguishable in mice genetically deficient in eNOS (NOS32/2; Fig 5, B). Similarly, the protective effect of LNAME in female mice (Fig 5, C, lower panel) was abolished when estradiol function was blocked by pretreatment with the ER antagonist ICI182,780 (Fig 5, C, upper panel). The effect of L-NAME on body temperature changes in the absence or presence of the ER antagonist (Fig 5, C) also correlated with corresponding effects on hematocrit values (ie, protective in the absence of ER antagonist and ineffective in the presence of ER antagonist; Fig 5, D, lower and upper panels, respectively) and behavioral changes, suggesting that the effect of eNOS inhibition does not reflect an action on thermal regulation33 but a general effect on the anaphylactic response. Collectively, the data provide evidence that estradiol, signaling through its receptors, upregulates eNOS expression and subsequent NO production, which are critically important for the increased vascular reactivity and worsening of the anaphylactic response seen in female mice.

DISCUSSION The increased incidence of anaphylaxis in women has been recognized in the clinic for years.34-36 Our study shows that

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FIG 3. The presence of female sex hormones leads to increased vascular permeability during anaphylaxis. A, Hematocrit values at baseline and after PSA in male (M), female (F), sham-operated, and ovariectomized (OVX) mice. B, Wet/dry lung weight ratios of male and female mice 2 hours after anaphylaxis. In Fig 3, A and B, there were 6 mice per group. C, Representative hematoxylin and eosin–stained lung slides from a male and female mouse 2 hours after IgE/antigen-induced PSA showing peribronchial edema (arrows) and red blood cells accumulated in the airspace (arrowheads). Scale bar 5 100 mm. *P < .05, **P < .01, and ***P < .001.

sex-specific differences in systemic anaphylaxis are replicated in mice and, as in human subjects, are not restricted to IgEmediated responses. The incidence, onset, and/or severity of many human diseases, including allergic diseases, such as asthma, show a sex bias that is likewise conserved across species.37,38 Although in some instances these sex differences have been linked to particular sex hormones that act by exacerbating or protecting against the disease, a direct demonstration of such a link and the specific mechanisms involved have mostly remained elusive.38 Here we provide evidence that a critical mediator exacerbating the anaphylactic response in female mice is the sex steroid estradiol. As shown by the data presented in this article, the presence of estradiol leads to upregulation of eNOS levels and activity, which are critical in determining the sex bias in anaphylaxis severity in this experimental model. Thus this study should help us to address sex disparities in anaphylaxis in human subjects, as well as provoke inspection of anaphylaxis linked to hormonal replacement therapy and to the menstrual cycle, and support the concept that the effectiveness of treatment strategies should also be evaluated by taking sex into consideration. The conclusion that the sex hormone estradiol potentiates anaphylaxis and is responsible for the sex differences in a mouse model of IgE-mediated PSA is supported by the findings that blocking its receptors or chronic sex hormone

depletion by using ovariectomy obliterated the exacerbated responses in female mice, an effect that was reverted in the latter after administration of exogenous estradiol. The differences in anaphylaxis severity between ovariectomized and sham-operated female mice were also apparent when anaphylaxis was induced by either FcgRIII cross-linking or histamine, which do not require FcεRI or mast cells.23 In addition, we found no evidence for an effect of estradiol on FcεRImediated mast cell responses, indicating that the estrogenmediated potentiation of anaphylaxis is subsequent to mast cell activation and occurs regardless of the trigger mechanism. This conclusion is in agreement with clinical reports describing a female preponderance not only in IgE-mediated but also in anaphylactic reactions evoked by certain drugs and radiocontrast media,3,5,6 which do not seem to involve FcεRI. Instead, estrogen-mediated sex differences in anaphylaxis are linked to the function of eNOS because they were abolished by pharmacologic inhibition or genetic deficiency of eNOS and because the beneficial effect of NOS inhibition in female mice was prevented by ER antagonists. This implies that the effect of estradiol takes center stage at the endothelial lining of vessels, where eNOS is predominantly expressed.39 The product of eNOS activity, NO, is an endogenous vasodilator40 that preserves endothelial function and homeostasis and thus protects against

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FIG 4. Estrogen regulates eNOS expression in vivo. Expression levels of eNOS and Ser1177-phosphorylated eNOS (p-eNOS) are shown in lung lysates from female (F), male (M), and ovariectomized (OVX) female mice at baseline and after IgE/antigen-induced PSA. A, Representative Western blots of lung lysates are shown. Numbers below indicate the fold increases in eNOS or p-eNOS band intensities (corrected to their respective b-actin loading control) compared with male mice. B, Band intensities in lung eNOS and p-eNOS (n 5 5 mice per group) are shown. *P < .05, **P < .01, and ***P < .001. No statistical differences were found in levels of eNOS or p-eNOS between male and OVX female mice.

endothelial dysfunction in diseases, such as hypertension, heart failure, diabetes, and atherosclerosis,41 all of which are generally less prevalent in women than in men.42 ERs are also associated with protective cardiovascular effects,43 and estrogens regulate the expression of eNOS production, NO production, or both44 in rat tissues45,46 and human endothelial cell lines.29,30 In agreement, we find that lungs and aortas from female mice express higher levels of eNOS, with concomitantly higher baseline phosphorylation in Ser1177 than their male or ovariectomized counterparts and that estradiol increases eNOS expression in mouse endothelial cells (see Fig E3, B). Preliminary results suggest that eNOS expression levels in primary human pulmonary artery endothelial cells (PAECs) from female donors are also higher than those in male donors (see Fig E3, C). Altogether, the data presented herein and elsewhere suggest that estradiol-mediated eNOS upregulation in female subjects is conserved across species.

NO is a known regulator of many processes involved in anaphylactic shock, such as vasodilation and vascular leakage.47 A study exclusively performed in female mice reported that eNOS deficiency protected against PAF- or antigen-induced shock,27 whereas in another study with male wild-type mice, the protective effect of NO in PAF-induced anaphylaxis was seemingly less pronounced.31 However, the relationship of NO and anaphylaxis to sex, as we examine here, was not directly investigated in these previous studies. Our results indicate that the protective effect of NOS inhibition during nonlethal IgE/ antigen-induced anaphylactic shock appears restricted to female mice. In human subjects the role of NO in anaphylaxis is not well understood, and its relationship with sex has not been explored. Based on our mouse study and given that NOS inhibitors, such as L-NAME, have helped patients with cardiogenic shock,48 we suggest that use of these inhibitors might deserve some consideration for the treatment of anaphylaxis in selected instances,

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FIG 5. eNOS activity is crucial for determining female susceptibility to anaphylaxis. A and B, Temperature changes during anaphylaxis induced by IgE/antigen in female and male mice pretreated with the NOS inhibitor L-NAME or vehicle (Fig 5, A) or in NOS32/2 female and male mice (Fig 5, B). C and D, Effect of LNAME on temperature (Fig 5, C) and hematocrit (Fig 5, D) changes during IgE/antigen-induced anaphylaxis in female mice pretreated with the ER antagonist ICI182,780 (upper panels) or vehicle (lower panels). In Fig 5, A-D, there were 5 to 8 mice per group. *P < .05, **P < .01, and ***P < .001.

such as in highly susceptible female subjects. Treatment with methylene blue, a dye that competitively inhibits guanylate cyclase, thus interfering with the vasodilatory actions of NO, has shown life-saving effects in female and male patients with severe anaphylaxis,49 supporting the possibility that NO inhibition could be explored as an option in certain cases of anaphylaxis regardless of sex. Further studies are clearly needed to determine the usefulness of such drugs in the management of patients with recurrent severe anaphylaxis. In the context of the known effect of estrogen on NO and cardiovascular health, our findings imply that although a higher availability of NO in female subjects might be beneficial for cardiovascular function,50 under circumstances of acute release of vasoactive mediators during an anaphylactic reaction, NO might predispose female subjects to greater and detrimental vascular responses. In addition, our results support consideration of whether estrogen-containing hormonal therapy and contraceptives should be examined for their consequences in women susceptible to anaphylaxis. Lastly, our study reinforces the concern that preferential use of male subjects in some animal models might overlook critical interactions between a particular gene or mutation and

female-predominant pathways (ie, the NO pathway) that might be dramatically consequential for sex-specific disease risk. Key messages d

The increased susceptibility to anaphylaxis in women noted in clinical studies is reproduced in a mouse model of systemic anaphylaxis, and the sex differences are dependent on estradiol.

d

The effect of estradiol on anaphylaxis does not relate to alterations in the responsiveness of mast cells but rather to enhanced vascular permeability, which is linked to estradiol-mediated upregulation of eNOS and NO production.

d

Our findings provide insight into the mechanistic basis of sex bias in anaphylaxis and infer that although increased eNOS action by estradiol might be beneficial for women in the context of cardiovascular disease, it could contribute to enhanced vascular responses during anaphylaxis.

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27. Cauwels A, Janssen B, Buys E, Sips P, Brouckaert P. Anaphylactic shock depends on PI3K and eNOS-derived NO. J Clin Invest 2006;116:2244-51. 28. Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, et al. Estrogen increases endothelial nitric oxide by a receptor-mediated system. Biochem Biophys Res Commun 1995;214:847-55. 29. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T. Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett 1995;360:291-3. 30. Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, Forstermann U. Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension 1998;31:582-8. 31. Cui H, Okamoto Y, Yoshioka K, Du W, Takuwa N, Zhang W, et al. Sphingosine1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice. J Allergy Clin Immunol 2013;132: 1205-14.e9. 32. Makabe-Kobayashi Y, Hori Y, Adachi T, Ishigaki-Suzuki S, Kikuchi Y, Kagaya Y, et al. The control effect of histamine on body temperature and respiratory function in IgE-dependent systemic anaphylaxis. J Allergy Clin Immunol 2002;110: 298-303. 33. Yang WW, Krukoff TL. Nitric oxide regulates body temperature, neuronal activation and interleukin-1 beta gene expression in the hypothalamic paraventricular nucleus in response to immune stress. Neuropharmacology 2000;39:2075-89. 34. Brown AF, McKinnon D, Chu K. Emergency department anaphylaxis: a review of 142 patients in a single year. J Allergy Clin Immunol 2001;108:861-6. 35. De Marco R, Locatelli F, Sunyer J, Burney P. Differences in incidence of reported asthma related to age in men and women. A retrospective analysis of the data of the European Respiratory Health Survey. Am J Respir Crit Care Med 2000;162:68-74. 36. Poulos LM, Waters AM, Correll PK, Loblay RH, Marks GB. Trends in hospitalizations for anaphylaxis, angioedema, and urticaria in Australia, 1993-1994 to 2004-2005. J Allergy Clin Immunol 2007;120:878-84. 37. Hayashi T, Adachi Y, Hasegawa K, Morimoto M. Less sensitivity for late airway inflammation in males than females in BALB/c mice. Scand J Immunol 2003;57: 562-7. 38. Ober C, Loisel DA, Gilad Y. Sex-specific genetic architecture of human disease. Nat Rev Genet 2008;9:911-22. 39. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109-42. 40. Loscalzo J, Welch G. Nitric oxide and its role in the cardiovascular system. Prog Cardiovasc Dis 1995;38:87-104. 41. Li H, Forstermann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000;190:244-54. 42. Yang S, Bae L, Zhang L. Estrogen increases eNOS and NOx release in human coronary artery endothelium. J Cardiovasc Pharmacol 2000;36:242-7. 43. Teede HJ. Sex hormones and the cardiovascular system: effects on arterial function in women. Clin Exp Pharmacol Physiol 2007;34:672-6. 44. Rosselli M, Imthurm B, Macas E, Keller PJ, Dubey RK. Circulating nitrite/nitrate levels increase with follicular development: indirect evidence for estradiol mediated NO release. Biochem Biophys Res Commun 1994;202:1543-52. 45. Caliman IF, Lamas AZ, Dalpiaz PL, Medeiros AR, Abreu GR, Figueiredo SG, et al. Endothelial relaxation mechanisms and oxidative stress are restored by atorvastatin therapy in ovariectomized rats. PLoS One 2013;8:e80892. 46. Reckelhoff JF, Hennington BS, Moore AG, Blanchard EJ, Cameron J. Gender differences in the renal nitric oxide (NO) system: dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition. Am J Hypertens 1998;11:97-104. 47. Brown SGA, Kemp SF, Lieberman PL. Anaphylaxis. In: Adkinson NF Jr, Bochner BS, Burks AW, Busse WW, Holgate ST, Lemanske RF, editors. Middleton’s allergy principles and practice. Philadelphia: Elsevier Saunders; 2013. pp. 1237-59. 48. Cotter G, Kaluski E, Milo O, Blatt A, Salah A, Hendler A, et al. LINCS: LNAME (a NO synthase inhibitor) in the treatment of refractory cardiogenic shock: a prospective randomized study. Eur Heart J 2003;24:1287-95. 49. Evora PR, Simon MR. Role of nitric oxide production in anaphylaxis and its relevance for the treatment of anaphylactic hypotension with methylene blue. Ann Allergy Asthma Immunol 2007;99:306-13. 50. Miller VM, Duckles SP. Vascular actions of estrogens: functional implications. Pharmacol Rev 2008;60:210-41.

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METHODS Mice and antibodies

Male and female wild-type C57Bl/6, BALB/c, and NOS32/2 mice (C57Bl/ 6 background; line no. 002684), as well as ovariectomized and sham-operated female mice, were obtained from the Jackson Laboratory. All mice were used for experiments at 10 weeks, except ovariectomized and sham-operated female mice, which were used at 12 to 14 weeks of age (2-4 weeks after surgery) or when using weight-matched mice, as indicated. The experiments with female mice and sham-operated female mice were conducted randomly at any stage in their estrous cycle (proestrus, estrus, metestrus, and diestrus). The estrous cycle in female mice repeats every 4 to 5 days, with estradiol levels peaking at the proestrus stage; however, estradiol levels in female mice are higher than in male or ovariectomized mice at any stage of the cycle. Mice were used in accordance with NIH guidelines and animal study proposal LAD2E approved by the NIH/National Institute of Allergy and Infectious Diseases’ Institutional Animal Care and Use Committee. Rabbit monoclonal anti-eNOS (#9586), rabbit monoclonal anti-Ser1177phosphorylated eNOS (#9571), and rabbit monoclonal anti-ERb (#8644S) were purchased from Cell Signaling (Danvers, Mass). Mouse monoclonal anti–b-actin was from Sigma-Aldrich (St Louis, Mo; clone AC-15). Biotinylated goat anti-rabbit IgG was purchased from Life Technologies (Grand Island, NY), horseradish peroxidase–conjugated secondary antibody was purchased from Thermo Scientific (Waltham, Mass), and IRDye-800–labeled streptavidin and secondary antibodies were purchased from LICOR Biosciences (Lincoln, Neb).

Systemic anaphylaxis For IgE-induced PSA, mice were sensitized intravenously with 3 mg of DNP-specific IgE (in 200 mL of PBS, clone H1-DNP-ε-26.82) and challenged 24 hours later with 200 mg of antigen intravenously (DNPHSA, 30-40 moles of DNP/mole of HSA; Sigma-Aldrich) in PBS. Anaphylactic responses were determined by measuring core body temperature every 5 minutes for 1.5 to 2 hours with implantable electronic transponders (IPTT-300; BioMedic Data Systems, Seaford, Del) inserted under the dorsal neck skin fold of anesthetized mice at least 18 hours before antigen challenge. Alternatively, anaphylaxis was induced by an intravenous injection of a bolus of histamine dihydrochloride (5 mmol in 200 mL of PBS, Sigma-Aldrich) or by 80 mg of anti-mouse CD16/32 (FcgRII/III) 2.4G2 clone (BioXCell, Kuala Lumpur, Malaysia) in PBS. All injections were performed on sedated mice (2% isoflurane/98% oxygen mix for 5 minutes in a closed chamber). Mice were injected intravenously with 2.5 mg of the NOS inhibitor L-NAME (Sigma-Aldrich) in 100 mL of PBS 1 hour before DNP challenge to investigate the involvement of NOS. Mice were killed 2 hours after induction of anaphylaxis. In some experiments mice were killed by means of CO2 inhalation 90 seconds after induction of anaphylaxis, and blood was immediately withdrawn by using cardiac puncture and collected in EDTA-containing tubes (BD Biosciences, San Jose, Calif). Plasma histamine and MCPT-1 levels were measured with specific ELISA kits (Beckman Coulter and eBioscience, respectively). TNF-a levels were also measured from plasma 90 seconds or 2 hours after induction of anaphylaxis by using ELISA (R&D Systems, Minneapolis, Minn). To determine hematocrit levels, blood was collected from the retro-orbital plexus 2 days before (baseline) and 2 hours after induction of anaphylaxis with heparinized microhematocrit tubes (Jorvet), and the ratio of red blood cell volume compared with total blood volume was determined with a hematocrit reader. For determination of lung fluid accumulation caused by plasma exudation, left lung lobes were excised after exsanguination of the mice 2 hours after antigen injection. Excised lungs were dabbed dry and weighed immediately (wet weight). After overnight drying at 558C, lungs were weighed again (dry weight), and the wet/dry weight ratio was calculated. The bronchi of the right lung lobes were instilled with 400 mL of buffered formalin, and lung lobes were excised, fixed, and embedded in paraffin. Histologic sections of the paraffin-embedded lungs were prepared and stained with hematoxylin and eosin (American Histolabs).

ER blockage and estradiol administration in female mice Female wild-type C57Bl/6 mice received 5 subcutaneous flank injections on alternate days with the ER antagonist ICI182,780 (fulvestrant, Tocris) to block estrogen binding to the ER. ICI182,780 was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL diluted to 2.5 mg/mL in sesame oil and injected into the mice at 20 mg/kg in PBS. Control mice were injected with the same proportions of DMSO/sesame oil in PBS (200 mL). Mice were sensitized with IgE 6 hours after the last injection and challenged the next day with antigen. Slow-releasing pellets (Innovative Research of America) containing either E2 (0.1 mg per pellet, 21-day release) or placebo (0.1 mg per pellet, 21-day release) were implanted subcutaneously in the dorsal area of ovariectomized female mice 2 weeks after surgery to restore estrogen levels in ovariectomized female mice. PSA was performed 2 weeks after implantation. Blood was collected 2 hours after anaphylaxis, and serum estradiol concentrations were measured with an estradiol ELISA kit (Calbiotech, Spring Valley, Calif).

Cell cultures and mast cell degranulation assays Murine mast cell progenitors were obtained from tibia and femur bone marrows, as previously described,E1 and differentiated in RPMI 1640 medium (Cellgro; Corning, Mannassas, Va) supplemented with 10% FBS, 4 mmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 25 mmol/L HEPES, 1 mmol/L sodium pyruvate, 1% nonessential amino acids, 50 mmol/L b-mercaptoethanol, and 30 ng/mL mouse recombinant IL-3 (PeproTech, Rocky Hill, NJ). Four- to 6-week-old BMMCs from mice were estrogen starved by culturing them for 48 hours in phenol red–free RPMI media (Quality Biological, Gaithersburg, Md) containing 10% charcoal/dextran-treated FBS (Atlanta Biologicals, Flowery Branch, Ga) and then sensitized overnight in cytokine-free phenol red–free RPMI media with 100 ng/mL anti-mouse monoclonal DNP-IgE (Sigma-Aldrich). Cells were washed and plated in 96well plates (2 3 104 cells per well). E2 (Sigma-Aldrich) was added at the indicated concentrations (0-3000 pg/mL in 0.1% DMSO/PBS) immediately before antigen challenge (1-100 ng/mL DNP-HSA). Degranulation was measured as b-hexosaminidase release into media 30 minutes after antigen challenge, as previously described.E1 In a control experiment mature BMMCs were preincubated with E2 for 1 week, 4 days, 2 days, 1 day, or 1 hour before antigen challenge, showing no significant differences in degranulation compared with simultaneous addition of E2 and antigen. For TNF-a release, E2 at the indicated concentrations (0-100 ng/mL 0.1% DMSO/PBS) was added to BMMC cultures before antigen challenge (100 ng/mL). After 6 hours at 378C, supernatants were collected, and TNF-a levels were determined by using ELISA (R&D Systems). Human PAECs from female or male donors were purchased from Lonza (Walkersville, Md) and maintained in supplemented EGM-2 medium containing 10% FBS, as recommended by the manufacturer. The cells were used for Western blot analysis at passages 3 to 5. Microvascular MLECs were purchased from Cell Biologics (Chicago, Ill) and cultured in supplemented mouse endothelial cell medium (#M1168) containing 5% FBS. At passage 6, cells were estrogen starved by culturing them for 24 hours in phenol red–free supplemented mouse endothelial cell medium (#M1168PF) containing 5% charcoal/dextran-treated FBS. Then E2 in DMSO (final concentration of 0.01%) was added to the phenol red–free culture medium at 0, 10, or 100 pg/mL. DMSO (0.01%) was used as a control. Forty-eight hours later, cells were lysed for Western blot analysis.

Western blot analysis Left lung lobes and thoracic aortas were removed from naive male, female, and ovariectomized female C57/Bl6 mice either before or 10 minutes after PSA and snap-frozen in liquid nitrogen until further processing. Tissues were homogenized in 700 mL (lung) or 400 mL (aortas) of cold lysis buffer (PBS with 10 mg/mL leupeptin, 10 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L orthovanadate, 0.5% sodium deoxycholate, and 1% Triton X-100) with a Precellys homogenizer (Bertin technologies,

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Montigny-le-Bretonneux, France) by using prefilled metal bead tubes (BioExpress, Kaysville, Utah; 23 15-second pulses) and centrifuged at 800g for 5 minutes to eliminate cell debris. Protein concentrations in the supernatants of the homogenates were determined. Boiling 2X NuPAGE LDS sample buffer (Life Technologies) was added to each sample, and they were sonicated for 1 minute. Confluent PAECs grown in 6-well plates or confluent E2-treated MLECs were rinsed with PBS and lysed by adding 2X boiling lysis buffer, as previously described.E2 For eNOS and phosphorylated eNOS detection, solubilized samples (25 or 12 mg per lane for lungs and aortas, respectively), or cells were resolved by means of electrophoresis in 4-12% NuPage Bis-Tris gels (Life Technologies) and transferred onto nitrocellulose membranes (Life Technologies). The membranes were blocked with 5% BSA and probed with anti-eNOS (1:1,000) or anti–Ser1177 phosphorylated eNOS (1:1,000) antibodies. Samples were probed for b-actin (1:10,000) to normalize for protein loading. The eNOS and Ser1177-phosphorylated eNOS immunocomplexes were detected by using a biotinylated secondary IgG antibody (1:1,000), followed by IRDye-800– labeled streptavidin (1:2,500). Bands were quantified with an Odyssey

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Infrared Imaging System (LICOR Biosciences). All antibodies were diluted in 5% BSA with 0.1% Tween-20 and IRDye-labeled streptavidin in 5% BSA with 0.1% Tween-20 and 0.02% LDS. For ERa detection, 1 3 106 BMMCs (5-week cultures) were rinsed with PBS and lysed by adding 2X boiling lysis buffer.E2 Cell lysates were resolved and transferred as described above, and membranes were blocked with 3% milk in TBS and probed with anti-ERa (1:1,000) for detection of protein bands.

REFERENCES E1. Kuehn HS, Beaven MA, Ma HT, Kim MS, Metcalfe DD, Gilfillan AM. Synergistic activation of phospholipases Cgamma and Cbeta: a novel mechanism for PI3K-independent enhancement of FcepsilonRI-induced mast cell mediator release. Cell Signal 2008;20:625-36. E2. Tkaczyk C, Metcalfe DD, Gilfillan AM. Determination of protein phosphorylation in Fc epsilon RI-activated human mast cells by immunoblot analysis requires protein extraction under denaturing conditions. J Immunol Methods 2002;268:239-43.

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FIG E1. A, Core body temperature decrease induced by antigen challenge in IgE-sensitized BALB/c female and male mice (left panel) or ovariectomized (OVX) and sham-operated female mice (right panel; n 5 6 mice per group). B, Changes in body temperature induced by IgE/antigen in weight-matched C57BL/6 female and male mice (n 5 5 mice per group). Inset, Body weights of female (F) and male (M) mice at the time of the experiment. The ages of female and male mice were 15 and 7 weeks, respectively. C, Serum E2 levels in sham-operated female mice and OVX female mice 2 weeks after implantation of E2-releasing (OVX1E2) or placebo-releasing (OVX) pellets (n 5 6 mice per group). D, Linear correlation between serum levels of estradiol in female mice 10 weeks of age and the temperature decrease measured 90 minutes after antigen challenge. **P < .01 and ***P < .001.

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FIG E2. A and B, TNF-a released into the circulation 5 minutes (Fig E2, A) or 2 hours (Fig E2, B) after challenging ovariectomized (OVX) or shamoperated female mice with antigen (n 5 6 mice per group). Mice were killed, and blood was collected by means of cardiac puncture. C, TNF-a release from BMMCs stimulated with antigen for 6 hours in the presence of different concentrations of E2, as indicated. Results are the average of 4 independent cultures. TNF-a production after E2 treatment in each experiment is represented as the fold change compared with IgE/antigenstimulated cells (second bar). No antigen (no Ag) indicates cells treated with IgE and no antigen, and control (Co) indicates untreated cells. TNF-a levels in plasma or supernatants from BMMCs were assessed by using ELISA.

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FIG E3. A, Expression levels of eNOS in aorta lysates from female (F), male (M), and ovariectomized (OVX) female. The graph represents expression levels of eNOS in aortas from 5 mice measured as the intensity of the eNOS bands relative to the corresponding loading control (b-actin). Blots represent 2 representative samples of aortas from male and female mice. B, Expression levels of eNOS in MLECs cultured for 48 hours in the presence of different concentrations of E2 (10 and 100 pg/mL, n 5 2 per condition). Blots show a representative sample for each condition. C, Quantification of eNOS expression levels in cultures of human PAECs derived from 2 male (M) and 2 female (F) donors. Blots show a representative sample of each group. All graphs represent expression levels of eNOS measured as the intensity of the eNOS bands relative to the corresponding loading control (b-actin). *P < .05.

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Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production.

Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias...
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