Extensive activation of mast cells is the major switch that triggers systemic anaphylaxis, resulting in the subsequent release of anaphylactic mediators into circulation. We previously demonstrated that rapid changes in oxygen tension lead to mast cell degranulation, and the released tryptase triggers retinal angiogenesis in a murine oxygen-induced retinopathy model. However, whether a rapid shift from hyperoxia to normoxia (relative hypoxic stress) is a risk factor for systemic anaphylaxis remains unknown. In this study, we demonstrated that the relative hypoxia stress induces systemic mast cell activation via transient receptor potential ankyrin 1 (TRPA1) channels, which immediately leads to hypothermia and increased vascular permeability in adult mice. Although mast cell–deficient or TRPA1-deficient mice did not exhibit anaphylactic symptoms following a rapid sift to normoxia, preinjection with bone marrow–derived cultured mast cells (BMCMCs) derived from wild-type TRPA1-expressing mice restored anaphylactic responses. In addition, we found that the rapid reductions in oxygen tension in a culture atmosphere triggered the degranulation of BMCMCs derived from wild-type TRPA1-expressing mice but not that of BMCMCs derived from TRPA1-deficient mice. In human LAD2 mast cells, the relative hypoxic stress led to the degranulation, which was suppressed by the addition of a TRPA1 inhibitor. Gradual reductions from hyperoxia to normoxia led to no anaphylactic symptoms. Our results demonstrated that TRPA1-triggered mast cell degranulation is a novel pathway that induces anaphylactic shock without Ag–Ab reactions. These findings introduce a potential role for oxygen in inducing mast cell–dependent anaphylaxis and highlight the need to reconsider chronic pure oxygen therapy for anoxic diseases.

This article is featured in Top Reads, p.2939

Anaphylaxis can be defined as a serious allergic reaction that is rapid in onset and may cause death (1). Abs and anaphylatoxin are widely known to be the first immunologic effectors of anaphylaxis, which is tightly linked with mast cells (25), basophils (6), neutrophils (7), and macrophages (8). In particular, IgE plays an important role in conferring immunologic specificity to mast cell activation (2). Upon exposure to bivalent or multivalent allergens, cross-linkage of IgE bound to FcεRI with high affinity rapidly induces the activation and degranulation of mast cells, leading to the subsequent release of various preformed and newly generated mediators, including histamine (9), mast cell tryptase (the most abundant serine protease) (2, 10), and platelet-activating factors (2) to strongly and potentially contribute to anaphylactic responses. This IgE-mediated response is categorized as the classical pathway of anaphylaxis (3). In fact, many studies that used several strains of mast cell–deficient mice confirmed a key role of mast cells in IgE-mediated anaphylaxis (1113). Thus, Ag exposure is regarded as the initial trigger for anaphylactic responses.

In contrast, various kinds of peptides associated with inflammation and drugs directly activate mast cells through human Mas-related G protein–coupled receptor (MRGPR) X2 (14), and IgG-mediated anaphylaxis occurs when Ag and Ab levels are both high (6). The IgE-independent mast cell–mediated anaphylactic pathways contribute to the development of allergic and inflammatory diseases. Recently, we demonstrated that a rapid shift from hyperoxia to normoxia (relative hypoxic stress) contributes to mast cell hyperactivation, eventually leading to oxygen-induced retinopathy in infant mice (15). It is well known that transient receptor potential ankyrin 1 (TRPA1), belonging to the TRP superfamily, is the Ca2+-permeable nonselective cation channel widely distributed in sensory neurons and in nonneuronal cells, including immune cells, mediates pain and inflammation (16, 17). In addition, TRPA1 has been reported to act as an oxygen sensor in neural cells (18). Therefore, we hypothesized that the relative hypoxic stress causes systemic mast cell activation, presenting as anaphylactic phenotypes, including hypothermia, systemic leakage of Evans blue dye, extravasation of IgG into the brain parenchyma, and high serum levels of histamine.

C57BL/6J and BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). Mast cell–deficient Cpa3Cre/+ (termed Cpa3Cre) mice were established by insertion of Cre recombinase into the gene encoding mouse mast cell Cpa3, based on previously described methods (11). Cpa3Cre mice completely lacked mast cells in the whole body. TRPA1-deficient Trpa1−/− mice (B6;129P-Trpa1tm1Kykw/J) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mast cell and TRPA1 double-knockout (Cpa3Cre;Trpa1−/−) mice were designed and generated in the laboratory of Veterinary Molecular Pathology and Therapeutics, Tokyo University of Agriculture and Technology. Male mice from age 10 to 12 wk were used. All animal experiments were conducted in accordance with the guidelines of and approved by the University Animal Care and Use Committee of the Tokyo University of Agriculture and Technology (approval numbers 24–93 and 28–84).

Wild-type (WT), Trpa1−/−, Cpa3Cre, and Cpa3Cre;Trpa1−/− mice were exposed to 50, 60, or 75% O2 for 4, 8, 16, 24, and 120 h in a sealed chamber (BioSpherix, Parish, NY). O2 and CO2 concentrations were then automatically regulated using a ProOx 110 controller (BioSpherix). After treatment, mice were maintained in the chamber under an ambient atmosphere. In some experiments, gradual shifts (50 and 40%) from 60% O2 to normoxia were carried out. Heparinized blood samples were isolated from the tail artery in mice exposed to individual oxygen concentrations for 1 h, and PO2 was measured using a blood gas and electrolyte analyzer (OPTI CCA-TS2; OPTI Medical Systems, Roswell, GA). Average levels of PaO2 were 324 mm Hg (60%), 256 mm Hg (50%), 160 mm Hg (40%), and 84 mm Hg (20%).

To detect the first-line phenotype of anaphylaxis, mice were anesthetized with 2% isoflurane inhalation in the hyperoxic condition. Afterwards, mice were moved to the normoxic condition, and core body temperature associated with systemic anaphylaxis was measured at 5 min, 4, 8, 16, 24, 72, and 120 h using a digital thermometer (Sato shoji, Kanagawa, Japan) using a mouse rectal probe (19).

Scratching frequency of mice was quantified using a SCLABA-Real system (Noveltec, Hyogo, Japan) for 30 min according to the manufacturer’s instruction (20).

Evans blue dye (0.5%; Sigma-Aldrich) was dissolved in PBS, and 100 μl of the solution was i.p. injected to mice at the end of hyperoxic stimulation. After 24 h, mice were sacrificed, and serum optical densities were measured at 630 nm (21).

According to previous studies, mouse whole brains were collected and fixed in 4% paraformaldehyde in PBS at 4°C for 6 h (22). After the embedding, leakage of plasma-derived IgG was detected in 30-μm cross sections with goat anti-mouse IgG Ab (1:500; BioLegend, San Diego, CA) following blocking of the nonspecific binding site in 5% BSA. The slices were incubated with HRP-conjugated donkey anti-goat secondary Ab (1:1000; Santa Cruz Biotechnology, Dallas, TX) after 24 h of the incubation with the primary Ab, then the positive reaction with 3,3′-diaminobenzidine substrates was detected.

Dorsal skins of mice were collected at 24 h after the mice were moved to the normoxic condition and fixed in 4% paraformaldehyde in PBS at 4°C for 6 h. Paraffin-embedded specimens were sliced into 3-μm-thick sections and stained with toluidine blue (pH 4.1) for 30 min. After washing briefly, sections were counterstained with 0.01% eosin G for 1 min (23). Intact and degranulated metachromatic cells between the epithelium and panniculus carnosus were counted under ×200 magnification at five randomly selected areas.

Murine mast cell protease-6 (USCN LIFE SCIENCE, Katy, TX), human mast cell tryptase β 2 (Cloud-Clone, Katy, TX), and histamine (LifeSpan BioSciences, Seattle, WA) were measured using each ELISA kit according to the manufacturer’s instructions.

Bone marrow cells were collected from femora and tibiae of WT and Trpa1−/− mice and were cultured in α-MEM with 1 × 10−4 M 2-ME, 10% heat-inactivated FCS, 10% pokeweed mitogen-stimulated spleen cell conditioned medium, 100 IU/ml penicillin, and 10 μg/ml streptomycin for 4–8 wk, as previously described (24). Bone marrow–derived cultured mast cells (BMCMCs) were incubated for 24 h in a sealed chamber under the 75% O2 condition. After 24 h of the provocation, BMCMCs were cultured in the normoxic condition for an additional 24 h. To detect FcεRI on BMCMCs, flow cytometry was performed. BMCMCs were preincubated with 10 μg/ml rat anti-mouse CD16/32 mAb (clone 93; BioLegend) for 5 min to block FcRIII/II and then incubated with 2 μg/ml PE/Cyanine7–conjugated Armenian hamster anti-mouse FcεRIα mAb (clone MAR-1; BioLegend) for 20 min. Washing with flow cytometry buffer, the cells were applied to the Gallios Flow Cytometer (Beckman Coulter, Indianapolis, IN) and analyzed using FlowJo software (BD Biosciences).

LAD2 mast cells, provided from National Institutes of Health (Bethesda, MD), were cultured at 37°C in α-MEM containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 ng/ml recombinant human stem cell factor (R&D Systems, Minneapolis, MN).

Transmembrane proteins of mast cells were isolated using a ProteoExtract Transmembrane Protein Extraction Kit (Merck) according to manufacturer’s instructions. Immunoblotting of TRPA1 was performed using rabbit anti-mouse TRPA1 Ab (1:1000; antibodies-online, Aachen, Germany) and HRP-linked anti-rabbit IgG (1:1000; Cell Signaling Technology, Danvers, MA). To detect loading controls, rabbit anti-mouse sodium potassium ATPase Ab (1:500; Cell Signaling Technology) was used after stripping at 50°C for 10 min. SDS-PAGE was performed using 10% gels, and the positive reaction was visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) (15). Mouse whole brain tissue lysate was used as a positive control. The immunocytochemistry of TRPA1 in BMCMCs and LAD2 cells was performed by immunoblotting using the same TRPA1 Ab. Briefly, 1 × 105 cells were washed twice in PBS and fixed in methanol for 7 min after cytocentrifugation at 600 rpm for 5 min. After blocking the nonspecific binding sites using 5% BSA, cells were incubated with the primary Ab (1:500) overnight at 4°C, followed by incubation with Alexa Fluor 594–conjugated goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific, Waltham, MA). Mounting was performed by ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific).

BMCMCs or LAD2 cells were cultured with or without 50 μM HC-030031, a TRPA1-specific inhibitor (Sigma-Aldrich) for 24 h in 75% O2 and then in 20% O2 for 24 h. To examine specificity of HC-030031, LAD2 cells sensitized with anti–4-hydroxy-3-nitrophenylacetyl (NP) human IgE mAb (clone JW8/1; MyBioSource, San Diego, CA) were activated with NP-BSA (Wako Pure Chemical, Osaka, Tokyo) in the presence of 50 μM HC-030031, according to the reported method (25). In the other experiment, BMCMCs were sensitized with 10 μg/ml anti-DNP mouse IgE mAb (clone SPE-7; Sigma-Aldrich), followed by stimulation with 100 ng/ml DNP-BSA (Cosmo Bio, Tokyo, Japan) (26). Degranulation of mast cells was quantified based on β-hexosaminidase release, using p-nitrophenyl-N-acetyl-β-D-glucosaminide dissolved in 0.1 M sodium citrate (Sigma-Aldrich) as a substrate. The positive reaction was terminated by the addition of 200 mM glycine solution after mixing the culture supernatants and cell lysate with the substrate. Absorbance was measured at 405 nm as previously described (26).

BMCMCs were obtained from WT or Trpa1−/− mice, and 1 × 106 cells were resuspended in 100 μl of α-MEM without FCS and were injected i.p. in mast cell–deficient Cpa3Cre mice before hyperoxic provocation, as previously described with slight modifications (15)

All data were compared by Mann–Whitney U test or Dunnett test, as described in the individual figure legends. All results are presented as mean ± SEM of values of at least two independent experiments. Two-sided significant tests were used, and a p value < 0.05 was considered statistically significant.

We examined whether relative hypoxic stress from hyperoxia to normoxia induces anaphylactic responses in adult mice. First, to investigate the duration of high O2 inhalation that is necessary for the development of anaphylaxis, C57BL/6J mice were exposed to 75% inhaled O2 for varying treatment periods, and rectal temperature and vascular permeability were measured at 5 min (peak of hypothermia, Supplemental Fig. 1A) and at 24 h after moving the animals to the normoxic (20% inhaled O2) condition, respectively. Rectal temperature was found to be markedly decreased in mice exposed to 75% inhaled O2 conditions for more than 16 h but not in control mice maintained under the normoxic condition (Fig. 1A). To detect vascular permeability, Evans blue dye was injected into the peritoneal cavities of the mice at the end of the 75% inhaled O2 period. As shown in Fig. 1B, Evans blue dye leakage was observed in mice exposed to 75% inhaled O2 for 16 h and was increased in a time-dependent manner. In addition, mice exhibited scratching behavior, which is an anaphylactic phenotype (1) (Supplemental Fig. 1B). Systemic inflammation, including anaphylaxis, is known to induce damage to the blood–brain barrier (19). Therefore, we performed IgG staining in the fixed-brain cross sections to evaluate extravasation following blood–brain barrier disruption. Mice exposed to 75% O2 for 24 h showed strong positive IgG staining as well as hypothermia and dye leakage (Fig. 1C). The above results demonstrated that relative hypoxic stress as a result of treatment with 75% O2 for more than 16 h increased the risk of inducing anaphylactic responses compared with the normoxic condition. Next, to define the threshold of the O2 concentration associated with anaphylaxis, mice were exposed to 50, 60, or 75% O2 conditions for 24 h. The anaphylactic phenotypes were observed when the mice were exposed to 60 or 75% O2 but not to 50% O2 (Fig. 1D, 1E). Thus, we concluded that hyperoxia treatment with 60% O2 for 16 h was the threshold required to trigger systemic anaphylaxis following a rapid shift to the normoxia condition. Mice maintained under 75% O2 for 24 h showed no anaphylactic phenotypes (data not shown). Although all the C57BL/6J mice survived under the relative hypoxic stress, BALB/c mice revealed severe hypothermia 5 min after being moved to the normoxic condition, and more than 33% of BALB/c mice died within 24 h (Supplemental Fig. 1C, 1D).

FIGURE 1.

Relative hypoxic stress (a rapid shift from hyperoxia to normoxia) induces systemic anaphylaxis. (A and B) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 75% O2 for varying treatment periods, respectively (n = 8 per group). Photographs show footpads of mice at 24 h in the normoxic condition following exposure to 75% O2 for 24 h. Blue footpads showing extravascular infiltration of dye is apparent in mice at 24 h after a rapid shift to normoxia (B). **p < 0.01 versus normoxic group, Mann–Whitney U test. (C) To evaluate blood–brain barrier disruption, cross-sectional analysis of the brain was carried out at 24 h after a rapid shift to normoxia. Paraffin-embedded sections were stained with anti-IgG Ab. A positive reaction is observed in mice exposed to relative hypoxia but not in mice maintained in normoxia. Scale bar, 1 mm. (D and E) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 50, 60, and 75% O2 for 24 h, respectively (n = 10 per group). Results are shown as mean ± SEM of values from three to four independent experiments. **p < 0.01 versus normoxic group, Mann–Whitney U test.

FIGURE 1.

Relative hypoxic stress (a rapid shift from hyperoxia to normoxia) induces systemic anaphylaxis. (A and B) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 75% O2 for varying treatment periods, respectively (n = 8 per group). Photographs show footpads of mice at 24 h in the normoxic condition following exposure to 75% O2 for 24 h. Blue footpads showing extravascular infiltration of dye is apparent in mice at 24 h after a rapid shift to normoxia (B). **p < 0.01 versus normoxic group, Mann–Whitney U test. (C) To evaluate blood–brain barrier disruption, cross-sectional analysis of the brain was carried out at 24 h after a rapid shift to normoxia. Paraffin-embedded sections were stained with anti-IgG Ab. A positive reaction is observed in mice exposed to relative hypoxia but not in mice maintained in normoxia. Scale bar, 1 mm. (D and E) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 50, 60, and 75% O2 for 24 h, respectively (n = 10 per group). Results are shown as mean ± SEM of values from three to four independent experiments. **p < 0.01 versus normoxic group, Mann–Whitney U test.

Close modal

Our previous findings suggested that the TRPA1 channel expressed on the surface of mast cells acts as the key sensor of relative hypoxic stimulation (15). Therefore, to define the role of mast cells and TRPA1 channels in the relative hypoxia-induced anaphylaxis, mast cell–deficient Cpa3Cre, TRPA1-deficient Trpa1−/−, and mast cell and TRPA1 double-knockout (Cpa3Cre;Trpa1−/−) mice were exposed to 50, 60, or 75% O2 conditions for 24 h, and the corresponding anaphylactic phenotypes were evaluated. All Cpa3Cre, Trpa1−/−, and Cpa3Cre;Trpa1−/− mice showed no changes in rectal temperature even after exposure to 75 or 60% O2 (Fig. 2A). Similarly, mast cell–deficient Cpa3Cre and/or TRPA1-deficient mice showed no significant changes in Evans blue dye leakage, extravasation of IgG (Fig. 2B, 2C), and scratching behavior (Supplemental Fig. 1B). Degranulated mast cells were observed in the dorsal skins of WT mice, whereas mast cells in TRPA1-deficient Trpa1−/− mice remained roughly intact (Fig. 2D, 2E). Mast cell–deficient Cpa3Cre and Cpa3Cre;Trpa1−/− mice completely lacked mast cells in their dorsal skins. In addition to the skin, in tracheobronchial tissues, degranulation of mast cells was observed in WT mice but not in Trpa1−/− mice (data not shown). The above findings demonstrated that the relative hypoxia-induced anaphylaxis was completely dependent on tissue mast cells and/or TRPA1 channels.

FIGURE 2.

Mast cell deficiency or TRPA1 deficiency prevents relative hypoxia-induced anaphylaxis. (A and B) Mast cell–deficient Cpa3Cre, TRPA1-deficient Trpa1−/−, Cpa3Cre;Trpa1−/−, and WT mice were moved from various hyperoxic conditions for 24 h to normoxic conditions. Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h after being moved to the normoxic condition, respectively. Hypothermia and extravasation were detected in WT mice exposed to 60 and 75% hyperoxia but not in mice lacking mast cells and/or TRPA1 (n = 9 per group). **p < 0.01 versus naive WT mice, Dunnett test. Photographs show footpads (B), brain (C), and dorsal skins (D) of mice transferred from 75% O2 conditions to normoxic conditions. Positive reactions showing extravascular infiltration of dye (B) and IgG infiltration (C) are seen in WT mice but not in mice lacking mast cells and/or TRPA1. Scale bar, 1 mm. (D and E) Dorsal skin tissues were stained with toluidine blue to identify mast cells. Mast cells were observed in WT mice and Trpa1−/− mice but not in Cpa3Cre and Cpa3Cre;Trpa1−/− mice. Arrowheads indicate degranulated mast cells (D). Scale bar, 30 μm. Mast cells with or without degranulation between the epithelium and panniculus carnosus were counted (n = 8 per group). Results are shown as mean ± SEM of values determined from four to five independent experiments. **p < 0.01 versus WT mice, Dunnett test.

FIGURE 2.

Mast cell deficiency or TRPA1 deficiency prevents relative hypoxia-induced anaphylaxis. (A and B) Mast cell–deficient Cpa3Cre, TRPA1-deficient Trpa1−/−, Cpa3Cre;Trpa1−/−, and WT mice were moved from various hyperoxic conditions for 24 h to normoxic conditions. Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h after being moved to the normoxic condition, respectively. Hypothermia and extravasation were detected in WT mice exposed to 60 and 75% hyperoxia but not in mice lacking mast cells and/or TRPA1 (n = 9 per group). **p < 0.01 versus naive WT mice, Dunnett test. Photographs show footpads (B), brain (C), and dorsal skins (D) of mice transferred from 75% O2 conditions to normoxic conditions. Positive reactions showing extravascular infiltration of dye (B) and IgG infiltration (C) are seen in WT mice but not in mice lacking mast cells and/or TRPA1. Scale bar, 1 mm. (D and E) Dorsal skin tissues were stained with toluidine blue to identify mast cells. Mast cells were observed in WT mice and Trpa1−/− mice but not in Cpa3Cre and Cpa3Cre;Trpa1−/− mice. Arrowheads indicate degranulated mast cells (D). Scale bar, 30 μm. Mast cells with or without degranulation between the epithelium and panniculus carnosus were counted (n = 8 per group). Results are shown as mean ± SEM of values determined from four to five independent experiments. **p < 0.01 versus WT mice, Dunnett test.

Close modal

Immunocytochemical and Western blot analyses were performed to evaluate TRPA1 expression levels examined on the transmembranes of BMCMCs obtained from WT mice and TRPA1-deficient Trpa1−/− mice. A moderately positive reaction was observed on the membranes of WT BMCMCs under normoxic conditions, whereas exposure to 75% O2 for 24 h induced a strong positive reaction (Fig. 3A). In contrast, no positive reaction was detected on the membranes of Trpa1−/− BMCMCs, even after exposure to 75% O2 (Fig. 3A). A functional TRPA1 molecule (110 kDa) was detected in the transmembrane lysates of WT BMCMCs; results revealed that TRPA1 signals were enhanced by treatment with 75% O2 for 24 h (Fig. 3B). Next, to elucidate the mechanisms by which fluctuations in oxygen levels induced mast cell degranulation via TRPA1, we assessed β-hexosaminidase (an enzyme contained in cytoplasmic granules) activity in the culture supernatants of BMCMCs derived from WT or Trpa1−/− mice. The protocol mimicked the in vivo experiments (a rapid shift from 75% O2 for 24 h to 20% O2 for 24 h). As shown in Fig. 3C, relative hypoxic stress induced the degranulation of WT BMCMCs; in contrast, the degranulation of Trpa1−/− BMCMCs was comparable with those of normoxia-cultured WT BMCMCs. In addition to the degranulation assay, mast cell tryptase and histamine levels in culture supernatants of WT BMCMCs were examined. Both parameters were detected at higher levels in WT BMCMCs, whereas relative hypoxic stress did not affect tryptase and histamine levels release from Trpa1−/− BMCMCs (Fig. 3D, 3E). We also performed experiments using human LAD2 mast cells. High O2 exposure enhanced expression levels of TRPA1 (Fig. 3F, 3G). In the culture supernatants obtained after the relative hypoxic stress, increased levels of β-hexosaminidase and tryptase β 2 were detected, but they were not detected in the presence of a TRPA1-specific inhibitor, HC-030031 (27) (Fig. 3H, 3I). There was no significant difference in the level of β-hexosaminidase from LAD2 cells stimulated with anti-NP IgE/NP-BSA between the presence and absence of HC-030031 (Supplemental Fig. 2A). Both WT BMCMCs and Trpa1−/− BMCMCs not only expressed FcεRI but also released β-hexosaminidase after stimulation with anti-TNP IgE/DNP-BSA (Supplemental Fig. 2B, 2C). The above results suggested that transmembrane TRPA1 expression in mast cells was upregulated by hyperoxic exposure and potentiated by the subsequent relative hypoxic stimulation, which in turn leads to their FcεRI-independent activation via TRPA1 channel opening.

FIGURE 3.

A rapid shift from the hyperoxic condition to the normoxic condition induces degranulation of BMCMCs via TRPA1. (A and B) TRPA1 immunohistochemical and Western blot analyses were performed on WT or Trpa1−/− BMCMCs incubated in 20 or 75% O2 for 24 h. Results are representative of four independent experiments. Scale bar, 50 μm. (B) 110 kDa TRPA1 molecules (upper bands) were detected in BMCMCs derived from WT mice but not in BMCMCs derived from Trpa1−/− mice. Mouse whole brain tissue lysate was used as a positive control, and sodium potassium ATPase was used as a control for one of the transmembrane proteins. (C) WT BMCMCs at 24 h after being moved from the 75% O2 condition to the normoxic condition showed marked β-hexosaminidase release (degranulation) (n = 10 per group). **p < 0.01 versus WT BMCMCs maintained in normoxic conditions, Dunnett test. (D and E) Mast cell tryptase and histamine levels of cultured supernatants in BMCMCs 24 h after being moved to the nomoxic condition were examined by an ELISA. WT BMCMCs exposed to relative hypoxia induced high levels of mast cell tryptase and histamine (n = 10 per group). Results are shown as mean ± SEM of values determined from three to five independent experiments. **p < 0.01 versus WT BMCMCs maintained in the normoxic condition, Dunnett test. (F and G) TRPA1 immunohistochemical and Western blot analyses were performed on human LAD2 mast cells incubated in 20 or 75% O2 for 24 h. Results are representative of four independent experiments. Scale bar, 50 μm. 110 kDa TRPA1 molecules were detected. (H and I) LAD2 cells moved from the 75% O2 condition to the normoxic condition showed high levels of β-hexosaminidase and mast cell tryptase β 2 but not in the presence of HC-030031 (n = 10 per group). Results are shown as mean ± SEM of values determined from two independent experiments. **p < 0.01 versus normoxia, Dunnett test.

FIGURE 3.

A rapid shift from the hyperoxic condition to the normoxic condition induces degranulation of BMCMCs via TRPA1. (A and B) TRPA1 immunohistochemical and Western blot analyses were performed on WT or Trpa1−/− BMCMCs incubated in 20 or 75% O2 for 24 h. Results are representative of four independent experiments. Scale bar, 50 μm. (B) 110 kDa TRPA1 molecules (upper bands) were detected in BMCMCs derived from WT mice but not in BMCMCs derived from Trpa1−/− mice. Mouse whole brain tissue lysate was used as a positive control, and sodium potassium ATPase was used as a control for one of the transmembrane proteins. (C) WT BMCMCs at 24 h after being moved from the 75% O2 condition to the normoxic condition showed marked β-hexosaminidase release (degranulation) (n = 10 per group). **p < 0.01 versus WT BMCMCs maintained in normoxic conditions, Dunnett test. (D and E) Mast cell tryptase and histamine levels of cultured supernatants in BMCMCs 24 h after being moved to the nomoxic condition were examined by an ELISA. WT BMCMCs exposed to relative hypoxia induced high levels of mast cell tryptase and histamine (n = 10 per group). Results are shown as mean ± SEM of values determined from three to five independent experiments. **p < 0.01 versus WT BMCMCs maintained in the normoxic condition, Dunnett test. (F and G) TRPA1 immunohistochemical and Western blot analyses were performed on human LAD2 mast cells incubated in 20 or 75% O2 for 24 h. Results are representative of four independent experiments. Scale bar, 50 μm. 110 kDa TRPA1 molecules were detected. (H and I) LAD2 cells moved from the 75% O2 condition to the normoxic condition showed high levels of β-hexosaminidase and mast cell tryptase β 2 but not in the presence of HC-030031 (n = 10 per group). Results are shown as mean ± SEM of values determined from two independent experiments. **p < 0.01 versus normoxia, Dunnett test.

Close modal

To verify the role of TRPA1-mediated mast cell activation in vivo, mast cell–deficient Cpa3Cre mice were i.p. injected with BMCMCs derived from WT or Trpa1−/− mice, after which anaphylactic phenotypes were assessed following a rapid shift from 75% inhaled O2 for 24 h to normoxia. Rectal temperature was markedly decreased at 5 min after a shift to the normoxia condition in Cpa3Cre mice injected with WT BMCMCs, whereas Cpa3Cre mice injected with Trpa1−/− BMCMCs did not show hypothermia (Fig. 4A). Vascular permeability was enhanced at 24 h after a shift to the normoxia condition in Cpa3Cre mice injected with WT BMCMCs (Fig. 4B), and IgG penetrated into the brain parenchyma (Fig. 4C). However, injection with Trpa1−/− BMCMCs did not affect vascular permeability (Fig. 4B, 4C). Serum levels of mast cell tryptase and histamine were increased at 24 h after a shift to the normoxia condition in Cpa3Cre mice injected with WT BMCMCs but not in mice injected with Trpa1−/− BMCMCs (Fig. 4D, 4E). The above findings clearly demonstrated that the mast cell/TRPA1 pathway is crucial to the development of relative hypoxia-induced anaphylaxis.

FIGURE 4.

Injection of WT BMCMCs restores relative hypoxia-induced anaphylaxis in mast cell–deficient Cpa3Cre mice. (A and B) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 75% O2 for 24 h, respectively. Cpa3Cre mice injected with BMCMCs derived from WT mice but not those injected with BMCMCs derived from Trpa1−/− mice induced hypothermia (A) and extravasation (B). n = 10 per group. **p < 0.01 versus Cpa3Cre mice treated with medium alone, Dunnett test. Photographs show footpads of mice at 24 h after a rapid shift to normoxia. Blue color showing extravascular infiltration of dye is apparent in mice injected with WT BMCMCs but not with Trpa1−/− BMCMCs (C). Extravasation of IgG in the brain was examined. A strong positive reaction was observed in the tissue sections of Cpa3Cre mice injected with WT BMCMCs. Scale bar, 1 mm. (D and E) Serum levels of mast cell tryptase and histamine were examined by ELISA. Mice injected with WT BMCMCs showed high levels of mast cell tryptase and histamine at 24 h after being moved to the normoxic condition (n = 10 per group). Results are shown as mean ± SEM of values determined from three to five independent experiments. **p < 0.01 versus normoxia WT mice, Dunnett test.

FIGURE 4.

Injection of WT BMCMCs restores relative hypoxia-induced anaphylaxis in mast cell–deficient Cpa3Cre mice. (A and B) Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h in the normoxic condition following exposure to 75% O2 for 24 h, respectively. Cpa3Cre mice injected with BMCMCs derived from WT mice but not those injected with BMCMCs derived from Trpa1−/− mice induced hypothermia (A) and extravasation (B). n = 10 per group. **p < 0.01 versus Cpa3Cre mice treated with medium alone, Dunnett test. Photographs show footpads of mice at 24 h after a rapid shift to normoxia. Blue color showing extravascular infiltration of dye is apparent in mice injected with WT BMCMCs but not with Trpa1−/− BMCMCs (C). Extravasation of IgG in the brain was examined. A strong positive reaction was observed in the tissue sections of Cpa3Cre mice injected with WT BMCMCs. Scale bar, 1 mm. (D and E) Serum levels of mast cell tryptase and histamine were examined by ELISA. Mice injected with WT BMCMCs showed high levels of mast cell tryptase and histamine at 24 h after being moved to the normoxic condition (n = 10 per group). Results are shown as mean ± SEM of values determined from three to five independent experiments. **p < 0.01 versus normoxia WT mice, Dunnett test.

Close modal

To examine whether a rapid reduction of the oxygen concentration was essential for induction of the anaphylactic shock, a gradual shift to normoxia after 60% O2 exposure for 24 h was performed in WT mice (Fig. 5A). The subsequent 50% O2 exposure for 1 h (group 2) showed hypothermia and Evans blue dye leakage that were comparable to those in 60% O2 exposure (group 1) (Fig. 5B, 5C). In contrast, in group 3 (50% O2 exposure for 1 h and later 40% O2 exposure for 1 h), the parameters were within normal levels.

FIGURE 5.

A gradual reduction in levels of oxygen avoids a risk of anaphylaxis. (A) Gradual shifts from 60% O2 conditions for 24 h to normoxic conditions were performed in WT mice. Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h after being moved to the normoxic condition, respectively. Hypothermia (B) and extravasation (C) were detected in WT mice exposed to 60% O2 for 24 h (group 1) and 60% O2→50% O2 for 1 h (group 2) but not in mice exposed to 60% O2→50% O2 for 1 h→40% O2 for 1 h (group 3). n = 7 per group. Results are shown as mean ± SEM of values determined from two independent experiments. **p < 0.01 versus group 1, Dunnett test.

FIGURE 5.

A gradual reduction in levels of oxygen avoids a risk of anaphylaxis. (A) Gradual shifts from 60% O2 conditions for 24 h to normoxic conditions were performed in WT mice. Rectal temperature and Evans blue dye leakage were measured at 5 min and 24 h after being moved to the normoxic condition, respectively. Hypothermia (B) and extravasation (C) were detected in WT mice exposed to 60% O2 for 24 h (group 1) and 60% O2→50% O2 for 1 h (group 2) but not in mice exposed to 60% O2→50% O2 for 1 h→40% O2 for 1 h (group 3). n = 7 per group. Results are shown as mean ± SEM of values determined from two independent experiments. **p < 0.01 versus group 1, Dunnett test.

Close modal

Overall, these results clearly demonstrated that a TRPA1-triggered mast cell–dependent pathway is crucial for the development of oxygen-induced anaphylactic shock in this mouse model. To our knowledge, the current study is the first to identify the pathological mechanisms underlying mast cell and TRPA1 channel-dependent anaphylaxis induced by relative hypoxia (a rapid shift from chronic hyperoxia to normoxia) of in mice. The upregulation of TRPA1 expression on the surface of mast cells after the hyperoxia explains the fact that at least 16 h of oxygen tension of 60% or above is required to prime mice in the model. When relative hypoxic stimulation was applied under the same conditions in the tests conducted in vivo, human LAD2 mast cells, as well as BMCMCs, were clearly activated in vitro, suggesting that human mast cells also are sensitive to relative oxygen stress. Because the degranulation of LAD2 cells was completely suppressed by a TRPA1 inhibitor, human mast cells may be activated via TRPA1 channels on their surface. TRPA1 channels have an oxygen/oxidant sensor function and are activated by reactive oxygen species (18, 28), suggesting the strong involvement of TRPA1 channels in the development of allergic and nonallergic inflammation (2932). In addition, scratching behavior promoted by itch sensations is one of the typical phenotypes of allergic diseases, including anaphylaxis. TRPA1 is downstream of the MRGPR family, which are activated by pruritogens released from tissue mast cells, possibly leading to histamine-independent itch (33). Although our study presents clear evidence that relative hypoxic stress directly activates murine and human mast cells via TRPA1 channels, we have to clarify the precise mechanism by which fluctuations in oxygen tension of the microenvironment result in degranulation of tissue mast cells in further experiments.

Typically, anoxic diseases are widely known as extreme forms of hypoxia caused by deficiency in oxygen supply (34), which can be a consequence of many conditions, including retinopathy of prematurity (15), acute high-altitude illness (35), breathing difficulties by chronic obstructive pulmonary diseases (36), asthma (37), or pneumonia (38). Pure oxygen therapy is recommended to alleviate these conditions. However, there are a certain number of unknown deaths after oxygen therapy. In the case of high-altitude illness, although high-altitude cerebral edema or high-altitude pulmonary edema is clearly supported by political emergency management, several patient cohorts experienced severe illness by an unknown cause (39). Some patients with severe asthma and pneumonia have the potential risk to influence systemic anaphylaxis, and pure oxygen therapy is performed to avoid serious anoxic damage in the tissue (40, 41).

The British Thoracic Society Emergency Oxygen Guideline issued in 2017 (42) describes that oxygen therapy is very effective as an emergency drug in saving life when the appropriate oxygen concentration is applied, but inadequate treatment with excessive oxygen gives often rise to serious risks, such as respiratory failure and reduced blood flow; they recommend that in most acute or chronic illness oxygen therapy should be reduced gradually as the patient recovers. In the multicenter cohort of adult patients admitted to the intensive care unit after resuscitation from cardiac arrest, exposure to hyperoxia (classified as PaO2 ≥300 mm Hg) is associated with increased mortality as compared with exposure to normoxia (classified as 60 < PaO2 <300 mm Hg), indicating that postresuscitation hyperoxia is harmful (43). Rapid reduction from 60% O2 (324 mm Hg PaO2), unlike 50% O2, (256 mm Hg PaO2) induced anaphylactic shock in mice (Fig. 1D, 1E), confirming that 300 mm Hg PaO2 may be the threshold required to trigger systemic anaphylaxis in humans. The current findings suggest that in chronic pure oxygen therapy an accident of a rapid shift from chronic hyperoxia to normoxia might lead to systemic anaphylaxis dependent on mast cell hyperactivation. Because a gradual shift from hyperoxia to normoxia was successful for avoiding anaphylactic shock, treatment with the optimal oxygen concentration for the optimal period can eliminate the risk of anaphylaxis during therapy. In addition, we have recently demonstrated that a mast cell stabilizer is effective for suppressing relative hypoxia-induced mast cell activation in mice (15). Therefore, our findings suggest that systemic anaphylaxis can be prevented by gradual reductions in O2 levels after chronic hyperoxia treatment and/or advanced treatment with mast cell stabilizers before pure oxygen therapy.

We thank Dr. Yukihiko Kitamura (Graduate School of Medicine, Osaka University) for valuable suggestions. We also thank Juri Toyama, Kaoru Karasawa, Mayumi Shibayama, Mayuko Nagasaki, and Naomi Noguchi (Tokyo University of Agriculture and Technology) for animal care. We are grateful to Thorsten B. Feyerabend and Hans-Reimer Rodewald (German Cancer Research Center, Heidelberg, Germany) for kindly providing the Cpa3Cre mice.

This work was supported by Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research: Start-up Grant 17H06669 (to K.M.), Early-Career Scientists Grant 19K15977 (to K.M.), S Grant 16H06383 (to H.M.), A Grant 19H00969 (to A.T.), and Fostering Joint International Research B Grant 18KK0191 (to A.T.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMCMC

bone marrow–derived cultured mast cell

NP

4-hydroxy-3-nitrophenylacetyl

TRPA1

transient receptor potential ankyrin 1

WT

wild-type.

1
Sampson
,
H. A.
,
A.
Muñoz-Furlong
,
R. L.
Campbell
,
N. F.
Adkinson
Jr.
,
S. A.
Bock
,
A.
Branum
,
S. G.
Brown
,
C. A.
Camargo
Jr.
,
R.
Cydulka
,
S. J.
Galli
, et al
.
2006
.
Second symposium on the definition and management of anaphylaxis: summary report--second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium.
J. Allergy Clin. Immunol.
117
:
391
397
.
2
Reber
,
L. L.
,
J. D.
Hernandez
,
S. J.
Galli
.
2017
.
The pathophysiology of anaphylaxis.
J. Allergy Clin. Immunol.
140
:
335
348
.
3
Galli
,
S. J.
2016
.
The mast cell-IgE paradox: from homeostasis to anaphylaxis.
Am. J. Pathol.
186
:
212
224
.
4
Strait
,
R. T.
,
S. C.
Morris
,
M.
Yang
,
X. W.
Qu
,
F. D.
Finkelman
.
2002
.
Pathways of anaphylaxis in the mouse.
J. Allergy Clin. Immunol.
109
:
658
668
.
5
Finkelman
,
F. D.
2007
.
Anaphylaxis: lessons from mouse models.
J. Allergy Clin. Immunol.
120
:
506
515, quiz 516–517
.
6
Finkelman
,
F. D.
,
M. V.
Khodoun
,
R.
Strait
.
2016
.
Human IgE-independent systemic anaphylaxis.
J. Allergy Clin. Immunol.
137
:
1674
1680
.
7
Francis
,
A.
,
E.
Bosio
,
S. F.
Stone
,
D. M.
Fatovich
,
G.
Arendts
,
Y.
Nagree
,
S. P.
Macdonald
,
H.
Mitenko
,
M.
Rajee
,
S.
Burrows
,
S. G.
Brown
.
2017
.
Neutrophil activation during acute human anaphylaxis: analysis of MPO and sCD62L.
Clin. Exp. Allergy
47
:
361
370
.
8
Beutier
,
H.
,
C. M.
Gillis
,
B.
Iannascoli
,
O.
Godon
,
P.
England
,
R.
Sibilano
,
L. L.
Reber
,
S. J.
Galli
,
M. S.
Cragg
,
N.
Van Rooijen
, et al
.
2017
.
IgG subclasses determine pathways of anaphylaxis in mice.
J. Allergy Clin. Immunol.
139
:
269
280.e7
.
9
Makabe-Kobayashi
,
Y.
,
Y.
Hori
,
T.
Adachi
,
S.
Ishigaki-Suzuki
,
Y.
Kikuchi
,
Y.
Kagaya
,
K.
Shirato
,
A.
Nagy
,
A.
Ujike
,
T.
Takai
, et al
.
2002
.
The control effect of histamine on body temperature and respiratory function in IgE-dependent systemic anaphylaxis.
J. Allergy Clin. Immunol.
110
:
298
303
.
10
Schwartz
,
L. B.
,
D. D.
Metcalfe
,
J. S.
Miller
,
H.
Earl
,
T.
Sullivan
.
1987
.
Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis.
N. Engl. J. Med.
316
:
1622
1626
.
11
Feyerabend
,
T. B.
,
A.
Weiser
,
A.
Tietz
,
M.
Stassen
,
N.
Harris
,
M.
Kopf
,
P.
Radermacher
,
P.
Möller
,
C.
Benoist
,
D.
Mathis
, et al
.
2011
.
Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.
Immunity
35
:
832
844
.
12
Oka
,
T.
,
J.
Kalesnikoff
,
P.
Starkl
,
M.
Tsai
,
S. J.
Galli
.
2012
.
Evidence questioning cromolyn’s effectiveness and selectivity as a ‘mast cell stabilizer’ in mice.
Lab. Invest.
92
:
1472
1482
.
13
Sawaguchi
,
M.
,
S.
Tanaka
,
Y.
Nakatani
,
Y.
Harada
,
K.
Mukai
,
Y.
Matsunaga
,
K.
Ishiwata
,
K.
Oboki
,
T.
Kambayashi
,
N.
Watanabe
, et al
.
2012
.
Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness.
J. Immunol.
188
:
1809
1818
.
14
Subramanian
,
H.
,
K.
Gupta
,
H.
Ali
.
2016
.
Roles of Mas-related G protein-coupled receptor X2 on mast cell-mediated host defense, pseudoallergic drug reactions, and chronic inflammatory diseases.
J. Allergy Clin. Immunol.
138
:
700
710
.
15
Matsuda
,
K.
,
N.
Okamoto
,
M.
Kondo
,
P. D.
Arkwright
,
K.
Karasawa
,
S.
Ishizaka
,
S.
Yokota
,
A.
Matsuda
,
K.
Jung
,
K.
Oida
, et al
.
2017
.
Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.
J. Clin. Invest.
127
:
3987
4000
.
16
Talavera
,
K.
,
J. B.
Startek
,
J.
Alvarez-Collazo
,
B.
Boonen
,
Y. A.
Alpizar
,
A.
Sanchez
,
R.
Naert
,
B.
Nilius
.
2020
.
Mammalian transient receptor potential TRPA1 channels: from structure to disease.
Physiol. Rev.
100
:
725
803
.
17
Sahoo
,
S. S.
,
R. K.
Majhi
,
A.
Tiwari
,
T.
Acharya
,
P. S.
Kumar
,
S.
Saha
,
A.
Kumar
,
C.
Goswami
,
S.
Chattopadhyay
.
2019
.
Transient receptor potential ankyrin1 channel is endogenously expressed in T cells and is involved in immune functions.
Biosci. Rep.
39
: BSR20191437.
18
Takahashi
,
N.
,
T.
Kuwaki
,
S.
Kiyonaka
,
T.
Numata
,
D.
Kozai
,
Y.
Mizuno
,
S.
Yamamoto
,
S.
Naito
,
E.
Knevels
,
P.
Carmeliet
, et al
.
2011
.
TRPA1 underlies a sensing mechanism for O2.
Nat. Chem. Biol.
7
:
701
711
.
19
Clay
,
C. D.
,
R. T.
Strait
,
A.
Mahler
,
M. V.
Khodoun
,
F. D.
Finkelman
.
2018
.
Anti-FcγRIIB mAb suppresses murine IgG-dependent anaphylaxis by Fc domain targeting of FcγRIII.
J. Allergy Clin. Immunol.
141
:
1373
1381.e5
.
20
Jang
,
H.
,
A.
Matsuda
,
K.
Jung
,
K.
Karasawa
,
K.
Matsuda
,
K.
Oida
,
S.
Ishizaka
,
G.
Ahn
,
Y.
Amagai
,
C.
Moon
, et al
.
2016
.
Skin pH is the master switch of kallikrein 5-mediated skin barrier destruction in a murine atopic dermatitis model.
J. Invest. Dermatol.
136
:
127
135
.
21
Jiang
,
Q.
,
R.
Oldenburg
,
S.
Otsuru
,
A. E.
Grand-Pierre
,
E. M.
Horwitz
,
J.
Uitto
.
2010
.
Parabiotic heterogenetic pairing of Abcc6-/-/Rag1-/- mice and their wild-type counterparts halts ectopic mineralization in a murine model of pseudoxanthoma elasticum.
Am. J. Pathol.
176
:
1855
1862
.
22
Dénes
,
A.
,
S.
Ferenczi
,
K. J.
Kovács
.
2011
.
Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood- brain barrier damage and brain oedema independently of infarct size.
J. Neuroinflammation
8
:
164
.
23
Furumoto
,
Y.
,
N.
Charles
,
A.
Olivera
,
W. H.
Leung
,
S.
Dillahunt
,
J. L.
Sargent
,
K.
Tinsley
,
S.
Odom
,
E.
Scott
,
T. M.
Wilson
, et al
.
2011
.
PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability.
Blood
118
:
5466
5475
.
24
Matsuda
,
H.
,
Y.
Kannan
,
H.
Ushio
,
Y.
Kiso
,
T.
Kanemoto
,
H.
Suzuki
,
Y.
Kitamura
.
1991
.
Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells.
J. Exp. Med.
174
:
7
14
.
25
Nagai
,
K.
,
T.
Fukushima
,
H.
Oike
,
M.
Kobori
.
2012
.
High glucose increases the expression of proinflammatory cytokines and secretion of TNFα and β-hexosaminidase in human mast cells.
Eur. J. Pharmacol.
687
:
39
45
.
26
Tanaka
,
A.
,
H.
Matsuda
.
2004
.
IgE crosslinkage of Fcepsilon receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells.
Cell. Immunol.
228
:
66
75
.
27
McNamara
,
C. R.
,
J.
Mandel-Brehm
,
D. M.
Bautista
,
J.
Siemens
,
K. L.
Deranian
,
M.
Zhao
,
N. J.
Hayward
,
J. A.
Chong
,
D.
Julius
,
M. M.
Moran
,
C. M.
Fanger
.
2007
.
TRPA1 mediates formalin-induced pain.
Proc. Natl. Acad. Sci. USA
104
:
13525
13530
.
28
Bessac
,
B. F.
,
M.
Sivula
,
C. A.
von Hehn
,
J.
Escalera
,
L.
Cohn
,
S. E.
Jordt
.
2008
.
TRPA1 is a major oxidant sensor in murine airway sensory neurons.
J. Clin. Invest.
118
:
1899
1910
.
29
Caceres
,
A. I.
,
M.
Brackmann
,
M. D.
Elia
,
B. F.
Bessac
,
D.
del Camino
,
M.
D’Amours
,
J. S.
Witek
,
C. M.
Fanger
,
J. A.
Chong
,
N. J.
Hayward
, et al
.
2009
.
A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma.
Proc. Natl. Acad. Sci. USA
106
:
9099
9104
.
30
Smith
,
P. K.
,
B.
Nilius
.
2013
.
Transient receptor potentials (TRPs) and anaphylaxis.
Curr. Allergy Asthma Rep.
13
:
93
100
.
31
Andrè
,
E.
,
B.
Campi
,
S.
Materazzi
,
M.
Trevisani
,
S.
Amadesi
,
D.
Massi
,
C.
Creminon
,
N.
Vaksman
,
R.
Nassini
,
M.
Civelli
, et al
.
2008
.
Cigarette smoke-induced neurogenic inflammation is mediated by alpha,beta-unsaturated aldehydes and the TRPA1 receptor in rodents.
J. Clin. Invest.
118
:
2574
2582
.
32
Engel
,
M. A.
,
A.
Leffler
,
F.
Niedermirtl
,
A.
Babes
,
K.
Zimmermann
,
M. R.
Filipović
,
I.
Izydorczyk
,
M.
Eberhardt
,
T. I.
Kichko
,
S. M.
Mueller-Tribbensee
, et al
.
2011
.
TRPA1 and substance P mediate colitis in mice.
Gastroenterology
141
:
1346
1358
.
33
Wilson
,
S. R.
,
K. A.
Gerhold
,
A.
Bifolck-Fisher
,
Q.
Liu
,
K. N.
Patel
,
X.
Dong
,
D. M.
Bautista
.
2011
.
TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch.
Nat. Neurosci.
14
:
595
602
.
34
Grossman
,
W. F.
,
F.
Alghoula
,
I.
Berim
.
2018
.
Anoxia (Hypoxic Hypoxia).
StatPearls Publishing
,
Treasure Island, FL
. Available at: https://www.ncbi.nlm.nih.gov/books/NBK482316/
35
Davis
,
C.
,
P.
Hackett
.
2017
.
Advances in the prevention and treatment of high altitude illness.
Emerg. Med. Clin. North Am.
35
:
241
260
.
36
Burki
,
T. K.
2017
.
Long-term oxygen in COPD.
Lancet Respir. Med.
5
:
13
.
37
Raedler
,
D.
,
B.
Schaub
.
2014
.
Immune mechanisms and development of childhood asthma.
Lancet Respir. Med.
2
:
647
656
.
38
Troy
,
L. K.
,
I. H.
Young
,
E. M.
Lau
,
T. J.
Corte
.
2016
.
Exercise pathophysiology and the role of oxygen therapy in idiopathic interstitial pneumonia.
Respirology
21
:
1005
1014
.
39
Kurtzman
,
R. A.
,
J. L.
Caruso
.
2018
.
High-altitude illness death investigation.
Acad. Forensic Pathol.
8
:
83
97
.
40
Harada
,
T.
,
A.
Yamasaki
,
T.
Fukushima
,
K.
Hashimoto
,
M.
Takata
,
M.
Kodani
,
R.
Okazaki
,
K.
Takeda
,
M.
Watanabe
,
J.
Kurai
,
E.
Shimizu
.
2015
.
Causes of death in patients with asthma and asthma-chronic obstructive pulmonary disease overlap syndrome.
Int. J. Chron. Obstruct. Pulmon. Dis.
10
:
595
602
.
41
Marshall
,
B. G.
,
V.
White
,
J.
Loveridge
.
2017
.
Breathlessness and cough in the acute setting.
Medicine (Baltimore)
45
:
86
90
.
42
O’Driscoll
,
B. R.
,
L. S.
Howard
,
J.
Earis
,
V.
Mak
;
British Thoracic Society Emergency Oxygen Guideline Group
; 
BTS Emergency Oxygen Guideline Development Group
.
2017
.
BTS guideline for oxygen use in adults in healthcare and emergency settings.
Thorax
72
(
Suppl. 1
):
ii1
ii90
.
43
Kilgannon
,
J. H.
,
A. E.
Jones
,
N. I.
Shapiro
,
M. G.
Angelos
,
B.
Milcarek
,
K.
Hunter
,
J. E.
Parrillo
,
S.
Trzeciak
;
Emergency Medicine Shock Research Network (EMShockNet) Investigators
.
2010
.
Association between arterial hyperoxia following resuscitation from cardiac arrest and in-hospital mortality.
JAMA
303
:
2165
2171
.

The authors have no financial conflicts of interest.

Supplementary data