Aging Increases the Severity of Anaphylaxis Through Changes in Mast Cell Numbers and Histamine Sensitivity

To the Editor,

Anaphylaxis is an acute and potentially fatal systemic allergic reaction which can affect people of any age [1]. Food‐induced anaphylaxis is mainly reported in children and young adults. However, as life expectancy increases, cases of food anaphylaxis are also now becoming more frequent among the geriatric population [1]. By contrast, venom‐ and drug‐induced anaphylaxis are much more frequent in the elderly [2]. Importantly, the risk for near‐fatal or fatal anaphylaxis is also markedly increased in individuals aged 60 and older [1].

It is generally accepted that food and venom anaphylaxis rely predominantly on IgE antibodies and activation of their high‐affinity receptor FcεRI on the surface of tissue mast cells (MCs) and blood basophils. Upon exposure to food or venom allergens, MC and basophil degranulation results in the release of both preformed and newly synthesized mediators, including histamine and tryptase [3]. Increased serum tryptase levels and accumulation of skin MCs with age have been suggested in humans [4, 5]. However, very few studies have focused on anaphylaxis in the elderly, and the mechanisms through which anaphylaxis severity is increased in this age group remain largely unknown. In particular, higher severity of anaphylaxis in older individuals could be due either to factors inherent to MCs (higher MC mass in the body and/or higher degree of MC activation) or to other factors (i.e., cardiovascular diseases). In this study, we aimed to identify factors in older individuals (human and mice) that could influence anaphylaxis frequency and outcome.

To confirm the age‐dependent increase in MC burden and anaphylaxis severity, we began by examining the number and distribution of MCs in 16 calf skin biopsies from healthy volunteers from the French INSPIRE cohort. We found that individuals aged 60 to 84 years had nearly twice as many tryptase⁺ skin MCs across the dermis than younger individuals (Figure 1a,b). We then conducted a retrospective analysis of 165 allergic patients who had experienced anaphylaxis. For 151 of these patients, we had information on clinical severity, which ranged from grade 1 to grade 4. We confirmed that individuals aged 60 to 92 years developed more severe anaphylaxis of grade 3 and anaphylactic shock (grade 4) compared to those aged 3 to 39 years (Figure 1c). We dosed baseline circulating levels of tryptase in the blood of these individuals, as serum tryptase is a well‐established clinical biomarker directly correlated with the number of MCs in humans. Importantly, patients with mastocytosis, hereditary alpha tryptasemia (HαT) or other mast cell‐related syndromes were excluded from our analysis, based on their medical records. Additionally, a pathologically reduced renal function (< 20% remaining function) was used as an exclusion criterion, as this can impact serum tryptase levels. However, no patients were excluded based on this. Regrettably, due to this being a retrospective study, we could not perform physiological renal function measurements. Consistent with our skin biopsy findings, individuals aged 60 to 92 years exhibited significantly more baseline tryptase compared to those aged 3 to 39 years (Figure 1d). Circulating levels of tryptase after anaphylaxis (Figure 1e) as well as the delta between levels at baseline and after anaphylaxis (Figure 1f) were significantly higher in aged patients. Although the causes of anaphylaxis were different in both age groups, with food‐related anaphylaxis being more frequent in younger individuals, and drug‐induced anaphylaxis more frequent in the elderly (Table S1), our data are indicative of an age‐dependent increased MC degranulation upon anaphylaxis.

FIGURE 1.

Mast cell density and anaphylaxis severity increase with age in humans. (a) Representative immunofluorescence staining of human tryptase (mast cell marker, yellow) in skin samples in 28‐year‐old and 76‐year old subjects from the INSPIRE cohort. (b) Mean mast cell density in skin samples from the INSPIRE cohort, separated by age groups of 20–29 year‐old, and 64–80 year‐old. (c) Severity of anaphylaxis in 3–39 year‐old and 60–92 year‐old patients admitted to the Toulouse University Hospital. (d–f) Baseline tryptase levels (d), tryptase levels after anaphylactic shock (e), and ∆ tryptase between baseline and peak tryptase levels in a cohort of patients admitted for anaphylactic shock in the Toulouse University Hospital (f), separated by age (3–39 year‐old, and 60–92 year‐old). Statistical analyses, unpaired two‐tailed Mann–Whitney test.

We then turned to mouse models to further phenotypically and functionally assess the effect of aging on MCs and basophils. We observed a clear age‐dependent increase in the number of skin and peritoneal MCs in naïve C57Bl/6 mice (Figure 2a–c), associated with significantly more surface IgE levels in 18‐month‐old (MO) versus 3MO skin MCs (Extended Figure 1a). Similarly, basophil numbers and surface IgE levels were also increased in 18MO mice compared to 3MO mice (Extended Figure 1b,c). This increase in surface IgE likely reflected an age‐dependent rise in circulating IgE levels, as we found ~6.5‐times more elevated total IgE in sera from 18MO mice versus 3MO mice (Extended Figure 1d).

FIGURE 2.

Mast cell density, anaphylaxis severity, and sensitivity to histamine increase with age in mice. (a) Representative toluidine blue staining for mast cells (indicated with red arrowheads) in 3 month‐old (MO) and 18 MO mice, and (b) mast cell density in sections of back skin samples from 3 MO to 18 MO mice. (c) Flow cytometry measurement of CD45⁺, ((CD3⁻/CD19⁻, Ly6G⁻, CD11b⁻) Lin^neg), CD117⁺ mast cell numbers in peritoneal lavage fluid. (d–i) Mouse models of anaphylaxis. (d) IgE‐induced passive cutaneous anaphylaxis (PCA) in mice sensitized i.d. with IgE anti‐DNP in the ear skin, followed by i.v. challenge with DNP‐HSA on the next day. Changes in ear thickness (∆ μm) were monitored over 1 h after challenge. (e,f) IgE‐induced passive systemic anaphylaxis (PSA) in mice sensitized i.p. with IgE anti‐DNP followed by i.p. challenge with DNP‐HSA on the next day. Changes in body temperature (e) and survival (f) were monitored over 1 h after challenge. (g–i) Mice were challenged i.p. with ciprofloxacin (g), histamine (h) or PAF (i) and changes in body temperature were monitored over 1 h. Data are pooled from two (d–g and i) or three (h) independent experiments, error bars indicate means ± SEM, using One‐Way ANOVA, with Šídák's correction for multiple comparisons, unpaired Student t test, or Kruskal‐Wallis' test with Dunn's correction for multiple comparisons, as appropriate. MO, month‐old; NS, not significant (p > 0.05).

We next performed in vivo anaphylaxis challenges to study the dynamics and intensity of the reaction in mice of different ages. We sensitized groups of mice ranging from 3 to 18 months with dinitrophenyl (DNP)‐specific IgE, followed by challenge with DNP to trigger passive cutaneous or systemic anaphylaxis (i.e., PCA or PSA, respectively). We observed a clear increase in ear swelling following IgE‐mediated PCA in 18MO mice as compared to 3MO mice (Figure 2d). We also measured an age‐dependent increase in immediate hypothermia, the main readout of PSA in mice, following systemic DNP challenge (Figure 2e). Moreover, while all 3 MO and 90% of the 6 MO mice survived, 70% of the 12 MO and all 18 MO mice died within 30 min after challenge (Figure 2f). Various drugs, including ciprofloxacin, can also directly induce MC degranulation and anaphylaxis through activation of the Mas‐related G‐protein coupled receptor member X2 (MRGPRX2 in human; MrgprB2 in mice) [3]. We found that the severity of ciprofloxacin‐induced anaphylaxis also increased in an age‐dependent manner in mice (Figure 2g).

Several factors can contribute to the increased severity of anaphylaxis in the elderly, including comorbidities, frailty, and changes in vascular permeability. Histamine is considered the main driver of MC‐mediated anaphylaxis in humans and mice [3]. We thus assessed whether aging could directly influence the sensitivity to histamine. We found a clear age‐dependent increase in immediate hypothermia following the injection of histamine in mice (Figure 2h). Besides histamine, platelet‐activating factor (PAF) is also considered a major mediator of anaphylaxis in mice, and potentially also in human [3]. Interestingly, we found no difference in hypothermia following PAF injection in 3MO versus 18MO mice (Figure 2i).

Altogether, our results demonstrate that aging increases the severity of both FcεRI‐ and MrgprB2‐mediated anaphylaxis in mice. While multiple factors likely contribute to the increased severity of anaphylaxis in the elderly, we found clear evidence that aging increases the number of MCs in different tissues (and to a lesser extent blood basophils), which should result in a marked increase in the overall histamine pool. It is generally accepted that allergen‐specific IgE levels decrease with age in humans. However, there are also indications that total IgE levels increase with age [6]. Here, we report that aging increases circulating and surface IgE levels in MCs and basophils in naïve mice born and housed in specific pathogen‐free (SPF) conditions. In addition, we demonstrated that aged mice also become much more sensitive to the effects of histamine, but not to PAF, which altogether could explain, at least in part, the increase in anaphylaxis severity observed in the elderly, by increased effector cell populations and function on the one hand, as well as increased sensitivity to histamine on the other. These findings support a clinical hypothesis in which an age‐dependent increase in anaphylaxis severity could result from the natural increase in MC numbers, intracellular MC mediator content, and the physiological sensitivity to histamine.

Author Contributions

J.B.J.K., A.L., W.P.M.W., N.S., T.V., P.A.A., B.T.‐E. generated the data; J.B.J.K., A.L., T.V., L.G., N.G., and L.L.R. analyzed and discussed results; J.B.J.K., A.L., N.G., and L.L.R. prepared the figures and wrote the manuscript. All authors were given the opportunity to read and make amendments to the manuscript.

Conflicts of Interest

L.L.R. is or recently was a speaker and/or advisor for and/or has received research funding from ArgenX, Novartis, Ceva, and Neovacs, and is an inventor on patents issued or pending relating to IgE detection and anti‐IgE therapies: EP2021/060829, EP20315224‐4, WO2019197607 (A1). N.G. is (or was) collaborating, consulting, or a member of the scientific advisory board for Genoskin (CSO, shareholder), Escient Pharmaceuticals, Aikium, CEVA, MaxiVax, Boehringer Ingelheim, Novartis, Sanofi, and ArgenX. The rest of the authors declare no conflicts of interest.

Supporting information

Data S1.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.