Anaphylaxis and desensitization differently activate and induce IL‐8 release by mast cells in a human peanut allergy in vitro model
Authors: Chiara Tontini, Rajia Bahri, Angela Simpson, Silvia Bulfone‐Paus
Journal: Allergy (2024)

While allergen immunotherapy (AIT) requires years to induce tolerance, rapid dose escalation protocols produce temporary allergen-specific refractoriness upon rechallenge,1 a process known as desensitization, involving effector cells crucial to the pathogenesis of allergy, namely mast cells (MCs). Correct timing and dose are fundamental to desensitize MCs, as described using mouse models,2, 3 human cell lines,3, 4 and tissue-derived MCs.5, 6 Nevertheless, the differences in activation between anaphylaxis and desensitization in human MCs remain to be elucidated, particularly late-phase activities (cytokine release, degranulation recovery kinetics), and when using human sera as source of allergen-specific IgE. To investigate functional differences in MC activation post-anaphylaxis/desensitization, we measured degranulation, kinetics of recovery and mediator release induced by a one-time anaphylaxis-like stimulation (anaphylaxis) or allergen escalation dose step-wise desensitization (desensitization) in vitro using peripheral blood-derived human MCs,7 and human IgE/anti-IgE (αIgE) or peanut (PN)-allergic sera/PN extract as stimuli (Tables S1–S3; Figures S1–S3). Upon αIgE stimulation, desensitized MCs showed decreased tryptase (−14.04 ± 3.99%, p = 0.0192, Figure 1A) and beta-hexosaminidase release (−40.4 ± 1.43%, p = 0.0013, Figure S.4A). At 1 h from initial activation, we could observe a significant reduction in CD63 expression (−23.73 ± 2.39 CD63 + %, p = 0.0035, Figure 1B) compared to anaphylaxis. Restimulated desensitized MCs showed little increase in CD63 expression, compared to non-restimulated (7.21 ± 1.87%, p = 0.0048), however still significantly below both anaphylaxis and positive αIgE activation control (Figure 1B), and not followed by significantly increased tryptase release (4.69 ± 5.329, p = 0.2723, Figure S4B). Twenty-four hours from initial activation, leftover expression of CD63 could be observed in non-restimulated anaphylaxis/desensitized MCs due to incomplete membrane recycling,3 although no further degranulation could be elicited upon restimulation in both anaphylaxis and desensitized MCs (p > 0.05, Figure 1C). MCs were completely refractory for at least 48 h, regardless of the initial stimulation modality. After 7 days degranulation was fully restored in both anaphylaxis and desensitized MCs (Figure 1C). During the refractory period MCs remained responsive to C3a, with no significant difference between initial stimulations (Figure 1D). Using PN we observed comparable activation/recovery to the αIgE activation model (Figure 1E,F). To investigate if refractoriness affected late-phase mediator release, we measured 10 cytokines/chemokines in cell supernatants at 20 h from initial activation. Desensitized MCs using αIgE showed significant reduction of IL-8, increased MIP-1α and MCP-1 release, with no difference in GM-CSF and IL-5 production, and remaining cytokines below the detection limit (Figure 2A and Supplemental Figure S5a). Reduced IL-8 production was further confirmed in cellular pellets harvested at 20 h from initial activation (Figure 2C). In the PN model, only IL-8 release by desensitized MCs was reduced compared to anaphylaxis (−43.13 ± 8.02%, p = 0.019) consistently with the αIgE model, despite anaphylaxis displayed increased serum-specific variation in IL-8 and MIP-1α production (Figure 2B, Supplemental Figure S5B,C). The variation in magnitude of IL-8/MIP-1α release did not correlate with CD63 expression, serum total IgE or Ara h 1/Ara h 2 specific IgE levels. To test a possible correlation between the intensity of MC activation and magnitude of IL-8 production, we performed surface CD63 and intracellular IL-8 staining in MCs treated with brefeldin A in the 6 h following the end of the αIgE activation protocol. While the percentage of IL-8+ MCs was not different between anaphylaxis and desensitized MCs, the normalized fluorescence intensity of IL-8 mirrored the findings at 20 h, and positively correlated with the percentage of CD63+ MCs, suggesting that IL-8 production is influenced by the intensity of initial activation (Figure 2D,E). This is the first attempt at elucidating late-phase post-activation differences between step-wise dose increase (desensitization) and one-time dose stimulation (anaphylaxis-like) using primary human MCs and sera of PN-allergic subjects as source of polyclonal IgE. In line with published literature,2-6 our findings suggest that anaphylaxis produces more potent activation than desensitization. However, we did not observe major differences in post-degranulation recovery between stimulation modalities, demonstrating that desensitization in itself is not the reason for the refractoriness observed in the days following the procedure, rather a mean to achieve low-level MC activation to a set allergenic dose in vivo. Refractoriness is likely due to antigen-IgE disassociation kinetics,6 IgE recycling/FcεRI signaling cascade reset, rather than persistence of cytoskeletal remodeling or exhaustion of the intracellular granule content, as degranulation using C3a is not significantly impaired. Besides, desensitization does not produce cross-inhibition to other allergens, nor other activating receptors, confirming the antigen-/receptor-specific nature of the observed effect.6 Among tested late-phase mediators, we demonstrated that only IL-8 was consistently influenced by the modality of initial activation, contrary to what observed in mouse models.2 This finding, however, parallels available literature from human MCs, showing that IL-8 is constitutively expressed at mRNA level by human MCs and displays dose- and time-dependent release upon stimulation with αIgE or substance P.8 However, several other factors could modulate IL-8 production beyond the initial stimulation modality (e.g., allergen-IgE affinity, formation of IgG-allergen immune complexes, presence of anti-IgE/FcεRI autoantibodies), as evidenced when using sera-based stimulation. Conversely, no difference was observed in cytokines produced de novo following activation, like GM-CSF and IL-5,9 hinting at an independent regulation of cytokine production from the degree of initial activation. Less is known on the regulation of the chemokines MCP-1 and MIP-1α in human MCs, except that both are also quickly released in response to activation with αIgE or cytokines, like IL-1β and IL-33.10 We found discrepancies in MCP-1 and MIP-1α release between αIgE and PN activation models, suggesting a possible influence of sera-based activation in reducing MIP-1α release during desensitization, although these results should be confirmed on a bigger cohort of PN-allergic subjects. Further studies are necessary to translate these findings to the immune response to AIT in vivo. Human peripheral blood-derived MCs exposed to increasing amounts of allergen over set intervals (desensitization) show similar stimulus-specific refractoriness and recovery to one-time allergen bolus dose (anaphylaxis-like), suggesting that activation is necessary to produce temporary antigen-specific refractoriness, which occurs independently of the initial activation modality. However, desensitization decreases the magnitude of overall activation and IL-8 release. CT, AS, RB and SBP designed the study. CT and RB performed the experiments. CT analysed the data and prepared the figures/tables. CT, RB, AS and SBP wrote the manuscript. Authors wish to thank Emily Leverrier for her help with processing beta-hexosaminidase samples, and Prof. Clare Mills (University of Manchester) for donating the peanut extract used for this study. The Flow Cytometry Facility equipment used in this study is supported by funding from the Lydia Becker Institute of Immunology and Inflammation, Wellcome Trust, and the University of Manchester Strategic Fund. We acknowledge the Manchester Allergy, Respiratory and Thoracic Surgery (ManARTS) Biobank for supporting this project and thank the study participants for their contribution. CT is funded by a UK Research and Innovation (UKRI) Medical Research Council Doctoral Training Partnership (MRC-DTP) studentship. SB-P and RB are funded by MRC (MR/S036954/1). AS is supported by the NIHR Manchester Biomedical Research Centre (NIHR203308). The authors have no conflict of interests to disclose for the present manuscript. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Figure S2. Figure S3. Figure S. Figure S5. Data S1. 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