Human neutrophils require short exposure to cytokines and allergen to become functional antigen‐presenting cells (2022)

High numbers of neutrophils accumulate in late-phase reactions (LPRs) of IgE-mediated allergy. We previously reported that neutrophils exposed to GM–CSF (granulocyte macrophage colony-stimulating factor), IL-3 (interleukin 3), and IFN-γ (interferon) for 18–24 h can present the prototypic major birch pollen allergen Bet v 1 to specific CD4+ T-cells, triggering their proliferation and cytokine production.1 The ex vivo detection of Human leukocyte antigen–DR (HLA–DR)-positive neutrophils in cutaneous allergic LPR1 together with the neutrophil-induced aggravation of allergen-induced T-cell-mediated airway hyperresponsiveness in a humanized mouse model2 tempted us to propose that neutrophils amplify allergic inflammation also by acting as antigen-presenting cell (APC). As the information on the timeline of this contribution is scarce, we aimed to study the periods of cytokine and allergen exposure essential for neutrophils to become APC by a sophisticated approach involving Bet v 1-specific T-cell lines (TCL) and autologous >99% pure neutrophils from birch pollen-allergic individuals (for details, see Appendix S1). Neutrophils were incubated with GM–CSF, IL-3, and IFN-γ for 1–12 h and after 18–24 h added to TCL without and with Bet v 1 (Figure 1). Cytokine exposure of only 1 h converted neutrophils into APC inducing allergen-specific proliferation of 7/8 TCL. After 3 h of cytokine-exposure, all TCL responded. In parallel, we observed that neutrophils displayed maximum HLA–DR expression after 1 h and significantly prolonged viability after 3 h of cytokine exposure (Figure S1). These results indicated that very few hours in the cytokine milieu of allergic LPR suffice to transform neutrophils into functional APC. We confirmed that sensitized primary skin mast cells produce GM-CSF and low amounts of IL-3 and IFN-γ following IgE-mediated degranulation (Table S1). As GM–CSF was the most prominent cytokine released by the mast cells and previously determined as a potent trigger of neutrophilic antigen-presenting activity,3 we analyzed its timeline of action on neutrophils. Again, only 1 h of exposure transformed neutrophils into functional APC (Figure 1B). Earlier work has demonstrated that GM–CSF is measurable 4–6 h after IgE-mediated degranulation of mast cells and peaks by 12 h.4 Furthermore, TNF-α was described to be released within minutes from intracellular stores of mast cells followed by de novo synthesis thereafter.5 Similar to GM–CSF, TNF-α promoted allergen-uptake and HLA–DR expression of neutrophils3 and was found in the supernatants of IgE-degranulated mast cells (Table S1). Neutrophils appear in LPR at around 4 h following allergen stimulation.6 Therefore, it is conceivable that they encounter these cytokines on their arrival and after 1 h, begin their conversion in APC, which is finished on the arrival of allergen-specific T-cells at 18–24 h after the mast cell degranulation.6 As it is very speculative to extrapolate the in vitro properties of single cytokines to the situation in vivo, we exposed neutrophils to the complex mixture of factors released from autologous allergen-activated basophils (for details, see Appendix S1). Supernatants of Bet v 1-stimulated but not of unstimulated basophils induced HLA–DR expression on neutrophils (Figure S2). Finally, we studied the period of allergen exposure required for T-cell activation and incubated neutrophils for 1–24 h with Bet v 1 before addition to specific TCL (Figure 2). Considering a SI >2 as positive, 2/3 TCL proliferated after a pulsing period of 3 h. All the TCL proliferated after allergen pulsing for 6 h. These results matched our finding that neutrophils rapidly process Bet v 1 into fragments encompassing relevant T-cell epitopes.1 Altogether, the here proposed chronology of neutrophilic action as APC makes their contribution to allergic inflammation plausible. Certainly, the situation in situ is more complex including parameters not considered by this study. Still, our results obtained with pure neutrophils, a single prototypic allergen, autologous allergen-specific T-cells and effector cells support that the HLA–DR-positive neutrophils detected in allergic LPR 20–24 h after allergen injection represent functional APC.1 DP and BB designed the experiments; DP and CK performed the experiments; AEB provided human skin mast cells; GJZ provided cytokine measurements; and DP and BB analyzed the data and wrote the manuscript. We dedicate this article to our dear colleague and friend Maria Gschwandtner who passed away unexpectedly. Rest in peace. This study was supported by the Austrian Jubiläumsfond, project ÖNB 17947, the Austrian Science Fund (FWF), projects P32953 and I4437, and by the Danube Allergy Research Cluster, Country of Lower Austria, and the Medical University of Vienna, Austria. The authors have nothing to disclose. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.