Identification of Mast Cell Progenitor Cells in the Airways of Individuals with Allergic Asthma

To the Editor:

Mast cells (MCs) are enigmatic innate immune cells that have classically been implicated in asthma as effector cells but more recently have been identified as key sources of type 2 (T2) cytokines and regulators of airway inflammation in asthma (1). MCs are a rare cell population in the airway wall, primarily located in the subepithelial space of healthy individuals but are shifted to the epithelial compartment in individuals with asthma (2). Despite their importance in asthma pathogenesis, the etiology of this trafficking to the epithelium is not well understood. Fate mapping studies in mice have identified MCs as yolk sac-derived cells that reside in locations outside of the bone marrow, circulate as MC progenitors (MCPs) and mature in the peripheral tissues (3). MCPs have been identified in peripheral blood of humans, where they are characterized by their expression of CD34, CD117, CD13 (4) and/or FcεRI (5) and have been linked to reduced lung function among individuals with allergic asthma (5). Prior studies have identified CD34+ progenitor cell populations in the airways of individuals with asthma both at baseline and following inhaled allergen challenge, and have characterized a portion of these progenitor cells as eosinophil progenitors (EoPs) based on expression of CD125 (IL-5Rα) (6, 7). However, the identity of the remaining CD34+ progenitor cell population is unknown, and no prior study has specifically evaluated for the presence of MCPs in the airways. Here we performed spectral flow cytometry on induced sputum samples obtained from five individuals with allergic asthma and one non-asthmatic individual with atopic disease, and for the first time in the literature have identified and characterized MCP populations in the airways.

Briefly, we recruited atopic individuals (defined by positive aeroallergen skin testing) with and without asthma who underwent spirometry, methacholine challenge testing, and induced sputum collection. Patient characteristics are detailed in Supplemental Table 1. The Institutional Review Board at the University of Washington (Seattle, Washington) approved the study protocol and all participants provided written informed consent. Dispersed induced sputum cells were labeled with antibodies to enumerate mature MCs, MCPs, and EoPs using antibodies to CD45, CD34, CD117 (c-KIT), CD13, FcεRI, and CD125 (IL-5Rα), after gating out cells expressing CD14 (monocytes and macrophages) and the lineage markers CD3 (T cells), CD19 (B cells), CD56 (NK cells) and CD15 (neutrophils). Cells were fixed and permeabilized to characterize intracellular staining for the T2 cytokines IL-5 and IL-13. A detailed description of sample processing and flow cytometry techniques are available in the Online Supplement.

We identified MCP and EoP populations in induced sputum from all six study participants (Figure 2A and 2B). MCPs were identified as progenitor cell populations (CD45+CD14-Lin-CD34+) with expression of CD117 and expression of CD13 and/or FcεRI while EoPs were identified as CD117− progenitor cells with surface expression of CD125 (Figure 1). Individual MCP populations were further characterized based upon their surface expression of CD13 and/or FcεRI. Although the composition of the CD117+ progenitor cell population varied significantly by study participant, CD13+FcεRI− cells represented the largest portion of the CD34+CD117+ cell population in the airways (47.1 – 94.7%) (Figure 2B; Supplemental Table 2). Additionally, there was significant heterogeneity in the intracellular staining for IL-5 and IL-13 amongst individual MCP populations and EoPs from each study participant but CD13+FcεRI+ MCPs had the highest median expression of IL-5 and IL-13 (Figure 2C; Supplemental Table 2). Finally, we were also able to identify mature MCs (CD45+CD14-Lin-CD34-CD117+FcεRI+) in the airway lumen of all study participants, although their frequency in comparison to MCPs varied significantly between subjects (Supplemental Table 3).

Figure 2.

Spectral flow cytometry results. (A) Total cell counts from induced sputum samples from six individuals, including total leukocytes (CD45+), total CD34+ progenitor cells (CD45+CD14-Lin-CD34+), total MCPs (CD45+CD14-Lin-CD34+CD117+ and surface expression of CD13 and/or FcεRI), and total EoPs (CD45+CD14-Lin-CD34+CD117−CD125+). Bars represent median values and interquartile ranges. (B) Percent composition of CD45+CD14-Lin-CD34+CD117+ cell population, based on surface expression of CD13 and FcεRI. (C) Percentages of individual CD45+CD14-Lin-CD34+ cell populations with positive intracellular staining for IL-5 and IL-13. Boxes represent the median value with interquartile ranges while the whiskers extend to include the minimum and maximum values.

Figure 1.

Spectral flow cytometry gating strategy of induced sputum samples. After removal of CD45+ cells expressing either CD14 or the lineage markers for B cells (CD19), T cells (CD3), NK cells (CD56), and neutrophils (CD15), we identified progenitor cell populations as CD45+ cells with surface expression of CD34 (CD45+CD14-Lin-CD34+). Mast cell progenitor cells (MCPs) were characterized by surface expression of CD117 and expression of either CD13 and/or FcεRI (CD45+CD14-Lin-CD34+CD117+ and surface expression of CD13 and/or FcεRI). Eosinophil progenitor cells (EoPs) were identified as CD45+CD14-Lin-CD34+CD117−CD125+. Individual CD34+ cell populations were further analyzed for intracellular staining with IL-5 and/or IL-13.

In summary, we successfully identified and characterized MCP populations in the airways of individuals with allergic disease. Total MCP cell counts were comparable to EoPs, and our results indicate that both progenitor cell populations may serve as unrecognized sources of T2 cytokines. We also identified significant heterogeneity in the surface expression of CD13 and FcεRI amongst the CD34+CD117+ cell population. Although a previous study found that CD13+FcεRI+ MCPs in the peripheral blood had significantly higher likelihood of developing into mature MC populations under ex vivo culture conditions in comparison to CD13+FcεRI− MCPs (5), our results demonstrate that CD13+FcεRI− cells are a common MCP subset in the airways. Additionally, the prevalence of MCPs in the airway lumen raises questions regarding whether these cell populations are also present in the airway tissue. Previous studies have identified MC populations in the airway wall by immunostaining with anti-tryptase antibodies (2, 8–10), but peripheral blood MCPs are also known to express tryptase and thus it is unclear if previous studies identifying tryptase-positive cells in specific airway tissues are staining mature MCs or distinct MCP populations. Finally, we acknowledge that our modestly sized study population limits our ability to make disease-specific conclusions regarding the relevance of individual MCP and EoP populations in asthma. However, our study reveals that MCPs are a significant portion of the airway CD34+ progenitor cell population and that both MCPs and EoPs represent a previously unrecognized source of T2 cytokines in the airways.