Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors

Abstract

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function.

Figure 1

Primary Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ blood cells constitute mast cell progenitors. (A) Enriched mononuclear cells from blood were analyzed with flow cytometry. (B-C) Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ cells were cultured in a myeloerythroid cytokine cocktail containing IL-3, IL-5, IL-6, IL-9, IL-11, FMS-like tyrosine kinase 3 ligand, stem cell factor, thrombopoietin, erythropoietin, and granulocyte macrophage–colony-stimulating factor for 7 days and analyzed with flow cytometry and May-Grünwald Giemsa staining. The results in panels B and C are representative of 3 independent experiments. The width of each photo corresponds to 50 μm. (D-E) Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ cells were cultured for a total of 17 to 19 days in a myeloerythroid cytokine cocktail, and enzyme histochemical staining of tryptase activity was performed to identify tryptase-containing cells. Red/brown color indicates positive staining. The results in panels D and E are representative of 3 independent experiments. The width of the photo in panel D corresponds to 160 μm, whereas the width of each photo in panel E corresponds to 40 μm. (F-G,I) Lin⁻ CD34^hi CD117^int/hi cells were analyzed with flow cytometry. Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ CD45RA⁻ CRTH2⁻ integrin β7⁻ (H) and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ (J) cells were sorted and cultured in a myeloerythroid cytokine cocktail for 7 days and analyzed with flow cytometry. The results in panels H and J are from 1 out of 2 independent experiments. The frequency of CD117⁺ cells (K) or CD117⁺ FcεRI⁺ cells (L) was analyzed after culturing subpopulations of Lin⁻ CD34^hi CD117^int/hi blood progenitors in a myeloerythroid cytokine cocktail for 7 days. The median values are shown. The differences between groups were evaluated using the 2-tailed Mann-Whitney U test. **P < .01.

Figure 2

Blood mast cell progenitors divide slowly. (A) Single Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ or Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ mast cell progenitors (MCp) were sorted into individual wells and cultured in a myeloerythroid cytokine cocktail. The number of cells in each well was counted after 7 days. (B) Cytocentrifuged and May-Grünwald Giemsa–stained cells from the wells in panel A were characterized based on morphology. At least 1 cell was identified from 25 of the wells in panel A. The Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ and the Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ cells were sorted in 1 experiment each, and the results from each experiment were pooled.

Figure 3

Primary blood mast cell progenitors have no or few granules. (A) Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ cells or Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ mast cell progenitors (MCp) were sorted and stained with May-Grünwald Giemsa. (B) Sorted mature lung mast cells stained with May-Grünwald Giemsa. (C) Sorted mature blood basophils stained with May-Grünwald Giemsa. The width of each photo in panels A-C corresponds to 40 μm. (D) Gating strategy for identifying mature lung mast cells. (E) Gating strategy for identifying mature blood basophils. Mature lung mast cells and mature blood basophils were sorted in 2 independent experiments. The photos of blood mast cell progenitors were representative of cells from 4 independent experiments, where either Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ or Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ cells were isolated.

Figure 4

Primary blood mast cell progenitors express mast cell–associated genes. Lin⁻ CRTH2⁺ FcεRI⁺ mature blood basophils (Ba) and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ blood mast cell progenitors (MCp) were analyzed using whole-transcriptome microarray analyses. The raw data were normalized using the robust multiarray method, and an empirical Bayes moderated t test was used to test for differentially expressed genes employing the computing language R (http://www.r-project.org) using the limma package available from the Bioconductor project (www.bioconductor.org). All analyses were performed according to the limma: Linear Models for Microarray Data User’s Guide (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). (A) Differences are shown on a log₂ scale. The mean values of each transcript from 3 blood donors are shown. *P < .05; ****P < .0001. (B) The results of a GSEA of the “biological process” Gene Ontology subcollection are shown for MCp. Only gene sets with a false discovery rate q value <.05 and an enrichment score >0.5 are shown.

Figure 5

The frequency of mast cell progenitors is increased in subjects with reduced lung function. The frequencies of Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ mast cell progenitors (MCp) were quantified from 24 blood donors by flow cytometry. (A) The subjects were grouped according to the classification in the MIDAS baseline study. (B) The asthmatic subjects, classified at the baseline study, were divided into 2 groups based on the prebronchodilator FEV1 values expressed as percent of the predicted at the time of blood mast cell progenitor analysis. (C) All subjects including healthy controls were divided into 2 groups based on the prebronchodilator FEV1 values expressed as percent of the predicted at the time of blood mast cell progenitor analysis. One of the healthy controls had developed asthma prior to our follow-up study when blood was sampled (shown as a triangle). One patient diagnosed with nonallergic asthma in the baseline study is shown as a square. Differences between groups were evaluated by 2-tailed unpaired Student t tests of the log-transformed data. The geometric mean values and all subjects are shown.