T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro

Abstract

Mast cells (MCs) arise in situ from circulating stem cell factor (SCF)-dependent committed progenitors (PrMCs) and accumulate at sites of allergic mucosal inflammation. We hypothesized that human (h)PrMCs and their mature counterparts might share overlapping patterns of chemokine and cytokine receptor utilization with eosinophils, basophils, and T helper type 2 (Th2) lymphocytes for their homing and allergy-associated hyperplasia. We have characterized committed hPrMCs and fully mature hMCs derived in vitro from cord blood for their functional responses to chemokine and cytokine agonists germane to allergic inflammation and for their maturation-related expression of the corresponding receptors. After 4 wk of culture in the presence of recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-10, the cells were characterized as hPrMCs based upon their uniform surface expression of c-kit and CD13, low-level expression of FcinRIalpha, absence of CD14 and CD16 expression, and immunoreactivity for MC chymase in >80%, and about half were immunoreactive for tryptase and metachromatic with toluidine blue. By week 9, the cells had matured into hMCs, identified by higher levels of c-kit, continued expression of CD13 and low-level FcinRIalpha, uniform toluidine blue metachromasia, and uniform immunoreactivity for both tryptase and chymase. The 4-wk-old hPrMCs expressed four chemokine receptors (CXCR2, CCR3, CXCR4, and CCR5). Each receptor mediated transient rapid calcium fluxes in response to its respective ligand. Both recombinant human eotaxin and stromal cell-derived factor 1alpha elicited chemotaxis of hPrMCs. Only CCR3 was retained on the mature 9-wk-old hMCs from among these chemokine receptors, and hMCs responded to eotaxin with a sustained calcium flux but without chemotaxis. The Th2 cytokines IL-3, IL-5, IL-6, IL-9, and granulocyte/macrophage colony-stimulating factor each augmented the SCF-dependent proliferation of hPrMCs and hMCs. In contrast, the prototypical Th1 cytokine, interferon gamma, suppressed SCF-driven proliferation of both hPrMCs and hMCs. Thus, throughout their development in vitro, hMCs obey SCF-dependent, cytokine-driven mitogenic responses that reflect a Th2-type polarization characteristic of allergy and asthma. Furthermore, committed hPrMCs have a unique profile of chemokine receptor expression from among reported hematopoietic cells, including CCR3, which is shared with the other cells central to allergic inflammation (eosinophils, basophils, and Th2 lymphocytes).

Figure 1

(A) Total cell numbers (•) and numbers of cells staining metachromatically with toluidine blue (▪) arising over 9 wk from cultures of 3 × 10⁷ cord blood mononuclear cells in the presence of SCF/IL-6/IL-10. (B) Progressive increase in the percentage of cells with toluidine blue metachromasia. (C) Percentages of cells with tryptase (▪) and chymase immunoreactivity (•) at weeks 4, 6, and 9 of culture. Results are the means ± SEM of three experiments.

Figure 2

Cytofluorographic characteristics of 4- (top row) and 9-wk-old (second row) cells, each representing uniform populations by light scatter (left). Representative histograms are shown for c-kit, CD13, Fc∈RIα, IL-3Rα, and β3 integrin. At week 6, two cell subpopulations are indicated by distinct groups that differ in terms of the height of SSC. Separate gating and analysis revealed that the population of lesser SSC (low granularity; third row) resembled the 4-wk-old cells in its surface phenotype, whereas the higher SSC population (high granularity; fourth row) resembled the 9-wk-old cells. Expression of each surface epitope is represented by the bold tracings, and the isotype-matched negative controls are represented by the lighter tracings. Results are representative of independent experiments performed with cells from three different donors.

Figure 3

FACSorting of 6-wk-old cells into low and high granularity populations by their light scatter profiles (A). Toluidine blue metachromasia (B, F), tryptase immunoreactivity (C, G), chymase immunoreactivity (D, H), and chloroacetate esterase activity (E, I) are depicted for the low granularity (left) and high granularity (right) populations. Dot plot density light scatter profile of cells derived after 2 wk of culture from the original sorted 6-wk-old populations (J) of low granularity (left) and high granularity (right). The results are representative of two experiments, and the fields depict the profile presented quantitatively in the text.

Figure 4

Incorporation of [³H]thymidine by 4-wk-old hPrMCs (A) and 9-wk-old hMCs (B) that were cultured for six subsequent days in the presence of the indicated concentrations of cytokines (ng/ml). Results for individual cytokines (left) are expressed as the means ± SEM of triplicates and are representative of experiments performed with the cells of three different donors. The results for comitogenic responses (center) are expressed as the percentage increases above the responses to SCF alone and are the means ± SEM for three independent experiments. IFN-γ–mediated suppression of SCF-driven proliferation of hPrMCs (A, right; means ± half range, n = 2) and hMCs (B, right; means ± SEM, n = 3) is expressed as the percentage of thymidine incorporation occurring in response to 100 ng/ml of SCF alone.

Figure 5

Reinduction of IL-3Rα on fully mature hMCs at week 9 of culture. hMCs were transferred to fresh medium containing SCF (100 ng/ml) alone (top row), SCF plus IL-3 (5 ng/ml; center row), or SCF plus IL-5 (5 ng/ml; bottom row). Cytofluorographic analysis was performed 1 wk later for the expression of c-kit (left column), IL-3Rα (center column), and IL-5Rα (right column). The results are representative of the two experiments performed.

Figure 6

(A) Cytofluorographic expression of CXCR2, CCR3, CXCR4, CCR5, and CD4 by hPrMCs at week 4 (top row) and by hMCs at week 9 (bottom row). Expression of each surface epitope is represented by the bold tracings, and isotype-matched negative controls are represented by the lighter tracings. Results are representative of three experiments with different donor cells (n = 2 for CXCR2). (B) CCR3 (left) and CXCR4 (right) mRNA expression determined by RT-PCR analysis at 3, 4, 6, and 9 wk of culture compared with the internal standard, G3PDH.

Figure 6

(A) Cytofluorographic expression of CXCR2, CCR3, CXCR4, CCR5, and CD4 by hPrMCs at week 4 (top row) and by hMCs at week 9 (bottom row). Expression of each surface epitope is represented by the bold tracings, and isotype-matched negative controls are represented by the lighter tracings. Results are representative of three experiments with different donor cells (n = 2 for CXCR2). (B) CCR3 (left) and CXCR4 (right) mRNA expression determined by RT-PCR analysis at 3, 4, 6, and 9 wk of culture compared with the internal standard, G3PDH.

Figure 7

Functionality of CXCR4 and CCR3. Both ETX (A, top) and SDF-1α (A, bottom) caused transient dose-dependent calcium flux of Fura-loaded hPrMCs (left panels) and promoted their directed migration (right panels). At week 9, ETX elicited marked, sustained calcium flux (B) without chemotaxis (not shown). The calcium flux assays depicted are representative of three independent analyses at both weeks 4 and 9. The chemotaxis assays were analyzed by counting the numbers of migrating cells per five high power fields (HPF) and are the means ± SEM for three experiments.

Figure 7

Functionality of CXCR4 and CCR3. Both ETX (A, top) and SDF-1α (A, bottom) caused transient dose-dependent calcium flux of Fura-loaded hPrMCs (left panels) and promoted their directed migration (right panels). At week 9, ETX elicited marked, sustained calcium flux (B) without chemotaxis (not shown). The calcium flux assays depicted are representative of three independent analyses at both weeks 4 and 9. The chemotaxis assays were analyzed by counting the numbers of migrating cells per five high power fields (HPF) and are the means ± SEM for three experiments.