American Society of Hematology

Figure 6

$Figure 6. Biologic activities of IL-33 on leukocyte subpopulations in PBMCs. (A) Basophils are the only direct target cells for IL-33 within the mononuclear leukocyte fraction. Freshly isolated PBMCs were stimulated for 10 minutes with IL-33 (50 ng/mL) or TNF-α (10 ng/mL) and analyzed for p38 phosphorylation by flow cytometry. The top panels show the gating of leukocyte subpopulations in total PBMCs stained with anti-CD3 and anti-CRTH2 Abs in the lymphocyte (left) and monocyte (right) light scatter gates: Ba, CRTH2high/CD3− basophils; Th2, CD3+/CRTH2+ Th2 cells; T, CD3+ total T cells; CD3−Ly, CRTH2−/CD3− cells in the lymphocyte scatter gate mainly composed of B cells and NK cells; Mo, CD3− cells in the monocyte scatter gate. Bottom panels: Histograms show overlays of phospho-p38 (P-p38) staining in the gated subpopulations in unstimulated (nil, shaded area) and stimulated (solid line) cells. Only basophils are activated by IL-33 while all other subpopulations, except basophils, respond to TNF-α stimulation to variable degrees. A representative experiment is shown. Identical results were obtained with cells from 10 different donors. (B,C) IL-33 moderately enhances anti-CD3/CD28–induced cytokine release in primary in vivo polarized Th2 cells. Sorted CD4+/CRTH2−/CCR5+ (Th1) and CD4+/CRTH2+/CCR5− (Th2) memory T cells were stimulated for 24 hours with plate-bound anti-CD3/CD28 mAbs (1 μg/mL each) in the presence of IL-33, IL-1β or IL-18 (all at 50 ng/mL) as indicated, and IL-4, IL-5, IL-13, and IFN-γ was measured in the cell supernatants. Panel B shows an example of CCR5/CRTH2 plots of the sorted Th1 and Th2 (left panels) cells and the cytokine profile in response to CD3/CD28-ligation (right panels; mean of 5 experiments). Panel C shows the effects of IL-33 on anti-CD3/CD28–induced secretion of IL-4, IL-5, and IL-13 by Th2 cells and of IL-33, IL-1β or IL-18 on IFN-γ release by Th1 cells. Shown are the mean of 5 independent experiments with cells isolated from different donors. Data from individual experiments and statistical analysis are shown in Figure S7.$

Biologic activities of IL-33 on leukocyte subpopulations in PBMCs. (A) Basophils are the only direct target cells for IL-33 within the mononuclear leukocyte fraction. Freshly isolated PBMCs were stimulated for 10 minutes with IL-33 (50 ng/mL) or TNF-α (10 ng/mL) and analyzed for p38 phosphorylation by flow cytometry. The top panels show the gating of leukocyte subpopulations in total PBMCs stained with anti-CD3 and anti-CRTH2 Abs in the lymphocyte (left) and monocyte (right) light scatter gates: Ba, CRTH2^high/CD3⁻ basophils; Th2, CD3⁺/CRTH2⁺ Th2 cells; T, CD3⁺ total T cells; CD3⁻Ly, CRTH2⁻/CD3⁻ cells in the lymphocyte scatter gate mainly composed of B cells and NK cells; Mo, CD3⁻ cells in the monocyte scatter gate. Bottom panels: Histograms show overlays of phospho-p38 (P-p38) staining in the gated subpopulations in unstimulated (nil, shaded area) and stimulated (solid line) cells. Only basophils are activated by IL-33 while all other subpopulations, except basophils, respond to TNF-α stimulation to variable degrees. A representative experiment is shown. Identical results were obtained with cells from 10 different donors. (B,C) IL-33 moderately enhances anti-CD3/CD28–induced cytokine release in primary in vivo polarized Th2 cells. Sorted CD4⁺/CRTH2⁻/CCR5⁺ (Th1) and CD4⁺/CRTH2⁺/CCR5⁻ (Th2) memory T cells were stimulated for 24 hours with plate-bound anti-CD3/CD28 mAbs (1 μg/mL each) in the presence of IL-33, IL-1β or IL-18 (all at 50 ng/mL) as indicated, and IL-4, IL-5, IL-13, and IFN-γ was measured in the cell supernatants. Panel B shows an example of CCR5/CRTH2 plots of the sorted Th1 and Th2 (left panels) cells and the cytokine profile in response to CD3/CD28-ligation (right panels; mean of 5 experiments). Panel C shows the effects of IL-33 on anti-CD3/CD28–induced secretion of IL-4, IL-5, and IL-13 by Th2 cells and of IL-33, IL-1β or IL-18 on IFN-γ release by Th1 cells. Shown are the mean of 5 independent experiments with cells isolated from different donors. Data from individual experiments and statistical analysis are shown in Figure S7.

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