Mice deficient in ST2, Rag1 or MyD88 or their control mice received intranasal PBS or Alternaria extract.
(A) One h (for IL-33) or 12 h (for IL-5, IL-6 and IL-13) after receiving PBS or Alternaria extract, BAL levels of IL-33 and IL-6 and lung levels of IL-5 and IL-13 were measured and compared between wild-type BALB/c and ST2^−/− mice (BALB/c background). Error bars represent the mean ± the SEM (5–12 mice per strain per treatment group). ** p<0.01 compared to wild-type mice receiving Alternaria by Mann-Whitney U test.
(B) One h (for IL-33) or 12 h (for IL-5 and IL-13) after receiving PBS or Alternaria extract, BAL levels of IL-33 and lung levels of IL-5 and IL-13 were measured and compared between wild-type C57BL/6 mice and Rag1^−/− mice (C57BL/6 background). Error bars represent the mean ± the SEM (4–8 mice per strain per treatment group).
(C) Lung levels of IL-5 and IL-13 and BAL levels of IL-33 were compared between wild-type C57BL/6 and MyD88^−/− mice (C57BL/6 background) 12 h (for IL-5) or 1 h (for IL-33) after receiving PBS or Alternaria extract. Error bars represent the mean ± the SEM (5–6 mice per strain per treatment group). ** p<0.01 compared to wild-type mice receiving Alternaria by Mann-Whitney U test.

(A) NHBE cells were stimulated with medium (upper panels), Alternaria extract (left and middle lower panels), or Poly I:C (right lower panel) for 4 h. Cells were stained and analyzed as in Figure 3. In upper row, left panel shows FITC-images with anti-IL-33, middle panel shows nuclear staining with DAPI (pseudocolored), and right panel shows overlay of left and middle panels. In lower row, left panel shows overlay of anti-IL-33 and DAPI images, middle panel shows overlay of anti-HMGB1 and DAPI images, and right panel shows overlay of anti-IL-33 and DAPI images. Results are representative of 4 independent experiments.
(B) NHBE cells were incubated with medium alone or Alternaria extract (50 μg/ml) for up to 8 h. IL-33 and IL-6 concentrations in the cell-free supernatants were analyzed by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * and ** p<0.05 and p<0.01 compared to cells stimulated with medium alone by Mann-Whitney U test, respectively.
(C) NHBE cells were stimulated with medium, Alternaria extract, Oriental cockroach extract, zymozan, poly I:C or LPS for 2 h (for IL-33 release, left panel) or 8 h (for IL-6 production, right panel). Cell-free supernatants were analyzed for IL-6 or IL-33 by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * and ** p<0.05 and p<0.01 compared to medium alone by Mann-Whitney U test, respectively.
(D) Culture supernatants from NHBE cells, which were treated with five freeze/thaw cycles (left lane) or stimulated with Alternaria extract for 2 h (right lane), were analyzed by immunoblot with anti-human IL-33. Results are representative of 3 independent experiments.
(E) NHBE cells were stimulated with medium or Alternaria extract for 2 h or treated with five freeze/thaw cycles or 1% NP-40. Cell-free supernatants were analyzed for LDH activity and for IL-33. Error bars represent the mean ± the SEM (6 independent experiments). ** p<0.01 compared to medium alone by Mann-Whitney U test.
(F) NHBE cells were incubated with medium or Alternaria extract for 2 or 8 h and then incubated with calcein AM and EthD-1 for 30 min. Upper panels show fluorescence images from confocal microscopy. Lower panels show intact (calcein AM positive and EthD-1 negative) and damaged (EthD-1 positive) cells in 5 randomly selected fields that were were counted and expressed as percent of intact or damaged cells/total cell number. Error bars represent the mean ± the SEM (3 independent experiments).

(A) NHBE cells were loaded with Fura-2-AM, preincubated with or without 300 μM oATP or 50 μM PPADS, and exposed to Alternaria extract (see arrows). Fluorescence and [Ca²⁺]_i in single cells were analyzed as in Figure 4. Left and middle panels show kinetic changes in [Ca²⁺]_i in one experiment from 50 randomly-selected cells, representative of 3 experiments. Right panel shows [Ca²⁺]_i before (basal) and 400 s after exposure to Alternaria. Error bars represent the mean ± the SEM (3 independent experiments with 50 cells each). ** p<0.01 compared to [Ca²⁺]_i after Alternaria exposure without oATP or PPADS by ANOVA.
(B) NHBE cells were fixed, permeabilized and stained with rabbit anti-P2X₇R or anti-P2Y₂R, followed by FITC-conjugated goat anti-rabbit IgG. After mounting and staining with DAPI, cells were visualized by confocal microscopy. Results are representative of 3 independent experiments. Scale bars indicate 20 μm.
(C) NHBE cells were transfected with siRNA against P2X₇R or P2Y₂R or control RNA and grown for 48 h. Expression levels of P2X₇R and P2Y₂R mRNA by NHBE cells were examined by real-time RT-PCR. In replicate wells, transfected NHBE cells were loaded with Fura-2-AM, and exposed to Alternaria (see arrows). Fluorescence and [Ca²⁺]_i in single cells were analyzed as in Figure 4. Left upper panels show expression of P2X₇R and P2Y₂R mRNA. Data were normalized to the amounts of P2X₇R and P2Y₂R mRNA in NHBE cells without siRNA transfection as 100%. Results are representative of 3 experiments. Left lower panels show kinetic changes in [Ca²⁺]_i in one experiment from 50 randomly-selected cells, representative of 3 experiments. Right panel shows [Ca²⁺]_i before (basal) and 400 s after exposure to Alternaria. Error bars represent the mean ± the SEM (3 independent experiments each with 50 cells). ** p<0.01 compared to [Ca²⁺]_i after exposure to Alternaria in the cells treated with control siRNA by ANOVA.

(A) BALB/c mice received intranasal PBS or Alternaria extract mixed with PBS, oATP or suramin. After 1 h, mice were killed, and IL-33 in BAL fluid supernatants was measured by ELISA. Error bars represent the mean ± the SEM (5 mice per group). * p<0.05 compared to mice receiving Alternaria without inhibitors by Mann-Whitney U test.
(B) BALB/c mice received intranasal PBS or Alternaria extract mixed with PBS, oATP or suramin. After 12 h, mice were killed, and IL-5 and IL-13 in lung homogenates were measured by ELISA. Error bars represent the mean ± the SEM (5 mice per group). * p<0.05 compared to mice receiving Alternaria without inhibitors by Mann-Whitney U test.
(C) Wild-type C57BL/6 mice, P2X₇^−/− or P2Y₂^−/− mice received intranasal PBS or Alternaria. After 12 h, mice were killed and IL-5 and IL-13 in lung homogenates were measured by ELISA. Error bars represent the mean ± the SEM (6 mice per group). * and ** p<0.05 and p<0.01 compared to wild-type mice receiving Alternaria by Mann-Whitney U test, respectively.

BALB/c or C57BL/6 mice received intranasal PBS, Alternaria extract or LPS. Mice were killed, and BAL fluids and lungs were collected. Kinetics of IL-5 and IL-13 levels (lung) and IL-33 and IL-1β levels (BAL) in BALB/c mice (A) and C57BL/6 mice (B) are shown. Error bars represent the mean ± the SEM (3 or 4 mice per treatment group per each time point). * and ** p<0.05 and p<0.01 compared to PBS-treated mice by Student’s t test, respectively.

(A) Nasal specimens from 12 normal individuals were stained with rabbit anti-human IL-33 or normal rabbit IgG. Results shown are representative. Insets show higher magnifications of the epithelium.
(B) NHBE cells were cultured, fixed, permeabilized and stained with rabbit anti-human IL-33 (top panels) or rabbit anti-HMGB1 (lower panels), followed by FITC-conjugated goat anti-rabbit IgG. After mounting and staining with a nuclear staining dye, DAPI, cells were visualized by confocal microscopy. Left panels show FITC-images with anti-IL-33 or anti-HMGB1; middle panels show nuclear staining with DAPI (pseudocolored); right panels show overlays of left and middle panels. Results are representative of 4 independent experiments. Scale bars indicate 20 μm.
(C) Immunoblot analysis of NHBE cell lysate (left lane) and cell culture supernatant (right lane) used anti-human IL-33. Results are representative of 3 independent experiments.
(D) Isolated CD4+T cells were cultured with DCs at a 1:10 DC:T cell ratio with medium alone or with a 10% freeze-thaw lysate of NHBE cells for 10 days. To block the IL-33/ST2 pathway, goat anti-human IL-33, goat anti-mouse ST2 or normal goat IgG were included. Concentrations of IL-5, IL-13, and IFN-γ were measured in supernatants by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * and ** p<0.05 and p<0.01 compared to the samples with lysate but without antibodies by Mann-Whitney U test, respectively.

(A) NHBE cells were treated with medium, Alternaria or Oriental cockroach extracts. The kinetics of ATP concentrations in cell-free supernatants were measured. Error bars represent the mean ± the SEM (4 independent experiments). * and ** p<0.05 and p<0.01 compared to medium alone by Mann-Whitney U test, respectively.
(B) NHBE cells were loaded with Fura-2-AM and exposed to Alternaria extract. Fluorescence in single cells was monitored and analyzed by inverted microscopy, as described in Materials and Methods. Upper panels show [Ca²⁺]_i in NHBE cell monolayer with pseudocolors (green<yellow<orange<red<white in the order of [Ca²⁺]_i). Left panel shows before exposure, and right panel shows 300 s after exposure to Alternaria. Lower panels use pseudocolors to show the kinetic changes in [Ca²⁺]_i in a randomly-selected NHBE cell. Images 1 through 6 were taken at 285 s, 293 s, 301 s, 317 s, 333 s and 347 s after exposure to Alternaria. Results are representative of 5 independent experiments.
(C) NHBE cells were loaded with Fura-2-AM, preincubated with or without EGTA, exposed to Alternaria extract (arrow), and 600 s later 10 μM UTP (arrow) was added to the cells with EGTA. Fluorescence and [Ca²⁺]_i in single cells were analyzed as described above. Left panel shows kinetic changes in [Ca²⁺]_i in one experiment from 50 randomly-selected cells, representative of 3 experiments. Right panel shows [Ca²⁺]_i before (basal) and 400 s after exposure to Alternaria. Error bars represent the mean ± the SEM (3 independent experiments each with 50 cells). ** p<0.01 compared to basal [Ca²⁺]_i by Student’s t test.

(A) NHBE cells were preincubated without (left panel) or with (right panel) 1 mM EGTA and stimulated with Alternaria extract for 4 h. The cells were stained with anti-human IL-33 and DAPI, and analyzed as in Figure 2. Panels show overlay of anti-IL-33 and DAPI images and are representative of 4 independent experiments.
(B) NHBE cells were preincubated with medium, EGTA, or BAPTA-AM and stimulated with medium or Alternaria extract for 2 h. IL-33 in cell-free supernatants was analyzed by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * and ** p<0.05 and p<0.01 compared to cells stimulated with Alternaria without inhibitors by Mann-Whitney U test, respectively.
(C) NHBE cells were treated with medium (left), ionomycin (middle), or thapsigargin (right) for 4 h. The cells were stained with anti-human IL-33 (upper panels) or anti-HMGB1 (lower panels) and DAPI, and analyzed as in Figure 2. Upper panels show overlay of anti-IL-33 and DAPI, and lower panels show overlay of anti-HMGB1 and DAPI. Results are representative of 4 independent experiments.
(D) NHBE cells were incubated with medium, ionomycin, or thapsigargin for 2 h. IL-33 in cell-free supernatants was analyzed by ELISA. Error bars represent the mean ± the SEM (4 independent experiments). * p<0.05 compared to cells incubated with medium alone by Mann-Whitney U test.
(E) NHBE cells were preincubated with medium, oATP, suramin or apyrase and stimulated with medium or Alternaria extract for 2 h. IL-33 in cell-free supernatants was analyzed by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * p<0.05 compared to cells stimulated with Alternaria without inhibitors by Mann-Whitney U test.
(F) NHBE cells were untreated (control) or transfected with siRNA against P2X₇R or P2Y₂R or control siRNA and cultured for 48 h. Cells were stimulated with medium or Alternaria extract for 2 h. IL-33 in cell-free supernatants was analyzed by ELISA. Error bars represent the mean ± the SEM (6 independent experiments). * p<0.05 compared to control siRNA-treated cells stimulated with Alternaria extract by Mann-Whitney U test.