MyD88 is required for IL-4 and IL-13 production. Wild-type C57BL/6 or MyD88−/− basophils were stimulated with IL-3 (10 ng/mL), alone or in combination with IL-18 (20 ng/mL) or IL-33 (10 ng/mL). IL-4 production (A) and IL-13 production (B) were determined by ELISA. Results are the mean of three experiments ± sem.

IL-4 and IL-13 production from bone marrow-derived basophils. Basophils from BALB/c and C57BL/6 mice, were stimulated with IL-3 (10 ng/mL) alone or in combination with IL-1β (10 ng/mL), IL-18 (20 ng/mL), or IL-33 (10 ng/mL) for 24 h. Supernatants were harvested and analyzed by ELISA to quantify IL-4 (A) and IL-13 (B). Results are the mean of three experiments ± sem.

IL-1 family receptor and cytokine mRNA expression in basophils. (A) RT-PCR detection of cytokine gene expression in mouse whole lung tissue, purified resting basophils, purified ionomycin (1 μm)-stimulated basophils, and mast cells. (B) RNA was isolated from bone marrow-derived basophils, and receptor mRNA levels were analyzed along with β-actin as a housekeeping gene by semiquantitative PCR. Results are the mean of three experiments ± sem.

IL-4 mRNA expression in cytokine-stimulated basophils. Basophils were stimulated with IL-3 (10 ng/mL) alone or in combination with IL-1β (10 ng/mL), IL-18 (20 ng/mL), IL-33 (10 ng/mL), or ionomycin (1 μm) for 15 min (A and C) or 2 h (B and C). IL-4 mRNA levels were analyzed along with β-actin as a housekeeping gene by semiquantitative PCR. IL-4 mRNA levels were normalized to β-actin and graphed as the percent increase relative to IL-3-only stimulation at 15 min (A and C) or IL-3-only stimulation at 2 h (B). A representative gel is shown in Supplemental Figure 4. Results are the mean of three experiments ± sem. Statistical significance data are reported for each time-point below the respective graph.

Phosphorylation of STAT3. (A) Basophils were stimulated for 15 min with the indicated cytokines. Western blot analyses of basophil whole cell lysates were carried out using antibodies to total STAT3 and phosphoserine-specific STAT3. Quantitation was performed by densitometry, and phospho-STAT3 was normalized to total STAT3. Fold-induction is relative to IL-3 stimulation alone. Blots are representative of three independent experiments, quantified in Supplemental Table 2B. (B) The fold-increase in phospho-STAT3 after 15 min cytokine stimulations is graphed relative to the fold-increase in IL-4 mRNA transcripts after 15 min cytokine stimulations. For each, the fold-increase value is relative to IL-3 stimulation alone. Data points are calculated using the mean value from three independent experiments. (C) Basophils were stimulated for 24 h with the indicated cytokines. Western blot analyses of basophil whole cell lysates were carried out using antibodies to total STAT3 and phosphoserine-specific STAT3. Quantitation was performed by densitometry, and phospho-STAT3 was normalized to total STAT3. Fold-induction is relative to IL-3 stimulation alone. Blots are representative of three independent experiments, quantified in Supplemental Table 2B. (D) Basophils from wild-type C57BL/6 or MyD88−/− mice were stimulated in the indicated cytokine conditions for 15 min, and then whole cell lysates were analyzed by Western blot. Bands were quantified densitometrically, and phospho-STAT3 was normalized to total STAT3. Fold-induction is relative to IL-3 stimulation alone. Blots are representative of three independent experiments.

Relative survival and Akt phosphorylation increases in the presence of IL-18 and IL-33. (A) Basophils were cultured in the indicated cytokine conditions and analyzed by trypan blue exclusion 24 h, 48 h, and 72 h post-FACS sort. Results are the mean of three experiments ± sem (*, P<0.05; **, P<0.01). Basophils were stimulated for 15 min (B) and 24 h (C) with the indicated cytokines. Western blot analyses of basophil whole cell lysates were carried out using antibodies to total Akt and phospho-Akt. Quantitation was performed by densitometry, and phospho-Akt was normalized to total Akt. Fold-induction is relative to IL-3 stimulation alone. Blots are representative of three independent experiments, quantified in Supplemental Table 2C.

IL-4 and IL-13 production from basophils is p38α-dependent. Basophils were stimulated for 15 min (A) and 24 h (B) in the indicated cytokines. Western blot analyses of basophil whole cell lysates were carried out using antibodies to total p38α and phosphotyrosine-specific p38 (p-p38α). Quantitation was performed by densitometry, and phospho-p38α was normalized to total p38α. Fold induction is relative to IL-3 stimulation alone. Blots are representative of three or four independent experiments, quantified in Supplemental Table 2A. (C) Basophils from wild-type (WT) C57BL/6 or MyD88−/− mice were stimulated in the indicated cytokine conditions for 15 min, and then whole cell lysates were analyzed by Western blot. Bands were quantified densitometrically, and phospho-p38 was normalized to total p38. Fold-induction is relative to IL-3 stimulation alone. Blots are representative of three independent experiments. (D) IL-4 and IL-13 production from basophils is reduced in the presence of the p38 inhibitor SB239063. Basophils were stimulated in the indicated cytokine conditions with or without SB239063 (500 nM). Results are the mean of three experiments ± sem (*, P<0.05).