(A and B) Fab, but not Fc, fragments bound to HRF. GST-mHRF was coated onto a 96-well ELISA plate. After blocking with 10% FCS, the plate was incubated with whole molecules (IgG) or with Fab or Fc fragments of the indicated IgGs. HRF-bound Fab was detected by incubation with HRP-conjugated goat anti-mouse κ chain antibody. HRF-bound Fc or IgG was detected with HRP-conjugated anti-IgG. (C and D) Competition by Fab, but not Fc, fragments for JK17 IgG binding to HRF. GST-mHRF was incubated with JK17 IgG in the presence or absence of Fab or Fc fragments at different molar ratios. Bound JK17 IgG was detected with HRP-conjugated anti-κ (C) or biotin-conjugated anti-IgG1 followed by streptavidin-HRP (D). (E and F) Competition by Fab, but not Fc, fragments for C38-2 IgE binding to HRF. GST-mHRF was incubated with C38-2 IgE in the presence or absence of Fab or Fc fragments at different molar ratios. Bound IgE was detected with biotin-conjugated anti-mouse IgE followed by streptavidin-HRP. *P < 0.05. All data are representative of 2 experiments.

(A) BMMCs were incubated with TAT-GST, GST, or GST-N19 protein for the indicated time periods at 37°C. Washed cells were fixed, permeabilized, and stained with anti-GST followed by Alexa Fluor 488–conjugated anti-mouse IgG. Nuclei were stained with DAPI. Fluorescence was observed by confocal microscopy. DIC, differential interference contrast. Original magnification, ×150. Percentages of the cells similar to the representative images are shown (≥150 cells scored). (B and C) BMMCs were incubated with 5 μg/ml of the indicated IgE and 100 μg/ml mHRF for 45 minutes (histamine release) or 20 hours (IL-6 production). (D and E) BMMCs were incubated with IgE and mHRF in the presence of 100 μM GST or GST-N19. *P < 0.05, **P < 0.01 versus respective control, Student’s t test. Data are representative of 3 (A) or 2 (B–E) experiments.

C57BL/6 mice were sensitized with OVA (10 μg) and i.n. challenged with OVA (20 μg) or PBS. Some mice were i.n. pretreated with GST or GST-N19 (400 μg) before every OVA challenge. 24 hours after the last challenge, mice were subjected to invasive lung function testing, and BAL fluids and lung tissues were collected. (A) Increased HRF amounts in the lung and sera of OVA-sensitized and -challenged mice. SDS-PAGE was performed on lung homogenates and serum samples, and HRF amounts of 3 mice were evaluated by immunoblotting. ERK1/2 expression was used as a loading control. (B) Total and specific immune cell numbers in BAL fluids. Eos, eosinophils; Neu, neutrophils; Lym, lymphocytes; MΦ, macrophages and monocytes. (C) H&E and periodic acid-Schiff (PAS) staining of lung tissues. Scale bars: 200 μm. (D) IL-5 and IL-13 in lung homogenates were measured by ELISA. (E) Airway resistance was measured using FlexiVent. *P < 0.05, **P < 0.01, ***P < 0.001. 3–6 mice were used for each cohort. All data are representative of 3 experiments.

(A) IgE molecules were incubated in GST-mHRF–coated wells. HRF-bound IgE was quantified by ELISA, as detected by color development with HRP. OD₄₅₀ values with GST-mHRF subtracted from those with GST control are shown. OD₄₅₀ ≤ 0.1 was used as an arbitrary cutoff value. Data represent at least 3 experiments. (B) IgEs were incubated with GST- or GST-mHRF–agarose beads. Bead-bound IgEs were pulled down. IgE and GST proteins were detected by immunoblotting. Lanes were run on the same gel but were noncontiguous (white lines). Representative of 2 experiments. (C) BMMCs preincubated with (black line) or without (gray shading) the indicated IgE (see Supplemental Table 3) were incubated with mHRF-His₆, and bound mHRF-His₆ was detected with rabbit anti-His tag antibody and Alexa Fluor 647–conjugated anti-rabbit IgG. HRF binding was detected by flow cytometry. Insets show IgE binding: the same cells were incubated with FITC-labeled anti-mouse IgE. Representative of 2 experiments. (D) HRF-bound IgGs were detected by ELISA. Representative of 3 experiments. HRF binding was independent of IgG isotype, as the tested IgG1, IgG2a, and IgG2b molecules contained both HRF-reactive and -nonreactive molecules. The K_D values for HRF binding were 0.685 μM (JK17), 2.78 μM (JK31), and 5.78 μM (JK96). Black bars, IgG1; white bars, IgG2a; gray bars, IgG2b.

(A) Scheme of full-length (FL) and truncated forms of GST-mHRF used for binding assays. Domain structures such as TCTP1–TCTP2 and H1–H3 are shown. (B) IgE-binding site mapping. C38-2 (5 μg/ml) and αOE (20 μg/ml) IgE molecules were incubated in wells coated with GST-mHRF. Bound Igs were detected by ELISA. <0.1, value too small to display. (C) Inhibition of HRF-Ig interactions by N19. IgE molecules were incubated in GST-mHRF–coated wells in the presence or absence of the indicated concentrations of competitors. After incubation, bound IgE was detected by ELISA. (D) Inhibition of HRF-Ig interactions by GST-H3. *P < 0.05, **P < 0.01. Data are representative of 2 (B), 5 (C), or 3 (D) experiments.

(A) IgE/HRF-induced acute PCA reactions. Mice were sensitized with the indicated IgEs. 24 hours later, Evans blue and mHRF-His₆ were injected in IgE-sensitized mice. After 30 minutes, dye leakage from the ears was measured. For controls, saline (Sal) and TNP₂₆-BSA (Ag) were injected in sensitized ears. Kit^{W–sh/W–sh} mice were used before or 6 weeks after engraftment of WT BMMCs by i.d. injection. Toluidine blue staining confirmed that the engrafted mice had numbers of mast cells similar to those of WT mice. (B–D) IgE/HRF-induced PCA LPRs. mHRF-His₆ was injected in IgE-sensitized ears, and LPR was analyzed by measurement of ear thickness at 6 hours. For controls, saline and TNP₂₆-BSA were injected. (C) C38-2 IgE–sensitized mice were pretreated with saline, GST, or GST-N19 (N19) before injection with mHRF-His₆. (D) LPR in FcεRIα^–/– mice. (E) Inhibition of IgE/antigen-induced PCA reactions by GST-N19. WT mice were sensitized overnight with the indicated IgEs. Left ears were injected with GST and right ears were injected with GST-N19, then TNP₂₆-BSA was injected in both ears. Ear thickness was measured. Insets show area under curve (AUC). *P < 0.05, ^#P < 0.01, ^§P < 0.001. 3–6 mice were used for each cohort. Data are representative of 2 (A–D) or 3 (E) experiments.

Naive WT C57BL/6 and mutant mice were treated i.n. with 40 μg mHRF-His₆ 3 times every third day. PBS served as a negative control. (A and B) HRF-induced lung inflammation required B and mast cells as well as FcεRI (and probably Fcγ receptors). BAL procedures were conducted 24 hours after the last HRF administration. Differential cell counting was performed on cytospin preparations stained with May-Giemsa. (C) Genes whose expression was up- or downregulated by HRF. Black symbols, ≤3-fold change; orange symbols, 3- to 5-fold change; red symbols, >5-fold change. Genes whose expression fluctuated ≤3-fold in WT, FcεRIα^–/–, and FcRγ^–/– mice are not shown in the plots for the mutant mice. Dashed and solid lines indicate 3- and 5-fold differences, respectively, in gene expression. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. Each cohort consisted of 3–5 mice. All data are representative of 5 (WT) and 2 (mutant) experiments.