(A) Localization of IgE and FcεR1α in CD68⁺ macrophage-rich areas in human atherosclerotic lesions. Original magnification, top panels: ×40, bottom panels: ×100. Adv, adventitia. (B) Localization of IgE and FcεR1α in α-actin–positive SMC-rich fibrous cap (top panels, ×100) and media (bottom panels, ×100). (C) Localization of IgE (×100) and FcεR1α (×400) in luminal ECs (CD31, ×100) (arrows). Antibody isotype control was used as a negative control (×100). (D) FcεR1α immunoblot analysis in human monocyte-derived macrophages (Mac) and in HuSMCs and HuECs treated without (–) and with (+) inflammatory cytokine IFN-γ. Each lane contains 20 μg cell lysate (top panel). Actin immunoblot ensured equal protein loading. Bottom panel: 2 μg of human macrophage lysate and 50 μg of HuEC and HuSMC lysates to enhance the detection of FcεR1α in SMCs and ECs. IFN-γ (20 ng/ml) induced FcεR1α expression in SMCs (lanes 2 and 3) and ECs (lanes 5 and 6).

Immunoblots to detect signaling molecule activation in IgE-treated macrophages at different times (50 μg/ml IgE) (A) or with different doses of IgE (15 minutes) (B). (C) Immunoblots to detect signaling molecule activation in IgE-treated macrophages from WT and Fcer1a^–/– mice. (D) Macrophage lysate JPM labeling to detect active cathepsins (Cat; arrowheads) in macrophages treated without (–) or with (+) IgE for 2 days. (E) WT macrophage phospho-p65 and phospho-JNK expression after 15 minutes of treatment with IgE, heated IgE, and LPS (100 ng/ml). (F) IgE-stimulated phospho-p65 and phospho-JNK expression in macrophages from different mice. (G and H) IgE-induced Il6 and MCP-1 mRNA (RT-PCR) and media protein (ELISA) levels in macrophages from different mice. (I) Immunofluorescence TUNEL staining of IgE-induced apoptosis (3 days) in macrophages from different mice. Left panels are representative images (original magnification, ×100). (J) Immunoblots to detect TLR4 and FcεR1α in macrophage total lysates before and after IgE stimulation (15 minutes). (K) Immunoprecipitation for FcεR1α or TLR4 followed by immunoblot analysis for TLR4 or FcεR1α in macrophages treated with and without IgE (15 minutes). The same immunoprecipitation (IP) antibody was used for immunoblot (IB) to ensure equal antibody precipitation. Except where indicated, 50 μg/ml of IgE or heated IgE was used for all macrophage stimulations. Total p38, p65, or β-actin immunoblots were used for protein loading controls. Data in G–I are mean ± SEM of 6–10 experiments. Asterisks indicate statistically significant differences; Mann-Whitney U test.

(A) Culture medium IL-6 in macrophages treated with and without IgE and different concentrations of NHE1 inhibitor EIPA. (B) IgE-induced macrophage apoptosis (TUNEL staining) and inhibition with different concentrations of EIPA. Data are mean ± SEM from 6 experiments. P < 0.05. Representative figures are shown (original magnification, ×100). (C) Immunoblot analysis to detect IgE-treated macrophage mitochondria cytochrome c (Cyt C) and cytosol Bax. X denotes a protein crossreacting with the cytochrome c antibody. (D) Macrophage cell death under different conditions. (E) NHE1 immunoblot in macrophages treated with IgE for different times. (F) RT-PCR to detect Nhe1 mRNA levels in atherosclerotic lesions from different mice. (G) pH change, IL-6 production, and cell death of macrophages from Nhe1^+/+ mice and Nhe1^+/– mice after 3 days of treatment without (Control) or with IgE. All experiments used 50 μg/ml mouse IgE (SPE-7). Actin immunoblot was used for protein loading control. Cell death was determined with a MTT assay on a 96-well plate with 5 × 10⁴ macrophages per well. Asterisks indicate statistically significant differences.

(A) IgE and TUNEL reactivity in adventitial microvessels (V, left 2 panels) and TUNEL activity in luminal ECs and fibrous cap SMCs (right 2 panels, corresponding to Figure 1, B and C, top panels) in human atherosclerotic lesions. Original magnifications, ×100, insets: ×400. (B) Phospho-p38 and phospho-JNK immunoblots in HuECs stimulated with IgE (15 minutes). (C) Cleaved caspase-3 (Casp-3) immunoblot in HuECs treated without (–) and with (+) IgE. (D) HuEC cell death induced with different concentrations of human IgE. (E) IgE-induced cell death of different numbers of HuECs. (F) Phospho-p38 and phospho-JNK immunoblots in HuSMCs after IgE stimulation at different time points. (G) BAX expression in HuSMCs treated with or without IgE. (H) HuSMC cell death after IgE treatment. Representative images are shown. Original magnification, ×40. (I) HuSMC cell death after treatment with different concentrations of IgE. (J) Phospho-p65 and phospho-ERK1/2 immunoblots in HuSMCs, treated with or without IgE for different times. (K) Culture medium IFN-γ, TNF-α, and IL-6 in HuSMCs, treated with or without IgE for 2 days. (L) Cathepsin JPM labeling of HuSMCs before and after IgE stimulation. Arrowheads indicate different active cathepsins. Except where indicated, 100 μg/ml human IgE was used for all HuEC and HuSMC experiments. Data in D, E, H, I, and K are mean ± SEM from approximately 6–10 experiments. Cell death data in D, H, and I were from MTT assays, and CyQUANT cell proliferation assay was used for E. Asterisks indicate statistically significant differences; Mann-Whitney U test.

(A) Serum IgE levels in Apoe^–/– mice before (–HFD) and after (+HFD) consumption of a high-fat diet for 12 weeks. (B) Thoracic-abdominal aorta lipid deposition (oil red O staining). Representative images are shown. (C) Intima area. (D) Media area. (E) Aortic arch atherosclerotic lesion macrophage content, CD3⁺ T cell content, IL-6–positive area, and MHC class II content. (F) Lesion TUNEL-positive apoptosis cells. (G) Brachiocephalic artery and aortic arch necrotic core (#) numbers. Representative aortic arch necrotic cores are shown (original magnification, ×40). The key in B applies to B–G. The number of mice per group is indicated in each bar. Data are mean ± SEM. Asterisks indicate statistically significant differences; Mann-Whitney U test.

IgE (original magnification, ×100), FcεR1α (×100), and TUNEL reactivity (×100) localization to CD68⁺ (×40, insets: ×100) macrophage-rich area in human atherosclerotic lesions. Serial sections from 2 representative lesions were used for immunostaining (A and B). (A) High levels of IgE and FcεR1α in macrophage-rich areas correlated with increased TUNEL-positive cells. (B) Low levels of IgE and FcεR1α in macrophage-rich areas correlated with few TUNEL-positive cells (arrows). (C) Fluorescence TUNEL staining detected human monocyte-derived macrophage apoptosis (fluorescent cells) after 3 days of stimulation with or without human IgE (50 μg/ml) or caspase inhibitor ZVAD-FMK (ZVAD) (20 μM). Representative figures are shown (×100). Data are mean ± SEM from 6 experiments using macrophages from the same donor. (D) IgE-induced macrophage RPMI medium pH reduction. Inhibition with NHE1 inhibitor EIPA (10 μM) blocked IgE-induced pH reduction. EIPA alone was used as experimental control. Data are mean ± SEM from 4 donors. *P < 0.0001 compared with untreated cells. (E) pH-dependent apoptosis (by TUNEL staining) of human macrophages from different donors. Each treatment used macrophages from 2 donors (A and B). *P < 0.04, pH 6.5 versus pH 7.5. (F and G) IgE-induced cell death (by MTT assay) and IL-16 production from macrophages from 4 donors (A–D). Data are mean ± SEM from 4 experiments in E–G. *P < 0.03, **P < 0.01. A final concentration of 50 μg/ml purified human IgE was used for all experiments.

pH change (A), cell death (B), and IL-6 production (C) in mouse macrophages treated with highly cytokinergic SPE-7 (50 μg/ml) or poorly cytokinergic H1 DNP-ε-206 (206; 50 μg/ml) with or without antigen (Ag; 10 ng/ml DNP-HSA). (D) Synergistic effect of SPE-7 (50 μg/ml) with LPS (100 ng/ml) or ox-LDL (50 μg/ml) in macrophage IL-6 production. Cell death and media IL-6 were determined by MTT assay and ELISA, respectively. Asterisks indicate statistically significant differences.