Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages

Abstract

Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.

Figure 1.

Redistribution of TLR4 in AMs after resuscitated hemorrhagic shock. AMs were isolated from sham animals or from animals that underwent hemorrhagic shock followed by resuscitation for 1 h (S/R). Where indicated, 0.5 g/kg NAC or 10 mg/kg Trolox was present during resuscitation (S/R+NAC, S/R+Trolox, respectively). Cells were allowed to adhere to coverslips, fixed, permeabilized, and stained with anti-TLR4. (A and D) Typical phase contrast and fluorescence microscopy images are shown. (B) Peripheralization of TLR4 was quantified by Scionimage software as described in Materials and methods. (C) Pictures in rows show confocal microscopic images of different focal planes of the same cell. Bars, 10 μm. (E) Specificity of the TLR4 antibody. Staining was performed with (left) or without (right) anti-TLR4. Both samples were stained with FITC-labeled secondary antibody.

Figure 2.

Resuscitated hemorrhagic shock increases TLR4 surface levels. AMs were stained live with anti-TLR4 and FITC-conjugated secondary antibody. Fluorescence was analyzed by flow cytometry as described in Materials and methods. (A) Profiles of fluorescence intensity of TLR4 staining (black line) or controls (secondary antibody only, solid). (B) Changes in TLR4 surface expression (mean channel fluorescence [MCF]) after the indicated treatments. Data are mean ± SEM. n = 4 animals per group. *, P < 0.05 for S/R versus all other groups.

Figure 3.

Oxidative stress induces increased TLR4 surface expression in RAW 264.7 cells. (A) RAW 264.7 cells, exposed to H₂O₂ for 1 h, were stained with anti-TLR4/MD2-FITC antibody. Representative fluorescence microscopy images (n = 4). (B and C) Dose and time dependence of the H₂O₂ effect. Cells were treated with the indicated concentration of H₂O₂ for 1 h (B) or with 100 μM H₂O₂ for the indicated time (C). TLR4 staining was analyzed using flow cytometry. In B, representative profiles of fluorescence intensity of TLR4 staining (gray solid) or unstained controls (black line) are shown (left panels). (D) RAW cells were treated with 50 μM menadione (Mena) or 100 μM H₂0₂ or 0.1 mM xanthine and 3 U/ml xanthine oxidase for 1 h. Surface TLR4 was measured as in B. The graphs show mean ± SEM of n = 3 (for D) or 4 (for B and C). *, P < 0.05 for points indicated versus control.

Figure 4.

Oxidant stress induces clustering of TLR4 in plasma membrane lipid rafts. (A). TLR4 and GM1 colocalization after oxidant stress. AMs were plated on coverslips and stained live at 4°C with rhodamine-CT_xB. The cells were then fixed, permeabilized, and stained with anti-TLR4. Representative images are shown of TLR4 (green), rhodamine-CT_xB staining (red), or the merged image. (B) RAW 264.7 cells were treated with 100 μM H₂O₂ for1 h. Where indicated, cells were depleted from cholesterol by incubating with 10 mM MβCD for 30 min before H₂O₂. Cells were stained and visualized as in A. (C) H₂O₂-induced increase in TLR4 surface level requires lipid rafts. RAW264.7 cells were treated with 100 μM H₂O₂ for 1 h. Where indicated, cells were depleted from cholesterol before or after treatment with H₂O₂. TLR4 expression was analyzed by flow cytometry. Data are mean ± SEM (n = 4 per group). *, P < 0.05 for H₂O₂, H₂O₂ then MβCD versus control and MβCD then H₂O₂. (D) Cholesterol depletion does not interfere with H₂O₂-induced signaling. Cells were treated with 100 μM H₂O₂ for 30 min and lysed. An equal amount of protein was loaded on SDS gels, and phospho-p38 was detected using Western blotting. Where indicated, cells were cholesterol depleted before H₂O₂ addition.

Figure 5.

Recruitment of TLR4 into lipid rafts by oxidant stress. RAW 264.7 cells, treated with 100 μM H₂O₂ for 1 h with or without MβCD pretreatment, were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation as described in Materials and methods. Fractions were analyzed by dot blotting using either CT_xB conjugated to horseradish peroxidase (GM1, top) or anti-TLR4 primary and peroxidase-coupled secondary antibody (bottom). Fractions 1–3 correspond to lipid rafts. Representative blots of three separate experiments are shown.

Figure 6.

FRET verifies oxidant-induced molecular interaction between TLR4 and GM1. (A) Controls for FRET. Untreated or H₂O₂-treated (100 μM for 1 h) RAW 264.7 cells were either left unstained or stained live at 4°C with rhodamine-CT_xB. Fluorescence of individual cells was determined using the excitation wavelength 480 ± 5 nm and emission was ≥590 nm as described in Materials and methods. The emission of all groups was normalized to the unstained untreated cells (100%). Data are mean ± SEM of n = 18 cells from three independent experiments. (B) H₂O₂ induces FRET between rhodamine-CT_xB (GM1) and anti–TLR4-FITC. RAW 264.7 cells were left untreated or treated with 100 μM H₂O₂ for 1 h and stained live at 4°C with rhodamine-CT_xB, anti-TLR4, and FITC-labeled secondary antibody. For control, identically treated cells were stained only with FITC-labeled secondary antibody. To obtain the value of FRET, non-FRET–related factors were corrected for as detailed in Results. Data are normalized to the untreated cells (100%) and are mean ± SEM of n = 24 cells, n = 4.

Figure 7.

Role of exocytosis in TLR4 translocation to the plasma membrane after oxidant stress. Raw 264.7 cells remained untreated (control) or exposed to DMSO vehicle or 1 μM jaspakinolide (JAS) (A and C) for 30 min or 10 μM BAPTA/AM for 10 min (B and D). Cells were then exposed to 100 μM H₂O₂ for1 h or 0.1 μg/ml LPS for 30 min, followed by staining live with anti–CD11b-FITC (A and B) or anti-TLR4/MD2-FITC at 4°C (C and D). Fluorescence was analyzed by flow cytometry. Data are mean ± SEM of n = 4 per group. *, P < 0.05 for groups indicated versus all other groups without an asterisk.

Figure 8.

Oxidant stress induces colocalization of TLR4 and MyD88 in the plasma membrane. (A) Oxidant-dependent colocalization of TLR4 and MyD88 after S/R. AMs were stained with anti-TLR4 and anti-MyD88 primary as well as the corresponding secondary antibodies. Representative images (n = 4 experiments) are shown of TLR4 staining (red), MyD88 staining (green), or merged images. (B) Colocalization of GM1 and MyD88 after H₂O₂ treatment. RAW 264.7 cells, treated with 100 μM H₂O₂ for 1 h, were stained live at 4°C with rhodamine-CT_xB, fixed, permeabilized, and stained with anti-MyD88 antibody as in A. Representative images (n = 3 experiments) of GM1 staining (red), MyD88 staining (green), or merged image. (C) Specificity of the anti-Myd88 antibody. RAW 264.7 cells were immunostained as in A. In the right image, the primary antibody was added in the presence of a specific blocking peptide (D). RAW 264.7 cells were exposed to 0.1 μg/ml LPS or 100 μM H₂O₂. Where indicated, cells were pretreated with 10 mM MβCD. Lipid raft fractions were isolated and analyzed by dot blotting using MyD88 primary and horseradish peroxidase–coupled secondary antibody. Fractions 1–3 correspond to lipid rafts.

Figure 9.

H₂0₂ treatment does not affect cholesterol depletion and repletion. (A) Cells were incubated with 10 mM MβCD for 30 min. Next, indicated samples were treated with 100 μM H₂0₂ for 1 h. Where indicated, cholesterol levels were restored using MβCD plus cholesterol. Total cellular cholesterol was measured. Data are normalized to control levels (100%). Data represent mean ± SE (n = 3 experiments). (B and C) Lipid raft integrity is required for oxidant-induced priming of LPS-stimulated NF-κB activation. (B) Cholesterol depletion inhibits and cholesterol repletion restores LPS-induced NF-κB nuclear translocation. RAW 264.7 cells were exposed to 0.1 μg/ml LPS for 15 or 30 min with or without cholesterol depletion. Where indicated, cholesterol levels were restored before the addition of LPS (MβCD+cholesterol). Cells were stained with anti-p65. NF-κB nuclear translocation was analyzed by fluorescence microscopy as described in Materials and methods. (C) Cholesterol depletion before H₂O₂ prevents LPS-induced early p65 translocation. Control or MβCD-treated cells were incubated with H₂O₂, followed by cholesterol repletion and addition of LPS where indicated. NF-κB nuclear translocation was analyzed as in A. Data are mean ± SEM of n = 3. *, P < 0.05 for groups indicated versus all other groups without an asterisk.