oxLDL antibody inhibits MCP-1 release in monocytes/macrophages by regulating Ca2+ /K+ channel flow

Abstract

oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP-1 release in monocytes/macrophages through their TLR-4 (Toll-like receptor 4) and ERK MAPK pathway and is calcium/potassium channel-dependent. Using blocking antibodies against CD36, TLR-4, SR-AI and LOX-1, only TLR-4 antibody was found to have an inhibitory effect and ERK MAPK-specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL-induced MCP-1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage-dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP-regulated potassium channel (K⁺_ATP ) inhibitor, inhibit oxLDL-induced MCP-1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel - inward rectifier channel (K_ir ) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL-induced Ca²⁺ /K_ir channel alterations. The interfering of oxLDL-induced MCP-1 release by its monoclonal antibody is through its FcγRIIB (CD32). Using blocking antibodies against FcγRI (CD64), FcγRIIB (CD32) and FcγRIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL-induced MCP-1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL-induced ERK MAPK activation and ensuing Ca²⁺ /K_ir channel alterations, and MCP-1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL-induced ERK MAPK pathway and consequent MCP-1 release.

Keywords: BI-204; Ca2+; FcgammarRIIB; MAPKs; MCP-1; atherosclerosis; inward rectifier K+ channel; oxLDL.

Figure 1

oxLDL induces monocyte/macrophage MCP‐1 release through TLR‐4‐ and ERK MAPK‐dependent pathways. Primary CD14⁺ monocytes were activated either by oxLDL‐containing human serum or foetal bovine serum (FBS) or by oxLDL antibody column‐treated control human serum (oxLDL(‐)). MCP‐1 release from CD14⁺ monocytes in culture medium was tested with ELISA. (A) Cells were pre‐treated with CD36, TLR‐4, SR‐AI and LOX‐1 blocking antibodies, respectively. ***P = 0.0001, one‐way anova. All data are shown as means ± S.D. (B) Cells were pre‐treated with inhibitors of JNK (SP600125), ERK (PD98059) and p38 (SB203580) MAPKs, respectively (n = 3). ***P = 0.0001, one‐way anova. All data are shown as means ± S.D.

Figure 2

Human serum‐induced MCP‐1 release is Ca²⁺/K⁺ channel‐dependent. Primary CD14⁺ monocytes were activated either by oxLDL‐containing human serum or by lipid‐depleted serum (Lipid(‐)). MCP‐1 released from the CD14⁺ monocytes into the culture medium was tested with ELISA. (A) CD14⁺ monocytes were pre‐treated with the potassium channel inhibitor glyburide (1 nM, 10 nM, 100 nM, 1 μM respectively) and then exposed to oxLDL‐containing human serum or control lipid‐depleted serum. ***P = 0.0001, one‐way anova. All data are shown as means ± S.D. (B) CD14⁺ monocytes were pre‐treated with the calcium channel inhibitor nifedipine (1 μM, 10 μM and 100 μM, respectively) and then exposed to oxLDL‐containing human serum or control lipid‐depleted serum (n = 3). **P = 0.0035, ***P = 0.0001, one‐way anova. All data are shown as means ± S.D.

Figure 3

oxLDL induces inward rectifier currents of K⁺ (Kir) channel closure in RAW264.7 cells. Representative traces of RAW264.7 cells current amplitude were documented when cells were exposed to foetal bovine serum (FBS, as control) (A) or when the K⁺ was replaced in the bath solution by CsCl to confirm that currents were coming from the potassium channels (B) or exposed to oxLDL (30 mg/ml) in the presence of 10% FBS (D). Current density (pA/pF) was recorded either when cells were exposed to FBS or after replacement of K⁺ in the bath solution by CsCl (C), or exposed to oxLDL (E) from a holding potential of −50 mV in 10 mV increments evoked at voltage steps from −170 mV to + 70 mV (mean ± SD from 5 to 10 cells).

Figure 4

oxLDL‐induced inward rectifier K⁺ channel closure is TLR‐4‐dependent. Current density (pA/pF) in RAW264.7 cells was documented when cells were exposed to foetal bovine serum (FBS, as control) or oxLDL (30 mg/ml) in the presence of 10% FBS, or in some cases pre‐treated with the blocking antibodies of TLR‐4 (8 μg/ml) (A), CD36 (8 μg/ml) (B) or LOX‐1 (8 μg/ml) (C). Histogram showing current density of cells specifically evoked at −100 mV when the cells were pre‐treated with these antibodies (D) (mean ± SD from 5 to 10 cells). *P = 0.028, one‐way anova. All data are shown as means ± S.D.

Figure 5

oxLDL‐induced inward rectifier K⁺ channel closure is ERK‐ and JNK MAPK‐dependent. Current density (pA/pF) in RAW264.7 cells was documented when the cells were exposed to foetal bovine serum (FBS, as control) or oxLDL (30 mg/ml) in the presence of 10% FBS, or in some cases pre‐treated with the p38 inhibitor SB203580 (25 μM) (A), the JNK inhibitor SP600125 (10 μM) (B) or the ERK inhibitor PD98059 (50 μM) (C). Histogram showing current density of cells specifically evoked at −100 mV when the cells were pre‐treated with these inhibitors (D) (mean ± SD from 5 to 10 cells). *P = 0.012, one‐way anova. All data are shown as means ± S.D.

Figure 6

oxLDL induces generation of [Ca²⁺]i oscillations in macrophages that is ERK‐ and JNK MAPK‐dependent. RAW264.7 cells were pre‐treated with the inhibitors PD98059, SP600125, SB203580 and nifedipine, respectively, and thereafter exposed to oxLDL (30 μg/ml) at time 0. Native LDL (nLDL, 30 μg/ml) was used as a control. [Ca²⁺]i oscillations were documented by Fluo‐4‐AM staining. (A) Fluorescence image of [Ca²⁺]i oscillations at time 0 and 2 min. later. (B) Percentages of [Ca²⁺]i oscillation‐positive cells [mean ± SD (n = 3)]. *P = 0.023, ***P = 0.0001, one‐way anova. All data are shown as means ± S.D.

Figure 7

BI‐204 inhibits oxLDL‐induced inward rectifier K⁺ channel closure through FcγRIIB. Current density (pA/pF) in RAW264.7 cells was documented when cells were exposed to foetal bovine serum (FBS, as control) or oxLDL (30 mg/ml) in the presence of 10% FBS, and in some cases pre‐treated with BI‐204 (4 μg/ml) (A), the CD32 antibody (3 μg/ml) (B), the CD16 antibody (3 μg/ml) or the CD64 antibody (3 μg/ml) (C). Histogram showing current density of cells specifically evoked at −120 mV when the cells were pre‐treated with these antibodies (D) (mean ± SD from 5 to 10 cells). **P = 0.0073, one‐way anova. All data are shown as means ± S.D.

Figure 8

oxLDL activates the ERK pathway through TLR‐4, and the recombinant antibody recognizing oxLDL inhibits human serum‐induced activation of the ERK pathway. CD14⁺ monocytes exposed to either foetal bovine serum (FBS, as control), oxLDL‐containing human serum, Cu²⁺‐oxidized LDL (oxLDL) or Fe³⁺‐oxidized LDL (minimal modified, mmLDL), and in some cases pre‐treated with BI‐204 or the control antibody (FITC‐8) (A), or the TLR‐4 blocking antibody or the control antibody (FITC‐8) (B and C) (n = 3). (D), (E) and (F) show normalized quantization of phosphorylation levels for proteins. Gels have been run under the same experimental conditions. ***P = 0.0001, one‐way anova. All data are shown as means ± S.D.

Figure 9

Schematic picture of the oxLDL regulation of MCP‐1 release, and the mechanism of the regulatory effect of oxLDL antibody. oxLDL‐induced MCP‐1 release from monocytes/macrophages is regulated by Ca²⁺ and K⁺ channels, and both of them are TLR‐4‐ and MAPK‐dependent. The oxLDL antibody inhibited oxLDL‐induced Kir channel closure and inhibited the oxLDL‐induced ERK activation through binding to FcγRIIB. Four kinds of Fcγ receptors have been identified: FcγRI, FcγRIIA, FcγRIIB and FcγRIII. FcγRI/III transduces activation signals through its immunoreceptor tyrosine‐based activation motif (ITAM) in cell membranes. Once activated, ITAM recruits spleen tyrosine kinase (SYK) and its downstream targets, such as phospholipase C (PLCγ), GTPase (Rho, Rac) and phosphatidylinositol 3‐kinase (PI3K), to activate macrophages. FcγRIIB transduces inhibitory signals through immunoreceptor tyrosine‐based inhibitory motif (ITIM), which recruits SH2 domain‐containing inositol phosphatase (SHIP), and specifically hydrolyses the PI3K product PIP3 to PIP2. We hypothesize that the oxLDL monoclonal antibody (BI‐204) inhibits oxLDL‐induced macrophage activation through its FcγRIIB by activation of MAPK phosphatase (MKP), which might specifically inactivate the ERK MAPK pathway.