Monocytic Cell Adhesion to Oxidised Ligands: Relevance to Cardiovascular Disease

Abstract

Atherosclerosis, the major cause of vascular disease, is an inflammatory process driven by entry of blood monocytes into the arterial wall. LDL normally enters the wall, and stimulates monocyte adhesion by forming oxidation products such as oxidised phospholipids (oxPLs) and malondialdehyde. Adhesion molecules that bind monocytes to the wall permit traffic of these cells. CD14 is a monocyte surface receptor, a cofactor with TLR4 forming a complex that binds oxidised phospholipids and induces inflammatory changes in the cells, but data have been limited for monocyte adhesion. Here, we show that under static conditions, CD14 and TLR4 are implicated in adhesion of monocytes to solid phase oxidised LDL (oxLDL), and also that oxPL and malondialdehyde (MDA) adducts are involved in adhesion to oxLDL. Similarly, monocytes bound to heat shock protein 60 (HSP60), but this could be through contaminating lipopolysaccharide. Immunohistochemistry on atherosclerotic human arteries demonstrated increased endothelial MDA adducts and HSP60, but endothelial oxPL was not detected. We propose that monocytes could bind to MDA in endothelial cells, inducing atherosclerosis. Monocytes and platelets synergized in binding to oxLDL, forming aggregates; if this occurs at the arterial surface, they could precipitate thrombosis. These interactions could be targeted by cyclodextrins and oxidised phospholipid analogues for therapy.

Keywords: CD14; LDL; TLR4; adhesion; atherosclerosis; monocyte; oxidation; phospholipid; raft; thrombosis.

Figure 1

U937 cell and blood monocyte adhesion to oxidised LDL in a static adhesion assay. (A) Adhesion of U937 cells to native LDL (nLDL), oxidised LDL (oxLDL) and fibronectin (FN); means of six experiments (expts). (B) Monocyte adhesion to nLDL and oxLDL, one of two similar expts. (C) Inhibition of U937 adhesion to oxLDL by control immunoglobulin (Ig) or CD14 antibody, means of four similar expts. (D) Inhibition of adhesion of monocytes by antibodies to CD14, malondialdehyde (MDA), and apolipoprotein B (apoB). One of two similar MDA expts. (E) Inhibition of U937 adhesion to oxLDL by anti-oxidised phospholipid antibody (EO6), one of three similar expts. (F) Inhibition of U937 adhesion to oxLDL by antibodies to CD14, Toll like receptor 4 (TLR4), MDA and apoB, one of three similar TLR4 expts. Control Ig in C, D and F is UPC10, in E it is MOPC104E. Figures in inhibitor and control Ig legends are concentrations in μg/mL. Statistical analysis was by paired t tests and ANOVA; $ p < 0.05, $$ p < 0.01 compared to (v) nLDL; ££ p < 0.01, v FN; and * p < 0.05 ** p < 0.01, *** p < 0.001 v Ig control of same concentration.

Figure 2

U937 cell and blood monocyte adhesion to heat shock protein 60 (HSP60) in a static adhesion assay. (A) U937 adhesion, inhibition by CD14, means of 3 expts. (B) Monocyte adhesion, inhibition by anti-CD14, means of four expts. UCHM1 antibody used in A an dB. (C) Monocyte adhesion, inhibition by anti-CD14 and HSP60 antibodies II-13 (neat supernatant and 1/10 dilution) and ML30; effects of the same antibodies on FN adhesion. One of two similar expts. (D) U937 adhesion, inhibition by TLR4 antibody HTA125, single expt. Control Ig is UPC10 in A, B and D, UPC10 and MOPC21 (dark blue) in C. Statistical analysis was by paired t tests and ANOVA, * p < 0.05, *** p < 0.001, **** p < 0.0001 v appropriate Ig control.

Figure 3

Inhibitory activity of membrane active agents on U937 adhesion. (A) The inhibition by raft disrupting agents nystatin (NST) and methyl β cyclodextrin (MCD) of adhesion to oxLDL at standard concentrations. (B) By NST and MCD to FN at standard concentrations. (C) By NST and MCD to HSP60 at low concentrations. (D) By dimethyl sulfoxide to HSP60, dilutions in assay stated. Concentrations of NST are given in μg/mL and MCD in mM. A total of three expts were done with each of oxLDL, FN and HSP60 as ligands. Statistical analysis by ANOVA, different from HSP60, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Variants on the adhesion assay. (A) Monocyte adhesion to HSP60 with cells suspended in 1% bovine serum albumin: inhibition by CD14 antibody or Ig control (UPC10). (B) Monocyte adhesion to lipopolysaccharide (LPS), comparison with FN or HSP60 as ligands. Statistical analysis by ANOVA; different from appropriate Ig control, **** p < 0.0001; different from HSP60, $$ p < 0.01; different from uncoated, ## p < 0.01.

Figure 5

Static adhesion of peripheral blood mononuclear cells to oxidised LDL in a flow chamber. The cells were introduced into a flow chamber coated with ligands, measured under static conditions, viewed by phase contrast microscopy and quantitated. Coating: (A) human recombinant galectin 9, (B) oxidised LDL, (C) native LDL. (D) Quantitation of adhesion by image analysis of cells per area. (E) Formation of mononuclear cell- platelet aggregates (arrows) on adhesion to oxLDL. Four similar flow chamber expts were done, each under flow and static conditions.

Figure 6

ELISA assay for the surface expression of oxidised lipids and HSP60 on HUVEC. (A) Cells cultured in 20% normal human serum with and without TNFα stimulation. (B) A second plate in same assay with culture in 20% FCS. Statistical analysis by ANOVA, different from appropriate MOPC21 (Ig) control, **** p < 0.0001. OD, optical density.

Figure 7

LDL, HSP60 and MDA in human arterial normal and atherosclerotic endothelium. (A) Native LDL in normal arterial wall, apoB antibody. (B) HSP60 in endothelium of atherosclerotic plaque, ML30 antibody. (C) MDA in atherosclerotic endothelium, MDA2 antibody. (D) Image analysis of MDA2 reactivity in atherosclerotic (lesions) and normal endothelium, analysis of 10 coronary or carotid arteries. Arrows—endothelium, arrowhead—probable mononuclear cell in traffic: ** p < 0.01, paired t test.