Anti-modified LDL antibodies, LDL-containing immune complexes, and susceptibility of LDL to in vitro oxidation in patients with type 2 diabetes.

We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential of IC, was significantly higher in macrophages incubated with red blood cell-adsorbed IC isolated from diabetic patients compared with IC isolated from control subjects (P < 0.03) or from patients with CAD (P < 0.04) and was strongly correlated with the content of apoB (r = 0.68, P < 0.001) and cholesterol (r = 0.61, P < 0.001) in IC. LDL from diabetic patients was more susceptible to oxidation in vitro, was significantly smaller, and contained significantly less alpha-tocopherol than LDL isolated from subjects in the other groups. In addition, the n-3 polyunsaturated fatty acid content of phospholipids and cholesteryl esters in LDL isolated from diabetic patients was significantly increased (P < 0.05) compared with that from patients with CAD or from control subjects. We postulate that LDL size, susceptibility to oxidation, and lipid fatty acid composition may play a critical role in the production of antibodies to oxidized LDL and consequently in the formation of LDL-containing IC in patients with type 2 diabetes.

Enhanced uptake and impaired intracellular metabolism of low density lipoprotein complexed with anti-low density lipoprotein antibodies.

We have previously shown that incubation of human macrophages with antigen-antibody complexes prepared with native human low density lipoprotein (LDL) and rabbit anti-LDL antibodies (LDL-ICs) results in an increased intracellular accumulation of cholesteryl esters (CEs) and induces a marked increase in the number of LDL receptors. To determine whether the increased CE accumulation in these cells occurred during incubation of the cells with LDL-ICs or whether it was secondary to the uptake of LDL by overexpressed LDL receptors, we incubated human macrophages with LDL-ICs for 22 hours, followed by incubation with native LDL for another 20 hours. We found that about 90% of the accumulated CEs could be accounted for by the first incubation with LDL-ICs. We then proceeded to show that the CEs accumulated during incubation of cells with LDL-ICs was secondary to enhanced uptake and impaired degradation of the LDL complexed with immunoglobulin G (IgG) (LDL-IC), which led to a marked intracellular accumulation of undergraded LDL (levels 199-fold higher than those obtained when the cells were incubated with the same concentration of native LDL not complexed with IgG). We have also shown that not all CEs accumulated in these cells were derived from accumulation of undegraded LDL and that some of them were derived from the reesterification of free cholesterol released during hydrolysis of LDL. LDL-ICs promoted increased CE accumulation and foam cell formation at concentrations as low as 25 micrograms/ml. To determine which receptors were involved in the uptake of LDL-ICs, we performed experiments in which the uptake of LDL-ICs was competitively inhibited with heat-aggregated gamma globulin, native LDL, beta-very low density lipoprotein, or acetylated LDL. Our results demonstrated that LDL-IC uptake was most effectively inhibited by heat-aggregated gamma globulin, partially inhibited by native LDL or by a monoclonal antibody to the LDL receptor, and not inhibited by acetylated LDL or beta-very low density lipoprotein. Thus, we conclude that the majority of LDL-ICs are taken up through Fc gamma receptors. Finally, we investigated whether the increase in LDL receptor expression was dependent on the receptor pathway used by the LDL-ICs, and we were able to demonstrate that when macrophages were incubated with LDL-ICs prepared with F(ab')2 fragments of the anti-LDL antibody, LDL receptor expression was not enhanced. Therefore, we postulate that the uptake of LDL-ICs through Fc gamma receptors results in an uncoupling of the normal regulation of the LDL receptor expression.

Erythrocyte-bound low-density lipoprotein immune complexes lead to cholesteryl ester accumulation in human monocyte-derived macrophages.

We have recently shown that incubation of macrophages with insoluble immune complexes (IC) containing low-density lipoproteins (LDL) leads to intracellular accumulation of esterified cholesterol (CE). This accumulation is associated with morphological transformation of the macrophages into "foam cells." In order to better characterize the conditions that lead to the uptake of LDL-IC and CE accumulation on macrophages, we studied the effects of soluble, insoluble, and red blood cell (RBC)-bound LDL-IC on macrophage lipid and lipoprotein metabolism. Using both apoB or IgG [anti-LDL] as the labeled moieties, we observed that the uptake of LDL-IC by human monocyte-derived macrophages (HMM) was markedly enhanced when the IC were adsorbed to RBC. Competition studies with unlabeled heat-aggregated IgG, native LDL, and acetylated LDL demonstrated that LDL-IC were ingested via the Fc receptor of HMM. The uptake of RBC-bound LDL-IC led to a marked intracellular accumulation of cholesteryl esters in HMM (78.4 +/- 1.7 vs 5.5 +/- 0.6 micrograms/mg cell protein; P less than 0.01) which apparently resulted from delayed degradation of the ingested LDL. Thus, it appears that the metabolism of LDL is altered when it is ingested as part of an antigen-antibody complex. These findings suggest that the formation of LDL-IC and their adsorption to red cells may play a significant role in the onset or in the evolution of human atherosclerosis.

Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. A possible mechanism of foam cell formation.

Human macrophages play a key role in atherogenesis and are believed to be the progenitors of the cholesteryl ester (CE)-laden foam cells present in early atherosclerotic lesions. Several mechanisms by which macrophages accumulate CE have been recently described. One involves a perturbation in LDL metabolism subsequent to macrophage activation. Thus, we decided to study the effect of macrophage activation by immune complexes on N-LDL metabolism. Initially, LDL-containing immune complexes (LDL-IC) were chosen, since increased plasma levels of these IC have been reported in patients with coronary heart disease. Human macrophages stimulated for 22 h with LDL-IC (250 micrograms/ml) and incubated afterwards for 20 h with 10 micrograms/ml 125I-N-LDL showed a six- and fourfold increase in the accumulation and degradation, respectively, of 125I-N-LDL over the values observed in nonstimulated cells. Scatchard analysis of 125I-N-LDL-specific binding suggests an increase (20-fold) in the number of LDL receptors in macrophages stimulated with LDL-IC. We studied other immune complexes varying in size and antigen composition. Some of the IC were able to stimulate, although to a lesser degree, the uptake of N-LDL by macrophages. Lipoprotein IC are more efficient and have the greatest capacity to increase N-LDL uptake and CE accumulation. We conclude that human macrophage activation by LDL-IC leads to an increase in LDL receptor activity and promotes in vitro foam cell formation.