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Antibodies to oxidized LDL (ox-LDL) and LDL-containing immune complexes (LDL-IC) have been reported to be associated with the presence or progression of arteriosclerosis. We screened for anti-modified LDL antibodies and isolated soluble IC by precipitation with 3.5% (w/v) polyethylene glycol (PEG) 6000 in two groups. The patient group was constituted by 16 insulin-dependent diabetes mellitus subjects free of macrovascular complications. The control group was constituted by 16 healthy, age-, gender-, race-, and body mass index-matched nondiabetic subjects. We detected anti-ox-LDL antibodies and anti-malondialdehyde-modified LDL antibodies with similar levels in patients and controls, while the levels of anti-glycated LDL antibodies were very low, but slightly higher in diabetics than in healthy controls. Isolated LDL-IC were adsorbed to red blood cells (RBC) and incubated with human macrophages for 18 hr at 37 degrees C. Under those experimental conditions, RBC-adsorbed IC are taken up by macrophages but the RBC remain intact and are not ingested. Slightly higher levels of cholesteryl ester (CE) accumulation were measured in macrophages incubated with RBC to which we adsorbed IC isolated from diabetics (15.4 +/- 2.5 micrograms/mg of protein, mean +/- SEM) than in macrophages incubated with IC isolated from controls (12.5 +/- 1.6 micrograms/mg of protein, mean +/- SEM), but the difference did not reach statistical significance. PEG-precipitable IC isolated from both normal and diabetic subjects led, in some instances, to the transformation of macrophages into foam cells. Significant correlations were observed between CE accumulation and the content of apo B (P < 0.0001), total cholesterol (P = 0.0004), IgG (P = 0.015), and IgA (P = 0.015) in the isolated IC. The correlation between CE accumulation and the content of apo B in isolated IC was stronger in diabetics than in the control group (r = 0.759 vs r = 0.500). Fractionation of isolated IC in immobilized protein A/G yielded immunoglobulin-rich fractions which contained cholesterol and IgG anti-ox-LDL antibodies. The cholesterol content of these fractions was significantly correlated (P = 0.001) with CE accumulation. In conclusion, both diabetics and normal individuals have circulating IC whose atherogenic potential appears to be related to the presence of LDL and antibodies of the IgG and IgA isotypes.

Human monocyte-derived macrophages are transformed into foam cells upon incubation with immune complexes containing low-density lipoprotein (LDL-IC), which are internalized predominantly through Fc gamma receptor-mediated phagocytosis. We investigated whether the FcR gamma-mediated ingestion of LDL-IC is associated with functional and metabolic activation of the ingesting cells. As end points we used the assay of released interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) and the reduction of nitroblue tetrazolium, which measures the respiratory burst. LDL-IC, added to the macrophages in concentrations known to induce intracellular accumulation of cholesterol esters and foam cell transformation, stimulated both the cytokine release and the respiratory burst more efficiently than control immune complexes. Time course studies of cytokine release and mRNA expression suggest that the synthesis and release of these two cytokines is under independent control. TNF alpha was released almost immediately after addition of LDL-IC to the macrophages, coinciding with increased early expression of TNF alpha mRNA, detectable 30 min after stimulation. In contrast, IL-1 beta was only increased in stimulated cell supernatants after 8 hr, and the onset of expression of IL-1 beta mRNA was also delayed in comparison to that of TNF alpha mRNA. We noted wide variations in the amounts of TNF alpha released by monocyte-derived macrophages from different donors. We also found that those macrophages which released higher levels of TNF alpha also took up higher amounts of 125I-labeled LDL, suggesting that the expression of LDL receptors by LDL-IC-stimulated macrophages is somehow linked to the degree of activation of these cells. Experiments using the measurement of the oxidative burst as end point corroborated that LDL-IC cause a general activation of macrophage functions. In conclusion, human macrophages are efficiently activated by LDL-IC, as reflected by the release of IL-1 beta and TNF alpha and by the release of oxygen active radicals. Thus, the presentation of LDL-IC to human macrophages induces a variety of metabolic and functional changes which are likely to contribute, directly or indirectly, to endothelial damage and progression of the atheromatous lesion.

We have studied the effects of dietary supplementation with fish oil on immunological parameters in a group of six normal volunteers, four of whom received a fish oil extract (total EPA dose of 2.4 g/day, which is on the lower range of clinically effective doses) for 6 weeks and two of which received a placebo (olive oil) for an identical period of time. Each volunteer was followed up for a period of 23 weeks after the dietary intervention was ended. All volunteers were boosted with tetanus toxoid (TT) at the onset of the trial. Several immune parameters were followed longitudinally, including NBT reduction and lysozyme release to test neutrophil function; lymphocyte subpopulations; mitogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and anti-CD3; IL-2 release after PHA and pokeweed mitogen (PWM) stimulation; immunoglobulin and anti-TT antibody (ATT) synthesis by stimulated lymphocytes; and serum levels of immunoglobulins and of ATT. No consistent changes were observed in neutrophil function tests, mitogenic responses to PHA and Con A, and lymphocyte subsets. The mitogenic response to anti-CD3 and the release of IL-2 after stimulation with PHA and PWM appeared reduced as a consequence of fish oil ingestion, and levels of serum immunoglobulins decreased in three of the volunteers receiving fish oil supplementation. The systemic humoral response after the TT booster appeared not to be influenced by the ingestion of fish oil. However, in those subjects who were given fish oil supplementation, the specific in vitro response of their peripheral blood lymphocytes to TT appeared to be compromised at Week 3. This could reflect the need for progressive accumulation of EPA in lymphocyte membranes for the suppressive effect to be detectable, but it could also reflect a differential sensitivity to the effects of fish oil of circulating B lymphocytes vs. bone marrow B lymphocytes. All the parameters apparently affected by fish oil ingestion were also affected by the incubation of normal lymphocytes with EPA in vitro. In conclusion, low doses of fish oil may have a mild immunosuppressive effect affecting both T and B cell functions. These observations stress the need for more extensive trials designed to determine whether immunosuppressive effects can be consistently elicited and for studies aimed at determining the mechanisms by which omega-3 fatty acids affect the immune system.

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.

Among the several pathological events that lead to the formation of an atheromatous lesion, endothelial cell damage, smooth muscle cell proliferation, and foam cell formation, are considered as particularly significant. In this review we summarize data suggesting that immunological and microbial factors may cause, directly or indirectly, these pathological events. Binding of immunocomplexes to endothelial cells, phagocytic cells, platelets, or erythrocytes could be the starting point for a variety of circuits leading to endothelial cell cytotoxicity and to the release of a variety of mediators, including cell proliferative factors. Endothelial cell toxicity could also be induced, directly or indirectly, by endotoxin; however, the possibility that endotoxin and other microbial factors may induce abnormalities in lipid metabolism at the monocyte/macrophage level which eventually result in intracellular accumulation of cholesterol (particularly if cholesterol levels are elevated) is specially attractive as a potential pathogenic mechanism. The various pathologic pathways discussed in this review appear plausible on the basis of our current knowledge and point to the need to investigate the potential role of infectious processes, autoimmune reactions, and administration of antigenic compounds as possible risk factors for the development of atherosclerosis.

Incubation of 0.5 nM platelet-activating factor (PAF) with 123 micrograms/ml C-reactive protein (CRP) for 60 min at room temperature in the presence of 2 mM calcium resulted in total inhibition of the platelet-aggregating capacity of 0.5 nM PAF. There was no inhibition of platelet aggregation if PAF and CRP were added to the platelets simultaneously, without prior incubation of the mixture, or if calcium was not present during the incubation of CRP and PAF. The inhibitory effect of CRP (123 micrograms/ml) was dependent upon the time of incubation with PAF (1 nM). We observed 0, 42, and 85% inhibition of platelet aggregation with incubations of 0, 20, and 120 min, respectively. The inhibitory effect of CRP was also concentration dependent. After incubations of 20 min at room temperature with 0.5 nM PAF there was no inhibition of PAF-induced platelet aggregation using 12.3 micrograms/ml CRP with the inhibition increasing to 73% using 123 micrograms/ml CRP. Pretreatment of the platelets with CRP (123 micrograms/ml) for 60 min before addition of PAF did not affect the induction of platelet aggregation by PAF. These observations indicate that CRP inhibits PAF and strongly suggest that binding of CRP to PAF, presumably to the phosphocholine moiety of PAF, is required for this inhibition.

Previous studies have demonstrated that eicosapentanoic acid (EPA) has anti-inflammatory properties in both humans and experimental animals and may also depress humoral immunity in experimental animals. Our investigations showed that the addition of eicosapentanoic acid to human peripheral blood mononuclear cell cultures inhibited B cell responses to mitogenic stimulation and depressed the expression of interleukin 2 receptors in pokeweed mitogen-stimulated lymphocytes. Neutrophils were also affected in their ability to release the contents of primary and secondary granules, particularly when stimulated with antigen-antibody complexes. Similar depressions of B cell responses and neutrophil functions were observed in a normal volunteer who ingested 6 g/day of a commercially available fish oil extract (equivalent to 2.1 g of EPA/day) during a 6-week period. Phagocytosis, enzymatic release, circulating immunoglobulin levels, and the response to tetanus toxoid both in vivo and in vitro were depressed during ingestion of fish oil. Most parameters showed a trend toward normalization 6 weeks after the suspension of fish oil supplementation. These effects of fish oil extracts and EPA on phagocytosis and humoral responses may be advantageously used in the therapy of chronic inflammatory diseases and autoimmune diseases but could be a cause for concern when these compounds are used for longer periods of time and with minimal medical supervision for the prophylaxis of atherosclerosis.