Gabriel Virella

Anti-oxidized low-density lipoprotein antibodies in patients with coronary heart disease and normal healthy volunteers.

SummaryWe have developed a solid-phase enzyme immunoassay for anti-oxidized low-density lipoprotein antibodies. Most sera showed some degree of non-specific binding to plates coated with oxidized low-density lipoprotein and the autoantibodies to oxidized low-density lipoprotein often appeared to have a relatively low affinity. To differentiate between specific and non-specific binding each sample was tested untreated and after absorption with oxidized low-density lipoprotein. The optical densities obtained with dilutions of the absorbed sample were considered to reflect non-specific binding and were subtracted from values obtained with identical dilutions of the unabsorbed sample, to yield corrected values from which the concentrations of anti-oxidized low-density lipoprotein antibody were calculated. Similar absorptions with native low-density lipoprotein and oxidized human serum albumin failed to induce a significant reduction in binding to immobilized oxidized low-density lipoprotein proving that the antibodies measured by this assay are primarily specific for oxidized low-density lipoprotein. We studied sera from two groups of individuals: (1) 33 subjects submitted to coronary angiography and split into two subgroups depending on the degree of coronary stenosis and (2) 64 healthy individuals also split into two subgroups according to lipid levels. Anti-oxidized low-density lipoprotein antibodies were detected both in patients and healthy individuals. Higher levels were detected in patients with moderate coronary disease and hyperlipemic healthy individuals, but the differences between patients and healthy volunteers or between their respective subgroups did not reach statistical significance. Our results suggest that autoantibodies to oxidized low-density lipoprotein are relatively frequent in both symptomatic and asymptomatic individuals. The investigation of their potential role as a risk factor will require mass screening and long-term follow-up.

The uptake of LDL-IC by human macrophages: predominant involvement of the FcγRI receptor

Abstract The incubation of human macrophages with antigen–antibody complexes prepared with rabbit anti-LDL and human LDL (LDL-IC) is followed by ingestion of those immune complexes (IC), massive cholesterol ester accumulation, cytokine release and overexpression of the LDL receptor. The massive accumulation of cholesterol esters and overexpression of the native LDL receptor are specifically induced by immune complexes containing native or modified LDL, but not by any other type of IC. We report the results of a series of experiments aimed at defining the receptor preferentially involved in LDL-IC uptake. Flow cytometry studies using CD16, CD32 and CD64 monoclonal antibodies showed a sharp reduction on the expression of CD64 (Fc γ RI) both by human monocyte-derived macrophages and THP-1 cells after incubation with LDL-IC, suggesting preferential engagement of this type of Fc receptor. Blocking experiments with aggregate-free IgG1 and CD32 monoclonal antibody confirmed that blocking Fc γ RI prevented both LDL-IC uptake and the upregulation of LDL receptors on THP-1 cells. In contrast, blocking Fc γ RII did not affect either the uptake of LDL-IC or the expression of LDL receptors on the same cells. The preferential engagement of Fc γ R-I by LDL-IC suggests a biological difference of LDL-IC relative to other types of IC and opsonized particles. The precise molecular mechanism(s) responsible for the paradoxical upregulation of LDL receptor after the uptake of LDL-IC remain to be elucidated.

ASSOCIATION BETWEEN IMMUNOGLOBULIN ALLOTYPES AND IMMUNE RESPONSES TO HÆMOPHILUS INFLUENZÆ AND MENINGOCOCCUS POLYSACCHARIDES

Serum samples were collected from 20 healthy White and 33 Black infants before and after immunisation with three doses of diphtheria-pertussis-tetanus vaccine and with one dose of Haemophilus influenzae type b polyribose phosphate vaccine and meningococcal group A and group C polysaccharide vaccines. Antibodies to these immunogens were measured and sera were allotyped for several Gm, A2m, and Km antigens. A highly significant association was found between the Km(1) allotype and the immune responses (difference between post-immunisation and pre-immunisation antibody levels) to H. influenzae and meningococcus C polysaccharides in the White children.

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-γ. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcγRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.

Risk Factors Related to Inflammation and Endothelial Dysfunction in the DCCT/EDIC Cohort and Their Relationship With Nephropathy and Macrovascular Complications

OBJECTIVE—Because endothelial cell dysfunction and inflammation are key contributors to the development of complications in type 1 diabetes, we studied risk factors related to endothelial dysfunction and inflammation (C-reactive protein and fibrinogen, soluble vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin, and fibrinolytic markers) in a subgroup of patients from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study cohort. RESEARCH DESIGN AND METHODS—We determined which of these risk factors or clusters thereof are associated with the presence of and subsequent development of nephropathy and macrovascular complications (reflected by carotid intima-media thickness [IMT]). RESULTS—After adjustment for conventional risk factors (age, sex, DCCT treatment group, diabetes duration, A1C, systolic blood pressure, waist-to-hip ratio, total and HDL cholesterol, and smoking status), fibrinogen remained strongly associated with progression of internal and common carotid IMT (P < 0.01) and soluble E-selectin had a strong association with nephropathy (P < 0.01). CONCLUSIONS—The best predictor for IMT progression in the DCCT/EDIC cohort was plasma fibrinogen, and the levels of soluble E-selectin discriminate patients with albuminuria better than conventional risk factors.

Immune Mechanisms of Atherosclerosis in Diabetes Mellitus

It was recently proposed that the increased levels of modified lipoproteins in diabetic patients may be responsible for the accelerated development of macrovascular complications associated with the disease. Modified lipoproteins are believed to induce the transformation of macrophages into foam cells and, in some cases, to induce endothelial cell damage. In addition, modified lipoproteins trigger an immune response leading to the formation of antibodies and then to the formation of LDL-containing immune complexes. In this review, we summarize the evidence linking LDL glycation and oxidation with intracellular accumulation of cholesterol esters and foam-cell formation, and we discuss their potential for inducing an autoimmune response and the formation of lipoprotein-containing immune complexes. The formation of LDL-ICs seems particularly significant, because these ICs are avidly taken up by macrophages through their Fc receptors and induce not only massive intracellular accumulation of CE but also a paradoxical increase in LDL-receptor expression. Our experimental data suggest that the uptake of LDL-IC is facilitated by RBC adsorption, in agreement with the role of RBC in the adsorption of circulating IC and their delivery to phagocytic cells. In addition, macrophages are activated when ingesting LDL-IC and release IL-1β and TNF-α, which can contribute to the initiation and progression of an atheromatous lesion by several mechanisms. Although it is difficult to envisage how LDL-IC could initiate an endothelial lesion, it is easy to speculate about their role as cofactors in the initiation and progression of the atherosclerotic process.

Immunosuppressive effects of fish oil in normal human volunteers: Correlation with the in vitro effects of eicosapentanoic acid on human lymphocytes

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.

Clinical significance of the humoral immune response to modified LDL.

Human low density lipoprotein (LDL) undergoes oxidation and glycation in vivo. By themselves, oxidized LDL (oxLDL) and AGE-LDL have proinflammatory properties and are considered atherogenic. But the atherogenicity of these lipoproteins are significantly increased as a consequence of the formation of immune complexes (IC) involving specific autoantibodies. OxLDL and AGE antibodies have been shown to be predominantly of the IgG1 and IgG3 isotypes. OxLDL antibodies are able to activate the complement system by the classical pathway and to induce FcR-mediated phagocytosis. In vitro and ex vivo studies performed with modified LDL-IC have proven their pro-inflammatory and atherogenic properties. Clinical studies have demonstrated that the levels of circulating modified LDL-IC correlate with parameters indicative of cardiovascular and renal disease in diabetic patients and other patient populations. The possibility that spontaneously formed or induced modified LDL antibodies (particularly IgM oxLDL antibodies) may have a protective effect has been suggested, but the data is unclear and needs to be further investigated.

Depression of humoral responses and phagocytic functions in vivo and in vitro by fish oil and eicosapentanoic acid.

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.

Immunological and microbiological factors in the pathogenesis of 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.