The Pathogenic Role of the Adaptive Immune Response to Modified LDL in Diabetes

The main causes of morbidity and mortality in diabetes are macro and microvascular complications, including atherosclerosis, nephropathy, and retinopathy. As the definition of atherosclerosis as a chronic inflammatory disease became widely accepted, it became important to define the triggers of vascular inflammation. Oxidative and other modifications of lipids and lipoproteins emerged as major pathogenic factors in atherosclerosis. Modified forms of LDL (mLDL) are pro-inflammatory by themselves, but, in addition, mLDLs including oxidized, malondialdehyde (MDA)-modified, and advanced glycation end (AGE)-product-modified LDL induce autoimmune responses in humans. The autoimmune response involves T cells in the arterial wall and synthesis of IgG antibodies. The IgG auto-antibodies that react with mLDLs generate immune complexes (IC) both intra and extravascularly, and those IC activate the complement system as well as phagocytic cells via the ligation of Fcγ receptors. In vitro studies proved that the pro-inflammatory activity of IC containing mLDL (mLDL-IC) is several-fold higher than that of the modified LDL molecules. Clinical studies support the pathogenic role of mLDL-IC in the development of macrovascular disease patients with diabetes. In type 1 diabetes, high levels of oxidized and AGE-LDL in IC were associated with internal carotid intima-media thickening and coronary calcification. In type 2 diabetes, high levels of MDA-LDL in IC predicted the occurrence of myocardial infarction. There is also evidence that mLDL-IC are involved in the pathogenesis of diabetic nephropathy and retinopathy. The pathogenic role of mLDL-IC is not unique to diabetic patients, because those IC are also detected in non-diabetic individuals. But mLDL-IC are likely to reach higher concentrations and have a more prominent pathogenic role in diabetes due to increased antigenic load secondary to high oxidative stress and to enhanced autoimmune responses in type 1 diabetes

Polyclonal Activation of Human Peripheral Blood B Lymphocytes by Formaldehyde-Fixed Salmonella paratyphi B: I. Immunoglobin Production without DNA Synthesis

Human peripheral blood B lymphocytes can be activated by pokeweed mitogen (PWM) 1, Staphylococcus aureus Cowan I, Epstein-Barr virus, or Nocardia water-soluble mitogen (1-4). Of these polyclonal activators, PWM and S. aureus have been shown to stimulate both B and T cells; furthermore, the activation of peripheral blood B cells by PWM appears to be T cell dependent (5, 6). Nocardia mitogen, Epstein-Barr virus, and S. aureus Cowan I have been shown to be relatively T cell-independent B cell activators (2-4). Several parameters can be used to evaluate the polyclonal activation of human peripheral blood B lymphocytes, including immunoglobulin (Ig) production in vitro, enumeration of Ig-secreting cells by reverse hemolytic plaque assay, and enumeration of intracytoplasmic Ig-containing cells in stimulated cultures. However, study of human peripheral blood B lymphocyte function in normal and disease states has been hampered by the failure of mature human B cells to respond to some classic activators of murine B cells, such as dextran sulfate, tuberculin (PPD), and Escherichia coli lipopolysaccharide (LPS) (7-10). Dextran sulfate activates relatively immature murine B cells and results predominantly in increased DNA synthesis; in contrast, PPD acts on relatively mature murine B cells and stimulates a marked increase in antibody production but only modest DNA synthesis (11). However, PPD and other polyclonal B cell activators (PBA) that activate mouse B cells do not have the same effects on human peripheral blood B lymphocytes. The ability to stimulate human B cells selectively would be of considerable value for studies of immune function. We describe here a new PBA, i.e., formaldehyde-fixed Salmonella paratyphi B, which activates human peripheral blood B cells to produce large amounts of Ig but does not stimulate DNA synthesis as measured by incorporation of tritiated thymidine. This PBA apparently acts on relatively mature B cells which differentiate into Ig-secreting cells without DNA synthesis. Furthermore, its action appears to be completely independent of T cells, since T-depleted cell populations respond well to formaldehyde-fixed S. paratyphi B

A Population-Based Serologic Survey of Immunity to Tetanus in the United States

Background. Vaccination rates are frequently considered a surrogate measure of protection. To provide more accurate estimates, serum levels of antibody against tetanus were measured as part of the third National Health and Nutrition Examination Survey (NHANES III), which studied a representative sample of the civilian, noninstitutionalized population of the United States. Methods. We measured tetanus antitoxin using a solid- phase enzyme immunoassay in serum samples from 10,618 persons six years of age and older who were examined during phase 1 of NHANES III in 1988 to 1991. Results. Overall, 69.7 percent of Americans six years of age and older had protective levels of tetanus antibodies (0.15 IU per milliliter). The rate decreased from 87.7 percent among those 6 to 11 years of age to 27.8 percent among those 70 years of age or older. Among children 6 to 16 years of age, 82.2 percent had protective levels of tetanus antibodies, with little variation according to race or ethnicity. More men than women were immune (79.0percent vs. 62.4 percent). Mexican Americans had a significantly lower rate of immunity (57.9 percent, P 0.05) than either non-Hispanic whites (72.7 percent) or non-Hispanic blacks (68.1 percent). Those with a history of military service, higher levels of education, or incomes above the poverty level were more likely to have protective antibody levels. Although the prevalence of immunity declined rapidly starting at the age of 40 years, most of the 107 cases of tetanus (with 20 deaths) reported in 1989 and 1990 occurred in persons 60 years of age or older. Conclusions. Despite the fact that effective vaccines against tetanus have been available since the 1940s, many Americans do not have immunity to tetanus, and the rates are lowest among the elderly. There is an excellent correlation between vaccination rates (96 percent) and immunity (96 percent) among six-year-olds. However, antibody levels decline over time, and one fifth of older children (10 to 16 years of age) do not have protective antibody levels

Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'

The role of the immune system in the pathogenesis of diabetic complications

The main causes of morbidity and mortality in diabetes are macrovasular and microvascular complications, including atherosclerosis, nephropathy, and retinopathy. As the definition of atherosclerosis as a chronic, smoldering, inflammatory disease has gained general acceptance, the attention of researchers has focused on the triggers of chronic vascular inflammation. The oxidation and other forms of modification of lipids and lipoproteins have emerged as a major pathogenic factor in atherosclerosis, with a significant interaction with the immune system. Modified lipoproteins by themselves are proinflammatory through the activation of the innate immune system as a consequence of the interaction with scavenger receptors and/or toll-like receptors expressed by a variety of cell types, including phagocytic cells and dendritic cells. A variety of modified forms of LDL (mLDL), including oxidized, malondialdehyde-modified, and Advanced Glycation End-product-modified LDL induce autoimmune responses in humans. Those modifications seem enhanced in diabetes, and the progression of atherosclerosis is accelerated in diabetic patients. The immune response to all forms of mLDL results in both activation of T cells in the arterial wall and in an autoimmune response characterized by the formation of IgG antibodies. Both arms of the immune response are believed to play a role in vascular inflammation. While the cell response is likely to activate resident macrophages, the humoral immune response results in the production of IgG antibodies that bind to specific epitopes in modified forms of LDL, generate immune complexes both intra- and extravascularly, and those complexes are able to activate the classical pathway of the complement system as well as phagocytic cells via Fc? receptors. In vitro studies suggest that the pro-inflammatory activity of immune complexes containing mLDL is several-fold higher than that of the modified LDL molecules by themselves. Clinical studies have provided significant support to the pathogenic role of immune complexes containing modified LDL in the development of atherosclerotic complications in patients with both type 1 and type 2 diabetes. At the same time, there is increasing evidence that the formation of immune complexes containing modified forms of LDL may also be involved in the pathogenesis of diabetic nephropathy and retinopathy. These are areas in which more research is needed to fully understand the pathogenic mechanisms activated by those immune complexes. Of interest is the fact that animal models have suggested the possibility of modifying the adaptive humoral immune response in ways that would result in slowing down, and perhaps prevent, the atherosclerotic process. This possibility is sufficiently alluring as to justify increased research efforts, both in animal models (including diabetic animals) and translational clinical studies. The manipulation of the T regulatory population is another area of potential translational impact, which has hardly been explored. Indeed at this point of time, what seems to be a high priority is an increased and open interchange of information among investigators, trying to reach a better general understanding and integration of knowledge generated from a variety of approaches and perspectives. This Research Topic provided an optimal platform for this open interchange of information. We encouraged interested scientists to submit mini-reviews, methods papers, review articles, perspectives and original research articles covering this topic in all its diversity to facilitate the communication of perspectives and new information between scientists interested in understanding the multiple implications of the involvement of the immune system in the pathogenesis of diabetic complications

Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N(ɛ)(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy

Oxidized LDL and AGE-LDL in circulating immune complexes strongly predict progression of carotid artery IMT in type 1 diabetes

OBJECTIVE: Over 90% of modified LDL in circulation is associated to specific antibodies circulating as part of immune complexes (IC); however, few studies have examined their relationship with cardiovascular disease. METHODS: We report the relationship between circulating concentrations of IC of oxidized LDL (oxLDL-IC), malondialdehyde-LDL (MDA-LDL-IC) and advanced glycation end products-LDL (AGE-LDL-IC) and progression of atherosclerosis over a 12 year period in 467 individuals with type 1 diabetes who participated in the Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) study. OxLDL-IC, AGE-LDL-IC and MDA-LDL-IC levels were measured at DCCT closeout. Internal carotid intima-medial thickness (IMT) was measured at EDIC follow-up years 1, 6 and 12. RESULTS: OxLDL-IC, AGE-LDL-IC and MDA-LDL-IC levels were significantly correlated with age, lipid levels, blood pressure levels and albumin excretion rates. Levels of oxLDL, AGE-LDL and MDA-LDL in isolated LDL-IC were highly inter-correlated (r=0.66 to 0.84, p<0.0001). After adjusting for cardiovascular risk factors individuals in the upper quartile of oxLDL-IC had a 2.98fold increased odds (CI: 1.34, 6.62) of having IMT ≥ 1.00 mm and had a 5.13-fold increased odds (CI: 1.98, 13.3) of having significant IMT progression, relative to those in the lowest quartile. Parallel odds ratios for AGE-LDL-IC were 2.95 (CI: 1.37, 6.34) and 3.50 (CI: 1.38, 8.86), while results for MDA-LDL-IC were 1.76 (0.87, 3.56) and 2.86 (1.20, 6.81). CONCLUSION: Our study indicates that high levels of oxLDL-IC and AGE-LDL-IC are important predictors of carotid intima-medial thickening in patients with type 1 diabetes