Circulating Reg1α proteins and autoantibodies to Reg1α proteins as biomarkers of β-cell regeneration and damage in type 1 diabetes

Type 1 diabetes is an autoimmune disease where β-cells are in a constant process of death and renewal. Reg genes play a role in β-cells regeneration. Reg proteins may be target of an autoimmune response in type 1 diabetes with consequent production of autoantibodies and failure of regeneration. The objective of this work was to characterize the role of Reg1α proteins and anti-Reg1α antibodies as biomarkers of β-cell regeneration and damage. Serum levels of Reg1α protein were investigated in 87 type 1 diabetic subjects (31 newly diagnosed and 56 long standing), 63 type 2 diabetic subjects, 39 subjects with systemic lupus erythematosus (SLE), a nonpancreatic autoimmune disorder, and 64 healthy subjects. The presence of anti-Reg1α antibodies and correlation with metabolic, immune, and genetic parameters were analyzed in diabetic subjects. Increased levels of Reg1α protein were observed in newly diagnosed (p=0.002), and long standing (p=0.001) type 1 diabetes patients and type 2 diabetic subjects (p<0.001). Anti-Reg1α antibodies were found in 47% of patients with type 1 diabetes. No correlation was found with metabolic, immune, and genetic parameters. Patients with SLE showed no increase in Reg1α protein. We report here for the first time raised serum Reg1α protein in type 1 and type 2 diabetes and anti-Reg1α antibodies in type 1 diabetes. Reg1α levels appear not to be influenced by genetic or metabolic control. These findings allow considering future studies on Reg1α protein and autoantibody as new tools in the evaluation and monitoring of β-cells regeneration and autoimmunity.     

Circulating oxidized LDL forms complexes with beta2-glycoprotein I: implication as an atherogenic autoantigen

Beta2-glycoprotein I (beta2-GPI) is a major antigen for antiphospholipid antibodies (Abs, aPL) present in patients with antiphospholipid syndrome (APS). We recently reported (J. Lipid Res., 42: 697, 2001; J. Lipid Res., 43: 1486, 2002) that beta2-GPI specifically binds to Cu2+-oxidized LDL (oxLDL) and that the beta2-GPI ligands are omega-carboxylated 7-ketocholesteryl esters. In the present study, we demonstrate that oxLDL forms stable and nondissociable complexes with beta2-GPI in serum, and that high serum levels of the complexes are associated with arterial thrombosis in APS. A conjugated ketone function at the 7-position of cholesterol as well as the omega-carboxyl function of the beta2-GPI ligands was necessary for beta2-GPI binding. The ligand-mediated noncovalent interaction of beta2-GPI and oxLDL undergoes a temperature- and time-dependent conversion to much more stable but readily dissociable complexes in vitro at neutral pH. In contrast, stable and nondissociable beta2-GPI-oxLDL complexes were frequently detected in sera from patients with APS and/or systemic lupus erythematodes. Both the presence of beta2-GPI-oxLDL complexes and IgG Abs recognizing these complexes were strongly associated with arterial thrombosis. Further, these same Abs correlated with IgG immune complexes containing beta2-GPI or LDL. Thus, the beta2-GPI-oxLDL complexes acting as an autoantigen are closely associated with autoimmune-mediated atherogenesis.     

A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that beta(2)-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for beta(2)-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by beta(2)-GPI and subsequently by anti-beta(2)-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both beta(2)-GPI and an anti-beta(2)-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-beta(2)-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with beta(2)-GPI. We suggest that autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL and Abs may be present in APS.     

The Coexistence of Antiphospholipid Syndrome and Systemic Lupus Erythematosus in Colombians

Objectives

To examine the prevalence and associated factors related to the coexistence of antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) in a cohort of Colombian patients with SLE, and to discuss the coexistence of APS with other autoimmune diseases (ADs).

Method

A total of 376 patients with SLE were assessed for the presence of the following: 1) confirmed APS; 2) positivity for antiphospholipid (aPL) antibodies without a prior thromboembolic nor obstetric event; and 3) SLE patients without APS nor positivity for aPL antibodies. Comparisons between groups 1 and 3 were evaluated by bivariate and multivariate analysis.

Results

Although the prevalence of aPL antibodies was 54%, APS was present in just 9.3% of SLE patients. In our series, besides cardiovascular disease (AOR 3.38, 95% CI 1.11–10.96, p = 0.035), pulmonary involvement (AOR 5.06, 95% CI 1.56–16.74, p = 0.007) and positivity for rheumatoid factor (AOR 4.68, 95%IC 1.63–14.98, p = 0.006) were factors significantly associated with APS-SLE. APS also may coexist with rheumatoid arthritis, Sjögren''s syndrome, autoimmune thyroid diseases, systemic sclerosis, systemic vasculitis, dermatopolymyositis, primary biliary cirrhosis and autoimmune hepatitis.

Conclusions

APS is a systemic AD that may coexist with other ADs, the most common being SLE. Awareness of this polyautoimmunity should be addressed promptly to establish strategies for controlling modifiable risk factors in those patients.     

Heparin inhibits the binding of beta 2-glycoprotein I to phospholipids and promotes the plasmin-mediated inactivation of this blood protein. Elucidation of the consequences of the two biological events in patients with the anti-phospholipid syndrome.

The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen recognized by the circulating autoantibodies in patients with the "anti-phospholipid syndrome" (APS). Although heparin is routinely used in the treatment and prophylaxis of APS patients, the primary heparin-binding site within beta2-GPI has not been identified. More importantly, how heparin exerts its beneficial effects in vivo in APS patients has not been deduced at the molecular level. Using an expression/site-directed mutagenesis approach, we now show that the positively charged site that resides in the first domain of beta2-GPI is not the primary heparin-binding site. Rather it is the second positively charged site located within the fifth domain of the protein that also binds to phospholipids. Lys(284), Lys(286), and Lys(287) in this domain are essential for the interaction of beta2-GPI with heparin. These data indicate that beta2-GPI binds to heparin in a relatively specific manner even though the affinity for the interaction is rather low. Lys(317) resides in the center of the high affinity phospholipid-binding site. Surprisingly, heparin at concentrations that can be achieved in vivo during anticoagulation therapy greatly enhances the plasmin-mediated cleavage of the Lys(317)-Thr(318) site in beta2-GPI. Because the cleaved form cannot bind to phospholipids effectively, the combined actions of heparin and plasmin result in a diminished ability of beta2-GPI to recognize phospholipids. This, in turn, decreases the prothrombotic activity of the endogenous circulating anti-beta2-GPI antibodies in the patients. Thus, heparin exerts its beneficial effects in APS patients by at least two distinct mechanisms.     

A protective role for innate immunity in systemic lupus erythematosus

Clinical and genetic studies in humans and animal models indicate a crucial protective role for the complement system in systemic lupus erythematosus (SLE). This presents a paradox because the complement system is considered to be an important mediator of the inflammation that is observed in patients with SLE. One current view is that complement provides protection by facilitating the rapid removal of apoptotic debris to circumvent an autoimmune response. In this Opinion article, I discuss an alternative model in which complement - together with other components of the innate immune system - participates in the 'presentation' of SLE-inducing self-antigens to developing B cells. In this way, the complement system and innate immunity protect against responses to SLE (self) antigens by enhancing the elimination of self-reactive lymphocytes.     

Cutting edge: protein phosphatase 2A confers susceptibility to autoimmune disease through an IL-17-dependent mechanism

The contribution of individual molecular aberrations to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease that affects multiple organs, is often difficult to evaluate because of the presence of abundant confounding factors. To assess the effect of increased expression of the phosphatase protein phosphatase 2A (PP2A) in T cells, as recorded in SLE patients, we generated a transgenic mouse that overexpresses the PP2Ac subunit in T cells. The transgenic mouse displays a heightened susceptibility to immune-mediated glomerulonephritis in the absence of other immune defects. CD4(+) T cells produce increased amounts of IL-17 while the number of neutrophils in the peripheral blood is increased. IL-17 neutralization abrogated the development of glomerulonephritis. We conclude that increased PP2Ac expression participates in SLE pathogenesis by promoting inflammation through unchecked IL-17 production and facilitating the development of end-organ damage.     

Protein-bound 4-hydroxy-2-nonenal: an endogenous triggering antigen of antI-DNA response

Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti-DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti-HNE titer than healthy individuals. To determine if a specific anti-DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti-DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i.e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.     

Serum protein oxidation and apolipoprotein CIII levels in people with systemic lupus erythematosus with and without nephritis

Increased oxidative stress is a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). This study compares serum protein oxidation levels in SLE patients without and with renal involvement (lupus nephritis); the latter have a significantly poorer prognosis. Similar increases in protein carbonyls and decreases in protein thiols were observed in both SLE groups compared to controls. Protein carbonyl distribution, determined by Western blotting of 2D gels, was similar in both SLE groups, suggesting factors other than oxidation also play a role in SLE complications. 2D electrophoresis examined the serum proteome further. Six proteins were significantly decreased in non-renal SLE patients compared to controls; five were identified by mass spectrometry, including one isoform of pro-atherogenic apoCIII. Total apoCIII levels (assessed by ELISA) in lupus nephritis patients were significantly elevated compared to controls or non-renal SLE patients. Thus, levels of oxidized proteins and apoCIII may be useful biomarkers in SLE studies.     

Immunization with an apoptotic cell-binding protein recapitulates the nephritis and sequential autoantibody emergence of systemic lupus erythematosus

The initial events predisposing to loss of tolerance in patients with systemic lupus erythematosus (SLE) are largely unknown, as are the events that precipitate the transition from preclinical to overt disease. We hypothesized that induction of murine SLE would require tipping the balance between tolerance and immunity in two ways: 1) an immunogen that could take advantage of apoptotic cells as a scaffold for epitope spread, and 2) an immune activator that would generate a strong and persistent T cell response to the inciting immunogen. We show that immunization of C57BL/6 and BALB/c mice with human beta(2)-glycoprotein I, an apoptotic cell-binding protein, in the presence of LPS induces a long-lived, potent response to beta(2)-glycoprotein I that results in epitope spread to multiple SLE autoantigens. SLE-specific autoantibodies emerged in a sequential manner that recapitulated the order seen in human SLE. Moreover, immunized mice developed overt glomerulonephritis closely resembling human lupus nephritis.     

Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.     

Systemic lupus erythematosus and the type I interferon system

Patients with systemic lupus erythematosus (SLE) have ongoing interferon-α (IFN-α) production and serum IFN-α levels are correlated with both disease activity and severity. Recent studies of patients with SLE have demonstrated the presence of endogenous IFN-α inducers in such individuals, consisting of small immune complexes (ICs) containing IgG and DNA. These ICs act specifically on natural IFN-α-producing cells (NIPCs), often termed plasmacytoid dendritic cells (PDCs). Given the fact that the NIPC/PDC has a key role in both the innate and adaptive immune response, as well as the many immunoregulatory effects of IFN-α, these observations might be important for the understanding of the etiopathogenesis of SLE. In this review we briefly describe the biology of the type I IFN system, with emphasis on inducers, producing cells (especially NIPCs/PDCs), IFN-α actions and target immune cells that might be relevant in SLE. On the basis of this information and results from studies in SLE patients, we propose a hypothesis that explains how NIPCs/PDCs become activated and have a pivotal etiopathogenic role in SLE. This hypothesis also indicates new therapeutic targets in this autoimmune disease.     

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.     

Aberrant phenotype and function of myeloid dendritic cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by a systemic autoimmune response with profound and diverse T cell changes. Dendritic cells (DCs) are important orchestrators of immune responses and have an important role in the regulation of T cell function. The objective of this study was to determine whether myeloid DCs from individuals with SLE display abnormalities in phenotype and promote abnormal T cell function. Monocyte-derived DCs and freshly isolated peripheral blood myeloid DCs from lupus patients displayed an abnormal phenotype characterized by accelerated differentiation, maturation, and secretion of proinflammatory cytokines. These abnormalities were characterized by higher expression of the DC differentiation marker CD1a, the maturation markers CD86, CD80, and HLA-DR, and the proinflammatory cytokine IL-8. In addition, SLE patients displayed selective down-regulation of the maturation marker CD83 and had abnormal responses to maturation stimuli. These abnormalities have functional relevance, as SLE DCs were able to significantly increase proliferation and activation of allogeneic T cells when compared with control DCs. We conclude that myeloid DCs from SLE patients display significant changes in phenotype which promote aberrant T cell function and could contribute to the pathogenesis of SLE and organ damage.     

Regulatory T cells and TH1/TH2 cytokines as immunodiagnosis keys in systemic autoimmune diseases

We assessed Helper T-cell involvement and possibilities to quantify the cell-based immune response in systemic autoimmune diseases (SAID) in 14 systemic lupus erythematosus (SLE) and 7 rheumatoid arthritis (RA) patients. The goals of investigation were T-CD4+/T-CD8+ ratio, regulatory T cells (Treg) status and TH1/TH2 serum cytokine profiles (IFN-gamma and IL-2, respectively IL-4 and IL-6). SLE group proved significant decreased average Treg value as compared to RA group and controls and showed significant low Treg incidence (86% patients). The distribution of high T-CD4+/T-CD8+ ratio registered no significant distinction among LES and RA groups. SAID patients presented low serum IFN-gamma (86% RA, 60% SLE), high IL-2 (57% RA) and high IL-6 (53% LES), but no significant IL-4 modification. We conclude that Treg percentage remains the only cellular criterion for SAID immune evaluation. In the same time, different secretion mechanisms seem to be involved in SAID, i.e. TH2 in SLE and TH1 in RA.     

Taming lupus-a new understanding of pathogenesis is leading to clinical advances

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the loss of tolerance to nuclear self antigens, the production of pathogenic autoantibodies and damage to multiple organ systems. Over the years, patients with SLE have been managed largely with empiric immunosuppressive therapies, which are associated with substantial toxicities and do not always provide adequate control of the disease. The development of targeted therapies that specifically address disease pathogenesis or progression has lagged, largely because of the complex and heterogeneous nature of the disease, as well as difficulties in designing uniform outcome measures for clinical trials. Recent advances that could improve the treatment of SLE include the identification of genetic variations that influence the risk of developing the disease, an enhanced understanding of innate and adaptive immune activation and regulation of tolerance, dissection of immune cell activation and inflammatory pathways and elucidation of mechanisms and markers of tissue damage. These discoveries, together with improvements in clinical trial design, form a platform from which to launch the development of a new generation of lupus therapies.     

New targeted therapies for treatment of thrombosis in antiphospholipid syndrome

Antiphospholipid (aPL) antibodies (Abs) are associated with thrombosis and pregnancy loss in antiphospholipid syndrome (APS), a disorder initially characterised in patients with systemic lupus erythematosus (SLE) but now known to occur in the absence of other autoimmune disease. There is strong evidence that aPL Abs are pathogenic in vivo, from studies of animal models of thrombosis, endothelial cell activation and pregnancy loss. In recent years, progress has been made in characterising the molecular basis of this pathogenicity, which includes direct effects on platelets, endothelial cells and monocytes as well as activation of complement. This review summarises the clinical manifestations of APS and current modalities of treatment, and explains recent advances in understanding the molecular events triggered by aPL Abs on target cells in coagulation pathways as well as effects of aPL Abs on complement activation. Based on this information and on additional scientific evidence using in vitro and in vivo models, new potential targeted therapies for treatment and/or prevention of thrombosis in APS are proposed and discussed.