Immunoglobulin A anti‐phospholipid antibodies in Swedish cases of systemic lupus erythematosus: associations with disease phenotypes,vascular events and damage accrual

Immunoglobulin (Ig) G‐ and IgM‐class anti‐cardiolipin antibodies (aCL) and lupus anti‐coagulant (LA) are included in the 1997 update of the American College of Rheumatology (ACR‐97) systemic lupus erythematosus (SLE) criteria. Despite limited evidence, IgA‐aCL and IgA anti‐β₂‐glycoprotein‐I (anti‐β₂GPI) were included in the 2012 Systemic Lupus International Collaborating Clinics criteria. The present study aimed to evaluate IgG‐/IgA‐/IgM‐aCL and anti‐β₂GPI occurrence in relation to disease phenotype, smoking habits, pharmacotherapy, anti‐phospholipid syndrome (APS) and organ damage among 526 Swedish SLE patients meeting ACR‐97. Patients with rheumatoid arthritis (n = 100), primary Sjögren’s syndrome (n = 50) and blood donors (n = 507) served as controls. Anti‐phospholipid antibodies (aPL) were analysed by fluoroenzyme‐immunoassays detecting aCL/anti‐β₂GPI. Seventy‐six (14%) SLE cases fulfilled the Sydney APS‐criteria, and ≥ 1 aCL/anti‐β₂GPI isotype (IgG/IgA/IgM) occurred in 138 SLE patients (26%). Forty‐five (9%) of the SLE cases had IgA‐aCL, 20 of whom (4%) lacked IgG‐/IgM‐aCL. Seventy‐four (14%) tested positive for IgA anti‐β₂GPI, 34 (6%) being seronegative regarding IgG/IgM anti‐β₂GPI. Six (1%) had APS manifestations but were seropositive regarding IgA‐aCL and/or IgA anti‐β₂GPI in the absence of IgG/IgM‐aPL and LA. Positive LA and IgG‐aPL tests were associated with most APS‐related events and organ damage. Exclusive IgA anti‐β₂GPI occurrence associated inversely with Caucasian ethnicity [odds ratio (OR) = 0·21, 95% confidence interval (CI) = 0·06–0·72) and photosensitivity (OR = 0·19, 95% CI = 0·05–0·72). Nephritis, smoking, LA‐positivity and statin/corticosteroid‐medication associated strongly with organ damage, whereas hydroxychloroquine‐medication was protective. In conclusion, IgA‐aPL is not rare in SLE (16%) and IgA‐aPL analysis may have additional value among SLE cases with suspected APS testing negative for other isotypes of aPL and LA.     

The Investigation on the Value of Repeat and Combination Test of ACA and Anti‐β2‐GPI Antibody in Women with Recurrent Spontaneous Abortion

Problem In order to investigate the value of anticardiolipin antibodies (ACA) and anti‐β₂‐GPI antibodies detection in screening autoimmune type recurrent spontaneous abortion and its clinic application in antiphospholipid syndrome diagnosis, we adopt repeat combined ACA and anti‐β₂‐GPI antibodies detection in this study. Method of study Sera were collected from patients and work‐up was done for detection of ACA and anti‐β₂‐GPI antibodies by enzyme‐linked immunosorbent assay (ELISA). The work‐up was done for detection of antibodies once in every 6 weeks for 14 times consecutively. Results The repeated and combined detection of ACA and anti‐β₂‐GPI antibodies detection could raise the positivity rate up to 21.8% (P < 0.05) in comparison with positive for ACA alone (14.1%), positive for anti‐β₂‐GPI alone (3.1%), and concurrently positive for both ACA and anti‐β₂‐GPI antibodies (4.6%). In 91 confirmed positive antiphospholipid antibodies (APA) patients, with more frequent screening for ACA and anti‐β₂‐GPI antibodies, more patients with APA were found. The positive rate of five and more screenings was over 81.32%, which was statistically significant (P < 0.05), in comparison with that of four or less screenings (68.13%). Conclusion Our data implied that it would be appropriate to take over five or more screenings of combined ACA and anti‐β₂‐GPI antibodies detection in suspect patients to facilitate the positive diagnostic rate for autoimmune type RSA.     

Anti‐citrullinated glucose‐6‐phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA‐DRB1 shared epitope alleles and disease activity

To identify and characterize anti‐citrullinated glucose‐6‐phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine‐bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG‐1–9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG‐1–9 were measured, and anti‐citrullinated α‐enolase‐1 (CEP‐1), ‐cyclic citrullinated peptides (CCP) and ‐GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA‐DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti‐CCG‐2, ‐4 and ‐7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1–99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti‐CEP‐1, ‐CCP and ‐GPI protein antibodies, respectively. Anti‐CCG‐2, ‐4 and ‐7 antibodies were correlated with anti‐CCP and anti‐CEP‐1 antibodies and with the presence of HLA‐DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti‐CCG‐2 and ‐7 but not of anti‐CEP‐1 antibodies. This is the first report documenting the presence of anti‐CCG antibodies in RA. Anti‐CCG‐2 and ‐7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.     

Toll-like receptor (TLR)-4 mediates anti-β2GPI/β2GPI-induced tissue factor expression in THP-1 cells

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti‐β₂‐glycoprotein I/β₂‐glycoprotein I complex (anti‐β₂GPI/β₂GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll‐like receptor 4 (TLR‐4) might act as an ‘adaptor’ for intracellular signal transduction in anti‐β₂GPI/β₂GPI‐induced TF expressing cells. In the current study, we investigated the roles of TLR‐4 and its related molecules, myeloid differentiation protein 2 (MD‐2) and myeloid differentiation factor 88 (MyD88), in anti‐β₂GPI/β₂GPI‐induced TF expressing human monocytic‐derived THP‐1 (human acute monocytic leukaemia) cells. The relationship of TLR‐4 and ANX2 in this process was also explored. Along with TF, expression of TLR‐4, MD‐2 and MyD88 in THP‐1 cells increased significantly when treated by anti‐β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD‐2 ligand, could inhibit the effects of anti‐β₂GPI/β₂GPI on TLR‐4, MD‐2, MyD88 and TF expression. Both ANX2 and TLR‐4 in THP‐1 cell lysates could bind to β₂GPI that had been conjugated to a column (β₂GPI‐Affi‐Gel). Furthermore, TLR‐4, MD‐2, MyD88 and TF expression was remarkably diminished in THP‐1 cells infected with ANX2‐specific RNA interference (RNAi) lentivirus (LV‐RNAi‐ANX2), in spite of treatment with a similar concentration of anti‐β₂GPI/β₂GPI complex. These results indicate that TLR‐4 and its signal transduction pathway contribute to anti‐β₂GPI/β₂GPI‐induced TF expression in THP‐1 cells, and the effects of TLR‐4 with ANX2 are tightly co‐operative.     

Serum antibodies to human leucocyte antigen (HLA)‐E,HLA‐F and HLA‐G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA‐F autoantibodies

T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over‐expression of human leucocyte antigen (HLA)‐Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA‐Ib (HLA‐E, HLA‐F and HLA‐G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA‐Ib and β2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex^®‐based flow cytometry. The values were expressed as mean florescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti‐HLA‐G IgG predominated over other HLA‐Ib antibodies, whereas SLE patients had a preponderance of anti‐HLA‐F IgG over the other HLA‐Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)‐2000, was reflected only in the levels of anti‐HLA‐F IgG. Anti‐HLA‐F IgG with MFI level of 500–1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti‐HLA‐F IgG were cross‐reactive with other HLA‐Ib alleles, their reactivity was reflected in the levels of anti‐HLA‐E and ‐G IgG. The prevalence of HLA‐F‐monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti‐HLA‐F IgG is possibly involved in the clearance of HLA‐F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti‐HLA‐Ib IgG should be considered as a biomarker in standard SLE diagnostics.     

Patients with inflammatory bowel disease may have a transforming growth factor‐β‐, interleukin (IL)‐2‐ or IL‐10‐deficient state induced by intrinsic neutralizing antibodies

Ulcerative colitis (UC) and Crohn's disease (CD) are considered to be immunologically mediated disorders that share certain features with murine models of colitis. Whether any of these models are physiologically relevant to the human condition remains controversial. The hypothesis is that increased amounts of antibodies neutralizing transforming growth factor (TGF)‐β, interleukin (IL)‐2 or IL‐10 create a relative immunodeficient state in inflammatory bowel disease (IBD) that predisposes to disease. To evaluate this, serum samples from patients with UC or CD and from normal healthy individuals were studied by enzyme‐linked immunosorbent assays. Antibodies recognizing TGF‐β were most prevalent in UC (P < 0·01); anti‐IL‐10 antibodies were elevated in CD (P < 0·05), while anti‐IL‐2 antibodies were the same for all three groups. Importantly, the percentage of IBD patients with at least one of the antibody levels greater than any control value was 30% for UC and 33% for CD. To verify the presence of these antibodies, immobilized TGF‐β was exposed to UC sera and the attached proteins identified by Western blot assay. The proteins proved to be exclusively immunoglobulin (Ig) G. To evaluate the neutralizing activity of these antibodies, cytokine‐specific IgG from subjects in each group of patients was incubated with TGF‐β, IL‐2 or IL‐10 before addition to a bioassay with changes in viability determined by a colorimetric analysis. Antibodies from most individuals in all three groups neutralized the action of each cytokine. This study shows that about one‐third of IBD patients may have a relative deficiency of TGF‐β, IL‐2 or IL‐10 due to an increase in neutralizing antibodies in their sera.     

Phospholipid specificity and requirement of β2-glycoprotein-I for reactivity of antibodies from patients with primary antiphospholipid syndrome

Some disease manifestations are associated with serum antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE) in what has been termed antiphospholipid syndrome (aPLS). There are patients with aPLS who do not have SLE or any other illness who have been grouped under the term primary antiphospholipid syndrome (PAPS). However, patients with diverse infections, notably syphilis, may have aPL but do not develop the associated clinical manifestations. This has been attributed, at least in part, to the immunochemical features of their aPL, including the requirement for β₂-glycoprotein-I (β₂GP-I) for binding of aPL to phospholipids, but these have not been studied in sera from patients with PAPS. By ELISA we studied 95 sera from 17 patients with PAPS and 100 sera from clinically normal individuals for IgG and IgM antibodies to the main anionic and zwitterionic phospholipids and their related compounds, phosphatidic acid (PA) and synthetic phosphorylcholine (PRC). β₂GP-I was present, either in newborn calf serum (NBCS) or purified, to block wells and to dilute samples, or was substituted by 0.3% gelatin. Inhibition studies with phospholipid micelles were used to confirm reactivities with the corresponding phospholipids. All 17 patients had IgG and 11 had IgM antibodies to cardiolipin. Antibodies to anionic phospholipids were primarily IgG whereas those to zwitterionic phospholipids were mainly, and often exclusively, IgM. We found a statistically significant difference in the mean levels of antibodies to all anionic phospholipids except aPTS, and to the haptene PA (P < 0.001) between patients and controls. The difference between levels of IgM antibodies to zwitterionic phospholipids was statistically significant with sphingomyelin (P < 0.001) and the haptene (P < 0.001). Levels of most IgG and most IgM aPL correlated significantly among them. The pattern and titers of reactivity are variable between patients, but stable within each patient. Requirement of β₂GP-I for this reactivity was not an all-or-nothing phenomenon in individual sera. In general, as in lupus sera, antibodies to anionic phospholipids require that this cofactor be present coating the ELISA plates, whereas those to zwitterionic phospholipids do not.It would appear that patients with PAPS have polyclonal mixtures of antibodies that react with various phospholipids and have different requirements for β₂GP-I for such reactivity.     

Correlation Between β2-Glycoprotein Antibodies and Antiphospholipid Antibodies in Patients with Reproductive Failure

PROBLEM: Antiphospholipid antibodies (APAs) are important in the etiology of reproductive failure. Studies have shown that binding proteins are necessary for the detection of APAs. One of these, β₂-glycoprotein, has been shown to be necessary for detection of anticardiolipin antibodies. It is felt that some APAs may be directed to the binding protein itself, or to a combination of the binding protein and phospholipid. METHOD OF STUDY: In this study, a comparison of APAs vs. anti β₂-glycoprotein antibodies was performed on the sera of 123 women younger than 40 years of age with a history of reproductive failure. Antibodies to six phospholipid epitopes, cardiolipin, phosphatidyle-thanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and phosphatidylserine, were measured. RESULTS: Of the 123 women tested, 33 had one or more positive immunoglobulin (Ig)G antibodies to phospholipids, of which 9 were to cardiolipin. However, only 1 of 123 women had IgG antibodies to β₂-glycoprotein and she was APA negative. Thirty-eight of 123 women had one or more IgM antibodies to phospholipids, with none directed to cardiolipin IgM. In contrast, only 8 of the 123 women had IgM antibodies to β₂-glycoprotein. Five of the eight patients had IgM APA; 4 of 5 had IgM antibodies to PE, 1 to PI. CONCLUSIONS: There is no correlation between β₂-glycoprotein antibodies and APA status in this population. To date, our most sensitive test for detecting phospholipid autoimmune-mediated in vitro fertilization failure still appears to be the ELISA assay for APA.     

Diagnostic and prognostic values of anti glucose-6-phosphate isomerase antibodies in community-recruited patients with very early arthritis

The objective of this study was to determine the diagnostic and prognostic values of antiglucose‐6‐phosphate isomerase (GPI) antibodies in patients with very early arthritis. Anti‐GPI antibodies were measured by ELISA using purified GPI from rabbit muscle in: (i) 383 sera from healthy blood donors (n = 120), well‐established rheumatoid arthritis (RA) (n = 99) and non‐RA differentiated arthritis (NRADA) (n = 164) patients; (ii) 195 sera obtained from community‐recruited patients with very early inflammatory arthritis (VErA cohort) that were studied for 1 year and classified as having RA (n = 116), NRADA (n = 41), and undifferentiated arthritis (UA) (n = 38) after the follow‐up period. The criterion for severity was the progression of radiographic damage. Prevalence of anti‐GPI antibodies was significantly higher in well‐established RA patients (45·4%) compared to healthy subjects (2·5%). Anti‐GPI antibodies were also present in sera from NRADA: systemic lupus erythematosus 53%, polymyositis 45·4%, adult‐onset Still's disease 44%, systemic sclerosis 42·8%, spondylarthropathies 25% and primary Sjögren’s syndrome 5·8%. No significant association was found between the presence of anti‐GPI antibodies and the 3 diagnostic groups from the VErA cohort. No correlation was observed between anti‐GPI and autoantibodies usually associated with RA. Anti‐GPI antibodies were not predictive of radiological progression in patients with very early arthritis. Thus, anti‐GPI antibodies are not useful for discriminating RA from non‐RA rheumatic diseases and do not constitute a predictive factor of structural damage.     

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.     

Effect of Antiphospholipid Antibodies on β2Glycoprotein I-Phospholipid Interaction

PROBLEM: β₂ glycoprotein I (β₂GPI) physiologically binds to negatively charged phospholipids (PLs) and is a natural regulator of the coagulation cascade. Thrombotic clinical complications and recurrent fetal loss associated with autoimmune antiphospholipid (aPL) antibodies are thought to be related to their binding to β₂GPI-PL complex and interference with the physiological function of β₂GPI. METHOD OF STUDY: To investigate the effect of aPL on β₂GPI-PL interaction, we studied the binding of biotinylated β₂GPI to cardiolipin (CL) by enzyme-linked immunosorbent assay (ELISA) in the presence and absence of purified aPL immunoglobulin G (IgG) antibodies. RESULTS: Adding five different aPL IgG antibodies with different levels of aPL activity isolated from the sera of five patients with aPL-associated recurrent fetal death greatly increased the binding of biotinylated β₂GPI to CL-coated plates. The optical densities (ODs) were 0.635, 0.890, and 1.265 in the presence of three aPL IgG antibodies, compared to 0.425 in the absence of aPL IgG. In contrast, normal human IgG had no enhancing effect. The OD was 0.480 and 0.425, respectively. The enhancement of β₂GPI binding to CL by aPL IgG correlated with the titers of aPL antibodies. The use of phosphate-buffered saline with increasing salt concentrations as a washing buffer for the ELISA resulted in more stable binding of β₂GPI to PL in the presence of aPL IgG. CONCLUSIONS: These findings suggest that the binding of autoimmune aPL antibodies to β₂GPI-PL complex results in abnormally tighter interaction between β₂GPI and PLs, which may lead to physiological dysfunction of β₂GPI as a regulator of coagulation.     

IgG autoantibodies against beta2-glycoprotein I complexed with a lipid ligand derived from oxidized low-density lipoprotein are associated with arterial thrombosis in antiphospholipid syndrome

We recently reported [J. Lipid Res. 42 (2001), 697; 43 (2002), 1486; 44 (2003), 716] that [beta2-glycoprotein I (beta2GPI) forms complexes with oxidized LDL (oxLDL) and autoantibodies against these complexes are present in patients with SLE and antiphospholipid syndrome (APS). The relationship of beta2GPI/oxLDL complexes and IgG autoantibodies against beta2GPI complexed with oxLig-1 (an oxLDL-derived ligand) with clinical manifestations of APS was studied in 150 APS and SLE patients. The beta2GPI/oxLDL levels of APS patients were similar to those of SLE patients without APS, but they were significantly higher than healthy individuals. There was no difference in the complex levels among the patients with arterial, venous thrombosis, or pregnancy morbidity. IgG anti-beta2GPI/oxLig-1 levels of APS were significantly higher than those of SLE without APS and healthy individuals. Further, antibody levels of APS patients with arterial thrombosis were significantly higher than those patients with venous thrombosis and pregnancy morbidity. Thus, oxidation of LDL leads the complex formation with beta2GPI in SLE and APS patients. In contrast, anti-beta2GPI/oxLig-1 autoantibodies were generated only in APS and were strongly associated with arterial thrombosis. These results suggest that autoantibodies against beta2GPI/oxLDL complexes are etiologically important in the development of atherosclerosis in APS.     

Anti-beta 2-glycoprotein I and antiphosphatidylserine antibodies are predictors of arterial thrombosis in patients with antiphospholipid syndrome

The predictive value (PV) and association of 4 antiphospholipid antibodies with clinical manifestations of the antiphospholipid syndrome (APS) were evaluated in 90 patients with systemic lupus erythematosus (SLE) and 100 with APS. Patients with APS were classified into arterial thrombosis, venous thrombosis, and pregnancy morbidity subgroups. IgG, IgM, and IgA anticardiolipin (aCL), antiphosphatidylserine (aPS), anti-beta 2-glycoprotein I (anti-B2GPI), and antiprothrombin (aPT) antibodies were determined by enzyme-linked immunosorbent assay. Individually, anti-B2GPI and aPS antibodies had the strongest PV for APS (86.4%-94.1%; P < .001) in patients with SLE. The PV for APS reached 100% when 2 or more antibodies were present. Similarly, anti-B2GPI and aPS antibodies had a stronger PV and association for arterial thrombosis (87%-95%; P < .001) compared with venous thrombosis (80%-92%; P = .01). Weak PV and association with pregnancy morbidity were seen with all antibodies. These results suggest an important pathogenic role of anti-B2GPI antibodies in arterial thrombosis. In addition, anti-B2GPI and aPS antibodies seem to provide the best diagnostic value for the laboratory assessment of APS.     

Validation of Autoantibody Assays in Type 1 Diabetes: Workshop Programme

Antiphospholipid syndrome (APS) was firstly described in systemic lupus erythematosus (SLE), but it was recognized also as a primary APS (PAPS) form. These forms are not always distinguishable, since they show some common clinical/serological manifestations. We actually may deal with: (1) patients initially classified as PAPS gradually developing SLE; (2) patients with SLE and associated APS, whose complications generally affect morbidity and mortality; (3) patients with SLE and positive antiphospholipid antibodies without APS manifestations; the relevant issue in such patients is to provide effective prophylaxis. The close relationship between PAPS and SLE is also supported by: (i) nuclear autoimmunity and (ii) complement activation at least in animal models of APS. Future studies on the genetic background and/or on regulatory suppressive mechanisms may clarify how and why PAPS can evolve into SLE.     

Guanine polynucleotides are self‐antigens for human natural autoantibodies and are significantly reduced in the human genome

In the course of investigating anti‐DNA autoantibodies, we examined IgM and IgG antibodies to poly‐G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20‐mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo‐nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti‐G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain‐associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti‐G20 antibodies; so natural anti‐G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti‐G20 antibodies in human health and disease and of runs of G20 in the human genome.     

Anti-β2-glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

Autoantibodies characteristic for anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti-β₂-glycoprotein I (β₂GPI) antibodies and anti-DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti-β₂GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti-β₂GPI antibodies to cell surface β₂GPI in monocytes and endothelial cells triggers the Toll-like receptor 4-myeloid differentiation primary response 88 (TLR)-4-MyD88) signaling pathway which leads to activation of p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinases (MEK-1/ERK) and/or nuclear factor kappa B (NF-κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR-4. Previously, we generated a mouse monoclonal anti-β₂GPI antibody WB-6 and showed that it induced a prothrombotic state – including TF expression on circulating monocytes – in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB-6 and resting monocytes, and found that WB-6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB-6 expressed TF and tumor necrosis factor (TNF)-α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM-I) and vascular cell adhesion molecule 1 (VCAM-I). These results suggest the possibility that a subset of anti-β₂GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor-mediated pathways, leading to produce proinflammatory and prothrombotic states.     

Immunoglobulin (Ig)G1 and IgG4 anti‐cyclic citrullinated peptide (CCP) associate with shared epitope,whereas IgG2 anti‐CCP associates with smoking in patients with recent‐onset rheumatoid arthritis (the Swedish TIRA project)

Given the possible importance of anti‐citrullinated peptide/protein antibodies (ACPA) for initiation and progression of rheumatoid arthritis (RA), extended knowledge about the different isotypes and subclasses is important. In the present study, we analysed the immunoglobulin (Ig)G subclasses regarding reactivity against cyclic citrullinated peptides (anti‐CCP) among 504 clinically well‐characterized patients with recent‐onset RA in relation to smoking habits, shared epitope (SE) status and IgA and pan‐IgG anti‐CCP antibodies. All patients, regardless of pan‐IgG anti‐CCP status, were analysed for IgG1–4 CCP reactivity. Sixty‐nine per cent were positive in any IgG anti‐CCP subclass, and of these 67% tested positive regarding IgG1, 35% IgG2, 32% IgG3, and 59% IgG4 anti‐CCP. Among ever‐smokers the percentages of IgG2 anti‐CCP (P = 0·01) and IgA anti‐CCP (P = 0·002)‐positive cases were significantly higher compared to never‐smokers. A positive IgG anti‐CCP subclass ‐negative cases. Combining SE and smoking data revealed that IgG1 and IgG4 anti‐CCP were the IgG anti‐CCP isotypes associated with expression of SE, although the lower number of patients positive for IgG2 or IgG3 anti‐CCP could, however, have influenced the results. High levels of IgG2 anti‐CCP were shown to correlate with expression of the ‘non‐SE’ allele human leucocyte antigen (HLA)‐DRB1*15. In conclusion, in this study we describe different risk factor characteristics across the IgG anti‐CCP subclasses, where IgG2 appears similar to IgA anti‐CCP regarding the predominant association with smoking, while IgG1 and IgG4 related more distinctly to the carriage of SE genes.     

Anti-β2 Glycoprotein I Antibodies Prevent the De-activation of Platelets and Sustain their Phagocytic Clearance

Exposure to phosphatidylserine (PS) tags dying and senescent cells for removal and identifies activated platelets. In this study we followed the fate of PS-exposing platelets in the presence of antibodies purified from Systemic Lupus Erythematosus (SLE) and primary Anti-phospholipid Syndrome (APS) patients' sera by β2GPI affinity chromatography. Thrombin-activated platelets exposed PS and associated to β2GPI. Both events were required for recognition by antibodies. Human monocyte-derived macrophages phagocytosed activated platelets only. Each macrophage internalized an average of 3.16±0.2 platelets after 60 min at 37°C. Phagocytosis did not increase after longer incubations (4.65±0.26 platelets internalized by each macrophage after 300 min). Recognition of platelets by anti-β2GPI antibodies significantly increased phagocytosis (P< 0.01). Upon withdrawal of thrombin, platelets downregulated PS (PS exposure t_1/2: 242 min) and the ability to be recognized by macrophages. Purified β2GPI bound to PS-exposing platelets (association t_1/2: 250 min). Phosphatidyl serine exposure and β2GPI association had virtually identical kinetics. Antibody binding prolonged the exposure of the β2GPI/PS complex (t_1/2: >1200 min). The ability to phagocytose opsonized platelets was accordingly sustained (5.3±0.2 opsonized platelets were internalized by each macrophage after 60 min and 9.4±0.3 after 300 min). Anti-β2GPI antibodies therefore poise activated platelets in a PS-exposing status, preventing the recycling of their function and favoring their phagocytic clearance.     

Tolerogenic dendritic cells specific for β2-glycoprotein-I Domain-I,attenuate experimental antiphospholipid syndrome

Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (β₂GPI) tDCs or β₂GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with β₂GPI or D-I or D-V derivatives. Our results revealed that β₂GPI related tDCs phenotype includes CD80^high, CD86^high CD40^high MHC class II^high. The miRNA profiling encompass miRNA 23b^high, miRNA 142-3p^low and miRNA 221^low. In addition the β₂GPI related tDCs showed reduced secretion of IL-1β, IL-12 and IL-23. D-I tDCs treatment was more efficient than β₂GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- β₂GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to β₂GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFβ. Tolerance was successfully transferred by Treg cells from the tolerized mice to β₂GPI immunized mice. We conclude that predominantly D-I-tDCs and β₂GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- β₂GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that β2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.