Stina Salomonsson

Expression of the B Cell‐Attracting Chemokine CXCL13 in the Target Organ and Autoantibody Production in Ectopic Lymphoid Tissue in the Chronic Inflammatory Disease Sjögren's Syndrome

Sjögrens syndrome is an autoimmune disease characterized by lymphocytic infiltrates resembling secondary lymphoid organs in salivary glands. In this study, we demonstrate the expression of the lymphoid tissue homing chemokine CXCL13 (BCA‐1/BLC), which has attracting properties for B cells and subsets of activated T cells, in salivary glands of patients with Sjögrens syndrome using immunohistochemistry and in situ hybridization. CXCL13 expression was primarily observed in epithelial cells in acini and ducts of inflamed glands while its receptor, CXCR5 (BLR‐1), was expressed on the infiltrating mononuclear cells. In addition, cells producing antibodies against one of the major autoantigens in Sjögrens syndrome, Ro 52, were identified at the periphery of the follicular infiltrates indicating that the ectopic lymphoid tissue is directly involved in the disease process. Identification of CXCL13 and CXCR5 in salivary glands suggests that the target organ plays an essential role in the inflammatory process by recruiting B and T cells. These results also provide a molecular mechanism by which lymphoid neogenesis and ectopic germinal centre formation might occur in the glands of these patients, which may be the key step in the development of the chronic inflammatory process in Sjögrens syndrome.

Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200–239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

T Cell Infiltrates in the Muscles of Patients with Dermatomyositis and Polymyositis Are Dominated by CD28null T Cells

Dermatomyositis and polymyositis are disabling rheumatic diseases characterized by an appreciable number of T cells infiltrating muscle tissue. The precise phenotype, function and specificity of these cells remain elusive. In this study, we aimed to characterize T cells in muscle tissue and circulation and to investigate their association to clinical phenotype. Twenty-four patients with dermatomyositis and 42 with polymyositis were screened for frequency of CD4+CD28null and CD8+CD28null T cells in peripheral blood by flow cytometry. Presence of these cells in inflamed muscle tissue from 13 of these patients was analyzed by three-color immunofluorescence microscopy. Effector functions, proliferation and Ag specificity were analyzed by flow cytometry after in vitro stimulation. The clinical relevance of CD28null T cells was analyzed by multiple regression analyses including six separate and combined disease variables. We demonstrate that muscle-infiltrating T cells are predominantly CD4+CD28null and CD8+CD28null T cells in patients with dermatomyositis and polymyositis. Muscle-infiltrating CD28null T cells were found already at time of diagnosis. Disease activity correlated with the frequency of CD8+ T cells in the inflamed muscles of polymyositis patients. Circulating CD4+CD28null and CD8+CD28null T cells were significantly more frequent in human CMV (HCMV) seropositive individuals, responded to HCMV Ag stimulation, and correlated with disease duration. These cells also display a proinflammatory cytokine profile, contain perforin and lack the costimulatory molecule CD28. Our observations imply that CD28null T cells represent clinically important effector cells in dermatomyositis and polymyositis, and that HCMV might play a role in propagating disease in a subset of patients.

Local production of Ro/SSA and La/SSB autoantibodies in the target organ coincides with high levels of circulating antibodies in sera of patients with Sjögren's syndrome

Objective: To analyze Ro and La autoantibody levels in the periphery and production in the target organ in patients with Sjögrens syndrome. Methods: The autoantibody production against the Ro and La proteins was investigated in 12 patients with Sjögrens syndrome. ELISA with recombinant antigens was used to determine levels in sera. The distribution of the antibody‐producing plasma cells in the target organ was studied by immunohistochemistry using biotinylated antigens. Results: All investigated patients with Sjögrens syndrome had detectable levels of Ro and La antibodies in sera, while local antibody‐production in the salivary glands was restricted to patients with high antibody levels. The autoantibody‐producing cells were identified at the periphery of the infiltrates and in interstitial spaces. Conclusions: The chronic inflammation of the salivary glands in Sjögrens syndrome may be a self‐sustaining process promoting autoantibody production in the target organ, possibly contributing to increased serum levels of autoantibodies.

Development of heart block in children of SSA/SSB-autoantibody-positive women is associated with maternal age and displays a season-of-birth pattern

Objective Congenital heart block may develop in the fetuses of Ro/SSA-positive and La/SSB-positive mothers. Recurrence rates of only 10–20% despite persisting maternal antibodies indicate that additional factors are critical for the establishment of heart block. The authors investigated the influence of other maternal and fetal factors on heart block development in a Swedish population-based cohort. Methods The influence of fetal gender, maternal age, parity and time of birth on heart block development was analysed in 145 families, including Ro/La-positive (n=190) and Ro/La-negative (n=165) pregnancies. Results There was a recurrence rate of 12.1% in Ro/La-positive women, and no recurrence in Ro/La-negative women. Fetal gender and parity did not influence the development of heart block in either group. Maternal age in Ro/La-positive pregnancies with a child affected by heart block was, however, significantly higher than in pregnancies resulting in babies without heart block (p<0.05).Seasonal timing of pregnancy influenced the outcome. Gestational susceptibility weeks 18–24 occurring during January–March correlated with a higher proportion of children with heart block and lower vitamin D levels during the same period in a representative sample of Swedish women and a corresponding higher proportion of children with heart block born in the summer (p<0.02). Maternal age or seasonal timing of pregnancy did not affect the outcome in Ro/La-negative pregnancies. Conclusion This study identifies maternal age and seasonal timing of pregnancy as novel risk factors for heart block development in children of Ro/La-positive women. These observations may be useful for counselling when pregnancy is considered.

A population-based investigation of the autoantibody profile in mothers of children with atrioventricular block.

The objective of the study was to investigate the antigen specificity and occurrence of individual autoantibodies in mothers of children diagnosed with atrioventricular (AV) block in a nation‐wide setting. Patients with AV block detected before 15 years of age were identified using national quality registries as well as a network of pediatric and adult cardiologists and rheumatologists at the six university hospitals in Sweden. Patients with gross heart malformations, surgically or infectiously induced blocks were excluded. Blood samples were obtained from the mothers and maternal autoantibody profile, including the occurrence of antibodies against Ro52, Ro60, La, SmB, SmD, RNP‐70k, RNP‐A, RNP‐C, CENP‐C, Scl‐70, Jo‐1, ribosomal RNP and histones was investigated in 193 mothers of children with AV block by immunoblotting and ELISA. Autoantibody reactivity was detected in 48% (93/193) of the mothers of children with AV block. In autoantibody‐positive mothers, the vast majority, 95% (88/93), had antibodies against Ro52, while 63% (59/93) had autoantibodies to Ro60 and 58% (54/93) had autoantibodies to La. In addition, 13% (12/93) of the autoantibody‐positive mothers had antibodies to other investigated antigens besides Ro52, Ro60 and La, and of these anti‐histone antibodies were most commonly represented, detected in 8% (7/93) of the mothers. In conclusion, this Swedish population‐based study confirms that maternal autoantibodies may associate with heart block in the child. Further, our data demonstrate a dominant role of Ro52 antibodies in association with AV block.

Upregulation of MHC class I in transgenic mice results in reduced force-generating capacity in slow-twitch muscle

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment, we used transgenic mice with inducible overexpression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast‐twitch) and soleus (slow‐twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared with control mice; however, when force was expressed per cross‐sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force‐generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. Muscle Nerve, 2008

Structurally Derived Mutations Define Congenital Heart Block‐Related Epitopes Within the 200–239 Amino Acid Stretch of the Ro52 Protein

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200–239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody–antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200–239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient‐derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI‐TOF‐MS demonstrate that the antibody recognition is dependent on a partly α‐helical fold within the putative leucine zipper of the 200–239 aa stretch and that the two human anti‐p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1‐like antibodies.

Maternal MHC Regulates Generation of Pathogenic Antibodies and Fetal MHC-Encoded Genes Determine Susceptibility in Congenital Heart Block

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1av1 (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1l). Different anti-Ro52 Ab fine specificities were generated in RT1av1 versus RT1l animals. Maternal and fetal influence was determined in an F2 cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1l than RT1av1 pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1l pups more frequently developed heart block than RT1av1 pups after passive transfer of RT1av1 anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52–positive pregnancies.

Autoantibodies Associated with Congenital Heart Block

Congenital heart block is the most severe manifestation of neonatal lupus syndrome. It is a passively acquired disease where transplacental passage of maternal autoantibodies is associated with irreversible damage of the foetal cardiac conduction system. It is well established that the condition, in the absence of structural abnormalities, is strongly associated with maternal autoantibodies to the Ro/La antigens. More specifically the disease has been closely linked to antibodies to the Ro52 component of the antigen complex. Congenital heart block constitutes a unique model where specific autoantibodies target and mediate organ‐specific disease. A wide panel of maternal antibodies has been discussed in literature in association with the disease and are described in this review.