ABSTRACT
Mothers giving birth to children with manifestations of neonatal lupus (NL) represent a unique population at risk for the development of clinically evident pathologic autoimmunity since many are asymptomatic and only become aware of anti-SSA/Ro positivity (anti-Ro+) based on heart block in their fetus. Accordingly, we hypothesized that the microbiome in saliva is associated with the development of autoreactivity and in some cases the progression in health status from benign to overt clinical disease including Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The study comprised a clinical spectrum of anti-Ro+ mothers, all of whom gave birth to a child with NL: 9 were asymptomatic or had an undifferentiated autoimmune disease (Asym/UAS) and 16 fulfilled criteria for SS and/or SLE. Microbial diversity was reduced across all levels from kingdom to species for the anti-Ro+ mothers vs healthy controls; however, there were no significant differences between Asym/UAS and SS/SLE mothers. Relative abundance of Proteobacteria and more specifically class Betaproteobacteria decreased with clinical severity (healthy controls <â¯Asym/UASâ¯<â¯SS/SLE). These ordered differences were maintained through the taxonomic hierarchy to three genera (Lautropia, Comamonas, and Neisseria) and species within these genera (L. mirabilis, N. flavescens and N. oralis). Biometric analysis comparing von Willebrand Factor domains present in human Ro60 with L. mirabilis proteins support the hypothesis of molecular mimicry. These data position the microbiome in the development of anti-Ro reactivity and subsequent clinical spectrum of disease.
Subject(s)
Antibodies, Antinuclear/immunology , Dysbiosis , Lupus Erythematosus, Systemic/congenital , Prenatal Exposure Delayed Effects , Salivary Glands/microbiology , Adult , Amino Acid Sequence , Autoantibodies/immunology , Autoimmunity , Biodiversity , Female , HLA Antigens/immunology , Humans , Infant, Newborn , Lupus Erythematosus, Systemic/etiology , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/therapy , Male , Microbiota , Peptides/chemistry , Peptides/immunology , Pregnancy , Young AdultABSTRACT
Linked Comment: Ultrasound Obstet Gynecol 2019; 54: 87-95.
Subject(s)
Antibodies, Antinuclear , Heart Block , Echocardiography , Female , Heart Block/congenital , Humans , PregnancyABSTRACT
OBJECTIVES: To evaluate the efficacy and safety of tabalumab, a human IgG4 monoclonal antibody that neutralises membrane and soluble B-cell activating factor (BAFF). METHODS: This randomised, placebo-controlled study enrolled 1124 patients with moderate-to-severe systemic lupus erythematosus (SLE) (Safety of Estrogens in Lupus Erythematosus National Assessment- SLE Disease Activity Index ≥6 at baseline). Patients received standard of care plus subcutaneous study drug, starting with a loading dose (240â mg) at week 0 and followed by 120â mg every 2â weeks (120 Q2W), 120â mg every 4â weeks (120 Q4W) or placebo. Primary endpoint was proportion achieving SLE Responder Index 5 (SRI-5) improvement at week 52. RESULTS: Clinical characteristics were balanced across groups. The primary endpoint was met with 120 Q2W (38.4% vs 27.7%, placebo; p=0.002), but not with the less frequent 120 Q4W regimen (34.8%, p=0.051). Although key secondary endpoints (time to severe flare, corticosteroid sparing and fatigue) were not met, patients treated with tabalumab had greater SRI-5 response rates in a serologically active subset and improvements in more stringent SRI cut-offs, SELENA-SLEDAI, Physician's Global Assessment, anti-double-stranded DNA antibodies, complement, total B cells and immunoglobulins. The incidences of deaths, serious adverse events (AEs), and treatment-emergent AEs were similar in the 120 Q2W, 120 Q4W and placebo groups, but depression and suicidal ideation, albeit rare events, were more commonly reported with tabalumab. CONCLUSION: SRI-5 was met with 120 Q2W and although key secondary endpoints were not met, numerous other secondary endpoints significantly improved in addition to pharmacodynamic evidence of BAFF pathway blockade. The safety profile for tabalumab was similar to placebo, except for depression and suicidality, which were uncommon. TRIAL REGISTRATION NUMBER: NCT01205438.
Subject(s)
Antibodies, Monoclonal/administration & dosage , B-Cell Activating Factor/antagonists & inhibitors , Lupus Erythematosus, Systemic/drug therapy , Adolescent , Adult , Aged , Antibodies, Antinuclear/blood , Antibodies, Monoclonal, Humanized , Autoantibodies/blood , B-Cell Activating Factor/administration & dosage , B-Lymphocytes/metabolism , Biomarkers/blood , Black People , Complement C3/metabolism , Complement C4/metabolism , Dose-Response Relationship, Drug , Double-Blind Method , Drug Administration Schedule , Female , Humans , Injections, Subcutaneous , Lupus Erythematosus, Systemic/ethnology , Male , Middle Aged , Severity of Illness Index , Treatment Outcome , Young AdultABSTRACT
Autoimmune congenital heart block (ACHB) is an immune-mediated cardiac disease included among the manifestations collectively referred to as neonatal lupus. The placental transference of maternal Ro/La autoantibodies may damage the conduction tissues during fetal development leading to blocking of signal conduction at the atrioventricular (AV) node in an otherwise structurally normal heart. Irreversible complete AV block is the main cardiac manifestation of ACHB, but some babies may develop endocardial fibroelastosis, valvular insufficiency, and/or frank cardiomyopathies with significantly reduced cardiac function requiring transplant. The severity of ACHB is illustrated by a global mortality rate of 20% and pacemaker rates of at least 64%, often within the first year of life. This review analyses the main complex and/or unusual clinical situations associated with ACHB, including unusual maternal immunological profiles, infrequent maternal autoimmune diseases, cardiac damage unrelated to AV block, fetal invasive management, late complications after birth, risk of congenital heart block (CHB) in ovodonation and in vitro fertilization techniques, the role of maternal features other than autoimmunity, the influence of the birth order or the risk of CHB in twins and triplets.
Subject(s)
Heart Block/congenital , Adult , Antibodies, Antinuclear/immunology , Autoantibodies/immunology , Autoimmune Diseases/immunology , Female , Heart Block/diagnosis , Heart Block/immunology , Humans , Infant, Newborn , Male , Pregnancy , Prenatal Diagnosis/methodsABSTRACT
OBJECTIVE: Anti-C1q has been associated with systemic lupus erythematosus (SLE) and lupus nephritis in previous studies. We studied anti-C1q specificity for SLE (vs rheumatic disease controls) and the association with SLE manifestations in an international multicenter study. METHODS: Information and blood samples were obtained in a cross-sectional study from patients with SLE (n = 308) and other rheumatologic diseases (n = 389) from 25 clinical sites (84% female, 68% Caucasian, 17% African descent, 8% Asian, 7% other). IgG anti-C1q against the collagen-like region was measured by ELISA. RESULTS: Prevalence of anti-C1q was 28% (86/308) in patients with SLE and 13% (49/389) in controls (OR = 2.7, 95% CI: 1.8-4, p < 0.001). Anti-C1q was associated with proteinuria (OR = 3.0, 95% CI: 1.7-5.1, p < 0.001), red cell casts (OR = 2.6, 95% CI: 1.2-5.4, p = 0.015), anti-dsDNA (OR = 3.4, 95% CI: 1.9-6.1, p < 0.001) and anti-Smith (OR = 2.8, 95% CI: 1.5-5.0, p = 0.01). Anti-C1q was independently associated with renal involvement after adjustment for demographics, ANA, anti-dsDNA and low complement (OR = 2.3, 95% CI: 1.3-4.2, p < 0.01). Simultaneously positive anti-C1q, anti-dsDNA and low complement was strongly associated with renal involvement (OR = 14.9, 95% CI: 5.8-38.4, p < 0.01). CONCLUSIONS: Anti-C1q was more common in patients with SLE and those of Asian race/ethnicity. We confirmed a significant association of anti-C1q with renal involvement, independent of demographics and other serologies. Anti-C1q in combination with anti-dsDNA and low complement was the strongest serological association with renal involvement. These data support the usefulness of anti-C1q in SLE, especially in lupus nephritis.
Subject(s)
Antibodies, Antinuclear/blood , Complement C1q/immunology , DNA/immunology , Lupus Erythematosus, Systemic/immunology , Adult , Case-Control Studies , Complement System Proteins/deficiency , Cross-Sectional Studies , Female , Humans , Lupus Erythematosus, Systemic/ethnology , Lupus Nephritis/ethnology , Lupus Nephritis/immunology , Male , Middle Aged , Proteinuria/blood , Rheumatic Diseases/immunology , Sensitivity and Specificity , Young AdultABSTRACT
Congenital complete heart block associated with transplacental passage of maternal autoantibodies reactive with SSA/Ro and SSB/La is a rare disease with significant fetal, neonatal, and childhood morbidity and mortality. We present the case of dichorionic, diamniotic twins (female twin A and male twin B) exposed to maternal Ro and La autoantibodies with different disease expression. Twin A (female) had Mobitz type I second degree atrioventricular (AV) block (Wenckebach); twin B (male) had normal sinus rhythm. Both twins had structurally normal hearts but demonstrated echocardiographic evidence of endocardial fibroelastosis (EFE). Following maternal dexamethasone 4 mg once daily, twin A reverted to sinus rhythm in utero; twin B remained in sinus rhythm throughout pregnancy. Echocardiograms after delivery demonstrated resolution of EFE in both fetuses, and EKGs confirmed sinus rhythm. However, at five months of age, Holter monitor demonstrated first degree AV block and intermittent Wenckebach in twin A. Twin B remains in sinus rhythm. This case is one of only three in the literature that describes Mobitz type I second degree atrioventricular block presenting in fetuses exposed to maternal SSA and SSB autoantibodies and is the first case that we have seen reported in twins. Importantly, this case also adds to the growing body of literature describing EFE as a presentation of neonatal lupus with or without conduction system abnormalities, emphasizes the spectrum of cardiac conduction abnormalities in neonatal lupus syndrome, and raises interesting questions about discordant disease expression in twins.
Subject(s)
Antibodies, Antinuclear/immunology , Atrioventricular Block/etiology , Lupus Erythematosus, Systemic/congenital , Pregnancy Complications/immunology , Atrioventricular Block/physiopathology , Dexamethasone/therapeutic use , Echocardiography , Female , Follow-Up Studies , Glucocorticoids/therapeutic use , Humans , Infant , Infant, Newborn , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/immunology , Male , Pregnancy , Twins , Young AdultABSTRACT
The Lupus Foundation of America (LFA) convened an international working group to obtain a consensus definition of disease flare in lupus. With help from the Paediatric Rheumatology International Trials Organization (PRINTO), two web-based Delphi surveys of physicians were conducted. Subsequently, the LFA held a second consensus conference followed by a third Delphi survey to reach a community-wide agreement for flare definition. Sixty-nine of the 120 (57.5%) polled physicians responded to the first survey. Fifty-nine of the responses were available to draft 12 preliminary statements, which were circulated in the second survey. Eighty-seven of 118 (74%) physicians completed the second survey, with an agreement of 70% for 9/12 (75%) statements. During the second conference, three alternative flare definitions were consolidated and sent back to the international community. One hundred and sixteen of 146 (79.5%) responded, with agreement by 71/116 (61%) for the following definition: "A flare is a measurable increase in disease activity in one or more organ systems involving new or worse clinical signs and symptoms and/or laboratory measurements. It must be considered clinically significant by the assessor and usually there would be at least consideration of a change or an increase in treatment." The LFA proposes this definition for lupus flare on the basis of its high face validity.
Subject(s)
Lupus Erythematosus, Systemic/diagnosis , Terminology as Topic , Acute Disease , Delphi Technique , Humans , InternationalityABSTRACT
The physicochemical properties of apparently acid-labile IFN-alpha from patients with SLE have been studied. The antigenicity, apparent molecular size, and isoelectric point of SLE IFN-alpha are indistinguishable from those of conventional, previously characterized, acid-stable subspecies of IFN-alpha. However, after partial purification by anion-exchange chromatography, SLE IFN-alpha no longer exhibits acid lability, suggesting that other plasma factor(s) are responsible for the acid lability of SLE IFN-alpha. Addition of SLE plasma, but not normal plasma, to conventional acid-stable IFN-alpha renders the exogenous IFN-alpha acid labile. Preliminary results demonstrate that an acid-dependent IFN-inactivating activity can be partially purified from SLE plasma by anion-exchange chromatography.
Subject(s)
Interferon Type I/blood , Lupus Erythematosus, Systemic/blood , Chemical Phenomena , Chemistry, Physical , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Drug Stability , Humans , Hydrogen-Ion Concentration , Isoelectric Point , Molecular Weight , Neutralization TestsABSTRACT
The 52-kD SS-A/Ro protein is one of the antigenic targets strongly associated with the autoimmune response in mothers whose children have manifestations of neonatal lupus. In addition to the cDNA clone we previously reported for the full-length 52-kD SS-A/Ro protein, an interesting MOLT-4 cDNA clone, p52-2, was found to have an internal deletion of 231 nucleotides including the domain encoding the leucine zipper motif. To further investigate the nature of this deletion, genomic DNA clones were isolated from a lambda FIXII library. The complete gene for the full-length 52-kD protein (alpha form, 52 alpha) spans 10 kb of DNA and is composed of seven exons. Exon 1 contains only the 5' untranslated sequence, while the translation initiation codon is located 3 kb downstream in exon 2, which also encodes the three zinc finger motifs. Exon 4 encodes amino acids 168-245, including the coiled coil/leucine zipper domain. Exon 7 is the longest and encodes the rfp-like domain and the 3' untranslated region. The cDNA p52-2 can now be accounted for as a product of alternative messenger RNA (mRNA) derived from the splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein (52 beta) with a predicted molecular weight of 45,000. An initial approach to identifying this alternatively spliced form in the human heart used a ribonuclease protection assay. Using an RNA probe corresponding to bases 674-964 of the full-length cDNA, two protected mRNA fragments were identified, a 290-bp fragment corresponding to expression of 52 alpha and a smaller fragment of 144 bp, the predicted size of 52 beta. Using reverse transcription followed by polymerase chain reaction, cDNAs from a 16-wk fetal heart, 24-wk heart, and adult heart were amplified with primers flanking exon 4. Two polymerase chain reaction products were observed in each tissue, one 1.0 kb likely representing 52 alpha and a second 0.78 kb, consistent with 52 beta. The 0.78-kb fragment identified in the 16-wk heart was cloned, and DNA sequencing confirmed the 52 beta type. Immunoprecipitation of in vitro-translated 35S-labeled 52 beta form was performed to evaluate the antigenicity of this novel form of 52-kD SS-A/Ro. 26 (87%) of 30 sera tested from mothers whose children were known to have neonatal lupus immunoprecipitated the 52 beta form.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Alternative Splicing , Autoantigens/genetics , Fetus/immunology , Leucine Zippers/genetics , Myocardium/immunology , RNA, Small Cytoplasmic , Ribonucleoproteins/genetics , Adult , Amino Acid Sequence , Autoantigens/immunology , Base Sequence , DNA, Complementary/genetics , Female , Gene Library , Humans , Infant, Newborn , Lupus Erythematosus, Cutaneous/congenital , Lupus Erythematosus, Cutaneous/genetics , Lupus Erythematosus, Cutaneous/immunology , Lupus Erythematosus, Systemic/congenital , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Molecular Sequence Data , Ribonucleoproteins/immunology , Sequence Analysis, DNA , Sequence Deletion , Sjogren's Syndrome/genetics , Transcription, GeneticABSTRACT
Neonatal lupus is a model of passively acquired autoimmunity whereby anti-SSA/Ro-SSB/La antibodies target the fetal heart and neonatal skin in a minority of cases. Since neuro-psychiatric impairment has been reported in humans and mice exposed prenatally to a variety of maternal autoantibodies including anti-Ro/La, this study was initiated to evaluate the potential neurotoxic effects of these specific autoantibodies and the overall frequency of autoimmune diseases, general health, and somatic growth of children with neonatal lupus and their unaffected siblings. In addition to the general health questionnaires maintained on family members enrolled in the Research Registry for Neonatal Lupus (RRNL), specific questionnaires related to neuro-psychiatric development were sent to all mothers whose children (both affected and unaffected) were older than 5 years of age. Controls consisted of healthy friends. Of 121 anti-Ro exposed children meeting the inclusion criteria, information was returned on 104 (33 cardiac manifestations of neonatal lupus, 20 rash, and 51 unaffected siblings) and 22 of the friend controls. The mean age of all of the children was 14.5 years (range 5-39). In total, 42 (40%) of the 104 anti-Ro exposed children were reported to have a neuro-psychiatric disorder, compared with 6 (27%) of the friend controls (p = 0.34). For 8 (24%) of the congenital heart block (CHB) children (6 boys, 2 girls) the mothers reported attention problems. Four, all boys, were on stimulants. Of the rash children, 4 (20%) (2 boys, 2 girls) had attention problems with one boy on Ritalin. Of the unaffected siblings, 9 (18%) (8 boys and 1 girl) had attention problems with 3 boys on stimulants. One (5%) of the control children (a girl) had attention problems, not requiring therapy. There was no statistical difference in attention problems between the groups (p = 0.120). Behavioral problems were present in all groups with no statistical differences noted. The prevalence of depression, anxiety, developmental delays, learning, hearing, and speech problems were not significantly different between groups. In the CHB children, one boy has nephrotic syndrome and one girl has psoriasis. In the rash children, one girl has juvenile rheumatoid arthritis. In the unaffected group there are five children with autoimmune diseases, two with inflammatory bowel diseases (one boy and one girl), one boy has a spondyloarthropathy, one girl has alopecia areata and one young woman has Antiphospholipid syndrome. In the control group one boy has Henoch Schonlein purpura. There were four cases of hypothyroidism, possibly secondary to Hashimoto's thyroiditis, three in boys with CHB and one in a girl with rash. None of the unaffected siblings or controls had hypothyroidism. Parental reporting of neuro-psychiatric abnormalities was high in anti-Ro exposed children regardless of the neonatal lupus manifestation. However, medication use was limited and although the frequency of this reporting was greater than friend controls, it did not reach significance.
Subject(s)
Antibodies, Antinuclear/immunology , Lupus Erythematosus, Systemic/immunology , Mental Disorders/etiology , Adolescent , Adult , Autoimmune Diseases/immunology , Case-Control Studies , Child , Female , Humans , Infant, Newborn , Infant, Newborn, Diseases , Lupus Erythematosus, Systemic/physiopathology , Male , Mental Disorders/epidemiology , Registries , Retrospective Studies , Ribonucleoproteins/immunology , Sjogren's Syndrome/immunology , Surveys and Questionnaires , Young AdultABSTRACT
One of the strongest associations with autoantibodies directed to components of the SSA/Ro-SSB/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring, an alarming prospect facing 2% of primigravid mothers with these reactivities. This risk is 10-fold higher in women who have had a previously affected child with CHB. Anti-Ro/La antibodies are necessary but insufficient to cause disease. In vitro and in vivo experiments suggest that the pathogenesis involves exaggerated apoptosis, macrophage/myfibroblast crosstalk, TGFbeta expression and extensive fibrosis in the conducting system and in some cases surrounding myocardium. A disturbing observation is the rapidity of disease progression, with advanced heart block and life-threatening cardiomyopathy observed <2 weeks from normal sinus rhythm. Once 3rd degree (complete) block is identified, reversal has never been achieved, despite dexamethasone. Current strategies include the evaluation of an early echocardiographic marker of injury, such as a prolonged PR interval and the use of IVIG as a preventative measure for pregnancies of mothers with previously affected children.
Subject(s)
Autoantibodies/immunology , Autoimmune Diseases/immunology , Fetal Heart/abnormalities , Heart Block/congenital , Heart Block/immunology , Antibodies, Antinuclear , Autoimmune Diseases/congenital , Female , Gestational Age , Heart Block/prevention & control , Humans , Male , Pregnancy , Risk Factors , Time Factors , Ultrasonography, PrenatalABSTRACT
OBJECTIVE: To evaluate autoimmune disease progression in asymptomatic and pauci-symptomatic mothers of children with neonatal lupus (NL). METHODS: Clinical information on mothers enrolled in the Research Registry for NL (RRNL) was obtained from medical records. Genotyping was performed for -308A/G tumour necrosis factor (TNF)alpha, 869T/C transforming growth factor (TGF)beta and -889C/T interleukin (IL)1alpha. RESULTS: Of the 321 mothers enrolled, 229 had at least 6 months of follow-up. Of the 51 mothers who were asymptomatic at the NL child's birth, 26 progressed: 12 developed pauci-undifferentiated autoimmune syndrome (pauci-UAS), 2 poly-UAS, 7 SS, 4 SLE and 1 SLE/SS. The median time to develop any symptom was 3.15 years. Of the 37 mothers classified as pauci-UAS at the NL child's birth, 16 progressed: 5 developed poly-UAS, 6 Sjögren syndrome (SS), 4 systemic lupus erythematosus (SLE) and 1 SLE/SS. Of the pauci-UAS mothers enrolled within 1 year, the median time to progression was 6.7 years. Four mothers developed lupus nephritis (two asymptomatic, two pauci-UAS). The probability of an asymptomatic mother developing SLE by 10 years was 18.6%, and developing probable/definite SS was 27.9%. NL manifestations did not predict disease progression in an asymptomatic mother. Mothers with anti-Sjögren syndrome A antigen (SSA/)Ro and anti-Sjögren syndrome B antigen (SSB)/La were nearly twice as likely to develop an autoimmune disease as mothers with anti-SSA/Ro only. Only TGFbetaT/T was significantly higher in SLE mothers compared to asymptomatic mothers (p = 0.03). CONCLUSIONS: Continued follow-up of asymptomatic NL mothers is warranted since nearly half progress, albeit few develop SLE. While the anti-SSB/La antibodies may be a risk factor for progression, further work is needed to determine reliable biomarkers in otherwise healthy women with anti-SSA/Ro antibodies identified solely because of an NL child.
Subject(s)
Lupus Erythematosus, Systemic/congenital , Mothers , Antibodies, Antinuclear/immunology , Autoantibodies/blood , Disease Progression , Female , Follow-Up Studies , Genotype , Humans , Infant, Newborn , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Polymorphism, Genetic , Registries , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to correlate with development of congenital heart block. The aim of the present study was to evaluate the clinical relevance and predictive value of p200-antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and American women were included in the study. Sera originated from 202 mothers with an infant affected by second- or third-degree atrioventricular block (AVB), 177 mothers with rheumatic disease having infants with normal heart rate and female blood donors (n = 136). A novel serological assay for Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3.8% respectively was developed. Mothers of children affected by AVB II-III had significantly higher p200-antibody levels than mothers with rheumatic disease having children with normal heart rate (P < 0.001). In the Swedish cohort, a distinction between foetuses with normal conduction, AVB I, AVB II and III was possible. A significant difference in anti-p200 levels between AVB I and AVB II-III groups compared with foetuses with normal conduction (P < 0.05 and P < 0.01) was observed. Using p200-antibodies as a second step analysis in Ro52-positive pregnancies increased the positive predictive value for foetal cardiac involvement (AVB I, II or III) from 0.39 (0.27-0.51) to 0.53 (0.37-0.68). In conclusion, Ro52 p200-antibodies may occur in women with unaffected children, but levels are significantly higher in mothers of children with congenital heart block and are suggested as a relevant marker in evaluating the risk for foetal AV block.
Subject(s)
Autoantibodies/blood , GTPase-Activating Proteins/immunology , Heart Block/congenital , Heart Block/immunology , Ribonucleoproteins/chemistry , Adult , Analysis of Variance , Atrioventricular Block/immunology , Autoantibodies/immunology , Biomarkers/blood , Case-Control Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Finland , Humans , Pregnancy , Risk Factors , Sweden , United StatesABSTRACT
The iC3b receptor (CR3) is required for neutrophil adhesive functions, including homotypic aggregation. Because stimuli that enhance neutrophil adhesion also induce up-regulation of surface CR3, it is widely held that these two responses are causally related. We have dissociated CR3 display (immunofluorescence) from CR3 function (aggregation). Neutrophils isolated at 4 degrees C and rewarmed to 37 degrees C up-regulated surface CR3 twofold, but did not aggregate. The kinetics of FMLP-induced CR3 up-regulation were discordant with those of aggregation. In the absence of extracellular divalent cations, CR3 expression increased twofold after exposure to FMLP, but neutrophils did not aggregate. FMLP elicited 3.5-fold more aggregation than the ionophore A23187, yet less than one-half as much CR3 up-regulation. 3 mM sodium salicylate inhibited aggregation 55 +/- 4%, but had no effect on CR3 up-regulation. Conversely, 1 mM tetracaine completely inhibited CR3 up-regulation, while significantly enhancing aggregation. Neutroplasts expressed CR3, but did not up-regulate the receptor; in contrast, FMLP induced CR3-dependent aggregation of neutroplasts. We conclude that, although constitutive surface CR3 is required for neutrophil aggregation, the up-regulation of CR3 is neither necessary nor sufficient to promote cell-cell adhesion.
Subject(s)
Cell Aggregation , Complement C3/metabolism , Neutrophils/metabolism , Receptors, Complement/metabolism , Antibodies, Monoclonal/physiology , Calcimycin/pharmacology , Cations, Divalent , Cell Aggregation/drug effects , Cell Membrane/metabolism , Cell Membrane/physiology , Cytoplasm/metabolism , Cytoplasm/physiology , Humans , Kinetics , Leukotriene B4/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/physiology , Receptors, Complement/drug effects , Receptors, Complement/immunology , Receptors, Complement 3b , Sodium Salicylate/pharmacology , TemperatureABSTRACT
It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.
Subject(s)
Antiphospholipid Syndrome/immunology , Apolipoproteins/physiology , Glycoproteins/physiology , Lupus Coagulation Inhibitor/analysis , Animals , Antibodies, Monoclonal/immunology , Humans , Immunoglobulin G/immunology , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred BALB C , beta 2-Glycoprotein IABSTRACT
Serum SS-A/Ro autoantibodies are commonly found in patients with Sjogren's syndrome, systemic lupus erythematosus, neonatal lupus, and subacute cutaneous lupus. Two proteins of 60 and 52 kD have been described as targets for these autoantibodies. To define the 52-kD component unambiguously, cDNA clones were isolated from human HepG2 and MOLT-4 cell cDNA libraries. The identity of cDNA was established by (a) the specificity of the antibody affinity purified from the recombinant protein, (b) the reactivity of the purified recombinant protein with prototype SS-A/Ro sera in immunoblot and ELISA, and (c) two-dimensional gel comigration of MOLT-4 cell 52-kD protein and the recombinant protein. A 1.9-kb cDNA encoded the complete 52-kD protein containing 475 amino acids (Mr 54,082). Putative zinc-finger domains and a leucine zipper motif were identified in the amino-terminal half of the 52-kD protein, implicating its possible association with DNA/RNA. Sequence homology detected between the 52-kD protein and human ret transforming protein, and mouse T cell gene expression down-regulatory protein rpt-1, may provide leads to the functional role of the 52-kD protein in addition to the possibility that these proteins might constitute members of a subfamily of finger proteins.
Subject(s)
Autoantigens/genetics , RNA, Small Cytoplasmic , Ribonucleoproteins , Zinc Fingers , Amino Acid Sequence , Autoantigens/analysis , Autoantigens/immunology , Base Sequence , Cloning, Molecular , DNA/analysis , DNA-Binding Proteins/analysis , Humans , Molecular Sequence Data , Recombinant Proteins/analysis , Transcription Factors/analysisABSTRACT
Mothers of children with neonatal lupus erythematosus (NLE) and heart block, as well as patients with Sjögren's syndrome (SS) and systemic lupus erythematosus, have serum autoantibodies that recognize SS-A/Ro autoantigens including the 60-kDa ribonucleoprotein. By yeast 2-hybrid screening, we identified a novel 75-kDa protein (pp75) that interacts with the carboxyl 70% of 60-kDa SS-A/Ro. The specificity of interaction was confirmed using mammalian 2-hybrid and chemical crosslinking studies. Immunoprecipitation with radiolabeled HeLa cell extracts showed that pp75 was phosphorylated and associated with 2 other phosphoproteins of 64 kDa. In Northern blot analysis, pp75 was expressed in all tissues analyzed; the highest expression was in the human heart. Based on immunofluorescence of transfected HeLa cells, pp75 is localized predominantly in the cytoplasm, an observation confirmed by immunohistochemistry in untransfected cells. Based on Western blot and ELISA assays, sera from 14 of 84 mothers of children with NLE recognized pp75, including 1 mother in whom anti-SS-A/Ro antibodies were not detected. In addition, sera from 5 of 80 patients with SS were positive for anti-pp75 antibody. Identification of molecular partners is a first step toward elucidating the functions and possible involvement in pathogenesis of long-recognized autoantigens such as 60-kDa SS-A/Ro, which are at present poorly understood.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Carrier Proteins , Mitochondrial Proteins , Phosphoproteins/chemistry , Phosphoproteins/genetics , RNA, Small Cytoplasmic , Ribonucleoproteins/immunology , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , COS Cells , Cloning, Molecular , Female , HeLa Cells , Humans , Lupus Erythematosus, Systemic/congenital , Lupus Erythematosus, Systemic/immunology , Maternal-Fetal Exchange , Models, Genetic , Molecular Sequence Data , Myocardium/metabolism , Phosphoproteins/immunology , Precipitin Tests , Pregnancy , Sjogren's Syndrome/congenital , Sjogren's Syndrome/immunology , Tissue Distribution , Transfection , Two-Hybrid System TechniquesABSTRACT
The molecular basis of autoantibody reactivity with components of the SSA/Ro-SSB/La particle exhibited by sera of mothers of infants with severe and permanent manifestations of neonatal lupus (NLE) was investigated using immunoblotting and immunoprecipitation. The characteristics of NLE that were studied included congenital complete heart block (CCHB), second degree heart block, and hepatic fibrosis. Antibodies specific for one or more components of the SSA/Ro-SSB/La particle were found in sera from all 20 mothers of permanently affected infants. However, no antibody specific for a single peptide of this particle was common to all sera. Using tissue extracts from a human cell substrate, 80% of these sera had antibodies to one or more components of the SSA/Ro particle demonstrable by immunoblotting. The predominant antibody response in the NLE group was to the newly recognized 52-kD SSA/Ro peptide component. In contrast, antibodies to the 60-kD SSA/Ro component although present, were the least represented and not significantly increased in frequency among mothers of these infants, compared with a group of 31 mothers with autoimmune diseases such as systemic lupus erythromatosus (SLE) but who had healthy offspring. Antibodies directed to the 48-kD SSB/La antigen were demonstrated in 90% of the NLE mothers often accompanying antibodies against the 52-kD SSA/Ro component. The combination of antibodies to 48- and 52-kD structures was significantly increased in the NLE group, with an odds ratio of 35. The type of cell or tissue substrate was shown to influence detectability of antibodies. The 52-kD SSA/Ro peptide and the 48-kD SSB/La peptide were abundant in cardiac tissues from fetuses aged 18-24 wk, further supporting the possible relevance of these peptides to heart block.
Subject(s)
Autoantibodies/analysis , Autoantigens/immunology , Heart Block/congenital , Lupus Erythematosus, Systemic/immunology , Pregnancy Complications/immunology , RNA, Small Cytoplasmic , Ribonucleoproteins , Enzyme-Linked Immunosorbent Assay , Female , Heart Block/immunology , Humans , Immunoblotting , Infant, Newborn , Myocardium/immunology , Precipitin Tests , Pregnancy , SS-B AntigenABSTRACT
Two forms of the human 52 kDa SS-A/Ro protein autoantigen, 52alpha and 52beta, are products of alternative mRNA splicing. The 52alpha form is ubiquitously expressed whereas 52beta, lacking the central leucine zipper domain, has been detected at higher levels than 52alpha during certain stages of fetal development. Because 52alpha has sequence similarity with macromolecules associated with transcriptional regulation and the two forms differ only in that 52beta does not contain the leucine zipper, their roles in protein dimer formation and in transcriptional activity were examined. Employing the yeast two-hybrid system, 52alpha was shown to interact with itself but not 52beta. The homodimerization of 52alpha was independently confirmed in gel filtration chromatography using in vitro cDNA template derived translation products and in HL-60 cell extracts; two peaks were observed corresponding to dimer and monomer of 52alpha, while in vitro the translation product of 52beta exhibited only a single monomer peak. In addition, dimer formation was also demonstrated in a chemical cross-linking experiment using HeLa cells transfected with 52alpha. To evaluate effects on transcription, eukaryotic expression plasmids encoding 52alpha or 52beta fused with the GAL4 DNA binding (DB) domain were co-transfected into 293 cells together with a luciferase reporter vector. A 6-fold increase in transcription activity of the reporter was detected with the GAL4-DB-52beta fusion constructs compared to GAL4-DB-52alpha or the empty vector control. We speculate that the ratio of cellular 52alpha and 52beta may play an important role in regulating gene expression as potential repressor and activator respectively.
Subject(s)
Autoantigens/chemistry , Leucine Zippers , RNA, Small Cytoplasmic , Ribonucleoproteins/chemistry , Autoantigens/genetics , Chromatography, Gel , DNA, Complementary/chemistry , Dimerization , Genes, Reporter , HeLa Cells , Humans , Luciferases/genetics , Recombinant Fusion Proteins/chemistry , Ribonucleoproteins/genetics , Transcription, Genetic , Transfection , Two-Hybrid System Techniques , beta-Galactosidase/analysis , SS-B AntigenABSTRACT
OBJECTIVES: The present study describes the demographics, mortality, morbidity and recurrence rates of autoimmune-associated congenital heart block (CHB) using information from the Research Registry for Neonatal Lupus. BACKGROUND: Isolated CHB detected at or before birth is strongly associated with maternal autoantibodies to 48-kD SSB/La, 52-kD SSA/Ro and 60-kD SSA/Ro ribonucleoproteins and is a permanent manifestation of the neonatal lupus syndromes (NLS). Available data are limited by the rarity of the disease. RESULTS: The cohort includes 105 mothers whose sera contain anti-SSA/Ro or anti-SSB/La antibodies, or both, and their 113 infants diagnosed with CHB between 1970 and 1997 (56 boys, 57 girls). Of 87 pregnancies in which sufficient medical records were available, bradyarrhythmia confirmed to be CHB was initially detected before 30 weeks of gestation in 71 (82%) (median time 23 weeks). There were no cases in which major congenital cardiac anatomic defects were considered causal for the development of CHB; in 14 there were minor abnormalities. Twenty-two (19%) of the 113 children died, 16 (73%) within 3 months after birth. Cumulative probability of 3-year survival was 79%. Sixty-seven (63%) of 107 live-born children required pacemakers: 35 within 9 days of life, 15 within 1 year, and 17 after 1 year. Forty-nine of the mothers had subsequent pregnancies: 8 (16%) had another infant with CHB and 3 (6%) had a child with an isolated rash consistent with NLS. CONCLUSIONS: Data from this large series substantiate that autoantibody-associated CHB is not coincident with major structural abnormalities, is most often identified in the late second trimester, carries a substantial mortality in the neonatal period and frequently requires pacing. The recurrence rate of CHB is at least two- to three-fold higher than the rate for a mother with anti-SSA/Ro-SSB/La antibodies who never had an affected child, supporting close echocardiographic monitoring in all subsequent pregnancies, with heightened surveillance between 18 and 24 weeks of gestation.