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Objective.Rheumatoid arthritis (RA) is more common in females, and although the cause of RA is unknown, it is characterized by the production of autoantibodies. The aims of this study were to determine whether RA-associated autoantibodies are more often found in females than males and to identify factors that influence the relationship between sex and seropositivity.Methods.Databases were searched and studies of RA (N ≥ 100) were included if they reported proportion of seropositive patients with RA by sex. Metaanalyses and metaregression were conducted using the random-effects model. Covariates regressed were smoking, age, BMI, Health Assessment Questionnaire–Disability Index (HAQ-DI), and the Disease Activity Score in 28 joints (DAS28).Results.Eighty-four studies with a total of 141,381 subjects with rheumatoid factor (RF) seropositivity and 95,749 subjects with anticitrullinated protein antibody (ACPA) seropositivity met inclusion criteria. The mean age of participants ranged from 37 to 68 years and the proportion of female subjects ranged from 9% to 92%. Results indicated that females were less likely than males to be seropositive: odds ratio (OR) 0.84 [95% CI 0.77–0.91] for RF and OR 0.88 [95% CI 0.81–0.95] for ACPA. BMI, smoking, mean age, DAS28, and HAQ-DI did not affect the relationship between sex and seropositivity.Conclusion.Although studies report that females have higher RA disease activity than males and that seropositivity predicts worse outcomes, females were less likely to be seropositive than males.

Objective: Antibodies to citrullinated and homocitrullinated (also known as carbamylated) proteins, specific for rheumatoid arthritis (RA), are associated with cardiovascular disease (CVD). Immune ...

Rheumatoid arthritis (RA) is a systemic musculoskeletal disease where immune dysregulation and subsequent autoimmunity induce significant synovial joint inflammation and damage, causing pain and disability. RA disease onset is promoted through multifaceted interactions between genetic and environmental risk factors. However, the mechanisms of disease onset are not completely understood and disease-specific treatments are yet to be developed. Current RA treatments include nonspecific disease-modifying antirheumatic drugs (DMARDs) that suppress destructive immune responses and prevent damage. However, DMARDs are not curative, and relapses are common, necessitating lifelong therapy in most patients. Additionally, DMARD-induced systemic immunosuppression increases the risk of serious infections and malignancies. Herein, we review the current understanding of RA disease pathogenesis, with a focus on T and B cell immune tolerance breakdown, and discuss the development of antigen-specific RA therapeutics that aim to restore a state of immune tolerance, with the potential for disease prevention and reduction of treatment-associated adverse effects.

IgA is the second most abundant antibody present in circulation and is enriched at mucosal surfaces. As such, IgA plays a key role in protection against a variety of mucosal pathogens, including viruses. In addition to neutralizing viruses directly, IgA can also stimulate Fc-dependent effector functions via engagement of Fc alpha receptors (FcαRI) expressed on the surface of certain immune effector cells. Neutrophils are the most abundant leukocyte, express FcαRI, and are often the first to respond to sites of injury and infection. Here, we describe a novel function for IgA:virus immune complexes (ICs) during viral infections. We show that IgA:virus ICs potentiate NETosis – the programmed cell death pathway through which neutrophils release neutrophil extracellular traps (NETs). Mechanistically, IgA:virus ICs potentiated a suicidal NETosis pathway via engagement of FcαRI on neutrophils through a toll-like receptor (TLR)-independent, NADPH oxidase complex-dependent pathway. NETs also were capable of trapping and inactivating viruses, consistent with an antiviral function.

Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex., Highlights • Splenocyte proliferation and antibody responses in DR4tg mice immunized with a homocitrullinated peptide were similar for females and males. • The cytokine profile of homocitrullinated peptide-immunized DR4tg mice was differentiated by sex: females had a higher ratio of IL-1α to IL-5. • Anti-homocitrullinated peptide splenocyte and antibody responses were more frequent in DR4tg than wild-type B6 mice.

Objective.Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone.Methods.Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED.Results.Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (rs) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide.Conclusion.Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice.

Introduction Periodontitis (PD) is an environmental risk factor for rheumatoid arthritis (RA), and there is increasing evidence that this is due to infection with Porphyromonas gingivalis. P. gingivalis expresses 3 candidate enzymes potentially capable of breaking tolerance to citrullinated proteins. These include a unique prokaryotic peptidylarginine deiminase (PPAD), which in concert with arginine gingipain (Rgp) citrullinates C-terminal peptides from known RA autoantigens. The third enzyme, P.gingivalis enolase (PgEnolase), induces autoimmunity in mice potentially via molecular mimicry with conserved regions on mammalian enolase. The aim of this study was to determine the contribution of PPAD, PgEnolase and Rgp to the induction of autoimmunity to citrullinated proteins. Materials and methods Cultured P. gingivalis (W83) was separated into cellular subfractions and probed with anti-PPAD, anti-Rgp and anti-PgEnolase antibodies by immunoblotting, and using a citrulline specific probe. C57BL/6 mice were immunised without adjuvant with purified recombinant PPAD, Rgp and PgEnolase, alone or in combinations. Serum taken at 10 and 28 days was used to measure antibodies to PPAD, PgEnolase, and their immunodominant citrullinated peptides CPP3 and CPgE2. ACPA were measured by anti-CCP2, and anti-CEP-1 ELISA. Results PgEnolase, PPAD, and Rgp were detected in the outer membrane of the bacterium together with the highest concentration of citrullinated proteins. In mice, PPAD elicited a robust antibody response indicating it was highly immunogenic without adjuvant. Immunisation with all 3 enzymes (PPAD, Rgp and PgEnolase) induced a significantly increased antibody response to PgEnolase and citrullinated peptides CPP3 and CPgE2 compared to any other group (p Conclusion PPAD, Rgp and PgEnolase are localised in the P. gingivalis outer membrane and are available for immune surveillance. In combination in vivo, they induce anti-P. gingivalis antibodies seen in RA and ACPA. The lack of citrulline specificity of antibody responses is comparable to serology of PD and early pre-symptomatic RA. Thus our model could be used to study tolerance breakdown in PD and pre-RA.

Objective First-degree relatives (FDRs) of rheumatoid arthritis (RA) patients sharing genetic and environmental risk factors for RA may represent a pre-RA state. Since anti–cyclic citrullinated protein/peptide antibodies (ACPAs) appear years before the onset of RA, the purpose of this study was to determine the prevalence of various ACPAs in FDRs of RA patients. Methods We evaluated 88 RA patients, 50 unaffected FDRs, and 20 healthy control subjects. Six different types of ACPAs were determined by enzyme-linked immunosorbent assay. Joint and periodontal disease symptoms were self-reported. Patients and FDRs were HLA typed for the shared epitope (SE) and the RA-protective alleles HLA–DRB*1301/1302. Results FDRs had a high prevalence of ACPAs (48%) as compared to controls (10%). Prevalence of the SE and smoking in FDRs was also high (62% and 49%, respectively). Of all of the ACPAs in the FDRs, 13 of 32 (41%) were of the IgA isotype. The most commonly expressed IgG ACPA targeted citrullinated vimentin, occurring in 20% of FDRs. The FDRs had an average of 1 type of ACPA, whereas the RA patients expressed a median of 5 different ACPAs. The only FDR to later develop RA expressed 4 different ACPAs. Joint and periodontal disease symptoms in the FDRs were significantly associated with smoking (OR 5.714 [95% confidence interval (95% CI) 1.151–28.3] and OR 12.25 [95% CI 2.544–58.99], respectively), but not with ACPAs. Conclusion The rate of ACPA positivity in unaffected FDRs of RA patients with a high prevalence of the SE and smoking was 48%, whereas ACPAs were rare in the healthy controls. ACPAs in the FDRs of RA patients was most commonly of the IgA isotype, but IgG ACPA targeting citrullinated vimentin was also frequently found.

The majority of rheumatoid arthritis (RA) patients express anti-citrullinated protein antibodies (ACPA). Unaffected first-degree relatives of RA patients (FDR) also express ACPA, commonly of the IgA isotype. IgG anti-homocitrullinated/carbamylated protein antibodies (AHCPA) have been detected in both RA and FDR. It is unknown whether other isotypes are expressed. We aim to investigate the AHCPA isotype profile in unaffected FDR of RA patients.The enrolled subjects were examined by a rheumatologist. FDR and healthy controls (HC) were excluded if they had swollen joints. Serum AHCPA targeting homocitrullinated fibrinogen was determined using enzyme linked immunoabsorbant assay (ELISA). FDR were genotyped for HLA-DR4 alleles encoding the shared epitope (SE).125 RA (35 probands), 61 FDR and 40 HC were included. 20% of FDR expressed IgG AHCPA, compared to 30% in RA patients and 5% in HC (p=0.0010 for RA vs. HC). Levels of IgG AHCPA in FDR were similar to RA. FDR rarely expressed IgM (8%) and did not express IgA AHCPA. 20% of RA and 13% of HC subjects expressed IgM, but very few expressed IgA AHCPA (7% in both groups). AHCPA expression in FDR was not significantly associated with joint symptoms, smoking or SE.IgG AHCPA is the most commonly expressed isotype in RA and FDR. The significance of IgG AHCPA in FDR is unclear as it was not associated with joint symptoms or other risk factors for RA. Longitudinal studies are needed to determine whether AHCPA is meaningful in populations at risk for RA.

Objective. To assess the role of T cells in the mouse model of citrullinated human fibrinogen - induced rheumatoid arthritis (RA) using CTLA-4Ig, an agent that blocks T cell costimulation, which is required for T cell activation. Methods. Humanized HLA-DRβ1*0401 - transgenic (DR4-Tg) mice were immunized with Cit - human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4-Tg mice were treated with CTLA-4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA-4Ig - treated or control mice via intraperitoneal injection into naive DR4-Tg mice. Recipient mice also received an intraarticular injection of Cit - human fibrinogen, unmodified human fibrinogen, or vehicle. Results. CTLA-4Ig - treated, but not human IgG1-treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA-4Ig, but not human IgG1, suppressed Cit - human fibrinogen - induced T cell activation, including citrulline-specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1 - treated arthritic mice caused arthritis in recipients, and this occurred when Cit - human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA-4Ig - treated mice were unable to transfer arthritis. Conclusion. Activated citrulline-specific T cells play a direct role in the development and progression of arthritis in this model of Cit-human fibrinogen-induced RA. © 2010, American College of Rheumatology.

Rheumatoid arthritis (RA) is a common autoimmune disease that afflicts the synovium of diarthrodial joints. The pathogenic mechanisms inciting this disease are not fully characterized, but may involve the loss of tolerance to posttranslationally modified (citrullinated) antigens. We have demonstrated that this modification leads to a selective increase in antigenic peptide affinity for major histocompatibility complex (MHC) class II molecules that carry the RA-associated shared epitope, such as HLA-DRB1*0401 (DR4). We describe the induction of arthritis in DR4-IE transgenic (tg) mice with citrullinated fibrinogen, a protein commonly found in inflamed synovial tissue and a frequent target of autoantibodies in RA patients. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a conspicuous polymorphonuclear cell infiltrate. Immunological analysis of these mice through T cell epitope scanning and antibody microarray analysis identified a unique profile of citrulline-specific reactivity that was not found in DR4-IE tg mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen, two conditions where arthritis was not observed. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules. JEM © The Rockefeller University Press.

Aggrecan has been implied as an autoantigen in rheumatoid arthritis (RA). Immunization with aggrecan induces arthritis in BALB/c (H-2d) mice but not in other strains of mice [e.g. C57BL/6 (H-2b)]. In humans, the strongest genetic association with RA is to the shared epitope (SE), and aggrecan peptides are predicted to bind to the SE. Therefore, we hypothesized that C57BL/6 mice transgenic (tg) for the RA SE (DR4 tg mice) may be susceptible to aggrecan-induced arthritis. C57BL/6 and DR4 tg mice were immunized with a mixture of SE-binding aggrecan peptides and tested for immune responses to the corresponding peptides as well as aggrecan. Sustained T- and B-cell immune responses to aggrecan and several of its peptides were detected in DR4 tg mice. C57BL/6 mice showed only transient T-cell responses to different immunizing peptides and little B-cell response. Therefore, an immune response to peptides of aggrecan can be induced experimentally in DR4 tg mice as anticipated from the predicted and actual binding affinities of these peptides for the RA SE. Failure to induce arthritis in these DR4 tg mice may be due to a lack of appropriate non-MHC genes. © 2007 The Authors.

Necrotizing pancreatitis is a severe form of pancreatitis and is associated with substantial morbidity and mortality. We report a case of necrotizing pancreatitis that developed following combined hepatitis A and B vaccination. No other causes of pancreatitis could be determined. Although confirming the diagnosis is challenging, 3 main factors suggest a possible link to the vaccine: the chronology of the events, the patient's human leukocyte antigen genotype and the incongruent immune response to the vaccine components. This report serves to alert physicians to the possible development of necrotizing pancreatitis after vaccination. © 2007 Canadian Medical Association or its licensors.

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4+ T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4+ T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.

Objective.Anticitrullinated protein/peptide antibodies (ACPA) are implicated in rheumatoid arthritis (RA) pathogenesis and linked to the shared epitope (SE). Citrulline modification is very similar to a different modified amino acid, homocitrulline. We investigated antihomocitrullinated protein/ peptide antibody (AHCPA) specificity for RA, whether ACPA were also able to bind homocitrullinated targets, and whether the SE could accommodate homocitrullinated peptide.Methods.Homocitrullinated fibrinogen was used to screen sera from patients with RA, psoriatic arthritis, and systemic lupus erythematosus, and healthy subjects for AHCPA using ELISA. Homocitrullination sites on fibrinogen were identified by mass spectrometry. ACPA were affinity-purified using a synthetic citrullinated peptide and tested for binding to homocitrullinated protein/peptide. Inhibition of antihomocitrullinated fibrinogen antibody binding was examined. Homocitrullinated peptide interaction with the SE was studied using computer modeling.Results.IgG antihomocitrullinated fibrinogen antibodies were found specifically in 49% of patients with RA. Enrichment of ACPA by affinity purification from 5 patients with RA also enriched AHCPA. Serum AHCPA was inhibited by citrullinated fibrinogen and more significantly by homocitrullinated fibrinogen. Computer modeling indicated that the SE could accommodate a homocitrullinated peptide without steric hindrance. Mass spectrometry identified that 89/103 lysines of fibrinogen could be homocitrullinated, and 5 peptides that could be both citrullinated and homocitrullinated and are predicted to bind the SE.Conclusion.Antihomocitrullinated fibrinogen antibodies are specific to RA. The presence of AHCPA coincides with ACPA, and AHCPA copurifies with ACPA in affinity purification and is inhibited by citrullinated and homocitrullinated antigens. Thus AHCPA and ACPA are frequently cross-reactive and homocitrullinated proteins/peptides may bind the SE.

Objective.Various cytokines have been implicated in the pathogenesis of rheumatoid arthritis (RA) and it is known that elevations in multiple cytokines occur prior to disease onset. The objective of our study is to determine whether the cytokine profile in unaffected first-degree relatives (FDR) of patients with RA is distinct from healthy controls.Methods.The sera of patients with RA, their unaffected FDR, and healthy controls were measured for 27 cytokines using a luminex-based multiplexed immunoassay. Subjects were also tested for rheumatoid factor and 6 different anticitrullinated protein/peptide antibodies (ACPA). Discriminant analysis was performed to define the cytokine profile of the 3 study groups.Results.Fourteen patients with RA, 37 unaffected FDR, and 27 healthy controls were enrolled. All patients with RA and 49% of FDR were ACPA-positive. Patients with RA had elevated levels of inflammatory cytokines compared to FDR and controls. Lower interleukin 13 (IL-13) levels were independently associated with RA. IL-4 was significantly lower in FDR compared to controls. Using discriminant analysis based on the levels of all cytokines measured, RA, FDR, and healthy controls could be distinguished with 91% accuracy. ACPA-positive subjects had higher levels of IL-9, but lower levels of IL-12p70 and macrophage inflammatory protein-1β. No individual cytokine was associated with ACPA-positivity in FDR, but the entire cytokine profile accurately distinguished ACPA-positive from ACPA-negative FDR.Conclusion.The cytokine profiles of patients with RA, healthy controls, unaffected ACPA-positive FDR, and ACPA-negative FDR appear to be distinct.

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC –II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms., Author Summary Streptococcus pyogenes is the most common cause of bacterial pharyngitis, also known as ‘strep throat’. However, this organism is also responsible for a range of other important human illnesses including necrotizing fasciitis and rheumatic heart disease (RHD). Indeed, complications from RHD and invasive infections by S. pyogenes are responsible for over one half million deaths per year in the world. S. pyogenes produces potent toxins called superantigens (SAgs), also known as the scarlet fever or erythrogenic toxins. SAgs have been studied for many years, yet we don't understand what purpose SAgs play in the life cycle of S. pyogenes. Rather than studying SAgs in the context of serious streptococcal disease, we studied the role of SAgs in a nasopharyngeal infection model. Our work demonstrates that for S. pyogenes to efficiently infect mice, the mice must express a human protein that is a receptor for the SAgs, and that S. pyogenes must produce SAgs. We further show that immunizing against SAgs prevents nasopharyngeal infection. This work demonstrates that SAgs are important factors for establishing infection by S. pyogenes and that SAgs may be potential candidates for inclusion within a S. pyogenes vaccine.

High titers of anti-citrullinated protein/peptide antibodies (ACPAs) have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of citrullinating enzymes and has been linked to RA. Therefore, our aim was to determine how transcription of PAD4 is regulated in the human myeloid lineage. We located the PAD4 transcription start site and promoter and phylogenetic comparisons of the area identified a 200. bp conserved region. Bioinformatics analysis predicted the presence of a NF-κB binding site and we tested this via luciferase assays. Intriguingly, mutation of the predicted NF-κB site significantly increased biological activity. We used RT-qPCR to quantify PAD4 expression in HL-60 cells treated with TNF-α to activate the canonical NF-κB pathway and found that PAD4 mRNA was reduced in response to TNF-α treatment. Finally, we used chromatin immunoprecipitation (ChIP) to determine NF-κB enrichment at the PAD4 promoter and the p50 subunit of NF-κB was more highly enriched than p65 at the PAD4 promoter. These results suggest that the p50 subunit of NF-κB may play a role in the repression of PAD4 transcription during inflammation. © 2013 Elsevier B.V.

B cells are stimulated by antigens or by polyclonal activators such as bacterial lipopolysaccharide (LPS) to produce antibody. In nonautoimmune strains of mice, LPS-stimulated antibody responses are inhibited by crosslinking the B cell antigen-receptor (BCR), while antigen-driven responses are shut down by cocrosslinking the BCR and the receptor for the Fc portion of IgG (FcγR). BCR signals are poor at shutting off LPS-induced antibody production, including antiss-DNA antibody production, in B cells from NZB, NZB/WF1, and BXSB lupus-prone mice but not MRL/lpr or NZW mice. In the current studies, the defect in NZB B cells was shown to be independent of T cells and macrophages. The inheritance pattern of resistance to BCR ligation of LPS-induced Ig production in BXSB mice could not be assigned to either founding strain. In New Zealand mixed (NZM) recombinant inbred mice, slightly but significantly more resistance was found in a line (NZM2410) that demonstrates a greater degree of clinical autoimmunity than another line (NZM64) with fewer autoimmune problems. The autoimmune defect is specific to BCR signals because inhibition of LPS activation by ligation of MHC class II occurs normally in NZB B cells. Bypassing the BCR by direct stimulation of second messengers with phorbol esters or ionomycin did not overcome the defect, suggesting that defects in downstream signaling events, rather than in the BCR mechanism itself, are responsible for the reduced ability to inhibit the LPS response in NZB B cells. The inability of the BCR signaling pathway to control LPS-induced Ig production in NZB mice was apparent at the level of Hμ-chain mRNA for secreted IgM. These results suggest that autoimmunity-associated B cell defects in BCR signaling and subsequent regulation of LPS-driven antibody responses have a number of inheritance patterns and involve downstream events in signaling pathways in B cells. The defect can result in aberrant regulation of Hμchain mRNA levels for secreted IgM production, and may be a predisposing factor in murine systemic autoimmune disease. © 1995 Academic Press. All rights reserved.

Heavy and light chain variable region nucleotide sequences were derived from 6 human hybridoma antibodies which bear characteristics of antibodies associated with the phospholipid antibody syndrome. All antibodies originated from non-autoimmune individuals and were polyspecific. Four of these reacted with cardiolipin (and other antigens) and three carried the H3 idiotype which is expressed on a high percentage of disease-associated anti-cardiolipin antibodies. This idiotype was localized to the lambda light chain of the H3 monoclonal antibody and found on two other antibodies which like H3 expressed Vλ4 or the related Vλ3 subgroup light chains. The H3 idiotype however did not define these subgroups nor was it required or sufficient for anti-cardiolipin activity. Anticardiolipin binding was found in VH1, VH3 and VH4 heavy chain families and in a Vk1 light chain. The D region was diverse in both length and gene usage. Although all cardiolipin binding antibodies showed little deviation from germline variable (V) gene sequences, where mutations occurred they tended to be replacement mutations and clustered in complementarity determining regions (CDR) suggesting these B cells were derived from antigen-driven responses. These results from our panel of hybridomas and their comparison to other human antibodies provide extensive information on the diversity of genetic elements which can be used by cardiolipin-binding antibodies. We also show gene sequences which encode the disease-associated H3 idiotype and its location on lambda light chains, which imply that some labda light chains may be preferentially utilized in auto-reactive hybridomas. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

First-degree relatives (FDRs) of rheumatoid arthritis (RA) patients sharing genetic and environmental risk factors for RA may represent a pre-RA state. Since anti-cyclic citrullinated protein/peptide antibodies (ACPAs) appear years before the onset of RA, the purpose of this study was to determine the prevalence of various ACPAs in FDRs of RA patients.We evaluated 88 RA patients, 50 unaffected FDRs, and 20 healthy control subjects. Six different types of ACPAs were determined by enzyme-linked immunosorbent assay. Joint and periodontal disease symptoms were self-reported. Patients and FDRs were HLA typed for the shared epitope (SE) and the RA-protective alleles HLA-DRB*1301/1302.FDRs had a high prevalence of ACPAs (48%) as compared to controls (10%). Prevalence of the SE and smoking in FDRs was also high (62% and 49%, respectively). Of all of the ACPAs in the FDRs, 13 of 32 (41%) were of the IgA isotype. The most commonly expressed IgG ACPA targeted citrullinated vimentin, occurring in 20% of FDRs. The FDRs had an average of 1 type of ACPA, whereas the RA patients expressed a median of 5 different ACPAs. The only FDR to later develop RA expressed 4 different ACPAs. Joint and periodontal disease symptoms in the FDRs were significantly associated with smoking (OR 5.714 [95% confidence interval (95% CI) 1.151-28.3] and OR 12.25 [95% CI 2.544-58.99], respectively), but not with ACPAs.The rate of ACPA positivity in unaffected FDRs of RA patients with a high prevalence of the SE and smoking was 48%, whereas ACPAs were rare in the healthy controls. ACPAs in the FDRs of RA patients was most commonly of the IgA isotype, but IgG ACPA targeting citrullinated vimentin was also frequently found.

Autoimmune thrombocytopenia has been attributed to the presence of antiplatelet autoantibodies which mediate platelet destruction. The derivation of these autoantibodies is presently unknown. While normal B cells do not produce these autoantibodies in vivo, it has been demonstrated in vitro by somatic cell hybridization that the B lymphocytes of non-thrombocytopenic individuals have the potential to produce antiplatelet autoantibodies. Antigen specificities of these antibodies are similar to those seen in autoimmune thrombocytopenic purpura and the lupus anticoagulant syndrome. The immunoglobulin V region genes encoding two such human monoclonal antiplatelet antibodies, and anti-GP IIb (STO 171) and an anti-phospholipid antibody (STO 103) derived from tonsillar lymphocytes of a non-thrombocytopenic male, have now been sequenced.These antiplatelet antibodies were found to be encoded by unmutated germline VH and VK genes. The third complementarity determining region (CDR3) of the genes encoding both of these antibodies have unique D regions with evidence of N-nucleotide additions, and the light chain genes show VK-JK junctional diversity. STO 103 is encoded by the VH4 V71-2 germline gene and a truncated JH4 gene. The light chain gene showed closest homology with the VK4 Humk18 gene and JK2 gene. STO 171 showed closest homology with the VH4.18 germline gene and had a complete germline JH6 gene. The light chain of STO 171 is encoded by the VK3 Humkv325 germline gene, which is also used by some rheumatoid factors and cold agglutinins, and a JK4 gene. Although these antibodies were not derived from circulating B cells or found to be actively producing antibody at the time they were harvested, it is possible that naturally occurring antibody producing B cells, similar to those represented here, are recruited for the development of pathogenic autoantibodies in immune thrombocytopenia. © 1994 Academic Press. All rights reserved.

Objective To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles. Methods Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE-transgenic mice and control mice (class II major histocompatibility complex-deficient [class II MHC -/-] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA. Results Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE-transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC -/- control mice. Conclusion This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti-CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA. © 2011 by the American College of Rheumatology.

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4 CD25 FoxP3 regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA. © 2012 Australasian Society for Immunology Inc.

GM 4672 is an IgGκ-producing lymphoblastoid cell line derived from a patient with multiple myeloma. It has been used by many laboratories as a fusion partner for the production of human-human hybridoma monoclonal antibodies. GM 4672 immunoglobulin variable region heavy and light chain family usage was originally assigned to VH1 and VK1, respectively. This assignment was based on the positions of [3H]leucine of the heavy and light chain proteins using the Edman degradation method. Using the polymerase chain reaction and variable region leader primers and constant region primers, we report here the immunoglobulin variable region gene sequence expressed by GM 4672. The VH region belongs to the VH4 family and is most homologous with the V71-2 (87.9%), Dk1, and JH4 germline genes. The entire heavy chain V region contained 41 mutations in 36 codons and included 11 N nucleotide additions flanking the D region. GM 4672 VK region contained a VK1 gene rearranged with a JK4 gene. The VK germline gene used by GM 4672 light chain was not identified but showed the most homology with Vb' germline gene (87.7%). When compared to Vb' and JK4 genes, there were 37 mutations in 30 codons with evidence of antigen selection as determined by the replacement to silent mutation ratio in the complementarity-determining regions. The high frequency of mutations in the V region genes of GM 4672 is comparable to the sequences of other myeloma proteins. © 1993 Butterworth–Heinemann.

Objective. To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA). Methods. Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anticyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients. Results. Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean ± SD 1.36 ± 0.43 versus 1.01 ± 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's ρ = 0.37, P < 0.0001). This correlation was higher in women (Spearman's ρ = 0.60, P < 0.0001) than in men (Spearman's ρ = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction. Conclusion. Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in auto-antibodies. © 2010, American College of Rheumatology.

Summary Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF’s iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

Objective.To evaluate the clinical, serologic, and MHC class II antigen characteristics of a group of patients with primary Sjögren’s syndrome (SS) and severe arthritis.Methods.A case-control study comparing 35 patients with primary SS: 17 with inflammatory arthritis, 18 without arthritis.Results.All patients fulfilled criteria for primary SS. There were no demographic or clinical features other than inflammatory arthritis, often erosive, that distinguished patients with arthritis from those without. All patients had anti-Ro/SSA autoantibodies, most had anti-La/SSB autoantibodies, and a high percentage of these patients had anti-citrullinated peptide antibodies absent in those without inflammatory arthritis. HLA typing revealed that most patients with anti-citrulline antibodies expressed MHC class II molecules with the shared epitope (SE). The presence of DRB1*0301 linked to the expression of anti-Ro/SSA autoantibodies did not influence the level or frequency of anti-citrulline antibodies in these patients.Conclusion.Severe arthritis with features resembling rheumatoid arthritis including erosive disease can occur in primary SS, particularly among those with anti-citrulline antibodies and the SE.

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome. © 2009 Nature America, Inc. All rights reserved.

Based on recent structural analyses of monoclonal autoantibodies, it appears that a number of these antibodies express germ-line immunoglobulin variable region (V) genes with little or no somatic mutation. In addition, our group and others have noted the identity or near identity of some autoantibody-associated V genes to V genes apparently expressed preferentially in the fetal pre-B cell repertoire. To extend these data, we now report that the heavy and light chain V genes of an anti-cardiolipin antibody derived from a healthy individual display 99% nucleotide sequence homology with V genes expressed in early B cell ontogeny. Sequence comparisons indicate the likely use of fetal-restricted V genes by this autoantibody. Taken together with other data on autoantibody V gene usage, these findings provide further evidence for overlap between the autoantibody-associated and early ontogeny expressed V gene repertoires and suggest that natural autoreactivity may be instrumental in the development and maintenance of the normal immune repertoire. © 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

Autoantibodies that bind citrullinated antigens are a sensitive and specific marker for rheumatoid arthritis (RA). While synthetic cyclic citrullinated peptides (CCP) are typically used to identify these antibodies, little is known about antibody reactivity to the predominant citrullinated protein found in the inflamed synovium, citrullinated fibrinogen (CitFib). We assessed the prevalence of anti-CitFib antibodies in patients with various rheumatic diseases.In total, 65 patients with established RA and 63 patients with other rheumatic diseases were tested for serum IgM rheumatoid factor (RF), IgG anti-CCP2, and IgG anti-CitFib antibodies. This cohort was used to determine optimal positive cutoff values for antibody reactivity to CitFib through receiver operating characteristic curve analysis. The specificity of these assays was confirmed with sera from 49 patients with psoriatic arthritis.Antibodies to both citrullinated antigens were identified in the majority of RA patients tested. The overall sensitivity and specificity of the assays were: CCP 82%, 96%, CitFib 75%, 98%, and IgM RF 80%, 64%, respectively. All but one patient that was positive for CitFib was also positive for CCP2, and close to half the RF-negative RA patients were positive for CitFib and CCP2.These results suggest that autoimmunity to CitFib is common in patients with RA and may play a role in disease pathogenesis.

2noted, in the pages of The Lancet, that Edmund F Biernacki (1866–1911) had described ESR a quarter of a century earlier, in 1894, and had provided a thorough protocol for its measurement, including pre-set time points at which to read it. 3 Observations of blood sedimentation during disease predating Biernacki’s report have been considered possible, but have never been documented. Here, we offer evidence for the discovery of ESR and its clinical link to inflammation one century before Biernacki’s work. The description is provided in detail by the British surgeon and anatomist John Hunter (1728–93, figure 1) in his posthumous A Treatise on the Blood, Inflammation, and Gun-Shot Wounds (figure 2). 4

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of ∼21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.

Collagen II arthritis (CIA) represents an animal model of human RA that can be induced in DBA/1J (H-2q) but not in C57BL/6 mice (H-2b). A vigorous CII specific CD4 Th1-cell response but not IgG2 anti-CII antibody or CIA could be induced in C57BL/6 mice made transgenic for the RA shared epitope DR4 (B1*0401). We developed CD4 Th1-cell clones specific for CII from these transgenic (tg) mice in order to determine if the adoptive transfer of these clones into syngeneic tg C57BL/6 recipients could induce CIA. Three bovine CII specific (bCII) CD4 Th1-cell clones and one T-cell line specific for an immunodominant region of bCII (p261-273) were generated. Among these only one clone that could up-regulate anti-CII, IgG2 antibody in the recipient mice was able to induce transient arthritis. However, this level of IgG2 anti-CII antibody was only one-third of that seen in CII immunized DBA/1J mice that develop persistent arthritis. These results confirm our previous observations that the induction of CIA requires a sustained IgG2 antibody response to CII, an effect difficult to achieve even in DR4 (B1*0401) tg mice reconstituted with CD4 Th1 cells. This suggests that a rate limiting step in the development of human RA among those individuals expressing the RA shared epitope may be the requirement to generate sustained levels of complement fixing antibody to arthritogenic antigens. © 2002 Elsevier Science Ltd. All rights reserved.

Rheumatoid arthritis (RA) is an autoimmune disease that is genetically associated with the MHC class II molecule HLA-DRβ1*0401 (DR4). In order to determine if this MHC can influence the immune response to the candidate autoantigen type II collagen (CII), we have studied collagen induced arthritis (CIA) resistant C57BL/6 mice, made transgenic (Tg) for human DR4. These DR4 Tg mice exhibited a strong T cell proliferative response to CII and its DR4 restricted peptide p261-273 after immunization with these antigens that was not seen in the C57BL/6 wild type mice. DR4 Tg mice also exhibited an increase in IFN-γ production in response to CII, indicating the activation of Th1 cells. While these Tg mice produced IgM anti-CII antibodies, they failed to produce a detectable level of IgG2a (Th1 type) anti-bCII antibody and did not develop CIA. This study shows that a Th1 type T cell response to CII can be established in CIA non-susceptible mice by introducing the human transgene, DR4. This T cell response, however, is not sufficient to induce an antibody isotype switch to IgG2a, nor is it sufficient for the induction of CIA. These results may help to explain why many individuals expressing HLA-DRβ1*0401 do not develop RA. © 2002 Elsevier Science Ltd.

Bacterial superantigens (SAgs) are pyrogenic exotoxins that cause oligoclonal T cell activation leading to toxic shock and death. The selective advantage of SAg expression by bacteria remains unknown. We hypothesized that the T cell response to SAg is modulated by the bacteria. If so, one should see intense T cell responses when SAg are introduced into the body alone, but not when the producing bacteria are also present. We examined the responses of primary T cells to a panel of pure staphylococcal SAg or in the presence of heat-killed bacteria. We found that the IL-2 response to SAg was inhibited by the presence of bacteria, while the TNF-? and IFN-? responses were not affected or were even increased. Such a modulation of T cell responses to SAg was mediated by TLR2/6 agonists but not by agonists of TLR1/2, TLR-9 or NOD proteins, involved activation of the NF-_B pathway, and was prevented by anti-TLR-2 blocking antibodies. At the cellular level, this modulation resulted from death of antigen-presenting cells (APCs) but not of T cells. Thus, we show that the response to SAg is modulated by TLR2/6 agonists from the bacterial cell wall. Such a modulation involves the induction of APC death that then limits the capacity of SAg to cause massive T cell activation. Our findings provide an explanation for the long-standing question of selective advantage of SAg expression, and point to novel therapeutic strategies for SAg-associated diseases.

Anti-Sm antibodies although highly specific for systemic lupus erythematosus can only be found in 10-25% of lupus patients and lupus-prone MRL/lpr mice. Molecular studies of these autoantibodies from mice have suggested that the anti-Sm response is Ag driven, its expression is controlled by stochastic events and may originate from the same B cell precursors as anti-DNA antibodies. However, relatively little information regarding the molecular characteristics of anti-Sm antibodies in man has been reported. We studied the V region genes of three IgM hybridoma monoclonal antibodies (BUD 45.12.8, BUD 114.4.11 and BUD 94.91.8) which were selected for Sm reactivity and derived from B cells of a healthy child. Two of these antibodies BUD 45.12.8 and BUD 114.4.11 also reacted with ssDNA, while the third (BUD 94.91.8) did not. Each of these anti-Sm/ RNP antibodies was encoded by different and predominantly unmutated Ig heavy chain germline genes (BUD 45.12.8 by VH3-23, DXP4 and JH4b; BUD 94.91.8 by VH3-33, D21-9 and JH6b; BUD 114.4.11 by VH 1-2, DK1 or DM1 or unknown D and JH4b) and light chain genes (BUD 45.12.8 by Humkv325 and Jκ2; BUD 94.91.8 by hsigg11150 (λIIIb) and Jλ2/3; BUD 114.4.11 by Humk18 and Jκ3). Many of these genes are also used by antibodies with other specificities including DNA. The two anti-Sm antibodies which also bound ssDNA shared an overall V region net positive charge, while the third antibody without ssDNA reactivity carried a negative V region net charge. These findings demonstrate that (1) normal individuals have the genetic potential to generate autoantibodies to Sm/RNP; (2) acquisition of Sm/RNP binding is not dependent on somatic mutations and (3) some human B cell clones exhibit specificity for Sm and ssDNA.

For strong inhibition of B cells, B-cell antigen-receptors (BCR) and Fc-receptors (FcR) are coligated. Of the many FcR, membrane-associated Fc{gamma}RIIb regulates negatively. Membrane Fc{gamma}RIIb isoforms consist of an extracellular (EC) ligand-binding region containing two immunoglobulin-like domains, a transmembrane (TM) region, and an intracytoplasmic (IC) region. Mouse Fc{gamma}RIIb receptors are encoded by fcgr2 and isoforms generated by alternate splicing of mRNA to give: (1) Fc{gamma}RIIb1 possessing a 47 amino acid membrane-proximal insert in the IC region, and (2) Fc{gamma}RIIb2 lacking this insert. The 47 amino acid insert binds the cytoskeleton to prevent localization of Fc{gamma}RIIb1 in endocytotic pits. The objectives of this study were: (1) to determine the cDNA and corresponding peptide sequences of membrane Fc{gamma}RIIb1 and -b2 isoforms from normal DBA/2 and from autoimmune NZB mice, and (2) to compare these sequences with those of BALB/c and A/WySn non-autoimmune mice, and (3) to identify changes in Fc{gamma}RIIb structure in NZB mice. 11 refs., 1 fig.

Objective. To investigate the structural basis for DNA binding of the natural human IgMλ monoclonal antibody KIM4.6. Methods. An IgMλ, non–DNA‐reactive variant hybridoma was derived during in vitro subcloning of the anti‐DNA antibody KIM4.6. The variable (V)‐region heavy (H) and light (L) chain genes expressed by the variant hybridoma were amplified by polymerase chain reaction, cloned, sequenced, and compared with those of the KIM4.6 parent and other DNA‐binding and non–DNA‐binding antibodies. Results. The VL chain of the variant was identical to that of KIM4.6. In contrast, the VH chain was completely different from the VH chain of the parent but was similar or identical, except in the diversity (D) and joining regions, to the VH chain of the systemic lupus erythematosus (SLE) IgG anti‐DNA antibody T14 and SLE IgM nephritogenic anti‐DNA antibodies NE‐1 and NE‐13. Conclusion. The expression of the KIM4.6 VL chain is not sufficient for DNA specificity. The VH chain and its D region play a key role in conferring DNA binding of the KIM4.6 anti‐DNA antibody. Copyright © 1995 American College of Rheumatology