Surface APRIL Is Elevated on Myeloid Cells and Is Associated with Disease Activity in Patients with Rheumatoid Arthritis.

OBJECTIVE: To assess surface APRIL (a proliferation-inducing ligand; CD256) expression by circulating myeloid cells in rheumatoid arthritis (RA) and to determine its relationship to disease activity. METHODS: Peripheral blood mononuclear cells (PBMC) and plasma were obtained from patients with RA and healthy donors. PBMC were stained for flow cytometry to detect surface APRIL and blood cell markers to identify circulating myeloid cell subsets. Based on CD14 and CD16 phenotypes, monocyte subsets described as classical (CD14+CD16-), intermediate (CD14+CD16+), and nonclassical (CD14loCD16+) were identified. Levels of surface APRIL expression were measured by flow cytometry and median fluorescence intensity was used for comparisons. Levels of soluble APRIL in the plasma were determined by ELISA. Disease activity was measured by the Disease Activity Score in 28 joints. RESULTS: In patients with RA, total myeloid cells showed expression of surface APRIL that correlated with disease activity and with plasma APRIL levels observed in these patients. In healthy donors, classical monocytes were composed of > 80% of circulating monocytes. However, in patients with RA, the intermediate and nonclassical subsets were elevated and made up the majority of circulating monocytes. In contrast to healthy donors, where high levels of surface APRIL were only observed in nonclassical monocytes, patients with RA showed high levels of surface APRIL expression by all circulating monocyte subsets. CONCLUSION: Surface APRIL is elevated in circulating myeloid cells in patients with RA where it is highly correlated with disease activity. Patients with RA also showed skewing of monocytes toward subsets associated with secretion of tumor necrosis factor-α and/or interleukin 1ß.

Differences in mouse and human nonmemory B cell pools.

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19(+)IgM(+) B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼ 8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

BRAF splice variants in rheumatoid arthritis synovial fibroblasts activate MAPK through CRAF.

Rheumatoid arthritis (RA) is a destructive polyarthritis in which synovial-like fibroblasts (SFs) invade and erode cartilage by expressing membrane-anchored type 1 matrix metalloproteinase (MT1-MMP). The mitogen activated protein kinase (MAPK) pathway is activated in RA SFs, but the mechanism of activation is unknown. Here we identify aberrant BRAF splice variants with deletions in both the kinase domain and RAS-binding domain (RBD) in SFs from the majority of RA patients and show that these BRAF splice variants constitutively activate MAPK through CRAF, increase expression of MT1-MMP, and enhance fibroblast invasion of collagen.

BRAF drives synovial fibroblast transformation in rheumatoid arthritis.

Synovial fibroblasts destroy articular cartilage and bone in rheumatoid arthritis, but the mechanism of fibroblast transformation remains elusive. Because gain-of-function mutations of BRAF can transform fibroblasts, we examined BRAF in rheumatoid synovial fibroblasts. The strong gain-of-function mutation, V600R, of BRAF found in melanomas and other cancers was identified in first passage synovial fibroblasts from two of nine rheumatoid arthritis patients and confirmed by restriction site mapping. BRAF-specific siRNA inhibited proliferation of synovial fibroblasts with V600R mutations. A BRAF aberrant splice variant with an intact kinase domain and partial loss of the N-terminal autoinhibitory domain was identified in fibroblasts from an additional patient, and fibroblast proliferation was inhibited by BRAF-specific siRNA. Our finding is the first to establish mechanisms for fibroblast transformation responsible for destruction of articular cartilage and bone in rheumatoid arthritis and establishes a new target for therapeutic intervention.

Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential.

BACKGROUND: This article reviews research conducted on the immunogenicity of the nucleosides of DNA, especially guanosine, the most immunologically active nucleoside. Discussed is the relationship between circulating antibodies to guanosine, their potential role in SLE disease activity, the binding properties of monoclonal antiguanosine antibody (4H2) compared to polyclonal antiguanosine antibodies in humans with SLE, cell membrane penetration by these antibodies and their interference with signal transduction possibly related to their binding to mitochondria and their apparent GTPase activity. METHODS: Enzyme-linked immunosorbent assay methodology was used to show clinical relationships between antiguanosine antibody levels and disease activity in SLE. These results are discussed along with methods of detecting cell penetration by this antibody using special staining techniques, laser-scanning microscope detection of mitochondrial localization, and interference of cAMP and pKA production/activation. Additionally, there is some discussion regarding the assay used to detect enzymatic activity of antiguanosine antibodies. RESULTS: Enhanced circulating levels of antiguanosine antibodies in patients with SLE correlate closely with SLE disease activity. Other factors are discussed that support the pathogenic potential of these antibodies, including their ability to penetrate lymphocytes, bind to mitochondria, inactivate mitochondrial function, interfere with signal transduction, and their potential enzymatically activity. CONCLUSIONS: Antiguanosine antibodies correlate with SLE disease activity and may be pathogenically important in SLE by interfering with signal transduction, inactivating mitochondrial and cell function in patients with SLE.

Development and evaluation of a patient self-report case-finding method for rheumatoid arthritis.

OBJECTIVE: To describe the development and evaluation of a patient self-report case-finding method for rheumatoid arthritis (RA) not dependent on direct contact with the treating physicians. METHODS: The American College of Rheumatology criteria for RA diagnosis were adapted for patient self-report using a questionnaire, and alternative scoring algorithms were evaluated to balance case-finding sensitivity and specificity. Positive rheumatoid factor tests were used to identify 1053 individuals in 2 large healthcare organizations; 440 agreed to receive study materials. Case-finding results were validated by medical record review (MRR) for a random sample of 90 patients. Three scoring algorithms were compared with MRR for likelihood of RA diagnosis. Cases not classifiable by algorithm were flagged and reviewed by 2 expert physicians for likelihood of RA diagnosis. RESULTS: Pilot testing demonstrated that patients comprehended the questionnaire and were willing to answer the questions. Completed questionnaires were returned by 265 (60%) of the 440 patients contacted. Following expert physician review of 16 flagged cases in the 90-patient MRR subsample, the most accurate scoring algorithm demonstrated 80% sensitivity, 67% specificity, 74% accuracy, and 77% positive predictive value for detecting early RA. CONCLUSION: The case-finding method represents a promising tool for identifying RA patients, with potential application in research and quality-assurance activities. RELEVANCE: This case-finding method should be useful in research and quality-assurance efforts requiring identification of RA patients treated by all types of providers in healthcare organizations in which centralized laboratory data are available.

Antiguanosine antibodies in murine and human lupus have the internal image of G-binding proteins.

OBJECTIVE: To examine the binding specificities of serum IgG antibodies of mouse and human origin directed against guanosine. The immunodominance of guanosine compared with the other nucleosides was established in the MRL/lpr murine model of systemic lupus erythematosus (SLE). Serum antiguanosine autoantibodies in human lupus correlate with nephritis and polyserositis in acute disease as well as in exacerbations of disease symptoms. METHODS: Antiguanosine autoantibodies obtained from humans with SLE were compared to a murine monoclonal antiguanosine antibody, 4H2. The fine specificity of the antiguanosine-binding site was determined by methylation of specific positions on the guanosine molecule and using defined analogs in competitive ELISA. RESULTS: Competitive inhibition assays revealed that serum antiguanosine antibodies bind across the 1 and 7 positions of the guanosine molecule (p < 0.01) and that an oxygen is necessary at position 6 in the molecule. 4H2 exhibited the same binding specificity for guanosine as human polyclonal antiguanosine antibodies, showing a conserved epitope across species. When the fine specificity was compared with known epitopes, the antiguanosine antibodies were found to have the internal image of a G-binding protein, identical to that of the Ha-ras oncogene product p21. CONCLUSION: The finding that antiguanosine autoantibodies vary directly with specific features of SLE, especially nephritis and polyserositis, suggests that they may contribute to the pathology of SLE. Our findings that antiguanosine antibodies have G-binding protein active site homology support the possibility that this species of antibody might interfere with cell signal transduction.

Abnormalities of serum antielastin antibodies in connective tissue diseases.

BACKGROUND: Antibodies (Abs) to alpha-elastin (elastin breakdown product) and tropoelastin (elastin precursor) are found in the serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs, peptides, and elastin metabolism were altered in autoimmune diseases by detecting a changing ratio of serum anti-alpha:tropoelastin Ab levels. METHODS: Serum from patients with a variety of connective tissue diseases, including 28 with systemic lupus erythematosus (SLE), 24 with scleroderma, 18 with rheumatoid arthritis (RA), 10 with polymyositis, and 39 with vasculitis, was compared with serum from 19 age-matched healthy subjects for levels of antitropoelastin and anti-alpha-elastin Abs. RESULTS: We found an increase in IgG anti-alpha-elastin and a decrease in antitropoelastin Abs in the sera of patients with scleroderma (p < .02 and .00005) and SLE (p < .006 and .011). There was also a marked increase in anti-alpha-elastin Abs in patients with polyarteritis nodosa (p < .0005) and decreases in antitropoelastin Abs in patients with RA (p < .05), polymyositis (p < .01), and a variety of other vasculidities (p < .0003). CONCLUSIONS: Abnormal variations in elastin metabolism may be detected in several connective tissue diseases by measuring ratios of alpha- and tropoelastin IgG Abs as markers of elastin degradation and synthesis.