Karl Skriner

Autoantigen microarrays for multiplex characterization of autoantibody responses

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

CD44 is a determinant of inflammatory bone loss

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell–matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44−/− human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

B and T Cell Responses to the Spliceosomal Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in Normal and Lupus Mice

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1–206 of A2 that contains most of the epitopes recognized by patients’ Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50–70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35–55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35–175 that generated an effective Th cell response with IL-2 and IFN-γ secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.

The concurrence of rheumatoid arthritis and limited systemic sclerosis: Clinical and serologic characteristics of an overlap syndrome

OBJECTIVE The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (IcSSc) were evaluated and compared with those of patients with IcSSc alone (n = 20) or with RA alone (n = 120). METHODS Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti-heterogeneous nuclear RNP (hnRNP)-A2/RA33 (anti-A2/RA33). RESULTS The 3 patients with RA developed IcSSc 11, 29, or 50 years after the onset of RA. Features of IcSSc were Raynauds phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti-A2/ RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti-A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to IcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity-purified antibodies, cross-reactivities between anti-centromere protein A (CENP-A) and anti-CENP-B antibodies with anti-A2/RA33 antigens were seen in the 2 anti-A2/RA33-positive patients. Such cross-reactivities were not found in IcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti-CENP-B reacted with the C-terminal region and anti-A2/RA33 with the second RNA binding domain in the N-terminal region of hnRNP-A2. CONCLUSION The RA-lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynauds phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross-reactivity of ACA with hnRNP-A2/RA33 in the sera of these patients.

Clinical and immunological aspects of autoantibodies to RA33/hnRNP-A/B proteins - A link between RA, SLE and MCTD

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins consisting of two adjacent RNA binding domains (RBD) within the N-terminal parts, whereas the C-terminal halves contain almost 50% glycine residues. These proteins, in particular A2/RA33, are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). In SLE anti-hnRNP antibodies frequently occur together with antibodies to U1 small nuclear RNP (U1-snRNP) and Sm, other proteins of the spliceosome. Preliminary epitope mapping studies have revealed major antibody binding sites in the RNA binding regions for all three diseases. Nevertheless, there is some indication of disease specific epitope recognition. Studies in animal models have demonstrated anti-RA33/hnRNP-A/B antibodies in lupus-prone mouse strains.Thus, autoantibodies to the spliceosomal hnRNP-A/B proteins are a common feature of RA, SLE, and MCTD. However, these diseases differ in their reactivities to other spliceosomal proteins, especially anti-U1 snRNP and Sm. Therefore, anti-RA33/hnRNP-A/B autoantibodies are not only valuable diagnostic markers but may also allow additional insights into the pathogenesis of rheumatic autoimmune diseases.

AUF1, the regulator of tumor necrosis factor α messenger RNA decay, is targeted by autoantibodies of patients with systemic rheumatic diseases

OBJECTIVE To investigate which members of the heterogeneous nuclear RNP (hnRNP) family are targeted by autoantibodies from patients with systemic rheumatic diseases. METHODS Using a semipurified preparation of natural hnRNP proteins, 365 sera from patients with rheumatic diseases and control subjects were screened by immunoblotting for the presence of autoantibodies. Bacterially expressed recombinant hnRNP D (AUF1) proteins were used for confirming the data obtained. Binding of RNA and autoantibody to AUF1 was investigated by gel retardation assays. Expression of AUF1 in cultivated cells and synovial tissue was analyzed by indirect immunofluorescence and immunohistochemistry. RESULTS Autoantibodies to AUF1 proteins were detected in 33% of patients with systemic lupus erythematosus, 20% of patients with rheumatoid arthritis, 17% of patients with mixed connective tissue disease, and <10% of patients with other rheumatic disorders. Epitope mapping studies showed the autoantibodies to be directed to conformational epitopes in the N-terminal RNA-binding part of AUF1. However, autoantibody binding did not interfere with RNA binding as assessed by gel-shift assays. Immunohistochemical studies revealed AUF1 to be expressed in the cytoplasm of RA synovial tissue as compared with nuclear staining in osteoarthritis and normal synovium, particularly in macrophages of the lining layer and in fibroblasts of the sublining areas. CONCLUSION These data identify AUF1 proteins as novel autoantigens in SLE and related autoimmune disorders. Because AUF1 proteins are major components of messenger RNA stability complexes, our findings suggest that these complexes form a novel macromolecular target structure for autoantibodies in rheumatic autoimmune diseases.

Characterization of autoreactive T cells to the autoantigens hnRNP-A2/RA33 and filaggrin in patients with rheumatoid arthritis and controls

In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

Characterization of RA33 (hnRNP-A2/B1)-autoreactive T cells in SLE-patients

SLE is a systemic autoimmune disease with distinct immunological characteristics including defective T cell functions, especially concerning IL2 production and proliferation. Furthermore, B-cell hyperactivity is observed leading to the formation of several characteristic autoantibodies (ab), among them ab to the heterogenous nuclear ribonucleoprotein A2/B1 (hnRNP/RA33). These antibodies are known to occur in over 20% of SLE patients. In order to elucidate the role of T cells and their influence in antibody production in SLE, we studied proliferation of PBMC to purified hnRNP-A2/B1 in 34 SLE patients and 21 healthy controls. While the stimulation indices (SI) in the healthy control group ranged from 0.5 to 3.5 (mean SI: 1.5 ± 0.9), the proliferative response of PBMC of the patient group ranged from 0.7 to 17 with a mean SI of 4.8 ± 4.0 (only 6 of 34 patients had an SI<2; P < 0.00004). We then proceeded to draw RA33-specific T cell clones (TCC) by cultivation and limiting-dilution cloning of T cell lines. The generated 30 TCC derived from SLE patients and 19 TCC from healthy controls did not reveal a significant difference in SI and produced either more IFNγ than IL4 or none of these cytokines at all, suggesting that these TCC were of T1 or T0, but not T2 phenotype. Interestingly though, while only 11% of healthy control patients showed a CD4-/CD8+ subtype and 16% displayed a CD4+/CD8+ phenotype, 37% of TCC derived from SLE patients were CD4-/CD8+ (and 20 % expressed CD4 as well as CD8). Our data reveal that more than 80 % of SLE-patients have a significant T cell reactivity (SI ≥ 2) to the nuclear protein hnRNP-A2/B1 indicating that the antibody response might be T cell driven. Furthermore, almost 60% of TCC derived from SLE patients were CD8+, which supports the importance of these T cells in SLE. Further studies will have to elucidate the pathogenetic implications of these findings.