Decreased levels of serum soluble complement receptor-II (CR2/CD21) in patients with rheumatoid arthritis

Objective. The soluble cluster of differentiation 21 (sCD21) represents the extracellular portion of the CD21 glycoprotein and is released by shedding from cell surfaces into plasma. Soluble CD21 binds complement fragments and activates monocytes through binding to membrane CD23. Elevated levels of sCD21 are found during Epstein-Barr virus EBV infections, B-cell lymphoma and other lymphoblastoid tumours. The present study was undertaken to investigate levels of sCD21 in rheumatoid arthritis. Methods. A specific enzyme-linked immunoassay was developed using sCD21, biochemically purified to homogeneity from human plasma as a standard for the determination of sCD21 concentration in patient sera. Peripheral blood B and T lymphocytes were isolated from healthy donors and rheumatoid arthritis patients and cultured, and supernatants were analysed for CD21 shedding. Results. The normal values of serum sCD21 in healthy individuals between 20 and 40 yr of age ranged from 100 to 477 ng/ml (median 292 ng/ml), decreasing with age but not differing with gender. In rheumatoid arthritis patients, sCD21 levels ranged from 50 to 300 ng/ml (median 182 ng/ml), did not differ with age and were independent of rheumatoid factor. Conclusions. In contrast to healthy donors, patients with rheumatoid arthritis have significantly lower sCD21 levels (P < 0.0001), independently of the age of the patients. Sorted B cells from rheumatoid arthritis patients released amounts of CD21 comparable with those of normal controls. Possible causes and consequences of the findings are discusse

Autoantikörper und das Komplementsystem--Bedeutung für die Pathogenese der Rheumatoiden Arthritis?

Spätestens seit dem Nachweis von Rheumafaktoren (1) als serologischem Merkmal der rheumatoiden Arthritis (RA) kennt man den Zusammenhang dieser Erkrankung mit Autoimmunphänomenen (2), und die rheumatoide Arthritis wird in der Regel den Autoimmunkrankheiten zugerechnet (3, 4). Neben den Rheumafaktoren wurden in den letzten Jahren auch viele weitere Autoantikörper als RA-assoziiert beschrieben (5). Autoantikörper sind die wohl am häufigsten beschriebenen Autoimmunphänomene bei der RA und zudem wichtige Hilfsgrößen in der Diagnostik. Dagegen wurde ihre Rolle in der Pathogenese bislang als eher gering eingestuft: Cytokine, Macrophagen und evtl. T-Lymphocyten sind Komponenten des Immunsystems, denen eher zentrale Positionen in der RA-Pathogenese eingeräumt werden (6)

Decreased levels of serum soluble complement receptor-II (CR2/CD21) in patients with rheumatoid arthritis

OBJECTIVE The soluble cluster of differentiation 21 (sCD21) represents the extracellular portion of the CD21 glycoprotein and is released by shedding from cell surfaces into plasma. Soluble CD21 binds complement fragments and activates monocytes through binding to membrane CD23. Elevated levels of sCD21 are found during Epstein-Barr virus EBV infections, B-cell lymphoma and other lymphoblastoid tumours. The present study was undertaken to investigate levels of sCD21 in rheumatoid arthritis. METHODS A specific enzyme-linked immunoassay was developed using sCD21, biochemically purified to homogeneity from human plasma as a standard for the determination of sCD21 concentration in patient sera. Peripheral blood B and T lymphocytes were isolated from healthy donors and rheumatoid arthritis patients and cultured, and supernatants were analysed for CD21 shedding. RESULTS The normal values of serum sCD21 in healthy individuals between 20 and 40 yr of age ranged from 100 to 477 ng/ml (median 292 ng/ml), decreasing with age but not differing with gender. In rheumatoid arthritis patients, sCD21 levels ranged from 50 to 300 ng/ml (median 182 ng/ml), did not differ with age and were independent of rheumatoid factor. CONCLUSIONS In contrast to healthy donors, patients with rheumatoid arthritis have significantly lower sCD21 levels (P < 0.0001), independently of the age of the patients. Sorted B cells from rheumatoid arthritis patients released amounts of CD21 comparable with those of normal controls. Possible causes and consequences of the findings are discussed

Reduced expression of the complement receptor type 2 (CR2, CD21) by synovial fluid B and T lymphocytes

The expression of CR2 (CD21) by synovial B and T lymphocytes of patients suffering from various forms of arthritis was analysed with cytofluorometry and with reverse transcriptase-polymerase chain reaction. CR2 (CD21) cell surface protein was detected in normal quantities on peripheral B cells, but was almost absent on synovial B lymphocytes of the same patients. This reduction was most severe in patients with rheumatoid arthritis, but also observed in all other cases. CR2 (CD21) did not reappear after in vitro culture. CR2 (CD21) mRNA was also strongly reduced in synovial B and T lymphocytes. Synovial fluid B lymphocytes were larger than peripheral blood B lymphocytes, while T cells from the same patients showed no size differences. We conclude that synovial fluid B lymphocytes have undergone an irreversible step towards terminal differentiation. The presence or absence of CR2 (CD21) mRNA in peripheral versus synovial T cells indicates that CR2 (CD21) is also differentially expressed by T lymphocytes

In silico Epitope Mapping of Glucose-6-Phosphate Isomerase: A Rheumatoid Arthritis Autoantigen

Rheumatoid arthritis-like symptoms can be initiated experimentally in naive K/BxN mice by simultaneously administering the two monoclonal antibodies 11H3 and 46H9. Both antibodies specifically recognize Glucose-6-Phosphate Isomerase (GPI), a known auto antigen in RA patients. Amino acid sequences of the Fv parts of the antibodies were determined by translating the respective hybridoma DNA sequences and served for threedimensional structure modeling of the paratope regions. In silico docking of both Fv antibody structure models to the X-ray structures of the homodimeric murine GPI as well as to the homodimeric human GPI predicted the murine epitope of the 11H3 antibodies to comprise partial amino acid sequences QRVRSGDWKGYTGKS (aa134-148) and AAKDPSAVAK (aa232-241), generating an assembled (conformational) epitope. The 11H3 epitope on human GPI encompasses the matching partial amino acid sequences QRVRSGDWKGYTGKT (aa134-148) and AAKDPSAVAK (aa232-241). The epitope of the 46H9 antibody was determined to consist of the partial murine GPI amino acid sequence RKELQAAGKSPEDLEK (aa446-461) and the human GPI amino acid sequence RKELQAAGKSPEDLER (aa446-461), respectively, resembling consecutive (linear) epitopes. The predicted epitopes were verified by mass spectrometric epitope mapping using synthetic epitope peptides. Peptide QRVRSGDWKGYTGKS[GSMSGS] AAKDPSAAK included a small spacer sequence in between the epitope sequences, mimicking the assembled epitope for the 11H3 antibody. The peptide RKELQAAGKSPEDLEK represented the consecutive epitope for the 46H9 antibody. The determined B-cell epitopes of GPI and their interactions with the monoclonal antibodies provide a detailed structural understanding of immunological disease onset mechanisms in a mouse model of rheumatoid arthritis

Hydrostatic pressure induces apoptosis in the human leukaemic T-cell line Jurkat via the mitochondrial pathway

We investigated the effect of pressure levels ranging from 80 to 500 bar on the proliferative capacity and viability of Jurkat leukaemic T cells. Pressurization at 360 bar induced apoptotic cell death as shown by apoptotic morphology after Hoechst staining, DNA fragmentation in the TdT-mediated dUTP nick end labelling-assay and cleavage of several caspase substrates. Cell death could be prevented by the general caspase inhibitor zVAD-fmk. Breakdown of the mitochondrial membrane potential and the release of cytochrome c provided strong evidence for an involvement of the mitochondrial pathway, whereas a central role of the death receptor pathway was excluded because caspase-8 was not significantly activated. Pressure incubation led to calcium influx after 5 min, and we hypothesize that calcium influx could be the primary trigger for pressure-induced apoptosis

Human IgG1/IgG3 cryoglobulin suggesting lack of allelic exclusion

Immunoglobulins undergoing cold-dependent precipitation are known as cryoglobulins. A type I cryoglobulin after Brouet et al. from serum of a patient with severe cutaneous vasculitis and membranoproliferative glomerulonephritis was purified by reversible temperature-dependent precipitation and analyzed using FPLC, Western blotting and peptide sequencing. The isolated cryoglobulin consisted of a single complex of a molecular weight of above 210 kDa observed under non-reducing conditions in SDS-polyacrylamide gel electrophoresis (PAGE). Under reducing conditions, this complex resolved into three bands, two of which were reminiscent of Ig heavy (HQ chains and one of Ig-light chains (LC). The FPLC-purified type I cryoglobulin showed reversible precipitation analyzed by spectrophotometry. Delineation of the peptides involved in complex formation by immunoblot analysis and peptide sequencing revealed IgG3-V(H)4/Igkappa-VkappaIII/JkappaII and IgG1/V(H)3 molecules with evidence of somatic mutation. Coomassie blue- staining suggested that molar amounts of the IgG3-heavy chain were much higher than that of the IgG1-heavy chain. Treatment with SDS and boiling did not disrupt the unusually high molecular weight Ig complex. Pre-treatment of the cryoglobulin in 6 M guadinium hydrochloride followed by gel filtration chromatography suggested covalent association of the IgG3, IgG1 and Igkappa molecules. Therefore, it might be that the cryoglobulin was produced by a single plasma B cell clone which passed immunological check-points in terms of B cell selection in the bone marrow in the absence of allelic exclusion, class switching and affinity maturation by somatic mutation. (C) 2003 Elsevier Science Ltd. All rights reserved

Mol. Immunol.

Immunoglobulins undergoing cold-dependent precipitation are known as cryoglobulins. A type I cryoglobulin after Brouet et al. from serum of a patient with severe cutaneous vasculitis and membranoproliferative glomerulonephritis was purified by reversible temperature-dependent precipitation and analyzed using FPLC, Western blotting and peptide sequencing. The isolated cryoglobulin consisted of a single complex of a molecular weight of above 210 kDa observed under non-reducing conditions in SDS-polyacrylamide gel electrophoresis (PAGE). Under reducing conditions, this complex resolved into three bands, two of which were reminiscent of Ig heavy (HQ chains and one of Ig-light chains (LC). The FPLC-purified type I cryoglobulin showed reversible precipitation analyzed by spectrophotometry. Delineation of the peptides involved in complex formation by immunoblot analysis and peptide sequencing revealed IgG3-V(H)4/Igkappa-VkappaIII/JkappaII and IgG1/V(H)3 molecules with evidence of somatic mutation. Coomassie blue- staining suggested that molar amounts of the IgG3-heavy chain were much higher than that of the IgG1-heavy chain. Treatment with SDS and boiling did not disrupt the unusually high molecular weight Ig complex. Pre-treatment of the cryoglobulin in 6 M guadinium hydrochloride followed by gel filtration chromatography suggested covalent association of the IgG3, IgG1 and Igkappa molecules. Therefore, it might be that the cryoglobulin was produced by a single plasma B cell clone which passed immunological check-points in terms of B cell selection in the bone marrow in the absence of allelic exclusion, class switching and affinity maturation by somatic mutation. (C) 2003 Elsevier Science Ltd. All rights reserved