Characterization of changes in IgG associated oligosaccharide profiles in rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis using fluorophore linked carbohydrate electrophoresis.

OBJECTIVE: To investigate fluorophore linked carbohydrate electrophoresis (FCE) as a method of analyzing serum immunoglobulin G (IgG) oligosaccharides in healthy individuals and those with rheumatic disease and compare with lectin binding assays of carbohydrate composition. METHODS: IgG was isolated from patients with rheumatoid arthritis (RA) (n = 21), ankylosing spondylitis (AS) (n = 20), psoriatic arthritis (PsA) (n = 20), and healthy adults (n = 36). IgG oligosaccharides were released enzymatically, fluorescently labelled using 8 aminonaphthalene-136 trisulfonic acid; and identification of the oligosaccharide bands was by stepwise enzymatic degradation. Comparison of FCE was made with lectin binding analysis in which the lectins Ricinus communis (RCA1) and Bandeiraea simplicifolia (BSII) were used to detect galactose (Gal) and N-acetylglucosamine (GlcNAc), respectively. RESULTS: Each disease could be differentiated from healthy adults on the basis of Band 1 asialodigalacto core fucosylated oligosaccharide (gf2) intensity (p = 0.001), but not from each other. Reduced levels of different sugars were associated with specific diseases: reduced gf2 with RA (p < 0.001), PsA (p < 0.001) and AS (p < 0.02), reduced Band 5 disialo-digalacto core fucosylated (a2f) oligosaccharide with AS (p < 0.001), reduced Band 6 disialo-digalacto (a2) oligosaccharide with AS (p < 0.001) and PsA (p = 0.021). All diseases were associated with a significant increase in Band 4 asialo-agalacto core fucosylated oligosaccharide (g0f) (p < 0.001). In RA, FCE band intensities correlated with sugar quantity when identified using lectin binding analysis (p < 0.003). In contrast, there was no correlation between the same bands in healthy individuals. CONCLUSION: FCE is an accurate method of analyzing IgG associated oligosaccharides and reveals unique band patterns or sugar prints associated with healthy adults and patients with RA, PsA, and AS, and comparison with lectin binding analysis suggests undetected RA glycoprotein structural differences. FCE has potential in the early diagnosis and differentiation of rheumatic diseases.

Molecular investigations implicate human endogenous retroviruses as mediators of anti-retroviral antibodies in autoimmune rheumatic disease.

Polymerase chain reaction using specific primers, failed to detect HTLV-I amplicons in patients with rheumatic diseases previously shown to possess antibodies to retroviral products. However, by employing broad spectrum oligonucleotide primers, 135 bp amplicons were generated from peripheral blood mononuclear cells and synovial fluid cells. Subsequent cloning and DNA sequencing revealed homology to a number of exogenous and human endogenous retroviruses (HERVs). Furthermore, in combining the presence of type B and C related endogenous retroviruses, a significant association (p=0.014) was apparent for chronic autoimmune rheumatic diseases as compared to controls. Reverse transcription polymerase chain reaction of RNA derived from patients, healthy controls and cell lines (U937, BJAB, human endothelial lung fibroblasts) demonstrated ubiquitous expression of HERV-K10 and RTVL-H2. Furthermore messenger RNA expression of HERV-K10 was enhanced in fibroblasts infected with human cytomegalovirus. It is plausible that subsequent production of HERV peptides could explain the presence of anti-retroviral antibodies in cohorts of patients with autoimmune rheumatic diseases.

Tissue-specific galactosyltransferase abnormalities in an experimental model of rheumatoid arthritis.

OBJECTIVE: To investigate whether the observed pathophysiological similarities that develop in both the collagen induced experimental model of arthritis (CIA) and rheumatoid arthritis (RA) are associated with similar glycosylation changes, and to evaluate possible differences in the relative activity of the glycosylation enzyme beta, 1-4 galactosyltransferase (GTase) within various tissues, and thus provide a new insight into the potential pathogenic mechanisms controlling glycosylation changes. METHODS: Lymphocytic membrane-bound GTase activity was examined in 30 mice with CIA, 30 age matched controls and 10 adjuvant treated non-arthritic DBA/1 mice. Tissue-specific changes were assessed by comparison of GTase activity in peripheral (P.GTase) and paired splenic lymphocytes. In addition, we also investigated the effect that these changes may exert on the overall extracellular level of this enzyme, by assaying serum GTase (S.GTase) activity in these and a further group of 27 arthritic and 20 control mice. To analyse this synthetic abnormality in greater depth and to investigate the relevance of these glycosylation changes to the pathogenesis of arthritis, we also examined the humoral regulatory component associated with this system by assaying for both anti-collagen as well as anti-GTase antibodies. RESULTS: The induction of arthritis in DBA/1 mice results in a marked reduction in P.GTase activity, compared with age-matched unimmunised mice and the adjuvant controls. In contrast to the P.GTase, splenic GTase activity was found to be similar in all the groups examined. Correspondingly, serum GTase activity was also found to be significantly lower in the collagen induced arthritic mice. This overall reduction in beta, 1-4 GTase activity reflects the clinical severity of arthritis and is associated with increased levels of naturally occurring anti-GTase antibodies. CONCLUSIONS: The GTase defect seen in the peripheral B and T cells in rheumatoid arthritis is also evident in the arthritic DBA/1 mouse model of RA. This may indicate a common pathological process in both rheumatoid disease and CIA, in which changes in glycosylation are dependent on the aberrant modulation of GTase in circulating, but not splenic lymphocytes. The relative expression and activity of glycosyltransferases within various tissues may not only contribute to immunoglobulin G (IgG) glycosylation changes, but perhaps also the aberrant expression of cell surface carbohydrates and thus cell trafficking.

Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods.

Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolialectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.

L-selectin expression on the surface of peripheral blood leucocytes from rheumatoid arthritis patients is linked to disease activity.

Recruitment of mononuclear cells from the circulation to sites of inflammation relies on migration across vessel endothelium. T and B cells, macrophages and neutrophils infiltrate synovial tissue of rheumatoid arthritis (RA) patients. The authors have analysed the numbers of circulating CD3+, CD19+ lymphocytes, monocytes, and granulocytes expressing adhesion molecules (L-selectin, CD44 and CD11a), together with levels of expression in RA patients compared to healthy individuals. Numbers of leucocytes expressing the adhesion molecules detected were similar in RA and control groups. Lower levels of expression of L-selectin on all cells were found in RA patients compared to controls. Expression of L-selectin on T and B cells was found to correlate with disease activity in RA. The authors have observed a characteristic pattern of adhesion molecule expression in RA patients, particularly when analysing the relationships between cells. The close regulation of these molecules between RA patients and healthy individuals is discussed.

Post-partum flare in MRL-lpr/lpr mice is associated with a parallel increase of N-acetylglucosamine on serum IgG.

Our objective was to investigate IgG glycosylation and disease activity post partum in the MRL-lpr/lpr mouse. Disease activity was monitored using bimalleolar ankle swelling. Levels of galactose and N-acetylglucosamine (GlcNAc) on IgG isolated from serum were measured using biotinylated lectins. Our results show that disease severity increased post partum. This post-partum flare correlated significantly with an increase in IgG GlcNAc expression. The disease severity increased post partum in the MRL-lpr/lpr mouse model similar to the changes seen in rheumatoid arthritis (RA).

A detailed lectin analysis of IgG glycosylation, demonstrating disease specific changes in terminal galactose and N-acetylglucosamine.

Serum IgG from rheumatoid arthritis patients contains a decreased number of oligosaccharide structures ending in galactose and thus there is an increase in N-acetylglucosamine as the terminal sugar, compared with healthy individuals. The relationship between these two sugars varies depending on the disease examined: IgG from patients with rheumatoid arthritis, juvenile onset chronic arthritis and Crohn's disease are at one extreme, and exhibit a reciprocal galactose:N-acetylglucosamine relationship, while Sjögren's syndrome and osteoarthritis IgG are at the other extreme, exhibiting a parallel increase in the expression of both galactose and N-acetylglucosamine. These results may occur as a consequence of more than one glycosylation site which is differentially glycosylated, but more likely by changes in the level of bisecting N-acetylglucosamine.

The relationship between exposed galactose and N-acetylglucosamine residues on IgG in rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and Sjögren's syndrome (SS).

The relationship between exposed galactose and N-acetylglucosamine on IgG in RA, JCA and SS was investigated. This was achieved using IgG isolated from serum where the levels of galactose and N-acetylglucosamine (GlcNAc) were detected using biotinylated lectins. Galactose and GlcNAc on IgG from patients with RA and JCA are inversely related, but in contrast, in SS, galactose expression on IgG decreased while GlcNAc expression remained similar to normal levels. Alterations in IgG glycosylation are closely associated with the development of adult and juvenile chronic arthritis and SS, but the changes involved are different in RA compared with SS, suggesting that the precise pattern of exposed sugars is associated with different rheumatological diseases.

Distinct oligosaccharide content of rheumatoid arthritis-derived immune complexes.

OBJECTIVE: To investigate the association between glycosylation and immune complex formation in various disease groups. METHODS: Immune complexes and IgG were isolated from serum and their carbohydrate content evaluated in a dot-blot assay using specifically binding lectins. RESULTS: Significantly more N-acetylglucosamine was detected in complexes from patients with rheumatoid arthritis (RA) than in those from patients with systemic lupus erythematosus, Crohn's disease, or infectious endocarditis, or from normal controls (P < 0.001). The immune complex concentration in the circulation was strongly associated with N-acetylglucosamine levels (P < 0.001 by chi-square analysis). CONCLUSION: The composition of immune complexes from RA patients is distinct in carbohydrate content from those found in other disease groups.

Polymerase chain reaction fails to incriminate exogenous retroviruses HTLV-I and HIV-1 in rheumatological diseases although a minority of sera cross react with retroviral antigens.

OBJECTIVES: To investigate the presence of antibodies to HTLV and HIV retroviral antigens in the rheumatological diseases rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), primary Sjögren's syndrome (pSS), and systemic lupus erythematosus (SLE), and to use polymerase chain reaction (PCR) to seek these exogenous retroviruses in proviral form in cellular DNA from these patients. METHODS: Thirty patients with active RA, 13 with PM, 14 with pSS and five with SLE were recruited and their sera tested for antibodies to HTLV-I in enzyme linked immunosorbent assay (ELISA) and Western blot analysis. Seropositivity to HIV-1 was also sought. DNA was extracted from peripheral blood lymphocytes, synovial tissue and muscle biopsies and tested by polymerase chain reaction using consensus primers for HTLV-I and HIV-1. RESULTS: In HTLV-I ELISA, nine rheumatological sera (4/30 RA, 3/13 PM/DM and 2/5 SLE patients) were considered positive; 14 from pSS patients and 30 from normal subjects were negative. In a control group which included osteoarthritis, Crohn's disease and bacterial endocarditis patients, only two of 80 proved positive in this system. Validation of these sera by Western blotting generally revealed weak reactivity against a variety of HTLV-I antigens. PCR of genomic DNA derived from patients' peripheral blood mononuclear cells did not reveal the presence of HTLV-I and HIV-1 target sequences. CONCLUSIONS: This study shows that PCR precludes HTLV-I and HIV-1 infection as causative agents in these rheumatological diseases although a minority of patients possess antibodies that are weakly cross-reactive with retroviral antigens.

The binding of synovial tissue-derived human monoclonal immunoglobulin M rheumatoid factor to immunoglobulin G preparations of differing galactose content.

There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation. We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactose-deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.

Differential B lymphocyte galactosyltransferase activity in the MRL mouse model of rheumatoid arthritis.

Oligosaccharides can be of fundamental importance to glycoprotein function. Glycosylation abnormalities are present in rheumatoid arthritis (RA) and may be associated with disease pathogenesis. To determine whether similar disease mechanisms occur in the MRL-1pr/1pr autoimmune arthritic mouse, studies on B lymphocyte galactosyltransferase (GTase) have been carried out. In MRL mice, a significant reduction in peripheral blood lymphocyte (PBL) GTase activity was found when compared to their paired splenic (SP) GTase activity (-69%, p = 0.002) and histocompatible non-autoimmune control CBA/Ca mice (-67%; p = 0.002). The changes in PBL GTase activity are similar to those found in RA and on further analysis, using mixing experiments in the presence of purified human milk GTase, this reduction was shown not to be due to the presence of a soluble intracellular GTase inhibitor. Furthermore when examining MRL derived hybridoma cells producing IgG, significantly reduced GTase activity was detected in the rheumatoid factor (RF) producing hybridoma cells compared to those secreting an irrelevant antibody (-21%, p < 0.05). Together these findings suggest that the glycosylation changes observed in this study, and those reported previously in RA, are tissue-specific, may result from cells trafficking from centres of disease activity and are not the result of direct enzyme inhibition. It is now important to further understand the mechanisms controlling glycosylation and relate disease associated changes with those occurring as part of normal cellular physiology.

Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles.

IgG from patients with rheumatoid arthritis (RA) is abnormally glycosylated in the Fc region, with sialic acid and galactose levels lower than normal. Protein G and DEAE purify populations which are differentially glycosylated. Significantly increased exposure of sialic acid was detected in normal IgG compared with that of RA IgG when ion exchange was used to prepare samples. However, when the same samples were prepared using protein G, no difference in the detection of sialic acid was seen between the two groups. When examining the heavy chain of IgG, more sialic acid, galactose and N-acetylglucosamine were detected in DEAE purified IgG compared with that prepared by protein G Detection of sialic acid and N-acetylglucosamine was also increased on light chains from IgG prepared by ion exchange chromatography. Since this occurs notably on rheumatoid light chains it would appear that this arrangement would contribute to the overall glycosylation changes in IgG. In the case of molecules lacking galactose the discrimination between the RA and normal IgG is significantly improved when ion exchange chromatography is used. Since differentiation between disease and normal groups relies on the purification technique used, we recommend that more than one method is employed before undertaking an analysis of glycosylation changes.

Lymphocytes from patients with rheumatoid arthritis produce agalactosylated IgG in vitro.

The percentage of oligosaccharide chains lacking galactose was measured in IgG obtained from pokeweed mitogen-activated cultures of blood lymphocytes from patients with rheumatoid arthritis and controls. Secreted IgG from rheumatoid arthritis lymphocytes was deficient in galactose compared with IgG from the lymphocytes of controls. This confirms that agalactosylation is a significant feature of the disease and demonstrates that it can occur at the B cell level and is not merely a post-secretory event.