Angela Bond

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.

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.

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

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.

THE ROLE OF ANTIGEN IN AUTOIMMUNE RESPONSES WITH SPECIAL REFERENCE TO CHANGES IN CARBOHYDRATE STRUCTURE OF IGG IN RHEUMATOID-ARTHRITIS

Abstract Evidence indicating an important role for antigen in the provocation of autoimmune responses is presented. Attention is especially focused on carbohydrate abnormalities in IgG in rheumatoid arthritis, since autosensitization to this molecule is thought to be of central importance in the pathogenesis of this disease. A higher percentage of Fcγ oligosaccharide chains in the serum IgG of patients with rheumatoid arthritis lack terminal galactose residues relative to age-matched controls. This does not appear to be a characteristic feature of chronic inflammatory diseases in general. A new, more rapid assay for agalactosyl chains is described and shown to give results comparable to the more conventional biochemical analysis. The defect probably arises from a reduction in activity of B-cell galactosyltransferases. The galactose changes may contribute to the autoantigenicity of IgG and could facilitate the self-association of IgG rheumatoid factors.

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.

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.

Immunofluorescent technique for the detection of monoclonal internal image anti-idiotypic antibodies of hepatitis B surface antigen

Monoclonal anti-idiotypic antibodies to HBsAg were screened by immunofluorescence for the presence of a subset behaving as the internal image of the original antigen. We describe the technique and the criteria fulfilled to establish that 2/6 monoclonals studied act as the internal images of the a determinant of hepatitis B surface antigen.

Changes in the glycosylation of IgG in the collagen-induced model of arthritis

It is now well established that rheumatoid arthritis patients have reduced levels of galactose on their immunoglobulin G (IgG) molecules compared with normal individuals. We have investigated whether, in an experimentally induced model of arthritis, similar glycosylation changes on IgG are to be found. Serum IgG was isolated from collagen-induced arthritic DBA/1 mice and a control group, and the glycosylation of the IgG in these preparations was compared using lectin blotting. The glycosylation of IgG in immune complexes was also analysed. Arthritic mice exhibited similar glycosylation changes on their IgG as observed for rheumatoid arthritis patients. On average, there was less galactose on the IgG from arthritic mice than from the control group, but this difference was of borderline significance. However, theN-acetylglucosamine content of IgG was significatly elevated in arthritic mice. There was no difference in the sialic acid content of IgG in the two groups. The results for immune complexes were similar to those obtained for serum IgG, but the data were limited by insufficient numbers. The similarity in glycosylation changes in collagen-induced arthritis and in patients with rheumatoid arthritis suggests that common pathogenic mechanisms may be involved.

The Role of Antigen in Autoimmune Responses with Special Reference to Changes in Carbohydrate Structure of IgG in Rheumatoid Arthritis

Evidence indicating an important role for antigen in the provocation of autoimmune responses is presented. Attention is especially focused on carbohydrate abnormalities in IgG in rheumatoid arthritis, since autosensitization to this molecule is thought to be of central importance in the pathogenesis of this disease. A higher percentage of Fcγ oligosaccharide chains in the serum IgG of patients with rheumatoid arthritis lack terminal galactose residues relative to age-matched controls. This does not appear to be a characteristic feature of chronic inflammatory diseases in general. A new, more rapid assay for agalactosyl chains is described and shown to give results comparable to the more conventional biochemical analysis. The defect probably arises from a reduction in activity of B-cell galactosyltransferases. The galactose changes may contribute to the autoantigenicity of IgG and could facilitate the self-association of IgG rheumatoid factors.