Frank C. Hay

Socioeconomic status, C-reactive protein, immune factors, and responses to acute mental stress

Low socioeconomic status (SES) is associated with increased risk of coronary heart disease and immune-related disorders. We hypothesised that SES would be inversely associated with the acute phase reactant C-reactive protein (CRP) and with circulating lymphocyte levels, and that lymphocyte responses to acute psychological stress would also vary with SES. CRP was obtained from 226, and lymphocyte counts from 127 healthy volunteers from the Whitehall II cohort, and SES was defined primarily by grade of employment. CRP concentration was greater in lower compared with higher SES participants (1.18+/-0.75 vs. 0.75+/-0.8 mg/l,p=.002) independently of sex, age, body mass, waist/hip ratio, smoking, alcohol, and season of the year. Similar differences were evident when SES was defined by income and educational attainment. Higher SES was also associated with lower total lymphocyte (p=.023), T-lymphocyte (p=.024) and natural killer (NK) cell counts (p=.006). Total, T- and B-lymphocyte, and NK cell counts increased with stress, but immune stress reactivity did not vary with SES. Post-stress recovery was delayed in women compared with men. The results suggest that moderate inflammation and immune activation may be processes through which lower SES increases disease risk.

Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis.

Autoantigens cross reactive with mycobacteria are implicated in the pathogenesis of adjuvant arthritis in the rat, and there are reports of changes in the immune response to mycobacteria in human rheumatoid arthritis (RA). We have therefore examined the IgM, IgG, and IgA antibody levels to crude mycobacterial antigens and to two recombinant mycobacterial heat shock/stress proteins (65 kD and 71 kD) in sera from patients with RA, systemic lupus erythematosus (SLE), and Crohns disease, and from healthy controls. IgA binding to the crude mycobacterial antigens was significantly raised in RA sera, though IgG and IgM binding tended to be lower than in controls. Both IgA and IgG binding to the heat shock proteins were significantly raised in the RA sera. Smaller significant rises in both classes were seen in sera from patients with SLE, and in the IgA class only to the 65 kD protein in Crohns disease. The rises in IgG and IgA antibodies to the 65 kD protein in RA were significantly higher than in the other diseases, however. It is interesting that this protein is the one responsible for adjuvant arthritis in the rat.

Analysis of glycosylation changes in IgG using lectins

A simple rapid assay based on the ability of lectins to bind carbohydrates has been developed to analyse changes in the oligosaccharide chains of IgG. Bandeiraea simplicifolia lectin and Ricinus communis agglutinin have been used to detect terminal N-acetylglucosamine and galactose moieties respectively in IgG using immunodot-blotting. IgG samples (approximately 1 micrograms) were dot-blotted onto nitrocellulose followed by boiling of the blots to expose the carbohydrate moieties. The blots were then treated with biotinylated lectins followed by either streptavidin-biotin-hydrogen peroxidase conjugate or 125I-labelled streptavidin. The colour was developed using chloronaphthol and the blots read on a densitometer. The labelled blots were cut and read on a gamma counter. The use of a monoclonal antibody to N-acetylglucosamine is also discussed. The results obtained using this method are comparable to those obtained by structural analysis.

Intra-articular and circulating immune complexes and antiglobulins (IgG and IgM) in rheumatoid arthritis; correlation with clinical features.

Solid phase radioimmunometric methods have been used to assay immune complexes and IgG and IgM antiglobulins in paired samples of synovial fluid and serum from patients with rheumatoid arthritis (RA) or osteoarthrosis. Over 60% of RA patients had some increase in complexes in their sera, while nearly 90% had synovial fluid complexes. Moreover, the levels of complexes within the joint were much higher than in the serum. Both IgG and IgM antiglobulins were raised in most RA patients.The levels of IgG antiglobulins--and to a less extent IgM antiglobulins--were nearly always higher in synovial fluid than in the corresponding serum sample.A strong correlation was found between the levels of immune complex and IgG antiglobulin. A marked association was seen between the presence of subcutaneous nodules and increased IgG antiglobulins.

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.

A critical evaluation of the magnetic cell sorter and its use in the positive and negative selection of CD45RO+ cells

In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and present a critical evaluation of its pitfalls and benefits. Satisfactory separation of lymphocytes into subsets with preservation of function can be achieved, but there are several drawbacks: in comparison with Dynal beads, MACS results in a higher cell loss due to the increased number of separation steps and requires depletion of plastic-adherent cells as these will engulf microbeads and contaminate the enriched fraction, and is more expensive. The advantage of MACS over Dynal beads is that the microbeads are biodegradable and do not interfere with proliferation assays: both the depleted and enriched fractions of cells can therefore be used in culture immediately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (greater than 98%), but a depleted fraction of greater than 90% purity could not be obtained even after running the same sample over three separating columns. Dynabeads, on the other hand, achieved 98% pure CD45RO-depleted fractions after three separation runs.

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.