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Biomedical subjects

I M Roitt

Publications and source records attributed to I M Roitt.

At least 37 records · Page 2Linked to original sources

IgG glycosylation in autoimmune-prone strains of mice.

The relationship between increased levels of IgG oligosaccharide chains lacking galactose (G0) and the development of rheumatoid arthritis is unclear. In order to further our understanding of the observed correlation between raised serum G0 and arthritis, we have studied G0 levels in arthritis-prone and non-susceptible (i.e. non-arthritis-prone) mice and the effects on G0 of mycobacterial antigens, which have been postulated to play a role in the early events leading to the development of arthritis. We have shown that different age-matched mouse strains have characteristic 'resting' levels of G0 which (in six out of seven strains of mice) increase with age. We have also shown that these increases can be enhanced by immunization of arthritis-prone strains of mice with an adjuvant containing mycobacteria (Freund's complete adjuvant (FCA)), suggesting that deflects in the ability to regulate these G0 changes may be related to susceptibility to arthritis.

Age Factors↗

Production of erythrocyte autoantibodies in NZB mice is inhibited by CD4 antibodies.

NZB mice spontaneously develop haemolytic anaemia as the result of production of erythrocyte autoantibodies. The mechanisms leading to breakdown in tolerance to erythrocyte autoantigens are unknown. Antibodies to CD4 have been successfully used to treat several murine models of autoimmune disease. In this study we injected NZB mice with non-depleting CD4 antibodies and were able to prevent and abrogate erythrocyte autoantibody production in young (Coombs' negative) and old (Coombs' positive) mice, respectively. Our data indicate the dependency of autoantibody production on CD4+ T cells. However, withdrawal of anti-CD4 antibodies resulted in the appearance of erythrocyte autoantibodies, showing that under these conditions we were unable to re-establish tolerance to autoantigens on erythrocytes using anti-CD4 treatment.

Animals↗

Active suppression induced by anti-CD4.

Treatment with a non-depleting monoclonal antibody to CD4 in the presence of mouse thyroglobulin (MTg) inhibits the development of murine autoimmune thyroiditis. This unresponsiveness was transferrable since such treatment generated a population of donor cells which could suppress the thyroiditis induced in lightly irradiated recipients by subsequent challenge with specific antigen. The suppression appears to be both antigen specific and antigen dependent and seems to discriminate between TH1 and TH2 helper subsets in that there is no significant effect on anti-MTg autoantibodies after challenge.

Animals↗

The regulation of autoimmunity through CD4+ T cells.

Our experiments imply that it is possible to use monoclonal antibody therapy to reestablish self tolerance to self antigens. This can be achieved by using a short course of an nd anti-CD4 antibody thus avoiding the problem of long term immunosuppression. The mechanism by which such a state of self tolerance is achieved remains to be clarified but possible mechanisms include deletion or anergy of autoreactive T cells or some form of suppression mediated through local cytokine production. As this antibody induced state of tolerance can be reversed in the NOD mouse by cyclophospamide deletion cannot be the method by which autoreactivity is prevented. The mixing experiments which have been described in the thyroiditis experiments strongly suggest that anery is not the mechanism. It therefore remains most likely that tolerance induced following administration of nd anti-CD4 is an active process maintained through the production of an inhibitory cytokine. This ability to reprogram the immune system using monoclonal antibodies makes it not beyond the realms of possibility that individuals suffering from IDDM may become tolerant of their beta cell antigens and thus be able to regenerate their own beta cell mass. If this could indeed occur it might mean that a lifetime of insulin injections and the development of the life threatening complications that may accompany a disease like IDDM may be avoided.

Animals↗

A thyroxine-containing peptide can induce murine experimental autoimmune thyroiditis.

A synthetic peptide based on a sequence containing thyroxine at position 2553 in thyroglobulin (Tg), and already shown to be recognized by two clonotypically distinct murine Tg autoreactive T cell hybridomas, can trigger primed lymph node cells to transfer thyroiditis to naive recipients. Donor lymph node cells could be prepared from mice immunized either with intact mouse Tg or with this peptide itself. After a second exposure to the priming antigen in vitro, both these populations induced 100% thyroiditis in recipient animals. The importance of the T4 residue in the development of disease was demonstrated by the failure of Tg tryptic peptides depleted of T4 to stimulate pathogenic effectors in vitro, even when the lymph node cells had been taken from mice primed with whole Tg. We conclude that this T4-containing 12mer sequence is a major thyroiditogenic epitope in CBA/J mice although we cannot exclude the possibility that there are other pathogenic epitopes present in the whole Tg molecule.

Animals↗

The forces driving autoimmune disease.

There are two classes of autoimmune disease, organ-specific and non-organ specific or systemic. That cells producing autoantibodies are selected by antigen is strongly suggested by the presence of mutations and high affinity antibody. T-cells are pivotal in all forms of autoimmunity as evidenced by the therapeutic benefit of anti-T-cell monoclonals such as anti-CD4, and the frequent development of high affinity IgG autoantibodies. The production of anergic T-cells by the use of non-depleting anti-CD4 in the presence of antigen is discussed with particular reference to its potential for immunological intervention in autoimmune disease. It is possible to identify T-cell epitopes in organ-specific autoimmunity using pathogenic T-cell clones or hybridomas to identify the peptide sequences which are reactive. Antigen-specific therapy may ultimately be based on such peptide epitopes. The specificity of the T-cells in systemic autoimmunity is still obscure, but there is some evidence that reactivity with certain germ-line idiotypes can lead to the development of systemic autoimmunity. The possibility of stimulating B-cells specific for auto-antigens such as DNA becomes feasible if a complex of antibody and DNA is taken up by these specific B-cells and processed idiotype is presented to T-helpers specific for those idiotype epitopes. Evidence is presented that there may be pre-existing defects in the target organ in certain organ-specific disorders, and the evidence for a glycosylation defect in the IgG in patients with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis also tend to have this glycosylation defect and this raises the possibility of an effect due to an environmental factor, such as a microbial infection. Molecular mimicry of autoantigens by microbes can stimulate autoreactive cells by their cross-reactivity. It is emphasized that cross-reaction which gives rise to the priming of autoreactive T-cells could give rise to the establishment of a chronic autoimmune state. In animals with normal regulatory immune systems, such induced autoimmunity is ultimately corrected and it is only in animals where there are defects in regulation, that autoimmunity persists. Thus, there are many factors giving rise to autoimmunity, and the diseases are rightly regarded as multifactorial in origin.

Amino Acid Sequence↗

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.

Adult↗

Changes in normal glycosylation mechanisms in autoimmune rheumatic disease.

To investigate potential mechanisms controlling protein glycosylation we have studied the interrelationship between lymphocytic galactosyltransferase (GTase) activity and serum agalactosylated immunoglobulin G levels (G(0)) in healthy individuals and patients with rheumatoid arthritis and non-autoimmune arthritis. In RA there was reduced GTase activity and increased G(0). A positive linear correlation between B and T cell GTase was found in all individuals. The relationship between GTase and G(0) was found to be positive and linear in the control population and negative and linear in the RA population. Sulphasalazine therapy maintained normal levels of GTase and caused a reduction in G(0) in the RA population. IgG anti-GTase antibodies (abs) were significantly increased in the RA population, whereas IgM anti-GTase abs were significantly decreased in both the RA and the non-autoimmune arthritis groups. These data describe a defect in RA lymphocytic GTase, with associated abnormal G(0) changes, which is corrected by sulphasalazine. A possible regulatory mechanism controlling galactosylation in normal cells is suggested, in which there is parallel control of B and T cell GTase. IgM anti-GTase abs may be integrated into this normal regulatory process. This is disrupted in RA, where the positive feedback between GTase and G(0) is lost and there is an associated increase in IgG anti-GTase abs, which may result from isotype switching as IgM anti-GTase abs are reduced. We suggest that these mechanisms are of relevance to the pathogenesis of RA, and that their manipulation may form part of a novel therapeutic approach.

Adult↗

Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells.

Although thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2. In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule. Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-gamma-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity.

Amino Acid Sequence↗

Changes in IgG glycoform levels are associated with remission of arthritis during pregnancy.

It was found that the percentage of IgG-associated agalactosyl N-linked oligosaccharides (G0) falls during normal human pregnancy and rises to values higher than before conception following delivery (n = 10, 39-55 days after delivery). Serial bleeds from a normal pregnant woman showed a fall in the percentage G0 during gestation and a rapid rise post-partum. A similar study on a pregnant arthritic woman with a pathologically elevated percentage G0 also showed a fall in percentage G0 during pregnancy and a rapid rise post-partum. The changes in IgG glycosylation in the pregnant arthritic woman occurred simultaneously with the pregnancy-induced remission and post-partum recurrence of disease. A further seven pregnant women with rheumatoid arthritis were studied and analysis of their G0 values pre- and post-partum confirmed the result. In a further series of experiments using an animal model of rheumatoid arthritis, DBA/1 mice with collagen-induced arthritis were found to have elevated G0 levels compared with control mice. The percentage G0 was found to fall simultaneously with pregnancy-induced remission to the same value as non-arthritic pregnant mice. Post-partum recurrence of arthritis in these mice was also accompanied by a simultaneous and rapid rise in percentage G0. Pseudopregnancy did not result in a change in the percentage G0, confirming the effect of true pregnancy. Since the proportion of agalactosyl IgG is abnormally high in the serum of patients with rheumatoid arthritis these changes in IgG glycoform levels, or the factors which control them, may be related to the mechanisms underlying remission of arthritis in humans during pregnancy.

Acetylglucosamine↗

Reduction in IgG galactose in juvenile and adult onset rheumatoid arthritis measured by a lectin binding method and its relation to rheumatoid factor.

Glycosylation changes in patients with juvenile chronic and adult onset rheumatoid arthritis have been studied using a novel binding method. Both these major types of arthritis showed decreased galactosylation of serum IgG, which confirms earlier studies using a different, more complex chemical method. No significant correlation between serum IgG, IgM, and IgA rheumatoid factors and age corrected G(o) (percentage of oligosaccharide chains lacking galactose) was found. The possibility that the less glycosylated IgG is preferentially confined to circulating IgM/IgG immune complexes cannot be excluded, however.

Adolescent↗

In vitro regulation of thyroglobulin (Tg) autoantibody production by Tg-specific T-cell lines and hybridomas.

To define the interactions between self thyroglobulin (Tg)-reactive T and B we co-cultured enriched B cells taken from rat or mouse Tg-primed mice with major histocompatibility complex (MHC) class II-restricted T-cell lines specific for iodinated determinants on self-Tg, or hybridomas derived from those lines. Using two clonally distinct T-cell hybridomas, ADA2 and CH9, in vitro help for Tg autoantibody responses was observed using mouse (M)Tg-primed B cells and a 100 ng/ml MTg challenge. Using rat Tg-primed B cells and the same conditions, only CH9 provided help, indicating that the fine specificity of B cells influences their ability to interact with specific anti-Tg T-cell clones. In contrast to T-cell hybridomas, their parent T-cell lines MTg9B3 and MTg12B suppressed Tg autoantibody responses in vitro, although they augmented bystander proliferation of unprimed B cells. The MTg12B cells also (i) diminished the survival of Tg-primed B cells, and (ii) inhibited the proliferation of an antigen-presenting B-cell hybridoma (LK35.2) in a cytostasis assay. These findings together support the view that their suppressive activity is mediated through cytotoxicity. While the role of class II-restricted cytotoxic cells in thyroid autoimmunity is unknown, the results suggest that such cells may act to suppress autoantibody responses as well as to mediate tissue damage to class II-expressing thyroid cells.

Animals↗

Cellular infiltration in induced rat thyroiditis: phenotypic analysis and relationship to genetic restriction.

We have investigated the responsiveness to thyroglobulin (Tg) plus complete Freund's adjuvant (CFA) and B. pertussis in a variety of inbred and MHC congenic strains of rats in terms of both Tg-autoantibody titres and histological thyroiditis index. Severity of thyroiditis was strongly Tg-dependent and closely related to the RT.1-MHC haplotype. Phenotypic examination of the inflammatory thyroid infiltrate using single and double indirect immunofluorescence techniques revealed a high proportion of macrophages and T lymphocytes, mainly of the cytotoxic/suppressor subset, in the high responder strains. Thyroid epithelial class II MHC expression although not prominent was strain-restricted and related to the amount of Ia+ leukocyte infiltrate.

Animals↗