Helper T cells in antibody-mediated, organ-specific autoimmunity.

The production of pathogenic autoantibodies in organ-specific autoimmune diseases is largely T cell dependent. For many of these diseases, the precise specificities and cytokine profiles of the T cells that respond to the corresponding autoantigens have now been identified. This knowledge has been exploited to treat some models of antibody-mediated autoimmunity using peptides corresponding to the dominant helper epitopes, giving impetus to the development of a similar approach in the equivalent human diseases.

Recombinant human interleukin-1 beta primes human polymorphonuclear leukocytes for stimulus-induced myeloperoxidase release.

Recombinant human Interleukin-1 (rhIL-1) beta was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhIL-1 beta and then stimulated with either heat-aggregated IgG (Hagg) or N-formyl-methionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 micrograms/ml Hagg, 2.5 X 10(-9) M FMLP) did not stimulate degranulation in the absence of rhIL-1 beta. However, such concentrations elicited marked degranulation from PMNs preincubated with rhIL-1 beta (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1 beta employed and on the period of incubation. In other experiments, the effect of rhIL-1 beta on the PMN oxidative response was determined. rhIL-1 beta did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1 beta may potentiate PMN degranulation, but not their oxidative response.

The isolation of immune complexes containing IgM rheumatoid factor and recovery of IgG rheumatoid factor from the complexes.

IgG with rheumatoid factor activity has been recovered from immune complexes containing IgM rheumatoid factor. This was done by passing serum from patients with rheumatoid arthritis through an immunoadsorbent column of the F(ab')2 fragment of rabbit anti-human mu chain. The recovered IgM was analysed by radioimmunoassays for IgM and IgG rheumatoid factors before and after sucrose density gradient centrifugation at neutral and acid pH. It is considered that the method may be generally applicable for the isolation of immune complexes containing IgM antibodies.

Low avidity as a cause of prozone phenomena.

The incidence of prozones was studied, under various conditions, in a model indirect haemagglutination system and in a radioimmunoassay--both assays depending on the use of anti-immunoglobulin (anti-Ig). It was found that prozones were correlated with the presence of low avidity antibodies, and that these could compete with high avidity antibodies for the limited amount of anti-Ig available. It is proposed that the relatively rapid dissociation of low avidity antibodies allows them to form immune aggregates with the anti-Ig. With increasing size these aggregates would become more susceptible to being washed off. In this way low avidity antibodies could occupy the anti-Ig, and yet be relatively ineffective, either for haemagglutination or for the binding of radioactively labelled (or fluorescein-labelled) anti-Ig to the antigen.

Preparation of covalent IgG complexes of defined size and their clearance from the circulation of mice.

Covalent complexes of various sizes were made by coupling the photosensitive hapten, NAP (4-azido-2-nitrophenyl), to rabbit IgG and reacting the conjugates with rabbit anti-NAP antibodies. The resultant oligomers were stable, being eluted as the same discrete peaks after chromatography on gels both before and after injection into mice. Tests of their biological properties showed they fixed guinea pig complement in vitro in a size-dependent manner. Normal mice and mice with chronic endogenous circulating complexes cleared the oligomers according to size in a manner best described by a two-component exponential curve. There was no difference in the clearance rates between the two groups of mice. The advantages of these model complexes for examining the effect of circulating complexes on the mononuclear phagocyte system are discussed.

The separation and identification by monoclonal antibodies of dog IgG fractions.

Four fractions of IgG from normal dog serum have been successfully isolated by gel filtration followed by protein A and protein G affinity chromatography using the fast protein liquid chromatography (FPLC) system. Protein A chromatography produced three peaks: peak 1 was fallthrough material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contained bound material eluting at pH 3.5. The three peaks were then subjected individually to protein G affinity chromatography. Peak 1 from protein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a single peak at pH 3.8, and was called peak x. Peak 3 from protein A chromatography emerged as two separate peaks (y and z) off the protein G column; peak y bound and eluted at pH 4.1, and peak z bound weakly to protein G and emerged as a broad band at pH 8. Peaks w, x, y and z have been named gamma w, gamma x, gamma y and gamma z, respectively, and there purified IgG fractions were used to immunize mice for the preparation of monoclonal antibodies (McAbs). To date, two sets of McAbs have been produced: one which recognizes an epitope present in both gamma w and gamma z fractions and another set of McAbs which recognizes an epitope in the gamma x and gamma y fractions.

Susceptibility to pristane-induced arthritis is altered with changes in bowel flora.

Pristane-induced arthritis (PIA) is unique among the animal arthritides in that a non-infectious, non-antigenic oil induces a chronic immune based arthritis with a prolonged delay between exposure to the inciting agent and development of the disease. Mice with pristane-induced arthritis have elevated T cell and humoral responses to the 65 kDa heat shock protein derived from Mycobacterium bovis (hsp65) and in common with several other models of autoimmune diseases the incidence of PIA is markedly suppressed by preimmunisation with hsp65 in Freund's incomplete adjuvant (Thompson et al. (1990) Eur. J. Immunol. 20, 2479). Recent studies have investigated how autoimmune reactions to heat shock proteins are involved in the development of arthritis. Arthritic CBA/Igb mice given pristane alone develop antibodies to both hsp65 and GroEl (bacterial 60 kDa heat shock proteins) and to hsp58 (the mammalian equivalent). Moreover, the splenic T cells of such mice proliferate vigorously in response to both bacterial and mammalian 60 kDa heat shock proteins. Remarkably, the anti-hsp65 antibody response in normal mice rises rapidly with age, directly correlating with the age related incidence of PIA. In addition, specific pathogen free mice (SPF) maintained in an isolator have negligible anti-hsp65 responses but these convert to positive responses if the animals are exposed to the open part of the animal facility (Thompson et al. (1992) Arthritis Rheum. 35, 139).(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative responses of T-cell lines grown from joint fluids of patients with rheumatoid arthritis and other arthritides.

T-cell lines were established by culturing, in the presence of IL-2, mononuclear cells obtained from synovial fluids (SF) of most (77%) patients with rheumatoid arthritis (RA). By contrast, SF from patients with osteoarthritis and crystal synovitis rarely yielded lines. The cell lines were identified as T-cells because they express the CD3 surface antigen and either CD8 or CD4. The ability of the T-cell lines to react with putative antigens was tested using autologous Epstein-Barr virus transformed B-cells (EBV-B) as antigen presenting cells. IgG failed to stimulate proliferation of any RA patients' T-cell lines while Type II collagen stimulated T-cell lines from one RA patient and from one with osteoarthritis. Some T-cell lines (17/26) took up more tritiated thymidine after three days culture with a mixture of autologous SF and irradiated autologous EBV-B than with either alone. The effect could not be ascribed to a mitogen in the RA SF as the fluids failed to stimulate blood mononuclear cells. We consider that RA SF contain a T-cell growth factor which can only exert its effect through an antigen presenting cell (EBV-B).

Antigenicity of rat erythrocyte glycophorins.

The relationship between rat red blood cell (RBC) glycophorins and the antigens recognised by anti-rat RBC antibodies was examined. Initially, murine monoclonal antibodies specific for surface epitopes on whole rat RBCs were tested for their reactivity with RBC membranes on Western blots and two were found which reacted with blotted antigens. These antibodies recognised two bands corresponding to the major PAS-stainable bands of rat RBC membranes (i.e., the glycophorins) and a number of minor bands, thus demonstrating that the bands are antigenically related. This band-pattern was remarkably similar to that obtained with mouse anti-rat RBC serum. Digestion with neuraminidase altered the electrophoretic mobility of most of the bands, providing additional evidence that they are sialoglycoproteins, although sialic acid was shown not to contribute to their antigenicity. The glycophorin nature of the major antigens was verified by reelectrophoresis and blotting of bands excised from SDS gels, which showed that they were interconvertible monomeric and dimeric forms of the same polypeptide chain. It is suggested that rat RBC glycophorins are a related family of sialoglycoproteins with the high molecular weight members being formed by dimerization of five lower molecular weight polypeptide chains in various combinations.

Interleukin-2 inhibitor in rheumatoid arthritis synovial fluid does not inhibit mononuclear cell responses to mitogens.

Synovial fluid (SF) from rheumatoid arthritis (RA) patients were tested for their ability to inhibit the proliferative responses of normal peripheral blood mononuclear cells (PBM) to mitogens and interleukin-2 (IL-2). SF significantly inhibited the responses to concanavalin A (CON A) and phytohaemagglutinin (PHA), but significantly enhanced the responses to IL-2. Similarly, SF mononuclear cells (SFM) were hyporesponsive to CON A and PHA compared with autologous PBM, but hyper-responsive to IL-2. It is concluded that an IL-2 inhibitor in RA SF is unlikely to be the cause of SFM hyporesponsiveness to mitogens.

Enhanced oxidative response of polymorphonuclear leukocytes from synovial fluids of patients with rheumatoid arthritis.

Polymorphonuclear leukocytes (PMN) isolated from the synovial fluid (SF) of patients with rheumatoid arthritis (RA) exhibited an enhanced oxidative response to heat aggregated IgG (Hagg) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as compared with either autologous or normal blood PMN. Normal blood PMN pretreated with synovial fluid showed a significantly increased response to FMLP which was unaffected by prior dialysis of SF. The degree of enhancement produced varied between SF's and was dependent on the period for which cells were incubated and on the concentration of SF used. In contrast there was no enhancement of the oxidative response when Hagg was used as a stimulus. Indeed, SF produced a dose dependent inhibition of Hagg stimulated superoxide production. These observations suggest that SF's from patients with RA contain factors which both enhance and inhibit the oxidative response of PMN depending on the subsequent stimulus.

Adoptive suppression of erythrocyte autoantibodies in Lyt-2+ depleted recipients.

Cells suppressing red blood cell (RBC) autoantibody responses were found to exert their effect in Lyt-2+ depleted recipients. Mice injected with monoclonal antibodies to Lyt-2 became deficient in Lyt-2+ cells as judged by indirect immunofluorescence. These mice and normal recipients were given either rat RBC primed spleen cells (suppressor cells) or normal spleen cells and challenged with rat RBC. The autoantibody response of both Lyt-2+ depleted and normal recipients was suppressed as compared with that of mice given normal spleen cells. It is suggested that an Lyt-1+ cell may be involved in the expression of suppression.

Serial changes in the galactosylation of autoantibodies and serum IgG in autoimmune haemolytic anaemia.

A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the C(H)2 domain oligosaccharide. The aims were to determine whether there are also persistently high levels of G0 autoantibodies or serum IgG in autoimmune haemolytic anaemia (AIHA), and whether any changes in galactosylation over time are related to the course of disease. Autoantibodies eluted from red blood cells, and serum IgG, were obtained from a patient with chronic AIHA over a 21 month period, and the degree of galactosylation measured using a lectin-binding assay. There were wide fluctuations in the galactosylation of autoantibody and serum IgG, but these changes were unrelated to the severity of the anaemia. The galactosylation of autoantibody and serum IgG varied independently, and the autoantibodies were preferentially G0 in comparison with serum IgG in only half of the serial samples. We conclude that AIHA differs from other, systemic autoimmune conditions in that high levels of G0 autoantibodies or serum IgG are not persistent, and that changes in galactosylation do not parallel the course of disease.

Cellular and humoral reactivity pattern to the mycobacterial heat shock protein hsp65 in pristane induced arthritis susceptible and hsp65 protected DBA/1 mice.

We have analysed the cellular and humoral immunity to the mycobacterial 65 kD heat shock protein (hsp65) in groups of DBA/1 mice with arthritis induced by intraperitoneal injection of the mineral oil pristane. Here we confirm that DBA/1 mice are highly susceptible to pristane induced arthritis (PIA) and demonstrate that the incidence of arthritis can be modulated by either pretreatment with low dose irradiation or by preimmunisation with recombinant hsp65. Global cellular responses to antigens such as BSA or type II collagen were not enhanced or impaired within groups of arthritic (A) or non-arthritic (NA) mice. However, the cellular response to hsp65 in arthritic animals preimmunised with the 65 kD antigen was significantly elevated in comparison to hsp65 preimmunised mice that were resistant to the induction of disease. On the contrary, the level of hsp65 specific antibodies was much high in NA animals than in PIA mice. CBA/Igb mice are partially susceptible to the induction of PIA. We have previously reported that arthritic CBA/Igb mice have both elevated cellular and humoral reactivity to hsp65. Although a central pivotal role for hsp65 has been postulated in autoimmune diseases these results indicate that there is no simple relationship between the pathogenesis of PIA and immune responses to hsp65.

Failure of blood mononuclear cells from human donors with autoimmune haemolytic anaemia to reconstitute severe combined immunodeficient mice.

The use of severe combined immunodeficient (SCID) mice to study humoral responses by peripheral blood mononuclear cells (PBMC) from patients with autoimmune haemolytic anaemia (AIHA) was assessed. Upon transfer to SCID mice, PBMC from normal donors and patients with autoimmune thyroid disease (AITD) produced substantial levels of immunoglobulin (Ig), detectable in the plasma of recipient SCID mice. In contrast, the majority of PBMC from AIHA donors did not produce Ig in recipient mice. The capacity of PBMC to reconstitute SCID mice was not related to the donor's age. In one case, remission of AIHA allowed the donor's PBMC to successfully reconstitute SCID mice, despite the fact that the donor had developed immune thrombocytopenic purpura (ITP). AIHA PBMC were viable by dye exclusion and contained cells in various states of activation, as judged by their IgG secretion profiles when cultured in vitro. The proportions of leukocytes in AIHA PBMC (T to B cell ratios, CD4+ to CD8+ cell ratios and monocytes) were highly variable compared to non-AIHA PBMC. To determine the effect of abnormal lymphocyte proportions on SCID reconstitution, depletion experiments were carried out on normal and AITD PBMC. This work demonstrated a requirement for high T cell numbers, especially CD4+ cells, and minimal B cell numbers for successful reconstitution. CD8+ depletion of PBMC led to increased levels of Ig production in some instances. It is considered that PBMC from AIHA patients have a defect different from that of other autoimmune disorders, which renders them incapable of reconstituting SCID mice.

Quantitation of erythrocyte-bound IgG subclass autoantibodies in murine autoimmune haemolytic anaemia.

A quantitative and sensitive cellular enzyme-linked immunosorbent assay was developed for determining the number of molecules of IgG of each subclass bound to the surface of murine red blood cells (RBC). To develop standard titration curves, RBC from normal mice were treated with tannic acid and coated with a known concentration of purified myeloma of each IgG subclass. The quantity of each subclass bound to the surface of erythrocytes was determined by calculating the protein concentration of the bound IgG, which was then converted into number of molecules of IgG/RBC. The assay was used to quantify the number of autoantibodies of all four IgG subclass bound to the erythrocytes of mice injected with rat RBC. Twenty one days after the first immunisation, a mean number of 84,000 molecules of IgG1/RBC were detected, which increased to 114,500 molecules/RBC on day 28. On days 56 and 96 the mean concentration of IgG1 remained high, however by day 110 the mean level of IgG1 had decreased slighty to 69,500 molecules/RBC. By contrast, the mean concentration of IgG2a autoantibodies was considerably lower throughout the experiment, starting at 40,200 molecules/RBC on day 21 and dropping to 2,500 molecules/RBC by day 110. The mean quantities of IgG2b and IgG3 autoantibodies were similar to each other, and intermediate between the levels of IgG1 and IgG2a autoantibodies.

Identification of an erythrocyte autoantigen using monoclonal autoantibodies induced by immunization of mice with rat erythrocyte.

An erythrocyte autoantigen has been identified by means of monoclonal autoantibodies raised by immunizing mice with rat red blood cells (RBC). The autoantibodies reacted with intact rat and mouse RBC as judged by a cellular radioimmunoassay, and with a 52K band on western blots of rat and mouse RBC. They did not react with intact sheep RBC or blotted sheep erythrocyte membranes. Although anti-rat erythrocyte antibodies did react with bands in the molecular weight region of 50-55 K (on blots of rat erythrocyte membranes), these bands were susceptible to neuraminidase digestion, thus distinguishing them from the 52 K band recognized by monoclonal autoantibodies. The implications of the above results for the known autoantibody specificity of suppression is discussed, and it is suggested that they favour the existence of autoantigen-specific suppressor cells.

Changes in the galactose content of IgG during humoral immune responses.

The Fc region of IgG bears two oligosaccharides of variable composition. The serum level of one variant which lacks terminal galactose and sialic acid (agalactosyl IgG) is raised in a number of autoimmune diseases and animal models thereof. Here it is shown that such changes in IgG glycosylation occur during non-pathological humoral immune responses. It was found that if specific pathogen free (SPF) CBA/Ca mice are transferred from a sterile to a conventional environment, their levels of total serum IgG rise whereas the degree of IgG galactosylation falls. Next, mice were immunised with bovine serum albumin (BSA) in incomplete Freund's adjuvant. As anti-BSA titres rose the antibodies became less galactosylated and later, as the titres fell, the antibodies became more galactosylated. By contrast, there was little or no variation in the relative galactosylation of total IgG. It is considered that the galactosylation of IgG antibodies varies during an immune response.

Relationship between interleukin 6, agalactosyl IgG and pristane-induced arthritis.

IL-6 titres in sera and peritoneal exudate fluids (PEF) derived from pristane injected DBA/1 and CBA/Igb mice were measured. Arthritic DBA/1 mice had significantly higher serum IL-6 titres than nonarthritic or normal mice at 160 days post pristane injection. By contrast, although both arthritic and non-arthritic CBA/Igb mice had higher serum IL-6 titres than normal mice, there was no significant difference in serum IL-6 titre between these two groups at day 200-230. In both strains of mice, the IL-6 titres in PEF were more than 10 times serum levels regardless of arthritis. As previously reported for CBA/Igb mice, agalactosyl IgG levels are raised in pristane injected DBA/1 mice and the percentage is higher in arthritic animals than that in non-arthritic mice. An association between serum agalactosyl IgG levels and PEF IL-6 in pristane injected DBA/1 was demonstrated. Moreover, the injection of recombinant IL-6 into normal mice increased their serum agalactosyl IgG levels. However, it is considered that IL-6 is not the only factor involved in the production of agalactosyl IgG.

Mediators of joint swelling and damage in rheumatoid arthritis and pristane induced arthritis.

Joint swelling and tenderness in rheumatoid arthritis (RA) probably result from IgG aggregates activating complement with the consequent attraction of polymorphonuclear leucocytes (PMNs) and the liberation of their granule enzymes such as kininogenases. By contrast IL-1 and TNF are the major stimulants of cartilage and bone loss although other agents contribute. The fundamental drive for the production of these mediators is unknown but a role for heat shock proteins is suggested from work on pristane induced arthritis.