Håkan Ström

Role of polyclonal activation in specific immune responses. Relevance for findings of antibody activity in various diseases.

The detection of antibodies of different specificities is used as a diagnostic tool in clinical medicine. Presence of autoantibodies is often taken as an indication of an autoimmune disease process. However, there is rarely an absolute correlation between the finding of such antibodies and specific autoimmune diseases. One such example of a good correlation between antibodies and diseases is the occurrence of acetylcholine receptor antibodies in patients with myasthenia gravis (MG). In this disease there is good reason to believe that the presence of these antibodies is of pathogenetic importance [2]. In experimental situations, immunization of rabbits with purified acetylcholine receptor material leads to the formation of specific antibodies and, in addition, to symptoms that are similar to those found in patients with MG [24]. However, the pathogenetic importance of antibodies of many other different specificities occurring in patients with different diseases is less well understood. As examples of this kind of antibody formation, we should like to bring attention to the formation of oligoclonal antibodies produced locally within the central nervous system (CNS) in patients with multiple sclerosis (MS) and to the production of anti-immunoglobulin antibodies (rheumatoid factor (RF)) which occurs in patients with rheumatoid arthritis (RA). In this article, we suggest that the presence of antibodies in the cerebrospinal fluid (CSF) of MS and the sera of RA patients does not necessarily indicate antibody production as a result of specific immunization but may be the result of polyclonal activation induced by ligands acting non-specifically on T and/or B cells. This does not imply that all instances of autoantibody formation can be considered in this context. The specificity repertoire at the level of antibody-forming B-cell precursors and the divergent specificities in produced autoantibodies will give important clues as to mechanisms of induction of immune responsiveness.

B Cell Differentiation Factor in Synovial Fluid of Patients with Rheumatoid Arthritis

In this paper we have summarized our findings on immune activity in patients with Rheumatoid Arthritis. RA is characterized not only by the formation of various autoantibodies but also of a hyperreactivity of the B cell system, shown as an increased DNA synthetic rate of blood non-T, non-monocytic lymphocytes as well as an increased number of actively antibody secreting cells both in the blood and the synovial fluid. Synovial fluid contains biological activity which synergizes with PWM for the induction of Ig-secreting cells in blood from healthy controls. The factor can also substitute for T cells in the PWM-induced antibody synthesis in vitro. This activity fits well with the finding that SF contains a factor which induces increased formation of IgG in LPS-pretreated mouse cell cultures. Experiments show that the factor leads to a preferential increase in the production of IgG2b antibody secreting cells. Therefore, we conclude that synovial fluid contains a B cell differentiating factor with a selective effect on the induction of a particular IgG subclass.

High Incidence of Spontaneous Ig-Producing Lymphocytes in Peripheral Blood and Synovial Fluid of Patients with Active Seropositive Rheumatoid Arthritis

Numbers of in vitro spontaneous IgG, IgM and IgA plaque‐forming cells (PFC) as assessed by a modification of the protein A haemolytic plaque assay were determined in the blood and synovial fluid of patients with seropositive rheumatoid arthritis (RA) and compared with those of control groups. The total numbers of PFC were significantly higher in the peripheral blood of patients with active seropositive RA than in that of normal controls. In addition, most B lymphocytes in the synovial fluid of patients with active seropositive RA were active immunoglobulin (Ig) producers, whereas synovial fluid lymphocytes from patients with inactive seropositive RA and seronegative arthritis were not. In general, IgA PFC were relatively high in blood, whereas IgG PFC dominated in the synovial fluid. IgM PFC appear to be relatively low in blood and synovial fluid. However, a relative increase of IgG PFC was noted in the peripheral blood of patients with active RA. To test for polyclonality of the increased Ig synthesis, we tested the sera of patients and controls for the presence of polyclonal antibodies against sheep erythrocytes (SRBC) and SRBC modified by fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP). No differences were observed with SRBC and TNP‐SRBC agglutinin titres between patients and controls, but patients with RA had higher titres of FITC‐SRBC agglutinins than normal sera. This finding supports the concept of a polyclonal nature of antibody production in RA patients.

Spontaneous DNA Synthesis in Rheumatoid Arthritis: Evidence of Enhanced Circulating Non‐T‐Cell Proliferation

3H‐thymidine incorporation in cultures of peripheral blood mononuclear cells from patients with seropositive rheumatoid arthritis (RA) and healthy controls was measured in vitro in the absence of added stimulants. A significantly higher level of spontaneous DNA synthesis was found in cultures of mononuclear cells from patients with clinically active RA than from patients with inactive disease and normal controls. This activity was more apparent in 24–h cultures than in 72–h cultures. There was no correlation between DNA levels and IgM rheumatoid factor (RF) litres. When T‐ and non–T–cell populations were separated and cultured simultaneously with unfractionated cells, only non‐T cells maintained high levels of DNA synthesis, and enrichment of surface membrane Ig+ (SmIg) cells was generally associated with enhancement of 3H‐thymidine incorporation. Furthermore, no difference was found in spontaneous DNA synthesis between cultures either containing or depleted of phagocytic cells. Moreover, the addition of graded numbers of autologous monocytes to highly purified T– and non–T–cell populations did not alter the background DNA synthesis. Thus, endogenously activated cells in RA patients are neither T lymphocytes nor monocytes. A regulatory influence by monocytes could not be demonstrated. It is suggested that cells actively engaged in DNA synthesis in RA blood are non–T cells in origin, most probably B lymphocytes.

Demonstration of a Helper Factor(s) with T‐Cell‐Replacing Activity in Synovial Fluid

Cell‐free synovial fluid (SF) obtained from patients with rheumatoid arthritis contains a helper factor(s) capable of augmenting the generation of plaque‐forming cells (PFC) in pokeweed mitogen (PWM)‐stimulated normal peripheral blood mononuclear cells (PBMC). This helper factor behaves like a polyclonal B‐cell activator, in that it triggers the formation of IgM, IgG. and IgA PFC. However, SF has little or no effect on the proliferation of PWM‐activaled PBMC. Furthermore, SF was capable of replacing T cells for PWM‐induced differentiation but not proliferation of enriched human blood B lymphocytes. No helper factor or T‐ccll‐replacing activity was found in SF from patients with traumatic synovilis. Fractionation of SF containing helper activity on staphylococcal protein A column indicated that the activity is induced by biologically active molecules distinct from materials that preferentially hind to protein A such as IgG immune complexes. We conclude that the present activity has striking similarities to the recently described B‐cell differentiation factor that is produced by specifically activated T‐cell lines in vitro.

Biological Characterization of T Cell-Replacing Factor in the Synovial Fluid of Rheumatoid Arthritis Patients

The synovial fluid of patients with rheumatoid arthritis (RA) contains a biologically active factor which has the ability to replace T cells for the induction of antibody secretion by human blood lymphoid cells stimulated by pokeweed mitogen (PWM) in vitro. This factor, which will be referred to as RA‐SF (synovial fluid), also has the capacity to act as a B cell‐stimulatory factor of mouse splenic lymphocytes in the presence of lipopolysaccharide (LPS). Using a test system developed for the definition of interleukin 4 (IL‐4), which is a B cell‐stimulating lymphokine which preferentially activates the synthesis of selected Ig classes in mouse lymphoid cells, we have shown that RA‐SF has properties similar to IL‐4 in that it induces differentiation of antibody secretion in the LPS‐pretreated mouse cell, but unlike IL‐4, which gives IgG1 and IgE, it selectively induces IgG2b synthesis. The present study demonstrates that RA‐SF has a biological activity that is reminiscent of other B cell‐stimulating mouse lymphokines, but it is biologically distinct from IL‐2, IL‐4, and IL‐5. Recent data also indicate that it is distinct from gamma interferon (IFN‐γ). Therefore, we conclude that the biological activity of RA‐SF has properties in common with a T‐cell replacing (TRF) and B‐cell differentiation factor (BCDF) and probably represents yet another biological activity which so far lacks an experimental counterpart. The relevance of this factor for autoantibody synthesis is discussed.

Polymorphism of the human immunoglobulin variable region segment V1-4.1

Department of Medical Chemistry, Faculty of medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan 2 Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606, Japan 3 Second Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan 4 Department of Clinical Immunology, Karolinska Institute at Huddinge Hospital, Novum. S-14t 57, Huddinge, Sweden 5 Klinische Forschergruppe ffir Rheumatologie, Medizinische Universit~tsklinik, Mooswaldallee 1-9, W=7800 Freiburg, Germany

HLA antigens and adverse drug reactions to sodium aurothiomalate and D-penicillamine in patients with rheumatoid arthritis

SummaryThe association between HLA antigens and adverse drug reactions (ADR), (e.g. proteinuria, haematological abnormalities, stomatitis, diarrhoea and dermatitis) in rheumatoid arthritis (RA) to sodium aurothiomalate (gold) and to Dpenicillamine (PA) were studied in 32 patients. Thirtyeight RA patients treated with gold and PA, and with no ADR to these drugs, were used as controls. The frequency of HLA B8 was significantly (p<0.05) increased among RA patients with ADR compared to plasma donors. DR3 was also significantly increased (p<0.05) in RA patients with haematological ADR compared to plasma donors. Haematological ADR occured significantly (p<0.05) more often in DR3 positive patients (55%) than among DR3 negative RA patients (27%).

Clinical Symptoms and HLA Antigens in a Family with Reiter's Disease

A clinical and immunogenetic study was performed on a three-generation family with Reiters disease (RD). Twelve of 56 members of the family (33 clinically examined) including one in-law, had symptoms of arthritis, urethritis, conjunctivitis, uveitis, and/or mucocutaneous manifestations, but only one had the complete triad of Reiters syndrome (RS). Radiographic sacro-iliitis was found in 7 individuals, and monoarticular onset was reported in 5 out of 7 with peripheral arthritis. HLA B27 was found in 26 of the 37 family members who were tissue typed (including one in-law). All individuals with RD were B27-positive. Seven different B27 phenotypes were identified. This finding suggests that RD is associated with the B27 antigen itself, and not to a gene closely linked to B27. From a pedigree analysis of this family an autosomal dominant inheritance with incomplete penetrance or multifactorial inheritance seemed the most probable alternatives. The family history is a useful adjunct in the diagnosis of RD.

Partial Biochemical Characterization and Purification of IgG2b Inducing Factor as a New Cytokine from Synovial Fluid of Patients with Rheumatoid Arthritis

Rheumatoid arthritis synovial fluid (RA‐SF) contains a novel biological activity, which selectively induces IgG2b antibody production in lipopolysaccharide (LPS)‐activated mouse spleen cells in vitro and in vivo. Our previous studies have shown that this activity is not functionally identical to otherwell‐known cytokines and interleukins. In this study we demonstrate the partial purification and biochemical characterization of the IgG2b inducing activity in RA‐SF. Biochemical characterization revealed that the IgG2b inducing activity in RA‐SF has the following properties: it is a protein, sensitive to pH > 11 and < 4, which is precipitated by 50% of saturated ammonium sulphate and has a molecular weight of 50–70 kDa; it binds to Cibacron‐blue and heparin and its activity is not mediated by immunoglobulins or immune complexes, which are present in RA‐SF. Biochemical characteristics of the IgG2b inducing activity also differ from other cytokines and interleukins. The term IgG2b inducing factor is proposed for this novel activity.