G. S. Panayi

RHEUMATOID ARTHRITIS: A DISEASE OF T-LYMPHOCYTE/MACROPHAGE IMMUNOREGULATION

In rheumatoid arthritis the synovial membrane has many of the characteristics of a hyperactive, immunologically-stimulated lymphoid organ. The basis of this hyperactivity is poorly understood. Highly specific antisera to human Ia-like (HLA-DR) antigens and monoclonal antibodies (OKT series) to various T-lymphocyte subsets were used to analyse both the normal and the rheumatoid synovium and to compare it with normal lymph nodes. In rheumatoid arthritis the synovium acquires an infiltrate with microanatomical similarities to the paracortical area of the lymph node. Large, very strongly HLA-DR-positive macrophage-like interdigitating cells form close contacts with the OKT4+ (inducer-type) T-cells, while the OKT8+ population (T-cells of suppressor-cytotoxic type) between the macrophage-OKT4+ cell clusters is scanty (T4/T8 ratio = 9:1). By contrast, in the lymph node there are more OKT8 T-cells interspersed between the HLA-DR+ interdigitating cells and OKT4+ cells (T4/T8 ratio = 2:1). The large interdigitating cells and the OKT4+ T-cell population may be mutually stimulatory. In the absence of efficient suppression this stimulation may lead to activation of B-lymphocytes and oligoclonal or polyclonal immunoglobulin synthesis, as is found in the synovial membrane in rheumatoid arthritis.

Cytokine expression in synovial membranes of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES--To compare, by immunohistochemistry, the cellular and cytokine profile in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes (SMs). Synovium was obtained at knee arthroplasty from 10 patients with RA and 10 with OA. METHODS--Synovial membranes were stained with a panel of monoclonal antibodies (MAb) to assess cytokine expression (IL-1 alpha, IL-1 beta, IL-6, GM-CSF, TNF-alpha and EGF) and the intensity of the mononuclear cellular infiltrate (MNC). RESULTS--Significantly greater percentages of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF and EGF cells were detected in all areas of the rheumatoid SMs when compared with osteoarthritic SMs. Five RA but only one OA SM demonstrated focal lymphoid aggregates. Lining layer thickening was noted in RA SMs only. The intensity of the MNC and number of blood vessels were greater in the RA group. CONCLUSION--The results suggest that the differences in cytokine production by RA and OA SMs are quantitative but that the greater thickness of the synovial lining layer and higher vascularity may be specific to RA.

HLA-DR antigens and toxic reaction to sodium aurothiomalate and D-penicillamine in patients with rheumatoid arthritis.

To investigate the possible relation between certain HLA antigens and toxicity during treatment with sodium aurothiomalate of D-penicillamine, we studied 91 patients with rheumatoid arthritis. Seventy-one had toxic reactions to either drug or both drugs; the remaining 20 took one of the drugs for at least six months, without toxicity. Nineteen of 24 patients in whom proteinuria developed were positive for HLA-B8 and HLA-DRW3 antigens; 14 of 15 episodes of aurothiomalate-induced proteinura and nine of 13 episodes of penicillamine-induced proteinura occurred in patients with these antigens. All 13 episodes of proteinuria in which urinary protein exceeded 2 g in 24 hours occurred in patients with DRw3. The relative risk of proteinuria during treatment with aurothiomalate is increased 32 times in patients who are HLA-DRw3 positive. No significant associations were found between any HLA antigen and development of skin rashes or hematologic complications. Toxicity during aurothiomalate or penicillamine treatment for rheumatoid arthritis may be under genetic control.

The Human Endoplasmic Reticulum Molecular Chaperone BiP Is an Autoantigen for Rheumatoid Arthritis and Prevents the Induction of Experimental Arthritis

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4+/−- and HLA-DR1+/+-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Immunohistological analysis of delayed-type hypersensitivity in man

Abstract BCG-immunized volunteers received intradermal injections of PPD to produce delayed-type hypersensitivity reactions. Skin biopsies were removed 12, 24, 48, and 72 hr after injection. Frozen sections of these biopsies and normal skin biopsies were examined with immunohistological techniques using heterologous and monoclonal antibodies to label specific cell populations. In some studies, these techniques were combined with cytochemical reactions to demonstrate acid phosphatase and adenosinetriphosphatase activity. Such combination analyses allowed the discrete identification of the mononuclear cells (lymphocytes and monocytes/macrophages) that constituted the DTH-associated infiltration. Lymphocytes of the inducer and cytotoxic/suppressor phenotype appeared in a ratio of 2:1 and some of the former (but not the latter) sub-population were seen to have migrated to the epidermis. Infiltrating monocytes/macrophages were identified by their “staining” with anti-HLA-DR antiserum and they could be divided into subpopulations using cytochemical techniques. The disposition of these cells one to another and their relationship to the Langerhans cells were also revealed. These observations are discussed in relation to existing histological descriptions of the DTH reaction, and implications relevant to underlying local cellular interaction are proposed.

Abnormal distribution of the helper-inducer and suppressor-inducer T-lymphocyte subsets in the rheumatoid joint

T lymphocytes can be divided into two main phenotypic populations, CD4 and CD8. These can be further subdivided into 2H4, 4B4, or UCHL1 subsets by appropriate monoclonal antibodies. We have investigated these subsets in the synovial fluid of patients with rheumatoid arthritis and have found (i) a virtual absence of CD4+ 2H4+ and the marked reduction of CD8+ 2H4+ T cells; (ii) a marked increase of CD4+ 4B4+ and CD8+ 4B4+ T cells; and (iii) a marked increase of CD4+ UCHL1+ and CD8+ UCHL1+ T cells compared with peripheral blood. Although the functions of the CD8 subsets are not known, the virtual absence of CD4+ 2H4+ suppressor-inducer T cells and the marked increase of CD4+ 4B4+ helper-inducer T cells and of CD4+ UCHL1+ memory T cells may help to explain the many known functional immunological properties of synovial T cells.

Genetic polymorphism of IL-12 p40 gene in immune-mediated disease.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3′ untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty’s syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95–4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

Pathogenesis of rheumatoid arthritis. The role of T cells and other beasts

The evidence coming from the different experimental approaches reviewed in this article strongly supports the hypothesis that RA is T-cell driven at all stages of the disease. Although the effector phases responsible for the events that lead to joint destruction involve several different cell types, cytokines, and other mediators, T cells still direct operations behind the scenes. Direct experimental proof of this proposition in patients is still lacking, but the development of nondepleting modulating CD4 monoclonal antibodies may provide new tools to test this hypothesis. In this respect, it is encouraging that using one such reagent, we have recently shown that not only did the activity of the disease improve but, more importantly, the inflammatory indices and production of non-T-cell cytokines were reduced. This is not to dissimilar from the results of experiments described in animals, where by blocking synovial T cells, the production of IL-1 beta and TNF alpha could be decreased by more than 90%. From this perspective, it may be predicted that by modulating T cells in the joint, it is possible to achieve our ultimate goal of permanently switching off the disease.

A ROLE FOR PURINE METABOLISM IN THE IMMUNE RESPONSE: ADENOSINE-DEAMINASE ACTIVITY AND DEOXYADENOSINE CATABOLISM

We have investigated a new hypothesis for the association between adenosine deaminase (A.D.A.) deficiency and immunodeficiency--namely, that deoxyadenosine rather than adenosine (an equally effective A.D.A. substrate) is toxic to proliferating cells of lymphoid origin. This possibility was explored in mitogen-stimulated lymphocytes cultured with a potent A.D.A. inhibitor, E.H.N.A. (erythro-9[2-hydroxy-3-nonyl] adenine) to simulate A.D.A. deficiency. In this in-vitro system deoxyadenosine was inhibitory at much lower and more physiological concentrations (1 mumol/1), compared with adenosine (100 mumol/1).

Intramuscular gold decreases cytokine expression and macrophage numbers in the rheumatoid synovial membrane.

OBJECTIVES--Cytokines, released from mononuclear cells (MNC) are mediators of joint destruction in rheumatoid arthritis (RA). The mechanisms of action of gold salts used in the treatment of RA are unknown. The aim of this study was to investigate cytokine expression and intensity of MNC infiltrate in the RA synovial membrane (SM) following treatment with sodium aurothiomalate (SAT). METHODS--Sequential blind needle biopsies were obtained at entry into the study and at two and 12 weeks after the start of SAT therapy in 10 patients with active RA. SMs were stained with a panel of monoclonal antibodies to assess cytokine expression (IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and GM-CSF). RESULTS--There was a significant decrease in IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha expression 12 weeks after treatment (p < 0.004, p < 0.002, p < 0.009 and p < 0.004 respectively). This was noted in the lining layer, the perivascular aggregates and the connective tissue areas. Detailed examination of the MNC infiltrate showed a significant reduction in inflammatory monocytes (MONO) in the lining layer at two weeks (p < 0.03). A decrease in the number of CD68+ macrophages (MAC) was noted in the perivascular and connective tissue areas at 12 weeks. No significant changes were observed in the number of T and B cells and blood vessels. CONCLUSION--The results suggest that gold may suppress RA disease activity by diminishing MONO and MAC numbers and consequently monokine production in the SM.