Clonality of T lymphocytes expanded with IL-2 from rheumatoid arthritis peripheral blood,synovial fluid and synovial membrane

The association of rheumatoid arthritis (RA) with particular MHC class II genes suggests that autoantigen-spccific T cell clones present in joints could be central to the pathogenesis of the disease. Previous investigations on the clonal diversity of T cells infiltrating the rheumatoid synovial membrane have yielded conflicting results. With the use of Southern blot analysis, we investigated the clonalily of rheumatoid T cell lines expanded from peripheral blood, synovial fluid and synovial tissue. From peripheral blood lymphocyte (PBL) of RA patients and healthy normal controls, we also checked the consequences of two different culture conditions on the clonality of these cell lines. From control PBL, we found that in vitro non-specific expansion of non-clonal T cell populations does not create artefactual clonal selection. However, growing T cells in ritro with IL-2 seems to be able to lead to preferential expansion of cells bearing IL-2 receptor (IL-2R). We identified such in vivo activated IL-2-sensitive T cell clones frequently in RA synovial tissue (8/13) and more rarely in synovial fluid and peripheral blood (3/12). One patient presents the same T cell receptor gene rearrangements in synovial membrane of two affected joints. In RA synovial tissue, the frequency of these IL-2-responsive T cells is most prevalent among actively inflamed membranes removed early in the disease process. The role and the relevance to the disease of these I L-2-responsivc T cells remain to be elucidated.     

Adhesion of peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells.

To determine whether adhesion of peripheral blood lymphocytes (PBL) of patients with juvenile rheumatoid arthritis (JRA) may be enhanced, adhesion of PBL of children with JRA, children with seronegative spondyloarthropathies (SSA), age-appropriate and adult controls, to human umbilical vein endothelial cells (HUVEC) was assessed in vitro. B and CD4 T lymphocytes in initial, adherent, and non-adherent cell fraction were identified by flow cytometry. B lymphocytes of all the younger subjects combined had a higher adherence to activated HUVEC compared with B lymphocytes of the adult donors. Except for greater adherence of HLA-DR+ CD4 T cells, lymphocytes of children with JRA showed no enhanced adhesion to either unactivated or activated HUVEC. The percentage of B cells adherent to activated HUVEC in each of the subject groups was 1.5-3.6-fold higher than adherent CD4 T lymphocytes. Surface analyses indicated higher percentages of CD49d (alpha 4)+ and CD29 (beta 1)+ CD4 T lymphocytes in adherent cells, but less of a differential in CD49 (alpha 4)+ and no difference in CD29 (beta 1)+ B lymphocytes. There were fewer Leu-8 (L-selectin)+ B and Leu-8+ CD4 T cells among adherent cells. The data suggest a greater adhesive capacity of B lymphocytes compared with CD4 T lymphocytes which is unrelated to disease, and the possibility that B lymphocytes may utilize adhesion molecules distinct from those of CD4 T lymphocytes. Only a small subset of T cells of patients with JRA may have an enhanced capacity for adhesion to endothelium.     

A highly polymorphic CA repeat marker at the human tumor necrosis factor alpha (TNFAα) locus

Tumor necrosis factor alpha (TNFα) in activated monocytes exerts cytotoxic activity and has a variety of other biological effects. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the gene located at 6p21.3. High heterozygosity (0.80) makes this polymorphism a useful marker in the genetic study of disorders affecting immunological response and cell differentiation. Received: June 2, 1998 / Accepted: June 24, 1998     

CD69-null mice protected from arthritis induced with anti-type II collagen antibodies

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Three Kinds of Foamy Cells in the Spleen: Comparative Histochemical and Ultrastructural Studies

By light and electron microscopy, we observed foamy cells in the spleens from a patient with hemolytic anemia due to red cell adenosine deaminase (ADA) overproduction, a patient with rheumatoid arthritis (RA) treated with gold, and patients with idiopathic thrombocytopenic purpura (ITP)

The foamy cells associated with red cell ADA overproduction were essentially similar to Gaucher-like cells described in patients with thalassemia, and it was suggested that the accelerated destruction of red cells was one of the factors responsible for the development of foamy cells. Foamy cells in ITP and RA were closely associated with an increased destruction of platelets in the spleen. Morphologic transitions between phagocytosed platelets and myelinlike materials were traced in these disorders. In RA, however, foamy cells were heterogeneous from an ultrastructural standpoint, with different cytoplasmic inclusions. In addition to myelinlike materials, dense bodies, vacuoles with flocculent materials, and gold were noted in most of foamy cells. As gold compounds are known to inhibit lysosomal enzymes, we surmise that an acquired disturbance in lysosomal digestion is partially responsible for the accumulation of intermediate metabolites.

In the pathogenesis of foamy cells associated with blood cell dyscrasia, the accelerated destruction of blood cells and/or acquired disorders in catabolic pathways within the macrophages are suggested to be the underlying mechanism of an intralysosomal accumulation of incompletely degraded cellular debris.     

Specificity and sensitivity of the IgM capture immunoassay: studies of possible factors inducing false positive or false negative results

The specificity and sensitivity of the IgM-capture immunoassay (IgM-CI) were evaluated for detection of rubella specific IgM and hepatitis B core (HBc) specific IgM. For rubella specific IgM, antibodies bound to the solid phase were detected by haemadsorption and for HBc specific IgM, by using HBc antigen (HBcAg) and radiolabelled IgG anti-HBc. Rheumatoid factor (RF) was found to interfere in the test for HBc specific IgM because IgM-RF bound to the solid phase reacted with aggregated radiolabelled HBc specific IgG. This false positive reaction did not occur when radiolabelled F(ab')2 was used instead of the whole IgG molecule. HBcAg purified from biological fluids might be coated with host IgG and under these conditions, HBcAg could react with RF. It was also demonstrated that high levels of IgG antibodies could interfere with IgG anti-mu coated-surface by means of non-immunological protein-protein interactions. In fact, IgG did not interfere in the rubella assay, whereas it did in the very sensitive anti-HBc test. To prevent this false-positive reaction, different dilution media were tested. Only the addition of non-specific IgG and fetal calf serum (FCS), to the dilution medium, seems to improve the specificity of the test. Furthermore, in order to decrease this non-specific IgG-IgG interaction and an occasional prozoning phenomenon, the dilution of serum to be tested was taken into account. Parameters considered to decrease sensitivity were also studied. RF, anti-F(ab')2 antibodies and non-specific IgM did not decrease significantly the sensitivity of the assay.     

Heat shock protein 70 gene polymorphisms in rheumatoid arthritis

A role for heat shock proteins (hsp) in rheumatoid arthritis has been suggested. In addition, the specific binding of human HSP70 protein to QKRAA and RRRAA motifs within the HV3 region of disease-associated DRB1*0401 and DRB1*1001 molecules, respectively, has been proposed as being relevant to rheumatoid arthritis. The purpose of this work was to analyze the influence of HSP70 gene polymorphism on the susceptibility to or severity of rheumatoid arthritis and to investigate the possible contribution of these HSP70 polymorphisms in determining HLA-DRB1*0401/*1001 disease association. The frequencies of the HSP70-1, HSP70-2 and HSP70-hom genotypes were analyzed by PCR-RFLP using BsrBI , Pst I and Nco I enzymes, respectively, in patients with heumatoid arthritis and in healthy controls. No significant differences were observed when HSP70 alleled istribution between the groups under study were compared. Moreover, we did not observe any significant difference in HSP70 allele frequencies between patients positive for HLA-DRB1*0401/*1001 alleles and matched controls. Our data indicate that HSP70 gene polymorphisms do not appear to be relevant in the susceptibility to or severity of rheumatoid arthritis.     

Cytokine profile in systemic lupus erythematosus,rheumatoid arthritis,and other rheumatic diseases

We investigated serum levels of interleukin-6 (IL-6), interferon-gamma (IFN-), and tumor necrosis factor alpha (TNF) from patients with systemic lupus erythematosus (SLE) and its various clinical manifestations of disease and from patients with rheumatoid arthritis (RA) and other rheumatic diseases. The serum levels of IL-6 and IFN- were highly elevated from patients with SLE associated with lymphadenopathy (LN) or nephrotic syndrome (NS). On the contrary, the serum levels of TNF were elevated from most patients with SLE associated with thrombocytopenia (TP). However, serum levels of TNF were in the normal range from patients with SLE associated with NS, LN, or central nervous system disease. Of interest, patients with SLE associated with humoral immunodeficiency disorder, hypogammaglobulinemia, had highly elevated levels of serum IL-6. The concanavalin A-stimulated mononuclear cells (MNC) of patients with SLE associated with TP secreted highly elevated levels of TNF compared to other patient groups. We suggest that abnormal production of various cytokines in SLE is an intrinsic defect of MNC and the immune system that may be the key element for a variety of clinical manifestations of this disease.     

Analysis of depressed cell-mediated immunity in asbestos workers

To explore the mechanisms of asbestos-related perturbations of the immune system, we evaluated the in vitro cell-mediated immunity of five asymptomatic asbestos workers with hypergammaglobulinemia and decreased T-cell numbers. These results were compared with those in 10 matched controls. Analysis of T-lymphocyte populations revealed decreased absolute numbers of OKT4+ (helper/inducer) T cells in the peripheral blood and phytohemagglutinin (PHA)-stimulated mononuclear cell cultures of the workers. When chrysotile asbestos was added to PHA cultures, expansion of OKT4+ cell populations was disproportionately inhibited in workers' cultures. Furthermore, control proliferative responses to PHA became indistinguishable from initial worker responses. These effects were incompletely explained by the cytotoxic effects of asbestos on cultured lymphocytes. We conclude that both in vivo and in vitro exposure of mononuclear cell populations to asbestos may lead to a diminution of helper-inducer T-cell numbers. In asbestos-exposed individuals, this latter lymphocyte subpopulation appears to be especially sensitive to in vitro asbestos exposure. Although the clinical implications of these findings are unclear, we hypothesize that many of the immunologic abnormalities that occur in asbestos workers could be explained by direct asbestos effects on the OKT4+ immunoregulatory population.