J. Thoen

Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjogren's syndrome

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2′ and Bm3δ–4δ), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38−IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38−IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sjögren’s syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.

Significantly Depressed Percentage of CD27+ (Memory) B Cells among Peripheral Blood B Cells in Patients with Primary Sjögren's Syndrome

CD27 has been found to be expressed on somatically mutated B cells and is thus a positive marker for memory B cells in peripheral blood (PB). Since abnormal immunogloblin (Ig) production is characteristic of the autoimmune diseases primary Sjögrens syndrome (pSS) and rheumatoid arthritis (RA), we have analyzed in detail the CD27 expression on PB B cell from these patient groups. Staining of PB B cells with monoclonal antibodies (MoAb) specific for CD19 and CD27 revealed a significantly depressed percentage of CD27+ PB B cells in patients with pSS (14.8 ± 1.6%) compared to both healthy donors (31.3 ± 4.7%, P = 0.005) and patients with RA (40.8 ± 4.1%, P = 0.0001). In addition, the percentages of both the IgD+CD27+ and the IgD‐CD27+ B‐cell subpopulations were significantly lower in pSS patients compared to RA patients and healthy donors. However, the relative proportion of IgD‐ and IgD+ cells among the CD27+B cells were almost the same for the three groups. Our data suggest a disturbance in the differentiation of peripheral B cells and possibly a bias towards plasma cell differentiation, resulting in a depressed percentage of CD27+ memory PB B cells in pSS. These results are potentially of pathological significance and of diagnostic value.

Tγδ Cells in Juvenile Rheumatoid Arthritis and Rheumatoid Arthritis

Using the anti‐TcRγ/δ‐1 monoclonal antibody and flow cytometry, we examined the number of Tγδ cells in paired samples of peripheral blood and synovial fluid or tissue from 24 children with juvenile rheumatoid arthritis (JRA). five adult patients with JRA, and 14 patients with rheumatoid arthritis (RA). No significant difference was found in the synovial compartment Tγδ values compared with the blood in JRA, adult JRA, or RA patients. Nor was any significant difference found in the peripheral blood or synovial compartment Tγδ values in any of the three patient groups compared with the peripheral blood of normal controls. However, seven of the children with JRA had very high Tγδ values in the synovial compartment while none of the normal children had high Tγδ values in the blood (P= 0.02. Fishers exact test). This may indicate a possible separate JRA patient group with high Tγδ levels in the synovial compartment. In six JRA patients further analysed for Tγδ subpopulations, a significant predominance of Vδ1 + cells was found in the synovial compartment compared with the corresponding peripheral blood samples (P<0 05. Wilcoxons signed test) and with peripheral blood of child controls (P<0 05, Mann Whitney U test). In these six patients, the Tγδ ‐cell expression of the very early activation antigen CD69 were significantly higher (P<0 05. Wilcoxons signed test) in the synovial compartment compared with the peripheral blood. Synovial Tγδ cells expressing HLA‐DR and interleukin 2 receptors could also be detected, in contrast to the peripheral blood in which no Tγδ cells expressing these antigens could be found. These data suggest that the synovial Tγδ cells had been activated in vivo.

Rheumatoid Synovial Dendritic Cells as Stimulators in Allogeneic and Autologous Mixed Leukocyte Reactions‐Comparison with Autologous Monocytes as Stimulator Cells

Dendritic cells were isolated from peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis and from peripheral blood of healthy blood donors on the basis of semiadherence to plastic surfaces, The cells were compared with autologous peripheral blood monocytes with respect to their stimulating capacities in allogeneic and autologous mixed leukocyte reactions (MLR), Dendritic cells from the various compartments stimulated allogeneic T cells 6‐14 times more than monocytes did. Dendritic cells also stimulated autologous T cells 10–24 times more than monocytes did. Evidence in favour of the dendritic cell as the major stimulating cell type in MLR was also found in mixed experiments in which various ratios of dendritic cells and monocytes were used as stimulator cells. Furthermore, the activating structures on the dendritic cells seem to be major histocompatibility complex class II antigens, since anti‐HLA‐DR antibodies inhibited the responses. The results, especially from the autologuus MLR, indicate that dendritic cells are important accessory cells for the various immune responses in rheumatoid inflammation.

Rheumatoid factors in primary Sjogren's syndrome (pSS) use diverse VH region genes, the majority of which show no evidence of somatic hypermutation

Rheumatoid factor (RF) is the most common autoantibody found in patients with Sjögrens syndrome (SS). To study the genetic origin and the mechanisms acting behind its generation we have characterized and sequenced the immunoglobulin VH genes used by 10 IgM RF MoAbs derived from peripheral blood of six female patients with pSS. We compared the structure of the RF immunoglobulin VH genes with those obtained previously from rheumatoid arthritis (RA) patients and healthy immunized donors (HID). VH1 and VH4 were each used by four RF clones, one clone was encoded by VH3 family gene and one by VH2 family gene. This distribution frequency was different from that observed in RA, where VH3 was the dominant family, followed by VH1. Eight different germ‐line (GL) genes encoded the clones and all of these genes were seen previously in RA and/or HID RF. Five clones rearranged to JH6, four rearranged to JH4 and one to JH5, in contrast to RF from RA and HID, where JH4 was most frequently used. D segment use and CDR3 structure were diverse. Interestingly, three out of four VH4 clones used the GL gene DP‐79 that was seen frequently in RA RF. The degree of somatic mutation in the pSS RF was very much lower than seen in RA and HID RF. All the pSS RF clones except three were in or very close to GL configuration. This indicates that there is little role for somatic hypermutation and a germinal centre reaction in the generation of RF from peripheral blood in pSS.

Effects of Piroxicam and D-Penicillamine on T Lymphocyte Subpopulations, Natural Killer Cells and Rheumatoid Factor Production in Rheumatoid Arthritis

The effects of piroxicam and D-penicillamine on T lymphocytes, NK cell activity and rheumatoid factor production as well as clinical parameters were studied in patients with rheumatoid arthritis. The level of total rheumatoid factor fell during treatment with D-penicillamine (p less than 0.02) and there was a positive correlation (K greater than 0.50, p less than 0.05) between this fall in rheumatoid factor and the improvement of several clinical activity parameters. No significant change was observed in the level of rheumatoid factor during treatment with piroxicam. Natural killer cell activity decreased from 21.1 +/- 2.5 to 15.8 +/- 1.9 after treatment with piroxicam for 3 weeks (p less than 0.05) as compared with changes in the controls. No change in natural killer cell activity was seen after treatment with D-penicillamine. Moreover, no significant changes in the numbers of T4+ and T8+ lymphocytes nor in the numbers of HLA-DR positive T cells were seen in the two treatment groups. Both laboratory and clinical activity parameters improved during the treatment with D-penicillamine, while only subjective parameters improved during treatment with piroxicam.

Macrophages and Dendritic Cells in Rheumatic Diseases

The normal synovial membrane is lined by a double layer of synovial lining cells, which form a multilayer in the rheumatoid synovial membrane. The synovial lining cells have previously been classified according to electron microscopic criteria as A and C cells, which have macrophage characteristics, and B cells, which have fibroblast characteristics. This classification has recently been confirmed using monoclonal antibodies specific for monocyte/macrophage and fibroblast antigens.

Phenotypes of T Lymphocytes from Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis: Evidence in Favour of Normal Helper and Suppressor Functions of T Lymphocytes from Patients with Juvenile Rheumatoid Arthritis

Lymphocytes from peripheral blood (PB) and synovial fluid (SF) from 21 patients with rheumatoid arthritis (RA) and 18 patients with juvenile rheumatoid arthritis (JRA) were studied with respect to T cell phenotypes using monoclonal antibodies in a rosette assay. The percentage of HLA-DR positive T cells was counted in PB and SF using indirect immunofluorescence. Suppressor cell activity of T cells from PB and SF was investigated by measuring the immunoglobulin production by pokeweed mitogen (PWM) stimulated B cells mixed with T cells at various ratios. The mean T4/T8 ratio was significantly lower in SF than in PB of both RA and JRA patients (p = 0.0062 and p less than 0.0001 respectively). The mean percentages of HLA-DR positive T cells were elevated in SF compared with PB in both patients groups (p less than 0.03 and p less than 0.04 in RA and JRA patients respectively). Mean suppressor cell activity and helper cell activity of T cells from SF and PB of JRA patients was normal. Thus there seems to be a dichotomy between the number of T8+ cells and suppressor cell function in mononuclear cells from SF of patients with JRA. This indicates that a considerable proportion of the T8+ cells in the SF do not have suppressor functions.

Inflammatory synovial T cells in different activity subgroups of patients with rheumatoid arthritis and juvenile rheumatoid arthritis

Mononuclear cells were eluted from synovial membranes of 39 patients with rheumatoid arthritis and 12 patients with juvenile rheumatoid arthritis. A considerable cell loss, about 50% or more, was seen during the various isolation steps. The CD4/CD8 ratio just after enzyme treatment (stage I) was significantly higher than at later stages, i.e. after removal of adherent cells (stage II, p less than 0.05) and after Isopaque Ficoll gradient centrifugation (stage III, p less than 0.01). This indicates a selective loss of CD4+ cells during isolation. In addition, stages I and II had higher CD4/CD8 ratios than peripheral blood of normal controls (p less than 0.01 and p less than 0.03), but not significantly higher than in peripheral blood of patients (p greater than 0.05). The CD4/CD8 ratio in eluted synovial membrane cells did not differ between patients with high and patients with low disease activity (p greater than 0.05). No correlation was found between any of the CD4/CD8 ratios and individual disease activity variables. Furthermore, a laboratory activity index and a disease outcome index were determined for each patient and no correlation was found between these indices and the CD4/CD8 ratios.

Effects of nonsteroidal antiinflammatory drugs on the immune network

The immune system is extremely complex. It comprises many different types of cells and their products. In patients with rheumatic diseases the immune system is activated and has disturbed regulation. It is also believed that immune reactions are involved in the pathogenesis of these disorders. Nonsteroidal antiinflammatory drugs (NSAID) have therapeutic effects on rheumatic diseases. These effects can all be explained by inhibition of prostaglandin production locally in the diseased joints, leading to reduced inflammation. Little or no effects on the number of circulating lymphocyte subpopulations or on peripheral blood mononuclear cell immune reactions can be seen after treatment of patients with rheumatoid arthritis (RA) with NSAID. The possibility, however, exists that immune reactions locally in the diseased joints are modulated by NSAID secondary to reduced prostaglandin production.