K. Waalen

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.

The Vδ Gene Usage by Freshly Isolated T Lymphocytes from Synovial Fluids in Rheumatoid Synovitis: a Preliminary Report

Taking advantage ofthe polymerase chain reaction we have studied the usage of variable delta‐(Vδ)) region genes in freshly isolated synovial fluid T cells from patients with rheumatoid synovitis. Amplified mRNA from one patient with rheumatoid arthritis (RA) was cloned into an Smaleleaved pUC19 vector and colonies were screened with probes for three of the known human variable δ‐gene families (Vδ1. Vδ2. Vδ3). Of 10 clones, seven used Vδl. two Vδ2 and one Vδ3. This pattern of distribution is different from that of normal peripheral blood, where approximately 60% of Tγδ) cells are reported to use the Vδ2 gene. Furthermore, Northern blot hybridization analyses of mononuclear cells from two additional synovial fluids derived from another patient with RA and one with juvenile rheumatoid arthritis (JRA) also showed significant hybridization only with Vδ I. In summary, these preliminary results suggest a usage of Vδ gene families in Tγδ lymphocytes in synovial fluid of rheumatoid patients different to that found in normal peripheral blood.

Spontaneous in vivo gene transcription of interleukin-2, interleukin-3, interleukin-4, interleukin-6, interferon-gamma, interleukin-2 receptor (CD25) and proto-oncogene c-myc by rheumatoid synovial T lymphocytes

Rheumatoid synovial T lymphocytes were investigaled for the presence of mRNA for the cytokines interleukin‐2, ‐3, ‐4, ‐6, interferon‐gamma, the interleukin‐2 receptor (CD25) and the proto‐oncogene c‐myc. The isolated RNAs were analysed by dot blot and Northern blot hybridization. Our results show that synovial T lymphocytes from patients with rheumatoid arthritis (n= 12) had spontaneous tn vivo gene transcription of interleukin‐2 (93%), interleukin‐4 (67%), interleukin‐6 (92%), interleukin‐2 receptor (92%) and the proto‐oncogene c‐myc (67%). Only a few of the RA patients had synovial T cells with increased expression of mRNA for interleukin‐3 (25%) and interferon‐gamma (25%). The amounts of mRNA for the various cytokines and activation molecules produced by the rheumatoid synovial T lymphocytes were in most instances comparable to those of normal peripheral blood T lymphocytes activated in vitro by the mitogen phytohaemagglutinin. The data thus indicate that the synovial T lymphocytes are activated in viov in the majority of rheumatoid arthritis patients.

Immunoregulatory T cell subsets and T cell activation in rheumatoid arthritis. A need for analysis on the clonal and molecular level.

ConclusionActivation of T cell may be accomplished following the interaction of the TCR-CD3 complex with antigen and MHC products. In vitro this may be replaced by antibodies to the TcR or CD3 complex which mimic ligand binding. So called “alternative pathways” may also trigger activation. Activational state may be measured by lymphokine production, proliferative capacity or by expression of activation antigens. By these criteria a proportion of T cells isolated from rheumatoid joints appear to have undergone an in vivo activation. Phenotypic analysis of the synovial T cells has also established that there is an unusual distribution of Tγδ and Tαβ cells and T cell subsets in many rheumatoid patients. As T cells play a central role in immunoregulation, further exploitation of these observations using T cell clones and molecular techniques will extend our understanding of the disease process. In particular, further knowledge is required on the possible role of Tγδ cells in RA, the clonality of the T cells, the possible use of “alternative” activation pathways, and ultimately, the specificity of these T cells.

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.

Dendritic Cells from Human Rheumatoid Synovial Inflammatory Tissue and Peripheral Blood as Accessory Cells in Mitogen Stimulation of T Lymphocytes.

Dendritic cells (DC) were purified from the peripheral blood (PB) of normal individuals and from the synovial fluid (SF) and synovial tissue (ST) of patients with rheumatoid arthritis. These cells are strongly HLA‐DR positive and lack B cell, T‐cell, and monocyte markers as well as Birbeck granules. The DC were compared with monocytes and non‐T cells from PB for their ability to act as accessory cells for T‐cell responses to concanavalin A (Con A) and phytohaemagglutinin (PHA). DC from PB, SF and ST were much more efficient accessory cells for the mitogenic responses than autologous monocytes from PB. The mean PHA responses in cpm obtained with DC from the various compartments were 4–20 times greater than the responses obtained with monocytes from PB. The Con A responses obtained when the various DC populations were used as accessory cells were 3–13 times greater than those obtained with monocytes from PB. The mitogenic responses seen wish monocytes were very low. The non‐T cells, which comprise a mixture of cells obtained after removal of T cells, also gave low T‐cell responses to PHA and Con A compared with DC as accessory cells.

Dendritic cells in rheumatoid inflammation

ConclusionsRheumatoid inflammatory synovial and blood DC seem to have the same characteristics as LDC. Thus, the cells are strongly positive for class II MHC antigens and the common leukocyte antigen, while they lack almost all other mononuclear cell markers. Rheumatoid DC also produce IL-1, a cytokine which has many different functions in the rheumatoid inflammation. IL-1 is involved in tissue destruction and in triggering of T lymphocytes as well as in the maturation of B lymphocytes. The rheumatoid synovial DC are potent accessory cells for T cell responses and probably play an important role for starting and perpetuating the chronic rheumatoid inflammation. To further characterize the DC, we need reliable methods for the purification of these cells. Thus, it is important to clone DC and to produce monoclonal antibodies specific for human blood and synovial DC surface antigens.

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.

Evidence for activation of rheumatoid synovial T lymphocytes--development of rheumatoid T cell clones.

Rheumatoid arthritis joints are infiltrated by numerous T cells. These T cells coexist in close proximity to HLA class I and class II bearing accessory cells. A great proportion of the rheumatoid T cells are activated in vivo as shown by their microscopic appearance, expression of surface activation antigens, spontaneous proliferation and their spontaneous production and consumption of interleukin-2 (IL-2). T cells from the rheumatoid lesions also express high levels of mRNA for IL-2, for IL-2-receptor and for other mediators. The expression of IL-2-receptors by activated synovial T cells make it easy to propagate them in IL-2 containing media which is the basis for the subsequent development of T cell clones. The rheumatoid T cells are hypo-responsive after stimulation with antigens and anti-CD3 antibodies which indicate that the CD3-TCR complex has been modulated in vivo.