Search

The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20-, CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction.

Rheumatoid factors (RFs) are autoantibodies directed against the Fc part of IgG. Considerable evidence exists that there are two classes of RFs, pathological and physiological. Whereas pathological RFs are associated with disease, physiological RFs are considered to be a normal component of the immune response. RF(+) precursor B cells present as part of the B cell repertoire of healthy individuals are held responsible for the production of physiological RFs, which is a transient phenomenon with a clear correlation with an initiating stimulus such as immunization or exposure to an infection. Here we demonstrate a difference in the regulatory control of total Ig and RF production by peripheral blood (PB) B cells of both healthy controls (HC) and patients with rheumatoid arthritis (RA). Highly purified B cells from HC and patients with RA were cocultured with T cells stimulated with immobilized anti-CD3 mAb. Similar to IgM production, IgM-RF production was shown to be dependent on CD40 cross-linking. However, activation of PB B cells in the CD40 system in the presence of IL-2, IL-4, IL-10, combinations of these cytokines or supernatant of anti-CD3-stimulated T cells failed to induce detectable IgM-RF, whereas total IgM production was considerable. From these results we conclude that conditions to activate physiological RF(+) B cells require additional contact besides CD40--CD40L interactions between T and B cells. Since the requirements for RF production were similar using PB B cells from HC and patients with RA it is suggested that the regulatory properties of RF(+) precursors in the PB B cell compartment is equal among these groups. Together, these results indicate that conditions for the induction of total Ig and physiological RFs are different.

Cytotoxic cells possess specialized granules which contain perforin and a group of serine proteinases termed granzymes. Granzyme-positive cells have been identified in synovial fluid and tissue of patients with RA, where they may play an important role as mediators of granule-mediated apoptosis, extracellular proteolysis, and cytokine induction. The aim here was to define further the involvement of cytotoxic cells in RA. Plasma and synovial fluid samples from the knee joint were obtained from 31 RA patients. The disease controls included 20 osteoarthritis (OA) patients and 10 reactive arthritis (ReA) patients. A recently developed capture ELISA was used to detect soluble granzymes A and B in all patients. Compared with OA and ReA disease controls, markedly increased levels of soluble granzymes A and B were detected in both plasma and synovial fluid of RA patients (P < 0.00001). When values for soluble granzymes A and B in plasma and synovial fluid were used simultaneously as independent variables, logistic regression analysis indicated that a diagnosis of RA could be predicted correctly in 84% of the RA patients and a diagnosis of non-RA in 90% of the controls. The markedly elevated levels of soluble granzymes A and B in plasma and synovial fluid of RA patients strongly suggest that cytotoxic cells are active participants in the pathogenesis of RA. Moreover, the results suggest that measurement of granzymes may assist the laboratory evaluation of patients with arthritis. Larger studies in patients with early disease may clarify the role of this test system in differential diagnosis.

Anaemia of chronic disease (ACD) is frequently found in rheumatoid arthritis (RA). In the pathogenesis of ACD both cytokines, such as tumour necrosis factor-alpha (TNF-alpha), IL-1 and IL-6 as well as a relative deficiency of erythropoietin (EPO), are thought to play a key role. In the present study the role of IL-6 in the pathogenesis of this anaemia was investigated. IL-6 was administered intraperitoneally to rats for 14 sequential days. It appeared that IL-6 was able to induce anaemia. No evidence for suppression of bone marrow erythropoiesis or enhanced sequestration of erythrocytes in the liver was found. However, decreased plasma and bone marrow iron contents were observed in anaemic rats. Blood loss in intestinal tissue was demonstrated using erythrocyte labelling with 99mtechnetium. Histologically this was associated with inflammatory cell infiltration, oedema and bleeding in the intestinal wall. In conclusion, IL-6 induced anaemia in rats. This anaemia was caused by intestinal blood loss.

Minocycline has been shown to have an anti-inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell-mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose-dependent inhibition of T cell proliferation and reduction in production of IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). Besides an inhibition of IL-2 production, minocycline exerted its effect on T cell proliferation by induction of a decreased IL-2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3-induced intracellular Ca2+ mobilization. Taken together, the results clearly indicate that minocycline has immunomodulating effects on human T cells.

Chloroquine (Chl) is an anti-rheumatic drug that is widely used in the treatment of rheumatoid arthritis (RA). It seems that T cells are important in the pathogenesis of RA, but it is not known whether Chl acts via inhibition of T cell function. We here present evidence that Chl, just like cyclosporine A (CsA), inhibits T cell proliferation as induced with immobilized alpha CD3 MoAb in a concentration-dependent manner, at least partly through interfering with the production of IL-2 protein and the induction of IL-2 mRNA. Furthermore, Chl impedes the responsiveness of T cell clones to IL-2 since (1) the inhibition of alpha CD3 MoAb-induced proliferation by Chl could not be reversed by rIL-2 and (2) Chl directly blocks IL-2-driven proliferation of cloned T cells. Chl appeared to interfere with the internalization (50% inhibition) and degradation (total blockade) of rIL-2. Finally, the combination of Chl and CsA synergistically inhibited T cell proliferation. We conclude that Chl may inhibit functional properties of human T cells, although the drug is 100- to 1000-fold less potent than CsA in inhibiting T cell proliferation and IL-2 production, respectively. It is speculated that the in vitro effects of Chl might be relevant in explaining the anti-rheumatic effect of this drug in patients with RA.

To investigate the origin of circulating rheumatoid factor (RF) and the relation between RF production at different sites in patients with rheumatoid arthritis (RA), mononuclear cells derived from bone marrow, synovium and peripheral blood of patients with RA were examined for the presence of plasma cells and for their capacity to produce RF and other immunoglobulins in vitro. Analysis of culture supernatants for the presence of immunoglobulins demonstrated that cells derived from bone marrow, synovium and peripheral blood were all found to be capable of producing every immunoglobulin and RF isotype investigated. No significant correlations were found between concentrations of immunoglobulin isotypes produced by cells derived from different sites of one individual. Significant correlations were found, however, between concentrations of RF isotypes produced by cells derived from the three sites. These results indicate that the production of RF in the different compartments is not an autonomously regulated process. Mononuclear cells derived from bone marrow were found to be able to produce RF in similar quantities to cells dissociated from synovial tissue. In combination with the fact that circulating immunoglobulins are produced mainly in the bone marrow, this observation suggests that bone marrow is also a major source of circulating RF.

In order to gain insight into the production and clinical significance of IgA rheumatoid factor (IgA-RF) in mucosal fluids of patients with rheumatoid arthritis (RA), we examined tear fluid, saliva and serum from 80 patients with RA. Significant correlations were found between IgA-RF levels in tear fluid and saliva (P = 0.002, r = 0.57), saliva and serum (P < 0.001, r = 0.79), and serum and tear fluid (P < 0.001, r = 0.31). No significant correlations were found between total IgA levels in these fluids. Comparison between circulating and mucosal IgA-RF levels after correction for total IgA, revealed that mucosal IgA-RF levels are on average 2.5 times higher than circulating IgA-RF levels. Analysis of IgA-RF specificity showed that lacrimal and salivary IgA-RF reactivity with various IgG subclasses is similar and differs from serum IgA-RF specificity. These results indicate local production of IgA-RF in salivary and lacrimal glands and support the view of a common origin of IgA-RF producing B cells present in mucosal tissues. Mucosal and circulating levels of IgA and IgA-RF were not associated with tests that quantify tear fluid production. This indicates that mucosal and circulating levels of IgA and IgA-RF in patients with RA cannot be regarded as markers for the development of secondary Sjögren's syndrome.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.

IgG anti-endothelial antibodies (AEA), as measured by ELISA or immunoblotting technique could be detected in serum samples of 56 out of 64 patients with SLE (88%) and mainly occurred in monomeric form. AEA were not cell specific, because the binding reactivity was absorbed partially by both fibroblasts and peripheral blood mononuclear cells. No correlation was found between the presence of AEA and anti-nuclear antibodies. Immunoblotting revealed reactivity of AEA against endothelial antigens ranging in size from 15 to 200 kD. AEA titres were significantly higher in patients with joint or skin abnormalities, compared with patients without these abnormalities. A significant correlation was found between nephritis in SLE and the presence of AEA reactivity against endothelial membrane antigens of 38, 41 and 150 kD. These data show that the pattern of AEA reactivity in serum of SLE patients is heterogeneous, and suggest that AEA against a limited number of antigens may be involved in the pathogenesis of nephritis in SLE.

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.

We investigated whether a decreased complement receptor expression or function of monocytes isolated from peripheral blood of 52 patients with rheumatoid arthritis (RA) and rheumatoid vasculitis (RV) could account for the previously observed diminished degradation of immune complexes by monocytes of patients with RA and RV. On average, monocytes from all patients expressed significantly less CR1, and degraded significantly less AC3b when compared with monocytes of healthy controls. In addition, monocytes from RV patients degraded significantly less AC3b when compared with monocytes from patients with RA. The expression of both CR1 and CR3 on monocytes of RV patients was lower compared with RA patients but this difference was only significant for CR3. No differences were found between AC3b degradation and the expression of CR1 and CR3 between patients with active and inactive RA. Using linear discriminant analysis on the variables AC3b, CR1 and CR3, 94% of the patients could be classified correctly as healthy controls, RA or RV, suggesting a true multi-variate relationship between these parameters and patients groups. Our results suggest that the diminished capacity of monocytes from RA patients to degrade AC3b is due partly to a decreased expression of CR1 and CR3.

IgG antibodies reactive with human umbilical vein endothelial cells were found in 19 out of 28 patients with rheumatoid vasculitis (RV), in four out of 24 patients with rheumatoid arthritis (RA), in seven out of 10 patients with systemic lupus erythematosus (SLE), but not in healthy donors. In four patients with RV who were followed longitudinally, regression of vasculitic episodes coincided with decreasing titres of anti-endothelial antibodies (AEA). Binding activity to endothelial cells was observed in intact IgG and F(ab')2 fragments of IgG. AEA activity was unrelated to antibodies against nuclear, blood group or major histocompatibility complex antigens and did not involve immune complexes. AEA activity was not specific for endothelial cells since the AEA-positive sera and the IgG fractions prepared from these sera also reacted with fibroblasts. Adsorption of positive sera and corresponding IgG fractions with endothelial cells decreased the IgG binding reactivity on both fibroblasts and endothelial cells. These findings show that RV patients have IgG-AEA, and suggest that these antibodies may play a role in the pathogenesis of the disease.

Receptors for the Fc part of IgG on polymorphonuclear cells (FcR) of patients with rheumatoid arthritis (RA), Felty's syndrome (FS) and healthy controls (HC) were studied by means of a rosetting technique with rabbit IgG coated ox erythrocytes. When cold isolated polymorphonuclear cells (PMN) were incubated at room temperature the percentage of rosette forming PMN (RF-PMN) from HC was more than twice that measured directly after isolation at 4 degrees C. The same phenomenon was observed for PMN from patients with RA although the RF-PMN increased by only 1/3. In contrast warming of PMN from patients with FS did not influence the RF-PMN. The presence of surface bound immunoglobulins and intracytoplasmic immunoglobulins was measured by immunofluorescence and appeared to be inversely related to the RF-PMN. A good correlation between the results of the C1q binding assay (C1qBA) and the immunofluorescence score was observed but no correlation existed between the C1qBA and the RF-PMN. These results indicate that the number of PMN expressing FcR in patients with RA and FS is decreased presumably because of the phagocytosis of immune complex like material. The decrease in the availability of FcR may influence the functions of the PMN.

The expression of receptors for the third component of complement (C3R) on polymorphonuclear cells (PMN) from patients with rheumatoid arthritis (RA) or Felty's syndrome (FS) and healthy controls (HC) was studied by means of a rosetting technique using ox erythrocytes coated with rabbit IgM and mouse complement. The percentage of rosette forming PMN (RF-PMN) was found to be increased in patients with FS (59%) and patients with RA (47%) compared to HC (18%), when measured directly after cold isolation of PMN. Incubation at 37 degrees C of cold isolated cells increased the percentage of RF-PMN in all three groups, although the values were significantly higher during the first 45 min of incubation in both patient groups. When incubated with serum from patients with FS at 37 degrees C, cold isolated donor PMN formed significantly more rosettes than after incubation with donor serum. This increase was shown to be due to the presence of circulating immune complexes in the patient sera and could be mimicked by incubating donor PMN with aggregated human IgG. As a result of these experiments it was demonstrated that the increased percentages of PMN with C3R in patients with FS or RA are most likely caused by a previous in vivo activation of PMN by immune complexes. The possible effect of such an increase in C3R expression in relation to PMN function in these patients is discussed.

The variability in response of peripheral blood mononuclear cells (PBMC) from 95 healthy donors to pokeweed mitogen (PWM) was investigated. Age, sex, and blood group of the donors, the numbers of PBMC recovered from 500 ml blood, the percentage of monocytes therein, the presence of antilymphocyte antibodies (L-Cyt), or of anti-cytomegalovirus (CMV) antibodies, as well as HLA-B8 and HLA-DR4 were evaluated for their relation with the level of blast cell formation, proliferative response, and immunoglobulin G (IgG) synthesis, induced by PWM. The results showed that increasing age of the donors and the presence of anti-CMV antibodies are significantly associated with low proliferative responses of PBMC, whereas the HLA-B8 antigen and female donor sex were found to be associated with high blast cell formation after PWM stimulation. High percentages of monocytes in isolated PBMC at the onset of stimulation were inversely associated with IgG production, whereas the HLA-DR4 antigen was associated with high levels of IgG production, induced by PWM. The observed relations indicate that for comparison of responses of unseparated PBMC to PWM between patients and healthy controls, age, sex, and laboratory data, including the HLA background of donors, may have to be taken into account.

In the present study, we tested mononuclear phagocyte system function in nine healthy controls and 15 SLE patients with complement activating 123I-labelled aggregates of human IgG (AIgG). Clearance half-time of AIgG was 26 +/- 8 min in controls, compared to 58 +/- 27 min in patients (P less than 0.005). Binding of AIgG to erythrocytes was significantly lower in patients, 9.3 +/- 8.1 vs 24 +/- 20% (P less than 0.05). The increase of C3a-levels in plasma was significantly lower in patients than in controls (P less than 0.05 at 3 and 8 min), suggesting less complement activation. Liver and spleen uptake of 123I-AIgG was measured with a gamma camera and expressed as liver/spleen uptake ratios. In patients, the liver/spleen uptake ratios were significantly higher than in controls at 15 min, 3.8 +/- 2.0 vs 2.31 +/- 0.7 (P less than 0.05), due to less splenic uptake of AIgG. Correlations between clearance half-time or liver/spleen uptake ratios and immune complex levels or disease activity were not found. This study indicates that clearance of soluble AIgG is abnormal in patients with SLE, due to decreased splenic uptake of AIgG.

Mononuclear phagocyte system (MPS) Fc-receptor function in 20 patients with seropositive rheumatoid arthritis (RA) was investigated using radiolabelled autologous erythrocytes coated with an average of 5,800 molecules of anti-rhesus IgG (E. IgG). Although clearance times (T1/2) of E. IgG tended to be longer in RA patients than those in healthy controls (46 +/- 6 min vs 38 +/- 5 min, mean +/- s.e.m., P = 0.38), this did not reach statistical significance. Liver spleen uptake ratios (LS ratios) were increased in patients with RA (13/100 +/- 1/100 vs 7/100 +/- 1/100, P less than 0.05). There was no correlation of T1/2 or LS ratios with articular disease activity, vasculitis, ESR, IgM containing immune complex levels or Clq-binding immune complex levels. Although Clq-binding immune complex levels were significantly higher in patients with vasculitis than in those without (P less than 0.01), T1/2 and LS ratios did not differ in these two groups of patients. The T1/2 and LS ratios of E.IgG did not reveal a defect in MPS Fc-receptor function and did not correlate with one of the above-mentioned clinical and immunological parameters. We suggest that in order to establish a possible defect in Fc-receptor function correlating with disease activity and immune complex levels in RA patients, soluble immune complexes or immune complex-like material should be used as probes.