Blood Of Rheumatoid Arthritis Patients Research Articles

The Antioxidant Status in Rheumatoid Arthritis.

In the present work, we studied the antioxidant status in the blood of a group (n=29) of rheumatoid arthritis (RA) patients and compared it with that of a control group (n=30). There was a significant increase (p<0.001) in the plasma lipid peroxide level, but no change in the plasma ascorbic acid level, when the RA group was compared with the control group. There was a significant decrease in reduced glutathione (GSH, p<0.01) and in glutathione peroxidase (GPX, p<0.05) and superoxide dismutase (SOD, p<0.001) activities in the hemolysate of RA patients when compared with the control group values. A good correlation was obtained between the parameters estimated in the hemolysate. This study reflects the oxidative stress as a result of altered antioxidant status in the blood of RA patients, which findings may have implications in the pathogenesis of the disease.

Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors.

We have used chimeric IgG antibodies and their genetically engineered variants prepared by a combination of site-directed mutagenesis and exon exchange to define the structure(s) on IgG recognized by monoclonal rheumatoid factor (RF) autoantibodies from rheumatoid arthritis (RA) patients. Nineteen RF produced by EBV-transformed cell lines from the synovium or blood of RA patients were analyzed. Their binding patterns differ significantly from those seen with RF obtained from patients with Waldenstrom's macroglobulinemia (WMac). Half of the RA-derived RF bound IgG1, 2, and 4, but not 3 (Ga specificity), the common pattern in WMac. However, heterogeneity in fine specificity within the Ga reactivity pattern was observed. Moreover, seven others bound all four IgG subclasses, a pattern observed for only one WMac-derived RF from a patient who also had RA. Three RF had subclass specificities unlike any observed with WMac-derived RF. Most RA-derived RF bound IgG at a discontinuous epitope comprised of residues from both the CH2 and CH3 H chain constant regions. However, unlike any WMac-derived RF, one RA-derived RF bound IgG in CH2, another in CH3, and a third at an undetermined site outside of the CH2-CH3 interface. Some RA-derived RF bound aglycosylated IgG4 less well than glycosylated IgG4, suggesting that the carbohydrate moiety was important in establishing their binding epitope in CH2. These studies demonstrate that the repertoire of RF expressed by RA patients contains some unique binding specificities for IgG epitopes not found among our panel of WMac-derived RF. Our results therefore call into question whether WMac-derived RF with their limited diversity are appropriate models for disease-related RF. In addition, RF with their multiple specificities can serve as probes of antibody structure.

Modulation of monocyte activation in patients with rheumatoid arthritis by leukapheresis therapy.

One of the hallmarks in rheumatoid arthritis (RA) is the intense activation of the monocyte-macrophage system. In the present investigation, the modulation of blood monocyte activation was studied with regard to the secretion of cytokines and inflammatory mediators, and to the expression of cytokine receptors. Patients with severe active RA underwent repeated leukapheresis procedures that removed all circulating monocytes. Highly enriched monocyte preparations from the first and third leukapheresis were studied. There were striking differences between the two monocyte populations. Cells obtained from the first leukapheresis constitutively released large amounts of prostaglandin E2 (PGE2), neopterin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). In particular, IL-1 beta and neopterin production were further enhanced by stimulation with either interferon-gamma (IFN-gamma) or TNF-alpha without a synergistic effect. In contrast, cells derived from the third leukapheresis procedure showed a close to normal activation status with only low levels of cytokine and mediator production as well as a reduced response to cytokine stimulation. The number of the receptors for IFN-gamma and TNF-alpha was not changed between first and third leukapheresis. However, TNF-binding capacity was only detectable upon acid treatment of freshly isolated monocytes. A further upregulation was noted upon 24 h in vitro culture, suggesting occupation of membrane receptors and receptor down-regulation by endogenously produced TNF-alpha. Northern blot analysis of cytokine gene expression was in good correlation with the amount of mediators determined on the protein level. These data indicate that cells of the monocyte-macrophage system are already highly activated in the peripheral blood in RA patients with active disease. These cells can be efficiently removed by repeated leukapheresis and are replenished by monocytes that have, with respect to cytokine and mediator production, a considerably lower activation status.

Integrins and other adhesion molecules on lymphocytes from synovial fluid and peripheral blood of rheumatoid arthritis patients.

Cell and matrix adhesion of lymphocytes participates in homing, migration and accumulation of these cells in inflamed tissues as well as in the generation of immune and inflammatory responses. In inflamed joints of rheumatoid arthritis (RA) patients, lymphocytes accumulate in the synovial membrane and the synovial fluid. In the present study we have analyzed the expression of integrins and other adhesion molecules in synovial fluid lymphocytes (RA-SFL) and paired peripheral blood lymphocytes (RA-PBL) from 21 RA patients by immunofluorescence flow cytometry. We have also investigated the expression of these adhesion molecules on peripheral blood lymphocytes obtained from 13 sex- and age-matched healthy controls (CO-PBL). RA-SFL, which consisted mostly of T cells, showed higher expression of the integrin subunits beta 1 (CD29), VLA-1 alpha, -3 alpha, -4 alpha, -5 alpha and -6 alpha when compared to RA-PBL. In turn, RA-PBL showed lower expression of these molecules than CO-PBL. The expression of the immunoglobulin-related molecules CD2, CD54 (ICAM-1) and CD58 (LFA-3) was higher on RA-SFL when compared to RA-PBL or CO-PBL, and similar results were obtained with the beta 2 integrin subunits CD11a and CD18. In contrast, L-selectin (LECAM-1) and ICAM-2 were expressed at much lower levels on RA-SFL than on RA-PBL or CO-PBL. CD44, a receptor for hyaluronic acid and collagen, was expressed by most RA-SFL, RA-PBL and CO-PBL cells but at higher density on RA-SFL. The results indicate that RA-SFL express a distinct array of adhesion molecules, similar to the one of memory T lymphocytes. This characteristic phenotype may contribute to the lymphocytic infiltration of the synovium and to the pathogenesis of RA.

Are lymphocytes a target for substance P modulation in arthritis?

The contribution of the neuropeptide substance P to the pathogenesis of rheumatoid arthritis (RA) has recently been suggested. The presence of immunoreactive substance P in the serum and joint fluid of RA patients was significantly increased compared with age-matched control patients. To investigate the ability of substance P to alter lymphocyte activity during the disease, lymphocytes were isolated from the synovial fluid and blood of RA patients and their ability to respond to substance P as measured by [ 3H]thymidine uptake was characterized. Upon exposure of RA synovial fluid and peripheral blood lymphocytes to various concentrations of substance P in vitro, no increase in proliferation was witnessed. To the contrary, control peripheral blood lymphocyte proliferation was significantly enhanced by various concentrations of substance P. However, synoviocytes from the joints of RA patients were responsive to substance P stimulation. These data suggest that substance P receptors may be desensitized on systemic and local lymphocytes in RA, or the proinflammatory activities of substance P may be mediated via the synovial membrane during chronic inflammation.

Formula omitted] T cells and their subpopulations in blood and synovial fluid from rheumatoid arthritis and spondyloarthritis

T cell receptor (TCR) γ δ bearing lymphocytes in peripheral blood and synovial fluid from rheumatoid arthritis (RA) and seronegative spondyloarthritides (SSA) were evaluated by means of double label immunofluorescence and cytofluorographic analysis. Three monoclonal antibodies (MAb) were used. TCR δ-1 against a common δ chain epitope, BB3 directed against the T cell subset whose TCR is encoded by the Vδ2 gene, and A13 directed against the Vδ1 subset. Peripheral blood Tγδ cells were significantly reduced compared to normal control blood in RA patients who had joint effusion but not in other RA patients. The decrease of Tγδ in the RA PB was mainly confined to the A13+ subset. In RA synovial fluid (RA SF) Tγδ cells were significantly increased compared to paired but not normal peripheral blood, the most significant being the increase of A13+ cells. In contrast, in patients with SSA, no change in Tγδ cells was observed on PB or SF. These data suggest that in RA, but not SSA, Tγδ cells migrate from PB to inflamed synovium and thus may be involved in the pathogenesis of RA.

Pre-B lymphocytes with intracytoplasmic μ chains in the peripheral blood of rheumatoid arthritis patients

A dysregulation of B-cell differentiation and activation has long been evidenced in rheumatoid arthritis (RA). Such analyses have, however, usually focused on the latest stages of B-cell development. Using a classical technique of immunofluorescence labeling on cytospins, we investigated the presence of peripheral pre-B lymphocytes in 92 RA patients and 23 controls. Cells with intracytoplasmic μ chains were evidenced in 58.7% of the RA patients studied, ranging between 0 and 30%, while small numbers of c-μ cells, never exceeding 6% of peripheral blood lymphocytes, were observed in 9 controls. Relationships between this feature and clinical or laboratory data were investigated, showing a negative correlation between the percentage of c-μ+ lymphocytes and Ritchie's index ( P = 0.05), the number of tender or swollen joints ( P = 0.05), erythrocyte sedimentation rate ( P = 0.005), seropositivity ( P = 0.05), and disease duration ( P = 0.01).

Depressed T-cell reactivity to recall antigens in rheumatoid arthritis

Reactivity toward soluble recall antigens (Candida albicans, cytomegalovirus, herpes simplex, streptokinase-streptodornase, and influenza) was determined in cultures of peripheral blood mononuclear cells from 41 rheumatoid arthritis patients (with clinically active as well as inactive disease) and from 28 controls. In the group with clinically active rheumatoid arthritis we found an increased incidence of "anergy," defined as nonreactivity to three or more antigens. In an attempt to explain this decreased antigen reactivity, the latter was correlated with peripheral blood lymphocyte subsets, as defined by two-color immunofluorescence with a panel of eight monoclonal antibodies. We found a significantly lower number of memory T4 cells (CD4+CD45RA-) and a significantly higher number of the CD3-CD57+ (nonspecific suppressor) cells and of CD3-CD56+/CD16+ (natural killer) cells in anergic RA patients. In the total group of rheumatoid arthritis patients, the antigen reactivity correlated positively with the percentage of memory T4 cells. Antigen reactivity was negatively correlated with the percentage of CD3-CD57+ cells and of the CD3- natural killer cells in peripheral blood. Our data suggest that a decrease in memory T4 cells and an increase in nonspecific suppressor cells may contribute to the impaired cellular immune function in peripheral blood of rheumatoid arthritis patients.

The metabolism leukotriene B 4 in synovial fluid and whole human blood

The metabolism in vitro of synthetic leukotriene B 4 (LTB 4) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB 4 and the percentage of the initial concentration of LTB 4 at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB 4 was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB 4 was metabolised more rapidly in the blood of rheumatoid arthritis patients that either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB 4 to albumin in vivo will in part determine the available concentration of LTB 4 in inflammatory lesions.

Antigen and unspecific mitogen stimulation of lymphocytes eluted from rheumatoid inflammatory tissue.

Lymphocytes were eluted from synovial tissues of 17 patients with classical rheumatoid arthritis, using a procedure previously reported. Stimulation was obtained with the unspecific mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A) as well as with purified protein derivative of tuberculin (PPD) and mitoycin-C-treated allogeneic lymphocytes, whereas candida antigen usually gave a low response. The pattern of reactivity to unspecific mitogens was similar to that obtained with lymphocytes from peripheral blood of rheumatoid arthritis patients. Two different PPD preparations usually gave transformation of the same magnitude as seen with PHA. This was in contrast to the reactivity of the peripheral blood lymphocytes. It could be demonstrated that the elution procedure initiated some degree of lymphocyte transformation, mainly potentiating the responses to PHA and Con A.