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Objectives: The contribution of IL-17-producing Th17 cells to the pathogenesis of T-cell-mediated inflammatory disorders such as RA and atopic dermatitis (AD) has to be viewed in relation to the role of Th1/Th2 cells and long-recognized key cytokines like TNF. We aimed to study the frequency and migration-associated phenotype of peripheral Th17, Th1 and Th2 cells in healthy individuals, RA and AD patients, and to study the influence of anti-TNF therapy in RA., Methods: Intracellular IL-17, IFN-γ and IL-4 production and CC-chemokine receptor CCR4 and CCR6 expression were analysed flow cytometrically in peripheral memory Th cells from healthy individuals, AD and RA patients. The latter were grouped by disease activity and presence or absence of adalimumab therapy. In RA patients initiating anti-TNF therapy, cytokine production by in vitro-stimulated peripheral mononuclear cells was measured by cytometric bead array., Results: The peripheral Th17 cell frequency is elevated in AD but not in RA. In RA, Th17 cells and IL-17 production increase after anti-TNF therapy, irrespective of disease activity. Th1 cells and IFN-γ production are elevated in remission and under anti-TNF therapy. CCR6 expression is up-regulated in Th17 cells, but RA patients in remission under anti-TNF therapy have significantly lower expression than those with active disease., Conclusions: The increase in peripheral Th17 cells in RA patients after anti-TNF therapy is accompanied by a decrease in Th17-specific CCR6 expression, which might prevent homing of these potentially pro-inflammatory cells to the synovium.

Objectives: TNF-alpha inhibition therapy affects the systemic immune response in rheumatoid arthritis by influencing T cell subtypes (Th1, Th2, Treg), but its effect on the intracellular signal transduction in T cells remains largely unexplored. Here we studied the activation of Th1-associated signalling molecule STAT4 and Th2-associated STAT6 in CD4+ T cells., Methods: Eight rheumatoid arthritis patients were studied before and after 12 weeks of adalimumab therapy and compared to 8 healthy individuals. Peripheral blood mononuclear cells (PBMC) were analysed flow cytometrically either directly after isolation or after 24 hours of anti-CD3/anti-CD28 stimulation, to determine spontaneous and IL-4/IL-12-induced STAT4 and STAT6 phosphorylation in CD4+ T cells. Cytokine production by stimulated PBMC was measured in the supernatant using a cytometric bead array. Non-parametric statistical tests were applied., Results: After adalimumab therapy, phospho-STAT6 increased, both in freshly isolated and anti-CD3/anti-CD28-stimulated CD4+ T cells. The STAT6 response to brief IL-4 stimulation did not change. In healthy individuals and adalimumab-treated patients, anti-CD3/anti-CD28 induced the phosphorylation of STAT4, but not in untreated patients. IFN-gamma production in untreated patients was significantly lower than in healthy individuals or adalimumab-treated patients. In contrast, the production of IL-4, IL-6 and IL-12 was not influenced., Conclusions: Adalimumab therapy increases Th2-associated STAT6 phosphorylation and restores the activation-induced STAT4 phosphorylation to the levels in healthy individuals. This advocates against a pro-inflammatory effect of Th1-associated STAT4 and might provide an explanation for the influence of TNF inhibition therapy on the systemic T cell response in rheumatoid arthritis.

Background: The therapeutic effect of TNFalpha inhibition in rheumatoid arthritis (RA) is accompanied by an altered peripheral T cell cytokine profile, but the underlying mechanisms are not well known. In CD4+ T cells, TNF signalling includes the p38 MAP kinase (MAPK) pathway, which is also involved in proliferation and production of IL-4 and IFNgamma., Methods: Phosphorylation of p38 MAPK was analysed flow cytometrically in peripheral blood mononuclear cells (PBMC) from healthy individuals and RA patients before and after adalimumab therapy. Cytokine production by CD3/CD28-stimulated PBMC was measured in the supernatant., Results: Despite a transient activation of p38 MAPK in response to cellular stress from the cell separation, a significant decrease of spontaneous p38 MAPK phosphorylation was observed after adalimumab, compared to RA patients with active disease. Brief stimulation with TNFalpha/IL-1beta significantly activated p38 MAPK after but not before adalimumab therapy. In CD3/CD28-stimulated PBMC, significantly less p38 MAPK activation and increased IFNgamma production were observed after adalimumab therapy., Conclusion: In rheumatoid arthritis, adalimumab therapy decreases the phosphorylation of p38 MAPK except for its response to TNF/IL-1, while enhancing the production of IFNgamma. This suggests that p38 MAPK is not directly involved in the effect of TNF inhibition on cytokine production.

Most cell surface markers for CD4(+)CD25(+) regulatory T cells (Tregs) are also expressed by activated non-regulatory T cells. Recently, CD127 down-regulation was found to identify functional Tregs in healthy individuals, but there are no data from patients with inflammatory conditions. We examined peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis patients with active inflammation and from healthy controls, and found that CD4(+) T cells contained an equal proportion of CD25(+)CD127(-)/low cells in both groups. In patients, not all these cells expressed intracellular FOXP3. Upon activation by anti-CD3/anti-CD28, PBMC rapidly down-regulated CD127, while FOXP3 up-regulation was transitory and occurred in fewer cells. The activated cells were not anergic to restimulation and had no suppressive effects. The distinct kinetics indicate that the FOXP3(-)CD127(-)/low cells in rheumatoid arthritis patients most likely represent activated non-regulatory T cells. This complicates the use of CD127 for identification of Tregs in inflammatory diseases.

The destruction of cartilage is an important characteristic of rheumatoid arthritis (RA). Immune complexes (IC) are usually found in high amounts in RA synovial fluids (SF) and in the superficial layers of RA cartilage. The objective of this study was to investigate if IC have a direct influence on proliferation, survival and production of nitric oxide (NO) of cytokine-activated chondrocytes. Primary bovine chondrocytes were incubated with cytokines (huIL-1alpha, bovIFN-gamma, huTNF-alpha) and IC containing precipitates of peripheral blood (PB) and/or synovial fluid (SF) of 14 RA patients, 5 osteoarthritis (OA) patients and 10 healthy age and sex-matched controls. After 48 h, chondrocyte viability, proliferation, apoptosis, NO production and oxygen radical levels were measured. Staining with May-Grünwald-Giemsa after incubation with IC of RA PB and SF, showed apoptotic chondrocytes with condensation of the nuclei. The proliferation rates of cytokine-activated chondrocytes, incubated with sera and SF IC of RA patients were significantly decreased compared to chondrocytes, incubated with sera and SF IC of OA patients and compared to sera of controls. Quantitative evaluation of apoptotic cells by annexin-V/propidium iodide and TUNEL assays revealed a significant increase after incubation with sera and SF IC of RA patients, compared to control sera and OAs sera and SF. In all TUNEL positive samples, active-caspase-3-positive cells were found. There was a significant increase of chondrocyte NO production, after incubation with SF IC of RA patients, compared to OA SF. These results support the hypothesis that IC, present in serum and SF of RA patients, have a profound influence on chondrocyte growth, NO production and apoptosis, contributing to cartilage destruction in RA.

Objective: To investigate whether anti-TNF therapy could have an effect on dendritic cells (DCs) and regulatory T cells in rheumatoid arthritis (RA) patients., Methods: A four-colour flow cytometric technique was used to measure CD4+CD25+ T cells i.e. CD4+CD25high+ (regulatory T cells) and CD4+CD25low+ (activated T cells)), DCs as well as the in vitro, intracellular, lipopolysaccharide-stimulated cytokine production of DCs., Results: Clinical and laboratory parameters of disease activity decreased after anti-TNF treatment. Before anti-TNF therapy, RA patients demonstrated a decreased count of Th2-promoting lymphoid DCs as compared to controls and after anti-TNF therapy this decrease was sustained. Intracellular cytokine production was only found in the myeloid DCs population and there was a higher production of TNF-alpha and IL1-b as compared to healthy controls. Treatment did not alter this cytokine production. Before anti-TNF therapy, the percentage CD4+CD25+ T cells was significantly elevated in RA patients than in healthy controls., Conclusion: These results demonstrate anti-TNF to be a potent anti-inflammatory drug, as mirrored by the decrease in clinical and biological parameters as well as the decrease in activated CD4+ T cells. However, in this study no demonstrable effect on DCs and regulatory T cells was found.

Introduction: It has been shown that T lymphocytes and monocytes/macrophages, producing pro-inflammatory cytokines, play a pivotal role in the pathophysiology of rheumatoid arthritis (RA). In recent placebo-controlled double-blind randomized studies, chimeric (human/mouse) tumour necrosis factor-alpha (TNFalpha) antibodies (cA2) proved to be very effective in improving clinical disease activity and reducing inflammatory parameters in RA., Objective: To investigate whether anti-TNFalpha therapy influences the in vitro intracellular cytokine production in peripheral blood monocytes and T lymphocytes of RA patients after one single (24 h) and multiple intravenous infusions (6 months)., Methods: An intracellular flow cytometric technique was applied to measure interleukin 1beta (IL-1beta), IL-6, TNFalpha, IL-10 and IL-12 in monocytes and IL-2, IL-4 and interferon-gamma in T lymphocytes of 15 patients, before, after 24 h and after 6 months of therapy with monoclonal chimeric anti-TNFalpha antibodies (3 mg/kg, bimonthly i.v.). All patients were on stable therapy with methotrexate (15-20 mg/week i.m.). Cytokine content in monocytes was measured directly after blood sampling (basal levels), after 8 h of culture (spontaneous production) and after 8 h of stimulation with lipopolysaccharides (LPS-stimulated production)., Results: Basal levels and production (after 8 h) of IL-1beta, IL-6 and TNFalpha were significantly decreased 24 h after the first administration of anti-TNFalpha (for IL-1beta P < 0.01; for IL-6 P < 0.01; for TNF P < 0.003) and after 6 months of therapy (for IL-1beta P < 0.02; for IL-6 P < 0.03; for TNFalpha P < 0.001). For IL-12, basal levels were significantly decreased 24 h and 6 months after the start of therapy with anti-TNFalpha antibodies (P=0.0001; P=0.003, respectively). In contrast, IL-10 production increased significantly after 24 h and after 6 months (P=0.02; P=0.01). The T(H2)/T(H1) cytokine ratio in CD4+ T cells was significantly increased after 24 h and after 6 months of anti-TNFalpha therapy (P=0.003; P=0.0007)., Conclusion: Anti-TNFalpha therapy might down-regulate the monocytic capacity to produce pro-inflammatory cytokines and induces a shift to a more pronounced anti-inflammatory T(H2) cytokine production.

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease with predominance of type I cytokine [interleukin 2 (IL-2), interferon-gamma (IFN-gamma)] production. In this prospective study, we evaluated the influence of longterm therapy with methotrexate (MTX) in combination with low dose corticosteroids on the type 1/type 2 cytokine balance in RA., Methods: Peripheral blood mononuclear cells were isolated from 10 controls and 20 patients with RA before therapy and after 12 mo of therapy with MTX in combination with low dose corticosteroids. Using flow cytometry, the intracellular production of IL-2, IFN-gamma, and IL-4 was measured in CD4+ and CD8+ T lymphocytes., Results: Compared with healthy controls, patients with RA before therapy showed an increased percentage of IL-2 positive CD4+ and CD8+ T cells (p = 0.002, p = 0.01, respectively). An increased percentage of IFN-gamma positive CD8+ T cells was found (p = 0.0006) compared with the control group. After 12 months of therapy, a significantly decreased percentage of IL-2 positive CD4+ T cells and IFN-gamma positive CD4+ and CD8+ T lymphocytes was observed (p = 0.0003, p = 0.0007, p = 0.001). The percentage of IL-4/IFN-gamma positive CD4+ and CD8+ T cells was significantly higher after 12 months of therapy (p = 0.01, p = 0.02). There was a positive correlation between the percentage of IFN-gamma positive CD4+ T cells and disease activity variables (Ritchie Index and number of swollen joints) in RA patients before therapy (r = 0.6, p = 0.04 and r = 0.4, p = 0.05)., Conclusion: Longterm therapy with MTX in combination with low dose corticosteroids for RA influenced the predominance of type 1 cytokines toward normalization of the cytokine balance in both CD4+ and CD8+ T lymphocytes.

Objectives: The aim of this work was to evaluate in a randomised double-blind prospective study the effect of pamidronate on intracellular monocytic cytokine profiles (IL-1, IL-6, TNF-alpha) and bone density in rheumatoid arthritis patients., Methods: Twenty rheumatoid arthritis patients were treated for one year with methotrexate and a low dose of prednisolone. Double blind randomisation was performed for either i.v. pamidronate (at 3-month intervals) or placebo. The effect of pamidronate was evaluated on intracellular cytokine profiles (IL-1, IL-6, TNF-alpha), disease activity and bone mass measurements. The human monocytic cell line THP-1 was used to evaluate in vitro apoptosis by pamidronate., Results: Spontaneous production of interleukin-1 beta by patient blood monocytes was lower in the pamidronate group and was associated with an increase in bone density of the spine after 12 months of therapy. In vitro a dose-related increase in pamidronate induced apoptosis was found in THP-1 cells., Conclusions: This prospective double-blind randomised study demonstrated that pamidronate therapy resulted in an increase of bone density despite treatment with steroids. This rise is associated with a suppression of interleukin-1 beta production in monocytes of patients treated with pamidronate. Our in vitro experiments suggest that this anti-inflammatory effect could be due to an increase in the apoptosis of monocytic cells.

Objective: To determine whether blood and synovial fluid (SF) immune complexes (IC) of patients with rheumatoid arthritis (RA) influence the production of nitric oxide (NO) and growth and viability of chondrocytes., Methods: IC were precipitated and IgM and IgG were determined in the precipitates by ELISA and nephelometry, respectively. Primary cultures of bovine articular chondrocytes were incubated with the IC precipitates. After 48 h NO was determined as nitrite. After 7 days, growth was determined by incorporation of tritiated thymidine and viability was detected by neutral red uptake., Results: Patient sera were positive in 8/12 for IgM IC and 9/12 for IgG IC, and 1/8 control sera was slightly positive for IgM IC. Seven of 12 SF samples were IgM IC and 10/12 IgG IC positive. With and without additional interleukin 1alpha (IL-1alpha) stimulation, NO production by chondrocytes was significantly higher with SF IC precipitates than with control serum precipitates (p = 0.03, p = 0.04, respectively). NO production by chondrocytes that were not stimulated with IL-1alpha was significantly increased with SF IC precipitates compared to RA serum IC precipitates (p = 0.03). SF IC significantly inhibited growth compared to control serum precipitates (p = 0.04) and RA serum IC (p = 0.012). Neutral red uptake by chondrocytes was significantly decreased when incubated with RA serum IC in comparison with control serum IC (p = 0.012) and SF IC (p = 0.006). With and without additional IL-1alpha stimulation, NO production by chondrocytes after incubation with SF derived IC was positively correlated to the Ritchie score (r = 0.8, r = 0.7, respectively) and the number of swollen joints (r = 0.8, r = 0.6, respectively)., Conclusion: These results support the hypothesis that, especially in active RA, SF derived IC stimulate NO production and inhibit chondrocyte growth.

Experimental data suggest that pro-inflammatory cytokines such as interleukin 1beta (IL-1beta), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) are important in the pathogenesis of osteoporosis in rheumatoid arthritis. Therefore we compared the production of these cytokines by monocytes in 10 rheumatoid arthritis patients and 10 controls. Cytokine levels in rheumatoid arthritis patients were related to disease activity parameters, bone mineral density (BMD) corrected for age and sex (Z scores) and osteocalcin as a laboratory parameter of bone remodelling. Cytokines were determined by a flow cytometrical technique. There was a tendency for higher IL-1beta levels in patients compared with controls. A positive correlation between erythrocyte sedimentation rate and spontaneous production of monocytic cytokines was found. Z scores of the lumbar spine showed a negative correlation with spontaneous production of IL-1beta and IL-6. Plasma osteocalcin levels were positively correlated with spontaneous production of IL-1beta, IL-6 and TNF-alpha. In conclusion, the correlation of the levels of these cytokines with parameters of bone metabolism and osteoporosis suggest that especially IL-1beta and IL-6 are associated with more pronounced osteoporosis in active rheumatoid arthritis., (Copyright 1999 Academic Press.)

Type 1 cytokines (a.o. IL-2 and IFN-gamma) play an important role in the pathogenesis of rheumatoid arthritis. On the other hand, IgE-mediated diseases such as allergic asthma and atopic dermatitis show a type 2 cytokine (amongst others IL-4 and IL-5) profile. This study examined simultaneously the intracellular production of IL-2, IFN-gamma, IL-4 and IL-5 in T-lymphocytes of patients with rheumatoid arthritis during treatment with methotrexate or salazopyrin, patients with allergic asthma or atopic dermatitis under stable treatment, compared to healthy controls.A three-colour flow cytometric analysis was used for cytokine detection in T-helper cells and T-suppressor/cytotoxic cells. Compared to controls, patients with symptomatic atopic dermatitis showed an increased number of IL-4-producing T-helper lymphocytes in basal circumstances (P=0.001), in contrast to asymptomatic allergic asthma patients. Compared to controls, rheumatoid arthritis patients, treated with salazopyrin, showed an increased number of IL-2-producing T-helper and T-suppressor/cytotoxic lymphocytes after in vitro stimulation with PMA and ionomycin (P=0.01). In contrast, rheumatoid arthritis patients, treated with methotrexate, a more potent disease modifying drug, did not show this type 1 cytokine profile. A positive correlation was found between the number of IFN-gamma producing T-helper cells and disease activity (Ritchie Index and number of swollen joints) in both rheumatoid arthritis patient groups. Active atopic dermatitis patients showed a type 2 cytokine profile, whereas stable asthma patients with lower disease activity did not show a predominance of type 2 cytokines. Rheumatoid arthritis patients under treatment with salazopyrin had a type 1 cytokine profile, which could not be demonstrated in patients treated with methotrexate. This imbalance between type 1 and type 2 cytokines in different immune mediated disorders can be related with treatment and the grade of disease activity. These results stress the need for further investigation of the influence of therapy on cytokine profiles., (Copyright 1999 Academic Press.)

There is evidence that neutrophil functions such as chemotaxis and oxygen radical formation are disturbed in rheumatoid arthritis (RA). Medication might also influence these functions. Cyclic formation and depolymerisation of actin microfilaments is crucial in cell motility, but this phenomenon has not been studied in RA. The aim of this study was to investigate basal and dynamic (formyl-methionyl-leucyl-phenylalanine (fMLP)-induced) neutrophil actin polymerisation in ten RA patients (a) during therapy with non-steroidal anti-inflammatory drugs (NSAIDS) and (b) after stopping NSAIDS> The results were compared with those of ten age-matched controls. Basal F-actin content in RA patients with NSAIDS was significantly lower than in RA patients without NSAIDS and controls: 35.5 (25.0-49.0), 50.5 (27.0-75.0) and 52.5 (32.0-85.0), respectively. Conversely, upon stimulation with fMLP, the actin polymerisation curve of RA patients with NSAIDS was higher than for RA patients without NSAIDS and controls. These results suggest that, in RA, the effects orf NSAIDS on neutrophil functions might be related to changes in the actin polymerisation-depolymerisation cycle.

To evaluate the role of interleukin-8 (IL-8) on the activation of neutrophils in rheumatoid arthritis (RA), we measured IL-8 and the adhesion molecules, L-selectin (CD62L), CD1 1b and CD18, on neutrophils in paired peripheral blood and synovial fluid of RA patients. Synovial fluid IL-8 levels were significantly increased compared to peripheral blood. L-selectin was split off and CD1 1b and CD 18 were upregulated on neutrophils in the synovial fluid. A positive correlation occurred between the IL-8 concentration and CD18 or CD1 1b densities on neutrophils in the synovial fluid (r = 0.75, P < 0.005 and r = 0.60, P < 0.05, respectively). Peripheral blood neutrophils of the patients were desensitised with IL-8 in vivo, as shown by the significantly lower L-selectin shedding after in vitro IL-8 stimulation: 1.6 times decrease for patients vs 3.2 for controls (P < 0.05). In conclusion, these results add further evidence for the role of IL-8 in the activation of neutrophils in RA.

Neutrophil infiltration in synovial fluid is an important step in inflammation characterizing rheumatoid arthritis (RA). In this study, the activation and functional state of neutrophils in the blood and synovial fluid of patients with rheumatoid arthritis were compared: mean density of neutrophil activation markers CD11b, CD18 and L-selectin was measured with a flow cytometer, and adhesion to chondrocytes using a fluorimetric assay. No significant differences between control and patient peripheral blood neutrophils were observed. When comparing neutrophils of patient peripheral blood with paired synovial fluid, an increase in CD11b (p = 0.008) and a decrease in L-selectin (p = 0.008) were measured. For neutrophils of control and patient peripheral blood, fMet-Leu-Phe stimulation induced upregulation of CD11b (resp p = 0.007 and p = 0.008) and CD18 (resp p = 0.005 and p = 0.01). In the synovial fluid, no significant increase in CD11b and CD18 could be induced with fMet-Leu-Phe. Percentages of adherent neutrophils were comparable between controls and patients, both in peripheral blood and synovial fluid. Adhesion to chondrocytes of peripheral blood neutrophils of patients was correlated with clinical (Ritchie) and biological (erythrocyte sedimentation rate) parameters (resp r = 0.67, r = 0.73). In conclusion, these results demonstrate that peripheral blood neutrophil adhesion to chondrocytes was correlated with active disease, and that synovial fluid neutrophils were activated in vivo. These findings provide further evidence for the contributing role of neutrophils in articular destruction in RA.

CD5+ and CD23+ lymphocytes were determined in peripheral blood of patients with rheumatoid arthritis (RA) (n = 56), systemic lupus erythematosus (SLE) (n = 20) and primary Sjögren's syndrome (pSS) (n = 21). No definite correlation between the numbers of CD5+ or CD23+ cells and most of the parameters of disease activity was found. A significant correlation (r = 0.59, p = 0.005) between rheumatoid factor levels and numbers of CD23+ cells was found in RA patients not taking disease modifying agents. For SLE and pSS no signification associations with autoantibody production were found. In conclusion, although CD5+ and CD23+ B cells might be important in the pathogenesis of autoimmune diseases, determination of these subsets in peripheral blood of RA, SLE or pSS patients provides no clear useful clinical information for the individual patient.

The synovial tissue of patients with rheumatoid arthritis (RA) is characterized by infiltration with inflammatory cells, mainly memory helper cells (CD4+CD29+). An important initiating step in tissue infiltration is the adhesion of peripheral blood lymphocytes to the vascular endothelium. Therefore, we studied lymphocyte-endothelium adhesion in 40 RA patients and in 19 controls by a sensitive fluorimetric assay, using human umbilical vascular endothelial cells. Furthermore, expression of adhesion molecules VLA (CD29) and LFA-1 (CD11a) on CD4+ and CD8+ T cells was determined. In order to evaluate the activation state of lymphocytes, the soluble interleukin-2 receptor (sIL2R) was measured. The relationship to disease activity was evaluated using the Ritchie articular index. RA patients had a higher percentage of CD4+ cells (p < 0.005) and a lower percentage of CD8+ cells (p < 0.001) than controls did. The CD4+CD29+/CD4+CD29- ratio and the CD8+CD29+/CD8+CD29- ratio were increased in patients with active RA (p < 0.01 and p < 0.05, respectively) and in patients with inactive disease (p = 0.09 and p < 0.005, respectively) compared with controls. LFA-1 (CD11a) was present on almost all T lymphocytes and its density did not differ between patients and controls. Serum levels of sIL2R were significantly higher in both patient groups compared with controls (p < 0.0005); patients with active disease showed significantly higher levels than patients with inactive disease (p < 0.05). Lymphocyte-endothelium adhesion was not increased in patients, although the expression of the adhesion molecule CD29 on T lymphocytes of RA patients was higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Beside lymphocytes and neutrophils, eosinophils are also involved in the inflammatory reaction in rheumatoid arthritis (RA). In this study, adhesion characteristics of peripheral blood eosinophils were studied in 43 RA patients and 19 controls, together with the expression of the beta2-integrin Mac-1 (CD11b/CD18). In addition, the production of oxygen radicals of isolated peripheral blood eosinophils and serum levels of eosinophil cationic protein (ECP) were measured in order to evaluate eosinophil activation. Adhesion of eosinophils to unstimulated human vascular endothelium was significantly higher in RA patients with active disease (n = 4) compared with controls (n = 14) (P < 0.005) and compared with patients with less active RA (n = 16) (P < 0.05). Nevertheless, the expression of the adhesion molecule Mac-1 (CD11b/CD18) was not increased in RA patients. ECP levels were higher in RA patients with active disease (P < 0.01). Release of oxygen radicals in response to phorbol stimulation was significantly elevated in active RA compared with controls (P < 0.05) and to less active RA (P < 0.05). We conclude that eosinophils of RA patients, especially those with active disease, are activated or at least primed and are involved in the inflammatory process in RA, analogous to the inflammation in asthma. The higher adhesion to inflamed endothelium is indicative of a higher infiltration in the joints, where tissue damage can be caused by toxic oxygen radicals and by granular proteins, such as ECP.

Neutrophil and monocyte activation by immune complexes containing IgE from serum and synovial fluid of patients with rheumatoid arthritis is reported. Activation of the inflammatory cells was measured by stimulation of the respiratory burst with production of intracellular hydrogen peroxide. Generation of hydrogen peroxide was analysed by a flow cytometric method, using the fluorochrome dichlorofluorescein. The technique was modified to allow measurement of cell activation of both neutrophils and monocytes by immune complexes in suspension. Ten of 14 polyethylene glycol precipitates from serum of patients with rheumatoid arthritis and 10/16 synovial fluids of these patients could activate neutrophils. A positive relation was found between the activation of neutrophils and the total concentration of immune complexes, the presence of IgG, and the presence of IgE in the immune complexes. Activation of monocytes was also shown, but to a lesser extent (8/14 rheumatoid serum samples and 8/16 rheumatoid synovial fluids activated monocytes). There was a weak correlation between the concentration of IgE immune complexes and the intensity of fluorescence measured in the monocytes.

The prevalence and composition of IgE-containing immune complexes in paired synovial fluid and serum of 42 patients with classical or definite rheumatoid arthritis were studied. IgE-containing immune complexes were found in 15/42 synovial fluids; 15 sera were also positive. The correlation between serum and synovial fluid complexed IgE levels was high (r = 0.77). The mean ratio of synovial fluid/serum levels was 1.96, i.e. significantly higher than 0.33, the synovial fluid/serum ratio for alpha-2-macroglobulin (molecular weight 820 kD), which was taken as high molecular weight control protein (p less than 0.0001). Apart from IgE in immune complex form, monomeric IgE was also significantly higher in synovial fluid compared to serum (ratio = 2.94). Other constituents which could be found in the immune complexes, i.e. anti-IgE antibodies, rheumatoid factors and anticollagen antibodies, were also higher in synovial fluid than predicted. Our results suggest intra-articular production of IgE-containing complexes in the synovial fluid, in addition to possible exudation of the complexes from the serum. These findings provide further evidence for the role of IgE-containing immune complexes in rheumatoid synovitis.

IgM-rheumatoid factor (RF) interference in the determination of total serum IgE and IgE-containing circulating immune complexes (IgE-CIC) was studied by inhibition experiments in vitro comparing a new ELISA technique free of IgM-RF interference with more widely used RIA methods. It was shown that a considerable overestimation of the IgE content in CIC can exist when high levels of IgM-RF are present in the same serum. The clinical part of this study revealed a dramatic fall in prevalence of IgE-CIC in patients with rheumatoid arthritis (RA) with the ELISA technique, compared with the more conventional RIA method (respectively 1/20 compared to 12/20 positive for IgE-CIC). In these patients, there was a good correlation between the level of IgM-RF and the amount of IgE detected in the CIC by the RIA method (r = 0.87) whereas the RF-interference free ELISA method showed no correlation between these two parameters (r = 0.06). Total serum IgE determination with a solid phase RIA was also influenced by IgM-RF interference, whereas the PRIST method was not affected by the presence of IgM-RF. In conclusion, in patients with rheumatic diseases, IgE-assays using polyclonal rabbit or sheep anti-IgE antibodies are not appropriate and monoclonal anti-IgE antibodies that have been proved not to interfere with IgM-RF should be advocated.

Biopsy specimens from apparently normal skin of 28 patients with classical or definite rheumatoid arthritis (RA) were examined for the presence of IgE deposition by a direct immunofluorescence technique. IgE deposition was found in 12 patients (43%) and in none of the 10 controls. This deposition was mainly localised on mast cells, and in three patients perivascular IgE staining was also noted. The skin from nine of the 12 patients also showed deposition of IgM and complement C3 or C4 factor, or both. All 12 patients with skin IgE deposition had raised levels of IgM rheumatoid factor (RF) in the serum. Nine of these also had IgE RF. IgE-containing circulating immune complexes (IgE CIC), raised serum IgE levels, and extra-articular (EA) manifestations were present in respectively 10, nine, and eight skin IgE positive patients. It is suggested that IgE and IgE CIC may be involved in the pathogenesis of RA and its EA manifestations.

IgE-containing circulating immune complexes (IgE-CIC) were determined with a 2.5% PEG-precipitation assay in 98 patients with classical or definite rheumatoid arthritis (RA). Of the 45 IgE-CIC positive sera, only 4 had elevated total serum IgE. IgE-CIC positive patients had more active disease than patients without IgE-CIC, as determined by their more swollen joints and higher Ritchie indices (p less than 0.04 and 0.02, respectively). Apart from IgE, other immunoglobulin isotypes, rheumatoid factor (RF) of the IgG-, IgA- and IgM-classes, C3 and antinuclear antibodies could be demonstrated in the IgE-containing PEG-precipitates. IgE-RF could not be demonstrated in serum or in IgE-CIC. Anti-IgE of the IgM-class (IgMaIgE) were frequently found (28/45 patients) in the IgE-positive PEG-precipitates. All 14 patients positive for IgGaIgE in the IgE-CIC were also positive for IgMaIgE in the CIC. As in the serum, there was a good correlation in the CIC between the level of IgGaIgE and the level of IgMaIgE (r = 0.64). The correlation between the respective levels of IgGaIgE and IgMaIgE in serum and in CIC was high (r = 0.93 and 0.79, respectively). On the other hand, only 1 patient was positive for IgAaIgE in the IgE-CIC. We conclude that IgE and aIgE of the IgM- and IgG-classes are frequently present in the immune complex form in RA and that they are correlated with the clinical activity of arthritis.