Relationship between α1-antitrypsin inactivation and tumor necrosis factor α concentration in the synovial fluid of patients with rheumatoid arthritis

Objective. To examine the relationship between α₁-antitrypsin (α₁AT) specific activity and tumor necrosis factor α (TNFα) concentration in synovial fluid from 48 patients with rheumatoid arthritis. Methods. The specific activity of α₁AT was calculated from the measurement of α₁AT concentration (by rocket immunoelectrophoresis) and elastase inhibitory capacity. TNFα was detected by enzyme-linked immuno-sorbent assay. Results. TNFα concentrations correlated with the extent of α₁AT inactivation. Conclusion. Our findings are consistent with a role of elastase in TNFα release within the inflamed joint.     

Regulation of tumor necrosis factor α–mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt

Objective

To determine if tumor necrosis factor α (TNFα)–driven proliferation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with up‐regulation of the activity of serine/threonine kinase B/Akt and with survival of RASF.

Methods

Staining of phosphorylated Akt was done using anti–phosphorylated Thr³⁰⁸ Akt antibody. Levels of phosphorylated Akt were analyzed by Western blot and Akt activity was analyzed using a kinase assay. TUNEL staining was used to analyze the cytotoxicity of TNFα treatment or TNFα combined with either the Akt activity inhibitor wortmannin, an adenovirus expressing dominant‐negative mutant (AdAkt‐DN), or an adenovirus expressing phosphatase and tensin homolog deleted on chromosome 10 (AdPTEN).

Results

The levels of phosphorylated Akt were higher in RASF than in osteoarthritis synovial fibroblasts (OASF), as demonstrated by immunohistochemical staining, immunoblot analysis, and an Akt kinase assay. The levels of phosphorylated Akt and Akt kinase activity were increased by stimulation of primary RASF with TNFα (10 ng/ml). Treatment of RASF with the phosphatidylinositol 3‐kinase inhibitor wortmannin (50 nM) plus TNFα resulted in apoptosis of 60 ± 8% (mean ± SEM) of RASF within 24 hours. This pro‐apoptosis effect was specific for Akt, since equivalent levels of apoptosis were observed upon TNFα treatment of RASF transfected with AdAkt‐DN and with AdPTEN, which opposes the action of Akt.

Conclusion

These results indicate that phosphorylated Akt acts as a survival signal in RASF and contributes to the stimulatory effect of TNFα on these cells by inhibiting the apoptosis response. This effect was not observed in OASF and may reflect the pathophysiologic changes associated with the proliferating synovium in rheumatoid arthritis.

     

Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor α

Objective. To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor α (TNFα) in the treatment of patients with rheumatoid arthritis (RA). Methods. Twenty patients with active RA were treated with 20 mg/kg of anti-TNFα in an open phase I/II trial lasting 8 weeks. Results. The treatment was well tolerated, with no serious adverse events. Significant improvements were seen in the Ritchie Articular Index, which fell from a median of 28 at study entry to a median of 6 by week 6 (P < 0.001), the swollen joint count, which fell from 18 to 5 (P < 0.001) over the same period, and in the other major clinical assessments. Serum C-reactive protein levels fell from a median of 39.5 mg/liter at study entry to 8 mg/liter at week 6 (P < 0.001), and significant decreases were also seen in serum amyloid A and interleukin-6 levels. Conclusion. Treatment with anti-TNFα was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNFα is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.     

Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor α

Objective. We have previously described the location of tumor necrosis factor α (TNFα)–producing cells in synovial tissue and cartilage–pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNFα, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. Methods. The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase–conjugated mouse anti–alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. Results. In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage–pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R–expressing cells were in the vicinity of TNFα-containing cells, and some TNFα-containing cells also expressed TNF-R. TNF-R–expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. Conclusion. These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNFα in this tissue. This study further supports the hypothesis that TNFα plays a major role in the pathogenesis of RA.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

Circulating soluble tumor necrosis factor receptors,interleukin-2 receptors,tumor necrosis factor α, and interleukin-6 levels in rheumatoid arthritis.

Objective. To assess whether circulating concentrations of soluble tumor necrosis factor receptors (sTNFR; p55 and p75), soluble interleukin-2 receptors (sIL-2R), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) reflect clinical response and whether changes are dependent on the drug used in rheumatoid arthritis (RA) patients taking methotrexate (MTX) or azathioprine (AZA). Methods. These cytokines and soluble receptors were assessed in 20 control subjects and serially for up to 48 weeks in 61 RA patients, by bioassay (IL-6) and immunoassays (sTNFR, sIL-2R, TNFα, and IL-6). Results. Concentrations of p55 and p75, sIL-2R, and TNFα (but not IL-6) were significantly higher in RA patients than in controls. Significant decreases in sIL-2R and p55 concentrations were associated with clinical improvement and were observed in patients treated with MTX, but not AZA. Both treatments induced decreases in IL-6 concentrations, but circulating AZA (or its metabolites) appears to interfere with the measurement of IL-6 bioactivity. TNFα and p75 levels did not show significant changes. Conclusion. Measurement of circulating sIL-2R, p55, and IL-6 may be useful in the evaluation of RA disease activity and response to therapy. Interference by circulating levels of drugs must be ruled out when bioassays are used to evaluate cytokine levels.     

Association of interleukin‐18 expression with enhanced levels of both interleukin‐1β and tumor necrosis factor α in knee synovial tissue of patients with rheumatoid arthritis

Objective

To examine the expression patterns of interkeukin‐18 (IL‐18) in synovial biopsy tissue of patients with rheumatoid arthritis (RA), and to determine whether expression of this primary cytokine is related to the expression of other cytokines and adhesion molecules and related to the degree of joint inflammation.

Methods

Biopsy specimens of knee synovial tissue either without synovitis (n = 6) or with moderate or severe synovitis (n = 11 and n = 12, respectively) were obtained from 29 patients with active RA. Paraffin‐embedded, snap‐frozen sections were used for immunohistochemical detection of IL‐18, tumor necrosis factor α (TNFα), IL‐1β, IL‐12, and IL‐17. Furthermore, adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E‐selectin, and cell markers CD3, CD14, and CD68 were stained.

Results

IL‐18 staining was detectable in 80% of the RA patients, in both the lining and sublining of the knee synovial tissue. IL‐18 expression in the synovial tissue was strongly correlated with the expression of IL‐1β (in the sublining r = 0.72, in the lining r = 0.71; both P < 0.0001) and TNFα (in the sublining r = 0.59, P < 0.0007, and in the lining r = 0.68, P < 0.0001). In addition, IL‐18 expression in the sublining correlated with macrophage infiltration (r = 0.64, P < 0.0007) and microscopic inflammation scores (r = 0.78, P < 0.0001), and with the acute‐phase reaction as measured by the erythrocyte sedimentation rate (r = 0.61, P < 0.0004). Interestingly, RA synovial tissue that coexpressed IL‐18 and IL‐12 demonstrated enhanced levels of the Th1‐associated cytokine IL‐17.

Conclusion

Our results show that expression of IL‐18 is associated with that of IL‐1β and TNFα and with local inflammation in the synovial tissue of patients with RA. In addition, synovial IL‐18 expression correlates with the acute‐phase response. These data indicate that IL‐18 is a primary proinflammatory cytokine in RA that drives the local production of IL‐1β and TNFα.

     

Lack of response to anakinra in rheumatoid arthritis following failure of tumor necrosis factor α blockade

Objective.

In patients with rheumatoid arthritis (RA) treated with tumor necrosis factor α (TNFα)– blocking therapy, there is heterogeneity of response. This raises the possibility that in certain circumstances, cytokines such as interleukin‐1 (IL‐1) may dominate the drive toward joint inflammation. This study was undertaken to investigate whether blocking the action of IL‐1 with an IL‐1 receptor antagonist (IL‐1Ra) is efficacious in patients with disease that did not respond to TNFα blockade.

Methods.

We identified 26 RA patients whose disease had failed to respond to TNFα‐blocking therapy, defined as failure to achieve or sustain a 20% improvement in disease activity according to the criteria of the American College of Rheumatology (ACR20 response). These patients were then treated with anakinra (100 mg/day subcutaneously) for 12 weeks, and their levels of response were assessed.

Results.

After 3 months of anakinra therapy, only 2 of 26 patients (8%) achieved an ACR20 response; none achieved an ACR50 or ACR70 response. A rise in the mean C‐reactive protein level and an increase in the mean swollen joint count were noted during the study period.

Conclusion.

This study demonstrates that patients with disease that fails to respond to TNFα blockade also do not respond to IL‐1Ra. These data do not provide evidence of a dominant role for IL‐1 in patients who do not respond to TNFα blockade, but they do not exclude a role for other proinflammatory mediators.