Using immunoaffinity‐purified polyclonal anti‐human recombinant tumor necrosis factor α (TNFα) F(ab′)2 fragments and immunohistochemical techniques, the cells that make TNFα were localized in the inflamed synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Anti‐TNFα antibody–stained cells were demonstrated in 9 of 11 RA and 2 of 4 OA but none of 5 normal synovial membranes examined. In RA, 26–64% of the lining layer cells were positive for TNFα. In the interaggregate area, 10–30% of the cells contained TNFα, often in a perivascular distribution, and up to 19% of the cells in lymphoid aggregates stained for TNFα. Some endothelial cells also stained with these antibodies. In OA tissues, the TNFα‐containing cells were found predominantly in the deeper layer. Cells containing TNFα were also found at the cartilage–pannus junction in all 4 RA specimens examined. Double immunofluorescence analysis demonstrated that most TNFα‐secreting cells in the RA synovial membrane expressed the monocyte/macrophage marker antigens CD11b and CD14, and a few expressed the T cell marker CD3. Our findings provide histologic evidence that TNFα is locally produced in the lining and deeper layers of the synovium by cells of the mónocyte/macrophage lineage, supporting its role in inflammation. Further, our findings demonstrate that TNFα is produced by cells at the cartilage–pannus junction, which could affect chondrocyte metabolism, leading to the cartilage degradation in RA.

Cytokine release at the cartilage/pannus junction (CPJ) may be involved in cartilage destruction and tissue repair in rheumatoid arthritis (RA). Tissue samples of CPJ from 12 RA patients were examined for the presence of cytokines using immunohistochemical techniques with immunoaffinity purified F(ab')2 antibodies raised against recombinant human cytokines. Twenty-four areas of distinct CPJ at which a discrete junction between cartilage and overlying pannus exists were observed. In all specimens, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha. IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta 1 were detected in cells in pannus particularly along the surface of cartilage and at the site of cartilage erosion. Double immunofluorescence staining showed that most cytokine containing cells also labelled with a macrophage marker (CD68). About 50% of blood vessel endothelial cells stained for GM-CSF. Twelve areas of diffuse fibroblastic CPJ, at which an indistinct margin is seen between cartilage and pannus were examined. At this site, TGF-beta 1 was the only cytokine detected in fibroblast-like cells. None of these cytokines were detected in synovial tissue at the normal synovium/cartilage junction. Chondrocytes from all 11 normal specimens as well as those from RA patients stained for IL-1 alpha, TNF-alpha, IL-6, GM-CSF and TGF-beta 1, especially those close to subchondral bone. However, IL-1 beta, interferon-gamma and lymphotoxin were not detected in either the normal synovium/cartilage junction or rheumatoid CPJ.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective To evaluate fenebrutinib, an oral and highly selective noncovalent inhibitor of Bruton's tyrosine kinase (BTK), in patients with active rheumatoid arthritis (RA). Methods Patients with RA and an inadequate response to methotrexate (MTX) (cohort 1; n = 480) were randomized to receive fenebrutinib (50 mg once daily, 150 mg once daily, or 200 mg twice daily), adalimumab (40 mg every other week), or placebo. Patients with RA and an inadequate response to tumor necrosis factor inhibitors (cohort 2; n = 98) received fenebrutinib (200 mg twice daily) or placebo. Both cohorts continued MTX therapy. Results In cohort 1, the percentages of patients in whom American College of Rheumatology 50% improvement criteria (ACR50) was achieved at week 12 were similar in the fenebrutinib 50 mg once daily and placebo groups, and were higher in the fenebrutinib 150 mg once daily group (28%) and 200 mg twice daily group (35%) than in the placebo group (15%) (P = 0.016 and P = 0.0003, respectively). Fenebrutinib 200 mg twice daily and adalimumab (36%) were comparable (P = 0.81). In cohort 2, ACR50 was achieved in more patients receiving fenebrutinib 200 mg twice daily (25%) than placebo (12%) (P = 0.072). The most common adverse events in the fenebrutinib groups included nausea, headache, anemia, and upper respiratory tract infections. Fenebrutinib had significant effects on myeloid and B cell biomarkers (CCL4 and rheumatoid factor). Fenebrutinib and adalimumab caused overlapping as well as distinct changes in B cell and myeloid biomarkers. Conclusion Fenebrutinib demonstrates efficacy comparable to adalimumab in patients with an inadequate response to MTX, and safety consistent with existing immunomodulatory therapies for RA. These data support targeting both B and myeloid cells via this novel mechanism for potential efficacy in the treatment of RA.

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.

Rheumatoid arthritis is a common cause of chronic disability for which current therapies are of limited value in controlling the disease process and outcome. Our initial approach to understanding the pathogenesis of RA and defining a novel therapeutic target was to investigate the role of cytokines by blocking their action with antibodies on cultured synovial-derived mononuclear cells in vitro. These investigations suggested that neutralization of TNF alpha with antibodies significantly inhibited the generation of other pro-inflammatory cytokines also over-produced, such as, IL-1, GM-CSF, IL-6 and IL-8. The implication that blockade of a single cytokine, TNF alpha might have far-reaching effects on multiple cytokines and thereby exert significant anti-inflammatory and protective effects on cartilage and bone of joints, was tested in arthritic DBA/1 mice immunized with collagen II. Impressive amelioration of joint swelling and joint erosions in this model encouraged clinical trials with a monoclonal anti-TNF alpha antibody. The cA2 chimeric anti-TNF alpha high-affinity antibody was initially tested in an open-label study at a dose of 20 mg/kg on 20 patients, with substantial and universal benefit. Subsequently, a randomized placebo-controlled double-blind trial was performed on 73 patients comparing a single intravenous injection of placebo (0.1% human serum albumin) with two doses of cA2. Using a composite disease activity index, at 4 weeks post infusion, 8% of patients receiving placebo improved compared with 44% receiving 1 mg/kg cA/2 and 79% receiving 10 mg/kg. Between 2 to 4 repeated cycles of cA2 were administered to 7 patients and all patients showed improvement of a similar magnitude with each cycle. These data support our proposition that TNF alpha is implicated in the pathogenesis of RA, and is thus a key therapeutic target. Monoclonal anti-TNF alpha antibodies control disease flares and are candidate agents for longer-term control of RA, although repeated therapy with cA2 is associated with anti-idiotypic responses in 50% of patients and a trend toward shortening of the duration of response. In the DBA/1 arthritic mice, synergy of action of anti-TNF and anti-CD4 is observed together with suppression of an anti-globulin response, indicating one way in which benefit might be augmented in the future.