Fionula M. Brennan

Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis.

The effect of tumour necrosis factor (TNF alpha) antibodies on synovial cell interleukin-1 (IL-1) production was investigated in 7 patients with rheumatoid arthritis and in 7 with osteoarthritis. Synovial cell IL-1 production was significantly reduced by anti-TNF alpha antibody in cultures from patients with rheumatoid arthritis, but antilymphotoxin antibody did not have this effect (except in 1 culture). In cultures from patients with osteoarthritis spontaneous IL-1 production was low, despite high concentrations of TNF alpha, and IL-1 production was not inhibited by anti-TNF alpha antibody. In rheumatoid arthritis, TNF alpha may be the main inducer of IL-1, and anti-TNF alpha agents may be useful in treatment.

Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.

Although there have been major advances in the treatment of rheumatoid arthritis with the advent of biological agents, the mechanisms that drive cytokine production and sustain disease chronicity remain unknown. Tenascin-C (encoded by Tnc) is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. Here we show that mice that do not express tenascin-C show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of tenascin-C promotes joint inflammation in vivo in mice, and addition of exogenous tenascin-C induces cytokine synthesis in explant cultures from inflamed synovia of individuals with rheumatoid arthritis. Moreover, in human macrophages and fibroblasts from synovia of individuals with rheumatoid arthritis, tenascin-C induces synthesis of proinflammatory cytokines via activation of Toll-like receptor 4 (TLR4). Thus, we have identified tenascin-C as a novel endogenous activator of TLR4-mediated immunity that mediates persistent synovial inflammation and tissue destruction in arthritic joint disease.

Cytokines in autoimmunity

The past few years have witnessed exciting developments regarding the role of cytokines in autoimmune diseases, particularly in rheumatoid arthritis and Crohns disease, with the demonstration that anti-TNF alpha therapy is clinically beneficial and provides reproducible results. Recent contributions to this field, derived from in vivo studies in animal models of autoimmunity, and increasingly from clinical trials, have greatly enhanced our understanding of this field.

Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex; reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis.

Experiments were designed to test the hypothesis that chronic exposure to tumor necrosis factor alpha (TNF) alters the function of activated T lymphocytes. Pretreatment of tetanus toxoid-specific T cell clones with TNF for up to 16 d impaired rechallenge proliferative responses to antigen in a dose- and time-dependent fashion. IL-2 and PHA responses were preserved. Prolonged treatment with TNF impaired production of IL-2, IL-10, IFN gamma, TNF, and lymphotoxin (LT) following stimulation with immobilized OKT3, and resulted in suboptimal expression of the IL-2R alpha chain (Tac) but not CD3, CD4, or HLA-DR antigens, when compared to untreated control cells. By contrast, pretreatment of T cells for prolonged periods in vitro with neutralizing anti-TNF monoclonal antibodies (mAb) enhanced proliferative responses, increased lymphokine production, and upregulated Tac expression following stimulation with OKT3. To determine whether TNF exerts immunosuppressive effects on T cells in vivo, we studied cell-mediated immunity in patients with active rheumatoid arthritis (RA), before and after treatment with a chimeric anti-TNF mAb. Treatment with anti-TNF restored the diminished proliferative responses of PBMC to mitogens and recall antigens towards normal in all patients tested. These data demonstrate that persistent expression of TNF in vitro and in vivo impairs cell-mediated immune responses.

Monoclonal anti-TNF-alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease

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.

Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production.

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Brutons tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

Cytokines and anti-cytokine biologicals in autoimmunity: present and future.

The increasing understanding of the role of cytokines in autoimmunity, and the observation that tumour necrosis factor alpha (TNFalpha) is central to the inflammatory and destructive process common to several human autoimmune diseases, has led to a new generation of therapeutics, the TNFalpha blocking agents. In this article, we review the current knowledge of the role of cytokines in autoimmunity as unravelled by studies both in the laboratory and the clinic. In addition, we discuss future prospects of the anti-TNFalpha therapy that may involve combination therapy with other anti-cytokine or anti-T cell biologicals, or the use of small chemicals targeting molecules involved in TNFalpha production such as NF-kappaB and p38 MAPK. The future developments of anti-TNFalpha and anti-cytokine therapy in general will be interesting.

Evidence that rheumatoid arthritis synovial T cells are similar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinase and nuclear factor kappaB pathways in tumor necrosis factor alpha production in rheumatoid arthritis.

OBJECTIVE To investigate the mechanism that leads to the spontaneous production of tumor necrosis factor alpha (TNFalpha) in rheumatoid arthritis (RA) synovial tissue. METHODS Normal blood monocytes were cocultured either with fixed activated T cells generated from normal blood or RA synovial T cells purified from synovium. TNFalpha production was measured in supernatants from these cocultures following blockade of the transcription factor nuclear factor kappaB (NF-kappaB) using adenoviral transfer of the inhibitor of NF-kappaB kinase alpha into the responding monocytes, or blockade of phosphatidylinositol 3-kinase (PI 3-kinase) using the inhibitory drugs wortmannin or LY294002. TNFalpha production was measured by enzyme-linked immunosorbent assay. RESULTS TNFalpha production in synovial tissue from patients with RA but not osteoarthritis was found to be T cell dependent. The RA synovial joint T cells resembled normal T cells that had been activated for 8 days using a cocktail of cytokines. These T cells, designated Tck (cytokine-activated T cells), and RA synovial T cells both induced TNFalpha production in resting monocytes in a cell-contact-dependent manner, which was abrogated by blockage of the transcription factor NF-kappaB but augmented if PI 3-kinase was inhibited. Normal blood T cells activated conventionally via the T cell receptor with crosslinked anti-CD3 antibody resulted in TNFalpha production from monocytes; this was unaffected by NF-kappaB blockade, but was inhibited in the presence of PI 3-kinase-blocking drugs. CONCLUSION These data provide strong evidence for the importance of T cells in inducing TNFalpha in chronic inflammatory rheumatoid tissue, and give insight into the mechanism whereby these T cells are activated in vivo. Furthermore, they indicate that production of TNFalpha in pathologic tissue is regulated differently from physiologic antigen-dependent TNFalpha production, which raises the possibility that selective inhibitors of TNFalpha in disease may be developed.

Bruton’s Tyrosine Kinase Is Required for TLR2 and TLR4-Induced TNF, but Not IL-6, Production

Bruton’s tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1β, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1β than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1β, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.

Blockade of IL-12 during the induction of collagen-induced arthritis (CIA) markedly attenuates the severity of the arthritis

The effect of blocking IL‐12, a potent inducer of interferon‐gamma (IFN‐γ) and promoter of Th1 cell responses, during the induction phase of CIA was investigated. Arthritis was elicited in male DBA/1 mice by immunizing with type II collagen (CII) in Freunds complete adjuvant. Neutralizing anti‐IL‐12 antibodies were administered twice weekly from CII immunization. It was found that administration of anti‐IL‐12 from immunization until the onset of clinical arthritis did not lower the incidence of arthritis, but dramatically attenuated the severity of the disease, both clinically and histopathologically. This regime was associated with reduced IFN‐γ levels produced by ex vivo CII‐stimulated draining lymph node cells, and with diminished spontaneous ex vivo production of tumour necrosis factor (TNF), IL‐6 and IL‐10 by freshly isolated synovial cells. Total anti‐CII antibody serum levels in these mice were lower than in the controls, but there was no change in the IgG2a/IgG1 ratio. These findings confirm that IL‐12 has a major role in the induction of murine CIA and suggests that this disease is propagated, in part, by cells of the Th1 phenotype.