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Biomedical subjects

F M Brennan

Publications and source records attributed to F M Brennan.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor-alpha, but not interleukin-10: possible relevance to pathophysiology of rheumatoid arthritis.

Previous studies in the laboratory have shown that the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The mechanisms involved in regulating monocyte/macrophage cytokine production are not yet fully understood, but are thought to involve both soluble factors and cell/cell contact with other cell types. We and others have previously demonstrated that T cells activated through the T cell receptor/CD3 complex induce monocyte TNF-alpha production by contact-mediated signals. In this report, we investigated further whether T cells activated by cytokines in the absence of T cell receptor stimulation also regulate monocyte cytokine production. T cells were activated in an antigen-independent manner using the cytokines interleukin (IL)-15 or IL-2 alone, or in combination with IL-6 and TNF-alpha. Subsequently, T cells were fixed and incubated with monocytes. Fixed, cytokine-stimulated T cells induced monocytes to secrete TNF-alpha in a dose-dependent manner, but did not induce secretion of IL-10, a potent endogenous down-regulator of TNF-alpha and other pro-inflammatory cytokines. Stimulation of monocyte TNF-alpha was markedly inhibited when T cells were physically separated from monocytes within the tissue culture well, confirming that T cell contact is necessary. T cell acquisition of monocyte-activating capacity was shown to be dependent on the period of cytokine stimulation, with T cells activated for 8 days more effective than T cells activated for shorter periods. Addition of interferon-gamma or granulocyte/macrophage colony-stimulating factor to the T cell/monocyte cultures enhanced T cell induction of monocyte TNF-alpha by threefold and ninefold, respectively. The results from this model of cognate interaction suggest that cytokine-stimulated T cells, interacting with macrophages in the rheumatoid synovial membrane, may contribute to the continuous excessive production of TNF-alpha observed in the RA joint, and to the imbalance of pro-inflammatory cytokines over anti-inflammatory cytokines.

Antigens, Surface↗
PubMed1997

Reduction of serum matrix metalloproteinase 1 and matrix metalloproteinase 3 in rheumatoid arthritis patients following anti-tumour necrosis factor-alpha (cA2) therapy.

Matrix metalloproteinase (MMP)-1 and MMP-3 levels were measured in serum samples from rheumatoid arthritis (RA) patients undergoing a double-blinded placebo-controlled trial with the chimaeric anti-tumour necrosis factor (TNF)-alpha antibody cA2. Both MMP-1 (P < 0.015), but to a larger extent MMP-3 (P < 0.001) levels were elevated in all RA patients prior to the commencement of the trial compared with normal control sera. Following cA2 therapy, MMP-1 and MMP-3 levels were assessed in the placebo, and 1 and 10 mg/kg cA2-treated groups at 7, 14, 21 and 28 days. In both the 1 and the 10 mg/kg cA2-treated groups, a significant decrease in serum MMP-3 levels at all time points was observed, reducing maximally to 41% of pre-infusion values at day 7. MMP-1 levels were also reduced, but less dramatically than MMP-3, to 85% of pre-infusion values after 14 days in the 10 mg/kg cA2 treated group. In a separate non-placebo-controlled study, we also evaluated the tissue inhibitor of metalloproteinase (TIMP)-1 levels in plasma following cA2 infusion. Pre-infusion TIMP-1 levels were above the normal control range, but were significantly reduced (P < 0.035) 14 days after infusion to 72% of pre-infusion values. This study confirms previous reports that MMP-3 levels are elevated and correlate with measures of inflammation in RA, and furthermore demonstrate that serum MMP-3 and MMP-1 levels are downmodulated following anti-TNF-alpha antibody therapy. Whilst serum MMP-3 levels correlated with C-reactive protein (CRP) both prior to and following anti-TNF-alpha antibody therapy, it remains to be demonstrated that serum MMP-3 and/or MMP-1 levels reflect the cartilage and bone resorptive processes which are evident in this disease.

Antibodies, Monoclonal↗
PubMed1997

TNF blockade in rheumatoid arthritis: implications for therapy and pathogenesis.

The role of the immune response in rheumatoid arthritis (RA) is a subject of debate, although it is widely believed to be a T-cell-driven disease. Progress is being hindered by lack of convincing evidence of a defined specific antigen initiating or perpetuating the response. Clinical trials using monoclonal antibodies directed against T-cell surface molecules such as CD4. CD5, and CD7 have thus far not provided evidence of efficacy. The negative data may reflect inadequate dosing or could suggest that indiscriminate depletion of T cells is insufficient by itself as a therapeutic strategy. Blocking proinflammatory cytokines (e.g. TNF alpha, IL-1) or augmenting anti-inflammatory cytokines (e.g. IL-10) offers an alternative approach to therapy. Clinical trials using monoclonal anti-TNF alpha have been particularly successful in controlling inflammation and markedly reducing acute phase proteins and cellular ingress. However, because disease invariably relapses, repeated therapy is necessary. Preliminary experience suggests that this is possible. Anti-TNF therapy for RA has defined a molecular target and new approach for treating immuno-inflammatory disorders.

Animals↗
PubMed1996

Interleukin-10 stimulation of phosphatidylinositol 3-kinase and p70 S6 kinase is required for the proliferative but not the antiinflammatory effects of the cytokine.

Interleukin-10 (IL-10) is a powerful suppressor of the proinflammatory monokine production by lipopolysaccharide-stimulated monocytes as well as a T- and B-cell growth cofactor. The signal transduction cascades initiated by IL-10 ligation to its cognate receptor remain to be elucidated. Here, we demonstrate that in both primary monocytes and the D36 cell line, IL-10 rapidly and transiently stimulated phosphatidylinositol 3-kinase activity associated with the p85 subunit of the enzyme. IL-10 also activated p70 S6 kinase in both cell types. The activation of both of these kinases was sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. The activation of p70 S6 kinase was also inhibited by the immunosuppressive drug rapamycin. Both rapamycin and wortmannin inhibited the IL-10-induced proliferation of D36 cells but in contrast had no effect on the antiinflammatory effects of the cytokine on lipopolysaccharide-stimulated monocytes. Similar results on D36 proliferation and lipopolysaccharide-stimulated monocyte inhibition by IL-10 were obtained with another phosphatidylinositol 3-kinase inhibitor, LY294002. This suggests that the activation of phosphatidylinositol 3-kinase and p70 S6 kinase is involved in the proliferative functions of IL-10 and that other as yet uncharacterized pathways affect the suppressive effects on monocytes, indicating that multiple and distinct signaling pathways mediate the various pleiotropic activities of IL-10. Furthermore, these findings suggest that it may be possible in the future to modulate the antiinflammatory effects of IL-10 for therapeutic benefit without disrupting other functions of the cytokine.

Androstadienes↗
PubMed1996

Paradoxical effects of a synthetic metalloproteinase inhibitor that blocks both p55 and p75 TNF receptor shedding and TNF alpha processing in RA synovial membrane cell cultures.

We have previously hypothesized that the pro-inflammatory cytokine TNF alpha has a pivotal role in the pathogenesis of rheumatoid arthritis (RA). It mediates its effects by cross-linking surface p55 TNF receptors (TNF-R), which can be proteolytically cleaved to yield soluble fragments. Upon binding TNF alpha soluble TNF-R (sTNF-R) can inhibit its function. We investigated the enzymatic nature of the proteases involved in TNF-R cleavage, and found that this process is blocked by a synthetic inhibitor of matrix metallo-proteinase activity (MMP), BB-2275. Inhibition of TNF-R cleavage was observed in a number of different cell types, as detected by retention of surface bound TNF receptor and by less sTNF-R released into the cell supernatant. The augmentation of surface TNF-R expression was of biological relevance as TNF alpha-mediated necrosis of human KYM.1D4 rhabdosarcoma cells was enhanced approximately 15-fold in the presence of BB-2275. The addition of BB-2275 to rheumatoid synovial membrane cell cultures totally inhibited MMP activity and also significantly reduced the levels of soluble TNF alpha (P < 0.006), p55 sTNF-R (P < 0.006), and p75 sTNF-R (P < 0.004). Paradoxically, despite the reduction in soluble TNF alpha levels, the production of IL-1 beta, IL-6, and IL-8, cytokines whose production was previously demonstrated to be inhibited by the addition of neutralizing anti-TNF alpha antibody were not down-regulated by BB-2275. These results raise the interesting possibility that a close relationship exits between the enzyme(s) which process membrane-bound TNF alpha, and those involved in surface TNF-R cleavage. Furthermore our observations suggest that hydroxamate inhibitors of MMP activity which block TNF alpha secretion and TNF-R cleavage may not modulate down-stream effects of TNA alpha, and as such suggest that the precise specificity of these compounds will be highly relevant to their clinical efficacy in inflammatory diseases.

Arthritis, Rheumatoid↗
PubMed1996

Cytokines in autoimmunity.

The past few years have witnessed exciting developments regarding the role of cytokines in autoimmune diseases, particularly in rheumatoid arthritis and Crohn's 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.

Animals↗
PubMed1996

Role of cytokines in rheumatoid arthritis.

Analysis of cytokine mRNA and protein in rheumatoid arthritis tissue revealed that many proinflammatory cytokines such as TNF alpha, IL-1, IL-6, GM-CSF, and chemokines such as IL-8 are abundant in all patients regardless of therapy. This is compensated to some degree by the increased production of anti-inflammatory cytokines such as IL-10 and TGF beta and cytokine inhibitors such as IL-1ra and soluble TNF-R. However, this upregulation in homeostatic regulatory mechanisms is not sufficient as these are unable to neutralize all the TNF alpha and IL-1 produced. In rheumatoid joint cell cultures that spontaneously produce IL-1, TNF alpha was the major dominant regulator of IL-1. Subsequently, other proinflammatory cytokines were also inhibited if TNF alpha was neutralized, leading to the new concept that the proinflammatory cytokines were linked in a network with TNF alpha at its apex. This led to the hypothesis that TNF alpha was of major importance in rheumatoid arthritis and was a therapeutic target. This hypothesis has been successfully tested in animal models, of, for example, collagen-induced arthritis, and these studies have provided the rationale for clinical trials of anti-TNF alpha therapy in patients with long-standing rheumatoid arthritis. Several clinical trials using a chimeric anti-TNF alpha antibody have shown marked clinical benefit, verifying the hypothesis that TNF alpha is of major importance in rheumatoid arthritis. Retreatment studies have also shown benefit in repeated relapses, indicating that the disease remains TNF alpha dependent. Overall these studies demonstrate that analysis of cytokine expression and regulation may yield effective therapeutic targets in inflammatory disease.

Animals↗
PubMed1996

Transgenic models for arthritis: useful clues to be gained?

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of unknown aetiology. Much of our understanding regarding the pathogenesis of this disease has been derived from studies in rodents induced to develop arthritis with injected agents. More recently with the advent of transgenic mouse technology, animal models of arthritis have been generated either by design or by serendipity, and have provided insight into the role of cytokines in particular in contributing to the pathogenesis of arthritis. This short editorial summarizes recent advances in this field and how this has increased our understanding of RA.

Animals↗
PubMed1995

TNF alpha is an effective therapeutic target for rheumatoid arthritis.

Rheumatoid arthritis is the most common of a number of diseases in which inflammation and tissue destruction is driven by an autoimmune process. Current therapy is inadequate, and this has prompted major research efforts, both in academia and industry, to understand more about the pathogenesis, and hence provide the rationale for new therapeutic strategies. Here we review our studies of cytokine expression and regulation in rheumatoid joints, which has culminated in demonstrating that TNF alpha blockade, using a chimeric (human IgG1/K, mouse Fv) anti-TNF alpha antibody, cA2, markedly ameliorates arthritis. This defines a therapeutic target for rheumatoid arthritis.

Animals↗
PubMed1995

Functional comparisons of different tumour necrosis factor receptor/IgG fusion proteins.

Nine different IgG fusion proteins and one non-fusion protein, all containing sequences from the extracellular domain of either of two human TNF receptors, were compared for their ability to bind and inhibit human TNF-alpha or TNF-beta. The fusion proteins differed with respect to TNF receptor type (p55 or p75 TNF receptor), receptor valency (one, two or four receptor domains per molecule), the presence or absence of a CH1 domain in the IgG constant region, and the proportion of the extracellular domain included in the construct. In vitro TNF binding assays and cytotoxicity assays indicated that, of the constructs that bound TNF, the greatest difference in affinity and neutralizing capability was between monovalent and bivalent receptor constructs. Differences were also noted between tetravalent and bivalent versions of p55 fusion proteins, as well as between a p75 fusion protein comprising the complete extracellular domain and one lacking the C-terminal 53 amino acids of the extracellular domain. p55 constructs that included only the second cysteine-rich domain (CRD) or only the second and third CRDs showed no TNF binding activity. The presence or absence of an IgG CH1 domain made no difference in the ability of fusion proteins to neutralize TNF-alpha or TNF-beta. Animal experiments comparing the tetravalent and bivalent p55 fusions and the effects of the CH1 domain did not show significant differences in their ability to protect mice from endotoxin-induced lethality, although the p55 fusion proteins appeared to be more protective than the p75 fusion proteins. Thus, this study has identified structural modifications to TNF receptor/IgG fusion proteins which have differing effects on their neutralizing ability towards TNF-alpha or TNF-beta.

Animals↗
PubMed1995

Cytokine expression in chronic inflammatory disease.

In chronic inflammatory diseases, typified by rheumatoid arthritis, we speculated that upregulation and/or disregulation of cytokine production in inflamed tissue might contribute both directly and/or indirectly to the pathology in the synovial joint tissue. This chapter summarises studies performed principally by our own group over the last 9 years or so, but also by others in the field who have investigated the expression of cytokines in RA. From our studies we identified one particular cytokine, tumour necrosis factor alpha (TNF alpha) as an important, 'pivotal', molecule in the disease process. This concept has led to the initiation and completion of the first successful clinical trials in RA patients to verify this hypothesis, using a neutralising antibody to TNF alpha.

Arthritis, Rheumatoid↗
PubMed1995

Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1 alpha and IL-1 receptor antagonist.

The IL-1 cytokine network in epidermal cells was studied in vitro, using the spontaneously transformed HaCAT human keratinocyte line. Intracellular (ic) IL-1 alpha and IL-1 receptor antagonist protein (IL-1Ra) following cell lysis were readily identified assayed using a capture ELISA; whereas in culture supernatants IL-1Ra was not detected, and IL-1 alpha was present at only very low levels. Confluent cultures of HaCAT cells were shown to provide optimal conditions for the study, since confluence increased the icIL-1Ra:IL-1 alpha ratio to a level as seen in vivo, which was independent of Ca2+ concentration in the culture medium. The IL-1Ra extracted from HaCAT cell lysates was functionally active, as demonstrated in the mouse thymocyte co-proliferation assay which could be blocked using a rabbit anti-IL-1Ra antibody. Transforming growth factor-beta (TGF-beta 1) stimulated a dose-dependent increase in HaCAT cell IL-1 alpha without changing IL-1Ra concentration, with a resultant reduction in the icIL-1Ra: IL-1 alpha ratio from 320:1 to 100:1. Similarly, TGF-alpha, interferon-gamma (IFN-gamma), IL-6, and tumour necrosis factor-alpha (TNF-alpha) substantially increased HaCAT cell IL-1 alpha, but had no effect on the IL-1Ra, with a concomitant reduction in the icIL-1Ra:IL-1 alpha ratio. In contrast to their effects on monocytes, IL-4 and IL-10 at biologically active levels had no effect on IL-1 alpha, IL-1Ra or the icIL-1Ra: IL-1 alpha ratio in confluent HaCAT cells. Hydrocortisone reduced IL-1 alpha to below the limit of sensitivity of the ELISA, and induced a small increase in IL-1Ra of questionable biological significance. Thus, regulation of the IL-1 cytokine network in keratinocytes involves modulation of icIL-1 alpha rather than of icIL-1Ra levels, and is markedly different from that noted in monocytes.

Calcium↗
PubMed1995

Beneficial effects of tumour necrosis factor-alpha (TNF-alpha) blockade in rheumatoid arthritis (RA)

The biological properties of TNF-alpha make it a candidate therapeutic target in RA. Our studies have demonstrated that TNF-alpha and its receptors are up-regulated and co-expressed in the synovium and cartilage-pannus junction of RA joints. Neutralizing TNF-alpha antibodies reduce the production of the many pro-inflammatory cytokines, including IL-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF), produced by mononuclear cells from RA in culture. When injected into DBA/1 mice with collagen-induced arthritis and TNF-alpha transgenic mice with arthritis, anti-TNF MoAbs decrease inflammatory damage of joints. Clinical trials employing cA2, a chimaeric anti-TNF-alpha MoAb, in open-label and randomized placebo-controlled studies have demonstrated a dose-dependent efficacy with impressive improvement in disease activity and acute-phase responses lasting several weeks. We conclude that TNF-alpha is a critical mediator of inflammation in RA, and is an important therapeutic target in this disease.

Antibodies, Monoclonal↗
PubMed1995

TNF inhibitors are produced spontaneously by rheumatoid and osteoarthritic synovial joint cell cultures: evidence of feedback control of TNF action.

We have proposed the hypothesis that tumour necrosis factor alpha (TNF-alpha) has a pivotal role in the pathogenesis of rheumatoid arthritis, based on in vitro observations that in RA synovial joint cell cultures removal of TNF-alpha, inhibited other potentially pathogenic cytokines such as the equally proinflammatory cytokine interleukin 1 (IL-1) and the macrophage activating factor, GM-CSF. Here we describe that in both rheumatoid (RA) and osteoarthritic (OA) synovial cultures there is a homeostatic mechanism to regulate the activities of TNF-alpha. This concept is based on several observations. First in these synovial joint cell cultures the substantial discrepancy between the levels of bioactive and immunoreactive TNF-alpha indicates the presence of an inhibitor. Second, TNF binding proteins are produced spontaneously, which are the soluble variants of surface p75 and p55 TNF receptor. The amount of soluble TNF receptors (sTNF-R) produced varied between cultures; p75 sTNF-R was more abundant than p55 sTNF-R (as detected by ELISA), and both were produced at higher levels by RA synovial joint cells in culture, compared to OA cultures. These TNF binding proteins act as endogenous inhibitors of TNF-alpha, since blocking their activity in synovial joint cell culture supernatants with MoAb to p55 and p75 sTNF-R enhanced their cytotoxic activity in the TNF bioassay. The regulation of production of these sTNF-R in synovial joint cell cultures is important as the balance between TNF-alpha and sTNF-R production may determine the outcome of the inflammatory process.

Arthritis, Rheumatoid↗
PubMed1995

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.

Animals↗
PubMed1995

Soluble TNF receptor production by activated T lymphocytes: differential effects of acute and chronic exposure to TNF.

Soluble tumour necrosis factor receptors (sTNF-R) are up-regulated at sites of chronic inflammation such as rheumatoid synovial joints. The p75 sTNF-R is the more abundant, suggesting an important role for this TNF inhibitor in regulating TNF bioactivity in vivo. As the precise cellular source of these soluble receptors is not known, we investigated the production and regulation of sTNF-R by T lymphocytes, an abundant cell type in inflammatory infiltrates, which upon activation express high levels of p75 surface receptors. Using panels of T-cell lines and clones expressing high levels of p75 TNF-R, we found that p75 sTNF-R production upon stimulation is a feature common to all subsets of T cells, including cells of the CD4-CD8- double negative phenotype expressing either alpha beta or gamma delta T-cell receptors (TCR). In contrast, levels of p55 sTNF-R were only detected when T cells were stimulated at higher densities and by potent mitogens such as phorbol 12-myristate 13-acetate (PMA). Detailed kinetic analyses revealed that the production of p75 sTNF-R was biphasic, the first phase was activation dependent, occurring in the absence of detectable TNF, while the second phase of p75 sTNF-R production was regulated by cytokines such as TNF. Unlike short-term exposure to TNF which enhances sTNF-R production in vitro and in vivo, prolonged exposure of T lymphocytes to TNF suppressed p75 sTNF-R (but not p55 sTNF-R) production in a dose- and time-dependent fashion. These results suggest that in patients with chronic inflammatory disease, which are exposed to augmented levels of bioactive TNF for prolonged periods, the production of p75 sTNF-R may be impaired.

Clone Cells↗