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Biomedical subjects

F M Brennan

Publications and source records attributed to F M Brennan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Immunoregulatory role of interleukin 10 in rheumatoid arthritis.

The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.

Arthritis, Rheumatoid↗
PubMed1994

Lymphotoxin acts as an autocrine growth factor for Epstein-Barr virus-transformed B cells and differentiated Burkitt lymphoma cell lines.

A critical event in B cell immortalization by Epstein-Barr virus (EBV) is the establishment of an autocrine loop where cells produce a growth factor which supports their own proliferation. We investigated the potential of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cell lines to produce and respond to the cytotoxins, tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT). Transformation in vitro of peripheral blood B cells by EBV from seven different donors resulted in spontaneous production of both LT (11,542 pg/ml +/- 7546, mean +/- SD) and, to a lesser extent, TNF-alpha (197 pg/ml +/- 174). Similarly BL cell lines derived from in vivo transformation which developed a 'LCL-like' phenotype in vitro (group III) produced more LT (1990 pg/ml +/- 1740) than the 'group I' BL cell lines (< 40 pg/ml LT) which had maintained the original BL biopsy cell phenotype in vitro. Transformation of peripheral blood B cells to generate LCL also resulted in an increase in surface p75 (p < 0.02) and to a lesser extent p55 (not significant, ns) TNF receptor (TNF-R) expression. Similar increases in surface TNF-R (p75 p < 0.02, p55 ns) were observed on the 'group III' BL cell lines compared with the 'group I' BL cell lines. Proliferation of an LCL and a 'group III' BL cell line in vitro was via an autocrine loop since inhibition of LT reduced proliferation. This proliferation could also be blocked in the presence of the antagonistic anti-p55 TNF-R antibody, H398, but not the antagonistic antibody anti-p75 TNF-R antibody UTR-1. Furthermore, proliferation could be induced with the p55 agonistic antibody, HTR-9. In contrast to these observations with p55 TNF-R antibodies, two out of six of the 'group III' BL lines (Jijoye and Oba) only expressed the p75 TNF-R and proliferation of these cells could only be blocked by the antagonistic anti-p75 TNF-R antibody UTR-1. These data suggest that LT is an autocrine growth factor for lymphoblastoid cells, and BL cell lines which display an LCL phenotype. Furthermore, although both TNF-R are increased on the surface of these cells, this autocrine growth signal is mediated principally through binding to the p55 TNF-R.

B-Lymphocytes↗
PubMed1994

Two inhibitors of pro-inflammatory cytokine release, interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes.

The biological activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha depends on the level of TNF-alpha itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF-alpha, the soluble p55 and p75 TNF receptors (TNF-R). Interleukin (IL)-10 and IL-4 are known to inhibit TNF-alpha production by monocytes. We, therefore, investigated the effects of IL-10 and IL-4 on the cell surface expression and release of TNF-R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF-alpha. Exposure to IL-10 (1-10 U/ml) for 24 or 48 h increased soluble p75 TNF-R expression and concomitantly reduced surface expression of p75 TNF-R. Further, IL-1 alpha-stimulated production of TNF-alpha was diminished by IL-10 and only a small proportion of this TNF-alpha was bioactive, consistent with increased production of inhibitory soluble TNF-R. IL-10 also induced down-regulation of surface p55 TNF-R on monocytes, and increased release of soluble p55 TNF-R. However, the expression of soluble p55 TNF-R was much lower than soluble p75 TNF-R, indicating that it contributed less importantly to neutralization of TNF-alpha under these conditions. Like IL-10, IL-4 supressed the release of TNF-alpha by monocytes. In contrast to IL-10, however, IL-4 (0.1-10 ng/ml) supressed the release of soluble p75 TNF-R from monocytes in a dose-dependent manner. Release of soluble p55 TNF-R was also supressed by IL-4. IL-10, therefore, reduces the pro-inflammatory potential of TNF in three ways: by down-regulating surface TNF-R expression whilst increasing production of soluble TNF-R and inhibiting the release of TNF-alpha itself. This suggests that IL-10 may be useful in the treatment of diseases where overexpression of TNF-alpha occurs.

Cells, Cultured↗
PubMed1994

TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.

KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.

Animals↗
PubMed1994

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.

Antibodies, Monoclonal↗
PubMed1994

TNF alpha as a therapeutic target in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with inflammatory manifestations in the peripheral synovial joints, which are infiltrated by activated T cells, macrophages, and plasma cells. We have investigated the role of cytokines in RA and have proposed that tumour necrosis factor has a pivotal role in the pathogenesis of this disease. This chapter describes those studies, which led to the first clinical trial in RA patients using a chimeric anti TNF alpha antibody. In addition to pro-inflammatory cytokine production, at sites of inflammation such as the RA synovial joint, there is also evidence for homeostatic immunoregulatory mechanisms which include the production of cytokine inhibitors, such as soluble TNF-R and the IL-1 receptor antagonist, and cytokines with immunoregulatory properties like IL-10. The evidence for these inhibitors in RA is presented, and the relevance of this homeostatic mechanism in relation to chronic inflammatory diseases is discussed.

Animals↗
PubMed1993

Elevated synovial fluid levels of interleukin-6 and tumor necrosis factor associated with early experimental canine osteoarthritis.

OBJECTIVE: To measure levels of cytokines, proteases, and glycosaminoglycans (GAG) in synovial fluid (SF) from the knees of animals with experimental osteoarthritis (OA) and from their contralateral (control) knees, and to compare and correlate these values with each other as well as with measures of proteoglycan synthesis in the corresponding articular cartilage. This study will help to identify cytokines of potential importance in the early stages of the development of OA. METHODS: OA was induced in 12 mature animals by sectioning the anterior cruciate ligament. After 3 months, SF from the operated and contralateral (control) knee joints was assayed for interleukin-6 (IL-6), tumor necrosis factor (TNF), IL-1, latent metalloproteinase, and sulfated GAG. Proteoglycan synthesis in the corresponding articular cartilage was also measured. RESULTS: IL-6 levels in SF from the operated joint compared with the control joint were significantly elevated in 11 of 12 animals. TNF levels were also elevated in 10 of 11 SF samples from operated joints, but to a lesser extent than those of IL-6. IL-1 and IL-1 inhibitors were undetectable in either the operated or control joint SF. The GAG concentration was elevated in SF from experimental OA joints. This elevation correlated with that of TNF, but not IL-6. There was no significant difference in the concentration of APMA-activatable metalloproteinase. The rate of proteoglycan synthesis was higher in the cartilage from the operated joint in 8 of 12 animals, and the mean rate of synthesis was significantly higher than in the control joint. There was a positive correlation between this increase in cartilage proteoglycan synthesis (operated versus control) and the increase in SF IL-6, but there was no correlation with the levels of TNF or GAG. CONCLUSION: This is the first study of SF levels of cytokines in early experimental OA. Our results show surprisingly high levels of IL-6 in operated joints, where the cytokine could act directly on the chondrocytes, and thus play a role in mediating their responses to cartilage injury.

Animals↗
PubMed1993

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

OBJECTIVE: To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor alpha (TNF alpha) in the treatment of patients with rheumatoid arthritis (RA). METHODS: Twenty patients with active RA were treated with 20 mg/kg of anti-TNF alpha 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-TNF alpha was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNF alpha is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.

Adult↗
PubMed1993

Differential regulation of tumour necrosis factor receptors (TNF-R) by IL-4; upregulation of P55 and P75 TNF-R on synovial joint mononuclear cells.

Interleukin 4 (IL-4) has previously been shown to downregulate the production of proinflammatory cytokines such as TNF-alpha, and hence has been considered to be a potential anti-inflammatory agent. In this study we have investigated the effects of IL-4 on the expression of both p55 and p75 TNF receptors (TNF-R) by flow cytometry and radioligand binding analyses and demonstrate that IL-4 downregulates both p55 and p75 TNF-R on HeLa and Jijoye cell lines in a dose dependent manner. IL-4 reduced the number of p55 TNF-R on HeLa cells from 6400 (Kd 5.1 nM) to 3900 (Kd 3.7 nM), and p75 TNF-R on Jijoye cells from 4800 (Kd 1.6 nM) to 3250 (Kd 1.5 nM). However, different effects were observed on peripheral blood mononuclear cells (PBMC). IL-4 inhibited the increase in p55 and p75 TNF-R on PBMC following adherence, whereas IL-4 upregulated p75 TNF-R expressed on PHA induced T cell blasts. To assess further the possible anti-inflammatory properties of IL-4, we studied its effects on synovial joint mononuclear cell cultures from 15 patients with inflammatory synovitis. In contrast to the differential effects of IL-4 on monocytes and T cells, IL-4 upregulated both p55 (P < 0.05) and p75 TNF-R (P < 0.005) on synovial joint cells in culture. IL-4 treatment caused a small decrease in levels of bioactive TNF-alpha in RA synovial culture supernatants, together with an increase in soluble p75 TNF-R levels although differences were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis↗
PubMed1993

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.

Humans↗
PubMed1992

Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases.

OBJECTIVE: Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis. METHODS: We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF) samples from patients with rheumatic diseases and compared their levels with levels of soluble interleukin-2 receptors (sIL-2R). Sensitive enzyme-linked immunosorbent assays (ELISA), specific for p55 and p75 sTNF-R and for sIL-2R, were used. RESULTS: Serum levels of p75 sTNF-R were 3-4-fold higher than levels of p55 sTNF-R, and both were significantly elevated in patients with osteoarthritis (OA) and rheumatoid arthritis (RA) compared with healthy controls. RA SF levels of sTNF-R were 4-5-fold higher than levels in serum, suggesting local production in the joint, and were significantly higher than levels in the SF of patients with seronegative arthropathy or OA. Furthermore, levels of p55 and p75 sTNF-R, but not sIL-2R or TNF alpha measured by ELISA, were increased in the SF of patients with clinically active RA. The soluble TNF-R in RA and OA SF were functional since they inhibited TNF activity in a cytotoxicity assay in proportion to the levels of inhibitor present. Evaluation of serially obtained serum samples suggested that sTNF-R may be a useful parameter for monitoring RA disease activity. CONCLUSION: Biologically active soluble TNF-R are up-regulated in patients with rheumatic disease and are produced locally in the joints. Measurement of serum levels of TNF-R may be useful for monitoring of disease, and determination of SF levels could be of diagnostic value.

Adult↗
PubMed1992

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 alpha.

OBJECTIVE: We have previously described the location of tumor necrosis factor alpha (TNF alpha)-producing cells in synovial tissue and cartilage-pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNF alpha, 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 TNF alpha-containing cells, and some TNF alpha-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 TNF alpha in this tissue. This study further supports the hypothesis that TNF alpha plays a major role in the pathogenesis of RA.

Arthritis, Rheumatoid↗
PubMed1992

Enhanced expression of tumor necrosis factor receptor mRNA and protein in mononuclear cells isolated from rheumatoid arthritis synovial joints.

We previously proposed the hypothesis that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) based on our observations that it is the dominant inducer of interleukin-1 (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in RA synovial joint mononuclear (MNC) cells in culture. Since TNF-alpha acts via two membrane receptors, we have extended those studies to investigate the distribution of the p55 and p75 TNF receptors (TNF-R) in RA tissue. Surface receptor expression was quantitated by flow cytometry using monoclonal antibodies specific to the p55 (HTR-9) and the p75 (UTR-1) TNF-R. Both receptors were significantly increased on MNC isolated from the synovial membrane of RA patients compared to normal or RA peripheral blood MNC. Interestingly, the p75 TNF-R was increased both on large monocytic/macrophage-type cells and CD3+ lymphocytes. Furthermore, there was a significant increase in the proportion of CD3+ cells in RA synovial fluid expressing the p75 TNF-R, compared to matched peripheral blood MNC. In contrast to RA synovial MNC, p75 or p55 TNF-R expression was not significantly increased in osteoarthritis synovial MNC. In addition, Northern blot analysis indicated abundant expression of both p55 and p75 mRNA in RA synovial joint MNC. This was in contrast to normal peripheral blood MNC cells which contained little or no constitutive TNF-R mRNA; following stimulation with phytohemagglutinin and IL-2, a rapid and transient expression of both receptor mRNA was induced. These results, therefore, indicate that in RA synovial joint tissue there is up-regulation of both p55 and p75 TNF-R mRNA and surface protein expression, and with the presence of TNF-alpha in RA tissues, these results provide support to our hypothesis that TNF-alpha is of critical importance in the pathogenesis of RA.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Cytokines in autoimmunity.

It is now generally accepted that many cytokines are involved in the pathogenesis of autoimmune disease, either directly by causing tissue destruction or indirectly through the activation of autoreactive and inflammatory cells. Thus, cytokines, such as tumor necrosis factor-alpha, are implicated in the pathogenesis of rheumatoid arthritis based on in vitro studies on synovial tissue from patients with rheumatoid arthritis, which suggest that the effects of tumor necrosis factor-alpha are amplified by its potential to induce other pro-inflammatory cytokines, such as interleukin-1 and granulocyte-macrophage colony-stimulating factor. Transgenic mouse technology has shown that mice expressing the human tumor necrosis factor-alpha gene develop a polyarthritis. Interleukin-2 has also been identified by transgenic technology as a cytokine involved in the pathogenesis of insulin-dependent diabetes mellitus through the activation and stimulation of growth of autoreactive T cells.

Animals↗
PubMed1992

Evaluation of the role of cytokines in autoimmune disease: the importance of TNF alpha in rheumatoid arthritis.

Cytokines and growth factors are involved in all important biological processes. Hence it is anticipated that they will be of importance in autoimmune disease. The pathogenesis of autoimmune diseases involves a number of stages, initiation, perpetuation and tissue damage, each of which involves different cell and molecular interactions. In this review, we will discuss an outline of the cytokine involvement in the various stages of autoimmune development, prior to focusing on the analysis of cytokines in rheumatoid arthritis. Cytokines exert their effect via high affinity cell surface receptors. Thus an understanding of cytokines involves the analysis of receptor expression, and also of cytokine inhibitors. Currently there is only adequate knowledge of these aspects in rheumatoid arthritis (RA), and as such the emphasis of this review is on RA. One of the major reasons for being interested in the role of cytokines in autoimmunity is to define possible therapeutic targets. There is now considerable evidence that TNF alpha is such a target in RA, and the effect of anti TNF alpha monoclonal antibody therapy in RA is discussed.

Animals↗