Clive Smith

Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region.

ABSTRACT Stimulation of the NF-κB pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide −863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-κB binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-κB site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-κB complex.

Evidence for a dual mechanism for IL-10 suppression of TNF-alpha production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-kappa B in primary human macrophages.

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-α production. The molecular pathways by which IL-10 inhibits TNF-α production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-α promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-α gene. When added, either with or after the stimulus, IL-10 required the 3′ untranslated region of the TNF-α gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5′ promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-α production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-κB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-κB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.

NF-κB-Inducing Kinase Is Dispensable for Activation of NF-κB in Inflammatory Settings but Essential for Lymphotoxin β Receptor Activation of NF-κB in Primary Human Fibroblasts

The transcription factor NF-κB is of major importance in the biology of pro-inflammatory cytokines, such as TNF-α and IL-1α, and thereby is intimately involved in the process of inflammation. Understanding the mechanisms by which NF-κB is activated in response to inflammatory stimuli has become a major goal of inflammation research. The discovery of NF-κB-inducing kinase (NIK) as a TNFR-associated factor-interacting enzyme and a potential activator of the IκBα-kinase complex appeared to have identified an important element of the NF-κB activition pathway, a view that was supported by several subsequent studies. However, recent experiments in the alymphoplasia (aly/aly) mouse, which has missense point mutation (G885R) in NIK, has challenged that view. The reasons for the discrepancy between the different studies is unclear and could be due to multiple factors, such as cell type, species of cell, or primary vs transformed cell lines. One system that has not been investigated is primary human cells. Using an adenoviral vector encoding kinase-deficient NIK, we have investigated the role of NIK in LPS, IL-1, TNF-α, and lymphotoxin (LT) βR signaling in primary human cells and TNF-α expression from rheumatoid tissue. These data show that, in the primary systems tested, NIK has a restricted role in LTβR signaling and is not required by the other stimuli tested. Also, there is no apparent role for NIK in the process of TNF-α production in human rheumatoid arthritis. These data also highlight the potential problems in extrapolating the function of signaling pathways between primary and transfected cell lines.

Is NF-kappaB a useful therapeutic target in rheumatoid arthritis?

There is increasing evidence that NF-κB is a major, if not the major transcription factor regulating inflammation and immunity. While this implies that blocking NF-κB might be therapeutically beneficial, it raises clear questions regarding the balance between efficacy and safety. In this brief review we discuss the effects of NF-κB blockade in rheumatoid arthritis, inflammation and immunity, and consider possible therapeutic targets within the NF-κB family.

Prospects for the development of small molecular weight compounds to replace anti-tumour necrosis factor biological agents

Tumour necrosis factor (TNF) blockade, achieved by the soluble receptors and antibodies, has been a major success in the treatment of rheumatoid arthritis (RA).1 The success of this treatment has also established biological agents as a new weapon in the armoury of therapeutic approaches to the treatment of autoimmune diseases. Established data and emerging evidence suggest that anti-TNF biological agents will have a therapeutic role in other autoimmune diseases. Infliximab is already licensed for Crohn’s disease and, recently, anti-TNF biological agents have shown major promise in spondyloarthropathies, psoriatic arthritis, and juvenile idiopathic arthritis.2–8 There is growing evidence that TNF blockade may be important in many other diseases, including sarcoidosis9 and psoriasis.10 Therefore, although the number of patients treated with the various anti-TNF biological agents is rapidly approaching half a million, there is no reason to suggest that the patient pool is anywhere near saturation. Rather, one can expect the number of patients with diseases likely to benefit from this form of treatment to increase. However, there are problems with anti-TNF biological agents that limit their use. From a safety perspective, although TNF blockade has not demonstrated the massive problems of susceptibility to infections once feared, there are clearly problems, especially with patients who have latent tuberculosis.11 There is also the constant problem associated with using proteins as drugs of their administration and dosing. However, by far the biggest constraint in using anti-TNF biological agents is the cost of treatment, which is of the order of

High-efficiency gene transfer into nontransformed cells: utility for studying gene regulation and analysis of potential therapeutic targets

15 000/patient/year. This has led to a relatively low uptake of the treatment in several European countries and to patients being described as “economic failures” in America. One must also add that the difficulty in producing proteins on a mass scale has led to a supply problem, especially for …

REPORTER GENE ASSAY DEMONSTRATES FUNCTIONAL DIFFERENCES IN ESTROGEN RECEPTOR ACTIVITY IN PURIFIED BREAST CANCER CELLS: A PILOT STUDY

Chapter summary The elucidation of the signalling pathways involved in inflammatory diseases, such as rheumatoid arthritis, could provide long sought after targets for therapeutic intervention. Gene regulation is complex and varies depending on the cell type, as well as the signal eliciting gene activation. However, cells from certain lineages, such as macrophages, are specialised to degrade exogenous material and consequently do not easily transfect. Methods for high-efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Significant research has been undertaken into the development of nonviral and viral vectors for basic research, and as vehicles for gene therapy. We briefly review the current methods of gene delivery and the difficulties associated with each system. Adenoviruses have been used extensively to examine the role of various cytokines and signal transduction molecules in the pathogenesis of rheumatoid arthritis. This review will focus on the involvement of different signalling molecules in the production of tumour necrosis factor alpha by macrophages and in rheumatoid synovium. While the NF-κB pathway has proven to be a major mediator of tumour necrosis factor alpha production, it is not exclusive and work evaluating the involvement of other pathways is ongoing.

Adenoviral Targeting of Signal Transduction Pathways in Synovial Cell Cultures

Tamoxifen has contributed to a dramatic reduction in breast cancer mortality and recent results indicate that aromatase inhibitors may further improve survival in some patients. Nevertheless, a substantial proportion of patients become resistant to treatment. To date, with the exception of estrogen receptor (ER) determination by ligand binding or immunohistochemical techniques, there has been no way of predicting which of several therapies is indicated in particular patients. We describe a novel assay using the adenoviral gene delivery system to assess ER function in breast cancer cells derived directly from patients. The purification and short‐term culture of these cells has been recently described by our laboratory. Adenovirus containing an estrogen‐regulated β‐galactosidase reporter gene (ERE‐lacZ) was constructed and used to test ER activity in breast cancer cells derived from 18 patients with primary and 16 patients with metastatic cancer, under varying treatment schedules. The adenoviral assay enabled ER activity to be readily determined in purified cells from primary breast cancers and secondary sites. Breast cancers cells could be categorized on the basis of ER activity in the absence of ligand, the presence of estrogen or anti‐estrogens. In primary breast cancers, our results correlated with ER determination by immunohistochemistry in 78% of cases. In patients who had become resistant to tamoxifen, however, we found some in whom reporter activity was stimulated by tamoxifen and others whose tumors were either still estrogen responsive or completely unresponsive, irrespective of the original ER content. Our findings indicate that this reporter assay could be useful in decisions regarding use of adjuvant endocrine therapies in breast cancer.

Distinct pathways of LPS-induced NF-kappa B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP.

Methods for high efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Adenoviruses have evolved to deliver their genes into cells with high efficiency and in recent years have been exploited as a gene transduction system. Prior to the discovery of adenoviruses, efficient expression of transgenes was only possible by cloning stably transfected cells; this was limited to cell lines and was not an option for primary cells. Here we describe a method of transgene expression, which enables previously untransfectable cells, such as primary myeloid cells or diseased synovium, to express protein at extremely high levels with nearly 100% of cells expressing the transgene. This allows us to examine the effect of target genes on signaling pathways in primary cells without the need for cell sorting or the simultaneous transfection of reporter genes. This is very important in studies of tissues such as rheumatoid synovium where sorting of cells will damage the biological value of the system.