Targeting adenosine receptors in the treatment of allergic rhinitis: a randomized, double-blind, placebo-controlled study.

BACKGROUND: There is evidence that adenosine plays a role in the pathogenesis of asthma and rhinitis; however, it is currently unclear whether adenosine receptors are useful therapeutic targets in the treatment of allergic airway diseases. OBJECTIVE: The study evaluated the efficacy of intranasal treatment with an adenosine A(2A) receptor agonist/adenosine A(3) receptor antagonist (50 micro g), administered twice daily for 7 days, to reduce nasal symptoms and release of inflammatory mediators following intranasal allergen challenge in patients with allergic rhinitis (AR). The compound was compared with twice-daily treatment with intranasal fluticasone proprionate nasal spray (FPANS) for 7 days. METHODS: A randomized, double-blind, double-dummy, placebo-controlled, three-way balanced, incomplete block, crossover study was conducted on 48 males with verified AR. Following intranasal challenge with either an extract from the house dust mite (HDM), Dermatophagoides pteronyssinus, rye grass or cat dander, nasal responses and the concentrations of albumin, tryptase, myeloperoxidase, eosinophilic cationic protein, epithelial neutrophil-activating protein-78 (ENA-78), IL-5 and IL-8 in nasal secretions were measured and treatment groups were compared. RESULTS: Drug improved nasal blockage but had no significant effect on rhinorrhoea, number of sneezes or peak nasal inspiratory flow measurements when compared with placebo. Drug reduced tryptase release after EAR but did not significantly reduce the levels of other mediators. CONCLUSION: A novel agonist/antagonist of adenosine A(2A) and A(3) receptors appears to have limited clinical benefit in both the early-phase and the late-phase response to intranasal allergen challenge. However, reduction of some pro-inflammatory mediators suggests that comparable, more selective compounds may have additional benefits meriting further investigation.

Transcription factors.

The regulation of gene expression by transcription factors is fundamental to the phenotype of all cells. The activated phenotype of cells engaged in inflammatory processes is characterized by induced expression of a diverse set of genes, including cytokines, enzymes and cell adhesion molecules. A relatively small number of inducible transcription factors, particularly NF-kappaB, AP-1, NFATs and STATs, are responsible for the expression of a wide variety of inflammatory phenotypic characteristics and therefore play a central role in the pathogenesis of rheumatic diseases. Each of these transcription factors can be modified by existing anti-rheumatic and anti-inflammatory drugs, although adverse effects and limited efficacy remain problems. The future development of therapeutic agents with specificity for transcription factors, especially NF-kappaB, might lead to safer and more effective treatment.

Synovial fluid induced nuclear factor-kappaB DNA binding in a monocytic cell line.

OBJECTIVE: To determine the effects of synovial fluids (SF) on DNA binding activity of transcription factor nuclear factor-kappaB (NF-kappaB) in the Mono Mac 6 monocytic/macrophage cell line as a model for the interaction between SF and synovial tissue macrophages in arthritis. METHODS: Mono Mac 6 cells were incubated with SF from the knee joints of human subjects with rheumatoid arthritis (RA), undifferentiated seronegative oligoarthritis, and osteoarthritis (OA). Nuclear extracts prepared from the Mono Mac 6 cells and RA synovial tissue were analyzed by electrophoretic mobility shift analysis (EMSA) for NF-kappaB DNA binding proteins. RESULTS: Induction of NF-kappaB DNA binding by the p65(RelA)/p50 heterodimer was observed in response to incubation of Mono Mac 6 cells with SF (20% in culture medium) from 5 of 8 subjects with RA, 4 of 5 with OA, and none of 3 with undifferentiated seronegative oligoarthritis. Incubation of SF with neutralizing antibodies against tumor necrosis factor-alpha (TNF-alpha), but not antibodies against interleukin 6 (IL-6), significantly reduced the induction of p65/p50 binding activity in SF from subjects with RA and OA. Unexpectedly, a slowly migrating SF inducible NF-kappaB-binding complex was observed in EMSA of Mono Mac 6 cells after incubation with SF from 5 of 8 RA and 2 of 5 OA subjects. The induction of this complex by SF was not affected by neutralization of TNF-alpha or IL-6 in SF, and the complex was not inducible by TNF-alpha, IL-1beta, TNF-alpha/IL-1beta, IL-6, platelet derived growth factor, lipopolysaccharide, or tetradecanoyl phorbol acetate. The slowly migrating complex could not be supershifted with antibodies against NF-kappaB, Jun, or the transcriptional coactivators p300 or CBP. A NF-kappaB-binding complex with similar slow mobility was observed in nuclear extracts prepared from fresh human RA synovial tissue. CONCLUSION: Biological activity of TNF-alpha in SF from RA and OA subjects is capable of inducing p65/p50 NF-kappaB DNA binding activity in macrophages. A property of SF that is independent of TNF-alpha and other cytokines is responsible for the induction of a novel slowly migrating NF-kappaB-binding complex. Soluble mediators in SF of subjects with RA and OA can therefore modulate binding of nuclear proteins to the NF-kappaB binding site in macrophages and may play a role in inflammatory gene expression in arthritis.

Rheumatic manifestations of hyperlipidaemia.

Familial hypercholesterolaemia is characterized by elevated serum cholesterol, tendon xanthomas, xanthelasmas, arcus corneae and premature atherosclerosis. Rheumatological manifestations include acute episodes of polyarthritis and tendinitis. Patients who are homozygous for familial hypercholesterolaemia have cardiovascular and rheumatological manifestations more frequently and at an earlier age than patients who are heterozygous.

Inhibition of transcription factors by anti-inflammatory and anti-rheumatic drugs: can variability in response be overcome?

1. The drugs used in the treatment of rheumatoid arthritis (RA) form a diverse group with unpredictable adverse effects, mostly weak efficacy and variable responses. Despite their differences, a common feature of many anti-inflammatory and disease-modifying anti-rheumatic drugs (DMARD) is inhibition of pro-inflammatory transcription factors, particularly nuclear factor (NF)-kappaB and activator protein (AP)-1. 2. The present brief review identifies those drugs capable of inhibiting transcription factors, particularly steroids, gold salts, D-penicillamine, cyclosporine A and possibly salicylates. 3. The newer biological inhibitors of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta are capable of indirect inhibition of NF-kappaB activation, although even with these potent agents the problem of variability in response has not disappeared. 4. The development of selective inhibitors of the transcription factor NF-kappaB should have the benefit of the anti-inflammatory drugs and DMARD, both new and old. 5. It is hypothesized that this strategy will overcome much of the variability in the therapeutic response and adverse effects that limit the usefulness of the existing drugs in the treatment of RA.

Transcription factors AP-1 and NF-kappa B: where steroids meet the gold standard of anti-rheumatic drugs.

The disease modifying anti-rheumatic drugs belong to a chemically heterogeneous group that includes inorganic salts (e.g. gold thiolates), plant derived products (e.g. chloroquine) and a drug metabolite (e.g. D-penicillamine). Understanding their mechanisms of action has long been clouded by numerous and conflicting theories. A wide variety of unpredictable adverse effects, extremely long half lives and lack of dose-response and concentration-response relationships have added to the confusion. Although they have no known high affinity receptors that might provide a clue to their mechanisms of action, the observation that gold(I) salts and penicillamine are reactive with thiols has suggested that there might be a mechanism in common between these two drugs. Recent advances in the function and chemistry of proteins involved in gene expression have indicated that thiol groups in the "pro-inflammatory" transcription factors AP-1 and NF-kappa B are targets for at least some of the therapeutic effects of disease modifying anti-rheumatic drugs. Developments in understanding the transcriptional effects of glucocorticoid and retinoid receptors indicate that they too act, at least in part, via inhibition of AP-1 and/or NF-kappa B activities. It is possible to develop a unifying hypothesis for the actions of this seemingly diverse group of drugs and make testable predictions for the development of more specific, and less toxic anti-inflammatory therapies.

Multinucleated cells in pigmented villonodular synovitis and giant cell tumor of tendon sheath express features of osteoclasts.

Pigmented villonodular synovitis (PVNS) and the histologically related lesion giant cell tumor of tendon sheath (GCTTS) are idiopathic, proliferative lesions that can induce osteolysis and formation of bone cysts. These lesions contain two predominant cell types: mononuclear polyhedral cells and multinucleated cells (MNCs). Previous studies demonstrated that the mononuclear cells exhibit phenotypic features consistent with derivation from a monocyte/macrophage lineage. The cell lineage of the MNCs and their relationship to osteoclasts are not known. To characterize the MNCs in these lesions and to establish the relationship of these MNCs to osteoclasts, histological sections from six cases of PVNS and two cases of GCTTS were studied. Mononuclear cells expressed CD14 and HLA-DR, in keeping with their relationship to cells of the monocyte/macrophage lineage. Characterization of the MNCs revealed features associated with an osteoclast phenotype. Seven of the eight specimens contained MNCs that were intensely tartrate-resistant acid phosphatase positive; approximately 5% of the mononuclear cells were tartrate-resistant acid phosphatase positive, and these tended to surround MNCs. MNCs in both lesions reacted strongly with the 23C6 monoclonal antibody that recognizes the alpha V beta 3 integrin (the vitronectin receptor), as did several mononuclear cells surrounding the MNCs. Most MNCs did not express CD14 or HLA-DR. Expression of receptors for calcitonin, a marker for osteoclasts, was detected on MNCs after incubation of sections with 125I-labeled salmon calcitonin and emulsion autoradiography. MNCs in four of six PVNS and two of two GCTTS samples demonstrated specific calcitonin binding. Expression of mRNA for calcitonin receptor was confirmed in all cases by reverse transcriptase polymerase chain reaction. These results demonstrate that MNCs in PVNS and GCTTS express phenotypic features of authentic osteoclasts and suggest that osteoclast-like multinucleated cells can arise in synovial soft tissues remote from bone.

D-penicillamine causes free radical-dependent inactivation of activator protein-1 DNA binding.

D-Penicillamine (beta, beta-dimethyl cysteine) is an antirheumatic thiol drug with a poorly understood mechanism of action. On the basis that gold(I) thiolates and D-penicillamine are both capable of forming stable bonds with endogenous thiols, we sought a common target of action. Cysteine residues in the basic DNA binding domains of Jun and Fos, members of the activator protein-1 (AP-1) transcription factor family, have been identified as likely targets for the therapeutic action of antirheumatic gold(I) thiolates. The current study demonstrates that AP-1 DNA binding is inhibited by D-penicillamine in the presence of Fenton reagents (Fe2+/EDTA and H2O2) but not with either agent alone. The effect is biphasic, with maximum inhibition in the concentration range of approximately 100-250 microM. Cysteine has qualitatively similar properties, although the effect is less pronounced. In contrast, glutathione and thiomalate do not inhibit AP-1 DNA binding, even in the presence of Fenton reagents. Mutant proteins were used to identify the cysteine residues within the DNA binding domains of Jun and Fos that are essential for the inhibitory action of D-penicillamine. The results suggest that D-penicillamine is distinguished from other thiols by its formation of sulfur-containing radicals capable of inhibiting AP-1 DNA binding by a mechanism involving the cysteine residues of Jun and Fos.

N-[4-hydroxyphenyl] retinamide in rheumatoid arthritis: a pilot study.

OBJECTIVE: To evaluate the efficacy and tolerability of N-[4 hydroxyphenyl] retinamide (4-HPR), a synthetic retinoid, in the treatment of rheumatoid arthritis (RA). METHODS: An uncontrolled, open clinical trial with synovial biopsy pre- and postmedication to evaluate the clinical effects of 4-HPR as well as its effects on metalloproteinase gene expression. RESULTS: Twelve patients with severe, longstanding RA were enrolled in this study. Six patients withdrew before study completion, 2 because of drug toxicity, 2 because of a flare of RA, and 2 because of intercurrent medical problems. No patient met predetermined Paulus criteria treatment response, and there was no improvement in the laboratory parameters, except for a modest decrease in C-reactive protein. No decrease in messenger RNA for the metalloproteinases collagenase and stromelysin was seen in the 2 patients in whom paired synovial biopsies were obtained. CONCLUSION: No beneficial clinical effect was observed with the retinoid 4-HPR in the treatment of severe, longstanding RA at the 300 mg/day dosage studied. The use of higher dosages is precluded by the observed toxicities. The effect of this drug in patients with early or mild disease was not studied. Although this particular retinoid was not effective in this pilot study, the use of other retinoids in RA should still be considered.

Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65.

OBJECTIVE: To identify the cells that express transcription factor NF-kappa B subunits p50 and p65 in synovial tissue from patients with rheumatoid arthritis (RA) and to correlate the distribution of p50 and p65 with CD14 (macrophage lipopolysaccharide receptor) and members of the AP-1 transcription factor family, Jun and Fos. METHODS: Immunohistochemistry was used to identify p50, p65, Jun and Fos in sections of synovial tissue from 13 patients with RA and 4 "normal" control subjects. Double staining for CD14 and each of the transcription factor subunits was performed. RESULTS: Subunits p50 and p65 were present in the nuclei of synovial cells in all 13 RA patients, with expression varying from rare cells to more than half of all cells. In most cases, nuclear p50 and p65 were present in approximately one-third of synovial lining cells and in a variable proportion of cells scattered throughout the sublining region, including the endothelium. The distributions of p50 and p65 were similar. Jun and Fos were present in the nuclei of a large proportion of synovial lining cells with significantly less expression elsewhere. In each case the Jun/Fos distribution was clearly different from the p50/p65 distribution, although there was significant overlap in many cases. Cells expressing CD14 were mostly Jun/Fos negative and were predominantly p50/p65 positive. There was negligible staining for p50 or p65 in the 4 normal control synovium samples. CONCLUSION: In most RA patients, the p50 and p65 subunits of NF-kappa B were present in the majority of CD14-positive cells within the lining and sublining regions and in a proportion of other cells throughout the synovium, including endothelial cells. NF-kappa B is likely to play an important role in the expression of macrophage-derived cytokines in rheumatoid synovium. Different but overlapping distributions of nuclear p50 and p65 versus Jun and Fos indicate separate or divergent mechanisms for the activation of NF-kappa B and the expression of AP-1 proteins in rheumatoid synovium.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Comparative effects of gold on the interactions of transcription factors with DNA.

We have previously hypothesized that a mode of action of the anti-rheumatic gold salt, aurothiomalate (AuTM), is the inhibition of DNA binding by transcription factors. Studies of the progesterone receptor (PR), which has a zinc finger structure in the DNA binding domain, were consistent with this hypothesis (1). Here we show that AuTM also markedly inhibits DNA binding by the transcription factor AP-1 and has less potent effects for AP-2, NF-1 and TFIID.

Oestrogen receptor gene structure and function in breast cancer.

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoR I, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.

Inhibition of DNA binding and transcriptional activity of a nuclear receptor transcription factor by aurothiomalate and other metal ions.

The antirheumatic gold salt aurothiomalate (AuTM) has cellular actions that are consistent with modulation of gene expression. We have tested the hypothesis that an important mode of action of AuTM is inhibition of binding of certain transcription factors to regulatory elements in DNA. The chemistry of transcription factors containing the zinc finger motif makes them candidates for such an interaction with AuTM. In this regard, the interaction of a steroid hormone receptor, the progesterone receptor (PR), with its DNA response element (PRE) was chosen as a suitable model. Nuclear extracts of T-47D human breast cancer cells rich in PR were incubated with radiolabeled PRE, and binding was determined by gel retardation assay. Preincubation of nuclear extract with AuTM caused a concentration-dependent inhibition of binding of PR to PRE (IC50, approximately 3 microM). Other metal ions inhibited binding at higher concentrations, in a rank order correlating with their binding affinity for thiols. Thiomalic acid had no effect in the absence of gold in this system. To test the effect of AuTM on PR-mediated transcription, we transfected the progestin-inducible expression vector pMSG-CAT into T-47D cells. Transfected cells were incubated in the absence or presence of AuTM and treated with the synthetic progestin ORG2058, to induce chloramphenicol acetyl transferase (CAT) activity. With 10 and 100 microM AuTM, there was inhibition to 67 +/- 3% (p = 0.012) and 42 +/- 8% (p = 0.008) of CAT specific activity, respectively, compared with controls. These results demonstrate that AuTM can regulate gene expression and that inhibition of binding of a transcription factor to its response element is a likely mechanism. This provides a molecular model for further study of the antirheumatic action of gold salts.

Coexistence of rheumatoid arthritis and a monoclonal CD4 T cell lymphoproliferative disorder.

A 33-year-old man developed seropositive rheumatoid arthritis (RA) followed soon after by a monoclonal lymphoproliferative disorder characterized by CD4 positive T lymphocytes in his peripheral blood, bone marrow and lymph nodes. Previous reports of polyarthritis in association with a T cell lymphoproliferative disorder have all involved the CD8 phenotype.