Marc Mathieu

Anti-inflammatory properties of desipramine and fluoxetine

BackgroundAntidepressants are heavily prescribed drugs and have been shown to affect inflammatory signals. We examined whether these have anti-inflammatory properties in animal models of septic shock and allergic asthma. We also analysed whether antidepressants act directly on peripheral cell types that participate in the inflammatory response in these diseases.MethodsThe antidepressants desipramine and fluoxetine were compared in vivo to the glucocorticoid prednisolone, an anti-inflammatory drug of reference. In a murine model of lipopolysaccharides (LPS)-induced septic shock, animals received the drugs either before or after injection of LPS. Circulating levels of tumour necrosis factor (TNF)-α and mortality rate were measured. In ovalbumin-sensitized rats, the effect of drug treatment on lung inflammation was assessed by counting leukocytes in bronchoalveolar lavages. Bronchial hyperreactivity was measured using barometric plethysmography. In vitro production of TNF-α and Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) from activated monocytes and lung epithelial cells, respectively, was analysed by immunoassays. Reporter gene assays were used to measure the effect of antidepressants on the activity of nuclear factor-κB and activator protein-1 which are involved in the control of TNF-α and RANTES expression.ResultsIn the septic shock model, all three drugs given preventively markedly decreased circulating levels of TNF-α and mortality (50% mortality in fluoxetine treated group, 30% in desipramine and prednisolone treated groups versus 90% in controls). In the curative trial, antidepressants had no statistically significant effect, while prednisolone still decreased mortality (60% mortality versus 95% in controls). In ovalbumin-sensitized rats, the three drugs decreased lung inflammation, albeit to different degrees. Prednisolone and fluoxetine reduced the number of macrophages, lymphocytes, neutrophils and eosinophils, while desipramine diminished only the number of macrophages and lymphocytes. However, antidepressants as opposed to prednisolone did not attenuate bronchial hyperreactivity. In vitro, desipramine and fluoxetine dose-dependently inhibited the release of TNF-α from LPS-treated monocytes. In lung epithelial cells, these compounds decreased TNF-α-induced RANTES expression as well as the activity of nuclear factor-κB and activator protein-1.ConclusionDesipramine and fluoxetine reduce the inflammatory reaction in two animal models of human diseases. These antidepressants act directly on relevant peripheral cell types to decrease expression of inflammatory mediators probably by affecting their gene transcription. Clinical implications of these observations are discussed.

Overexpression of the human glucocorticoid receptor α and β isoforms inhibits AP-1 and NF-κB activities hormone independently

Abstract. The human glucocorticoid receptor isoforms GRα and GRβ are generated by alternative splicing. Upon hormone binding, GRα regulates positively or negatively transcription. In particular, it represses numerous genes encoding pro-inflammatory mediators by inhibiting the transcription factors activator protein (AP)-1 and nuclear factor (NF)-κB. The observation that GRβ, which does not bind the hormone, may act as a dominant negative receptor is subject to controversy. Because GRβ must be more abundant than GRα to act as such, we evaluated the relative amounts of GRα and GRβ in COS-1, A549 and HeLa cells using a monoclonal antibody that recognises the two isoforms equally well on western blots. Messenger RNA levels of GRα and GRβ were compared by reverse transcriptase polymerase chain reaction analysis. To gain insight into the possible function of GRβ, we examined the ability of overexpressed GRβ to alter transcription of glucocorticoid, AP-1 and NF-κB inducible reporter genes using transient transfection in COS-1 and A549 cells. Subcellular localisation of GRβ was determined in A549 cells by immunofluoresence microscopy. Data indicate that GRα is the predominant endogenous isoform in A549 and HeLa cells. GRβ became the major form after transfection with the corresponding expression vector and translocated into cell nuclei even in the absence of hormone. Overexpression of GRβ inhibited glucocorticoid-induced transcription markedly in COS-1 cells but weakly in A549 cells. We found that GRβ did not act as a dominant negative modulator of GRα for repression of AP-1 and NF-κB activities. In fact, both GRβ and GRα inhibited hormone-independently these activities by 25–60%. This property was not shared by the closely related mineralocorticoid receptor. Our results suggest that overexpression of either GRα or GRβ may have an anti-inflammatory effect.

Stable transfection of the estrogen receptor cDNA into Hela cells induces estrogen responsiveness of endogenous cathepsin D gene but not of cell growth

Cathepsin D, a lysosomal protease, is induced by estrogens in hormone responsive breast cancer cells, by progesterone in normal endometrium and expressed at high constitutive levels in estrogen receptor (ER)-negative cells. To investigate whether ER is the only transacting factor missing in ER negative cells to obtain estrogen regulation, we transfected an ER cDNA expression vector (HEO) into ER-negative Hela cells and showed that it could recover estrogen sensitivity for cathepsin D gene expression but not for cell growth regulation. These results show i. that the expression of an endogenous gene in humans can be stimulated by estradiol after ER supplementation, indicating that Hela cells contain sufficient amounts of the other transcription factors required for cathepsin D estrogen inducibility; ii. that cathepsin D expression is stimulated by estrogens in this cervix cancer cell line, as it is in the ER-positive breast cancer cells, which contrasts with its regulation by progesterone in normal endometrial cells.

Correlation between different gene expression assays designed to measure trans-activation potencies of systemic glucocorticoids

The glucocorticoids (GC) betamethasone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone and triamcinolone acetonide are currently used in the treatment of inflammatory diseases. Through a process called trans-activation, GC activate gene expression and produce various physiological and pharmacological effects. In particular, by inducing gluconeogenic enzymes, long-term GC treatment may cause diabetes. Using three different assays, we have extensively compared the capacity of the above GC to activate gene expression. trans-Activation of a GC inducible luciferase gene was assessed in HeLa and A549 cells after stable and transient transfection, respectively. In hepatoma tissue culture cells, we measured trans-activation of the endogenous gene encoding tyrosine aminotransferase, a gluconeogenic enzyme. Half-maximal effective concentrations of GC were determined by dose-response analyses. Results obtained with these assays were highly correlated and GC were ranked in three groups according to their trans-activation potency: betamethasone, dexamethasone, and triamcinolone acetonide > methylprednisolone and prednisolone > hydrocortisone. Potencies were not strictly related to receptor binding affinities and not significantly affected by the amount of endogenous GC receptor.

Is there a role for glucocorticoid receptor beta in asthma

Glucocorticoids (GCs) are routinely used as anti-inflammatory drugs in the treatment of asthma. They act through binding to glucocorticoid receptor α (GRα), which represses numerous genes encoding pro-inflammatory mediators. A hormone binding deficient GR isoform named GRβ has been isolated in humans. When overexpressed by transfection, GRβ may function as a dominant negative modulator of GRα. However, to act as such, GRβ has to be more abundant than GRα, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. Moreover, the dominant negative effect was not confirmed by independent laboratories. In GC-resistant asthmatics, GRβ was expressed by an increased number of peripheral blood mononuclear cells (PBMCs), airway T cells, and cells found in skin biopsies of tuberculin responses. However, the relative amounts of GRα and GRβ in these cells were not determined. In GC-dependent asthmatics, PBMCs expressed GRα predominantly. No cells containing higher levels of GRβ than GRα have yet been reported in asthmatics. Even if the existence of such cells is demonstrated, the role of GRβ in asthma will remain a matter of controversy because functional studies have given discrepant data.

Histamine H1-receptor antagonists inhibit nuclear factor-kappaB and activator protein-1 activities via H1-receptor-dependent and -independent mechanisms

Background Histamine H1‐receptor antagonists are used to relieve the symptoms of an immediate allergic reaction. They have additional anti‐inflammatory effects that could result from an inhibition of the transcription factors activator protein‐1 (AP‐1) and nuclear factor‐kappa B (NF‐κB). The implication of the H1‐receptor in these effects is controversial. Diphenhydramine is a first‐generation H1‐receptor antagonist while mizolastine and desloratadine are second‐generation compounds. Mizolastine is also an inhibitor of 5‐lipoxygenase (5‐LO), an enzyme that has been involved in NF‐κB activation.

Fluticasone propionate and mometasone furoate have equivalent transcriptional potencies

Background Glucocorticoids exert their anti‐inflammatory effects mainly through transrepression of the transcription factors activator protein‐1 (AP‐1) and nuclear factor‐kappa B (NF‐κB). Certain adverse effects of glucocorticoids are mediated through gene transactivation. Fluticasone propionate (FP) and mometasone furoate (MF) are the most recently developed topical glucocorticoids for the treatment of airway disorders. Their relative capacities to repress AP‐1 and NF‐κB activities are not known and comparison of their transactivation potencies has given unclear results.

Cytokine-Induced CEACAM1 Expression on Keratinocytes Is Characteristic for Psoriatic Skin and Contributes to a Prolonged Lifespan of Neutrophils

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell-surface glycoprotein, belonging to the carcinoembryonic antigen family, expressed by human neutrophils, epithelial cells, activated T and NK cells. CEACAM1 is expressed as a cell-surface molecule with different isoforms or can be secreted as a soluble protein. Here, we show that keratinocytes in the outer epidermal layer of psoriatic skin express CEACAM1, unlike those in healthy skin or in cutaneous lesions of patients with atopic or nummular dermatitis. Stimulation of primary human keratinocytes or in vitro reconstituted epidermis with culture supernatants of activated psoriatic lesion-infiltrating T cells, IFN-gamma or oncostatin M, but not IL-17, induced the expression of transcripts for the CEACAM1-long and -short isoforms and cell-surface CEACAM1, whereas soluble CEACAM1 was not produced. The uppermost layers of the epidermis in psoriatic lesions also contain neutrophils, a cell type with inflammatory and antimicrobial properties. Coculture of CEACAM1-expressing keratinocytes or CHO transfectants with neutrophils delayed spontaneous apoptosis of the latter cells. These results show that cytokine-induced cell-surface expression of CEACAM1 by keratinocytes in the context of a psoriatic environment might contribute to the persistence of neutrophils and thus to ongoing inflammation and the decreased propensity for skin infection, typical for patients with psoriasis.

Glucocorticoid insensitive asthma: a one year clinical follow up pilot study

BACKGROUND Glucocorticoid resistant or insensitive asthmatic subjects are usually defined as patients whose baseline pre-bronchodilation forced expiratory volume in one second (FEV1) of less than 70–80% predicted improves significantly in response to β2 agonists but by less than 15% following 1–2 weeks of 40 mg prednisolone daily. Since there is little long term clinical information on these patients, a one year prospective study was performed to assess whether glucocorticoid sensitivity may vary over time. METHODS Nineteen severe asthmatic subjects were studied and received 40 mg prednisolone daily for seven days. Prednisolone was given for a further seven days in glucocorticoid insensitive asthmatics and then stopped. Patients were followed up for one year and the glucocorticoid test was repeated on five patients in each group six months later. RESULTS Eleven patients were classified as glucocorticoid insensitive and eight as glucocorticoid sensitive on day 7. The demographic characteristics of the patients were similar in both groups. Four glucocorticoid insensitive patients became responsive after one further week of prednisolone treatment. Six months later, four of five glucocorticoid sensitive patients and three of five previously glucocorticoid insensitive patients were glucocorticoid sensitive. CONCLUSIONS Glucocorticoid sensitivity varies over time.

Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells

Background: Due to their ability to differentiate into chondrocytes, mesenchymal stem cells (MSCs) are candidates for cartilage repair. Results: During chondrogenic differentiation of MSCs, angiopoietin-like 4 (ANGPTL4) triggers degradation and reduced synthesis of the cartilage matrix. Conclusion: ANGPTL4 promotes cartilage matrix remodeling. Significance: In the perspective of MSC-based cartilage engineering, inhibiting ANGPTL4 expression or action could help to stabilize cartilage formation. Mesenchymal stem cells (MSCs) are considered for cartilage engineering given their ability to differentiate into chondrocytes. Chondrogenic differentiation of MSCs is currently triggered by micromass culture in the presence of a member of the TGF-β superfamily. However, the main constituents of the cartilaginous matrix, aggrecan and type II collagen, are degraded at the end of the differentiation process through induction of matrix metallopeptidase (MMP)13. We hypothesized that MSCs undergoing chondrogenic differentiation produce an intermediate cytokine that triggers this matrix remodeling. Analysis of transcriptomic data identified angiopoietin-like 4 (ANGPTL4) as one of the most strongly up-regulated gene encoding a secreted factor during TGF-β-induced chondrogenesis. To gain insight into the role of ANGPTL4 during chondrogenesis, we used recombinant ANGPTL4 as well as a RNA interference approach. Addition of exogenous ANGPTL4 during the course of TGF-β-induced differentiation reduced the mRNA levels of aggrecan and type II collagen, although it increased those of MMP1 and MMP13. Accordingly, deposition of aggrecan and total collagens was diminished, whereas release of MMP1 and MMP13 was increased. Conversely, transfection of MSCs with an siRNA targeting ANGPTL4 prior to induction of chondrogenesis increased expression of type II collagen and aggrecan, whereas it repressed that of MMP1, MMP3, and MMP13. A neutralizing antibody against integrin αVβ5, a known receptor for ANGPTL4, mimicked some of the effects observed after siRNA-mediated ANGPTL4 silencing. Our data provide evidence that ANGPTL4 promotes cartilage matrix remodeling by inhibiting expression of its two key components and by up-regulating the level of certain MMPs.