Corinne Henriquet

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.

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.

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.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.