Activation of nuclear factor-κB by lipopolysaccharide in mononuclear leukocytes is prevented by inhibitors of cytosolic phospholipase A₂

Abstract

In monocytes, lipopolysaccharide induces synthesis and activity of the 85-kDa cytosolic phospholipase A₂. This enzyme releases arachidonic acid and lyso-phospholipids from membranes which are metabolized to eicosanoids and platelet-activating-factor. These lipid mediators increase activity of transcription factors and expression of cytokine genes indicating a function for cytosolic phospholipase A₂ in signal transduction and inflammation. We have shown previously that trifluoromethylketone inhibitors of cytosolic phospholipase A₂ suppressed interleukin-1β protein and steady-state mRNA levels in human lipopolysaccharide-stimulated peripheral blood mononuclear leukocytes. In this study, the subcellular mechanisms were analyzed by which trifluoromethylketones interfere with gene expression. We found that they reduced the initial interleukin-1β mRNA transcription rate through prevention of degradation of inhibitor-κBα. Consequently, cytosolic activation, nuclear translocation and DNA-binding of nuclear factor-κB were decreased. Trifluoromethylketones ameliorate chronic inflammation in vivo. Thus, this therapeutic potency may reside in retention of inactive nuclear factor-κB in the cytosol thereby abrogating interleukin-1β gene transcription.

Introduction

Phospholipases A₂ hydrolyze glycerophosphatides into lyso-phosphatides and arachidonic acid representing the rate-limiting step in the synthesis of eicosanoids and are involved in the formation of platelet-activating factor. Phospholipases A₂ are implicated in digestion, membrane remodelling, blood coagulation and signal transduction and in pathophysiological states of inflammation, cancer and autoimmune diseases. In mammals, multiple isoforms exist where among the 85-kDa cytosolic Ca²⁺-dependent type IV phospholipase A₂ is responsible for agonist-induced receptor-mediated intracellular arachidonic acid generation (Murakami et al., 1998). Human monocytes treated with antisense oligonucleotides against cytosolic phospholipase A₂ mRNA (Roshak et al., 1994) and macrophages from knock-out mice exhibit a decreased arachidonic acid release and lipid mediator production. Furthermore, these mice exert a diminished acute allergic response (Uozumi et al., 1997). Concordantly, inhibitors of arachidonic acid metabolism like non-steroidal-(NSAID) and cytokine-suppressive-antiinflammatory drugs (CSAID®) abrogate cytokine production in vitro and prevent inflammation in vivo (Boehm et al., 1996; Osnes et al., 1996). Collectively, these data suggest that cytosolic phospholipase A₂ plays a priorized role in inflammation.

This isoform is expressed in platelets, granulocytes, monocytes, tissue macrophages, mast cells, fibroblasts, epithelial and endothelial cells and is activated by diverse proinflammatory stimuli like bacteria, cytokines, growth factors, kinins, complement and immune complexes (Clark et al., 1995; Murakami et al., 1997b). In peripheral blood mononuclear cell populations monocytes but not B- or T-lymphocytes are the major source for arachidonic acid release (Camussi et al., 1995; Surette et al., 1996). Lipopolysaccharide binding to CD14 on monocytes triggers rapid Ca²⁺-dependent translocation of the cytosolic phospholipase A₂ from the cytosol to membranes. Mitogen-activated protein kinases and protein kinase C further activate the enzyme by phosphorylation. Within hours de novo synthesis of cytosolic phospholipase A₂ mRNA and protein is also induced (Clark et al., 1995; Murakami et al., 1997b). The lipid derivatives generated upon cytosolic phospholipase A₂ activation either bind in an auto- or paracrine fashion to G-protein-coupled receptors on plasma membranes inducing, e.g., bronchoconstriction, vasopermeability and chemotaxis or transfer signals as intracellular `second messengers' (Serhan et al., 1996). Thus, arachidonic acid, platelet-activating factor and leukotriene B₄ enhance whereas prostaglandin E₂ decreases activity and synthesis of the transcription factors nuclear factor-κB, nuclear factor-interleukin-6 (CCAAT/enhancer binding protein-β) and activation protein-1 (c-Jun/c-fos) (Brach et al., 1992; Camandola et al., 1996; Danesch et al., 1994; Kravchenko et al., 1995; Rizzo et al., 1995; Rossi et al., 1997). Thereby, lipid metabolites control the expression of genes for cytokines (Chaughey et al., 1997; Gormand et al., 1996; Poubelle et al., 1991; Rola-Pleszczynski and Lemaire, 1985; Rola-Pleszczynski and Stankova, 1992; Spangelo and Jarvis, 1996; Zhong et al., 1995), adhesion molecules (Milam et al., 1991) and tissue proteases (Mehindate et al., 1995) which are responsible for induction and maintenance of inflammation.

The trifluoromethylketone analogues of arachidonic acid (C20:4-COCF₃), γ-linolenic (C18:3-COCF₃) and linoleic acid (C18:2-COCF₃) are specific, reversible inhibitors of the cytosolic phospholipase A₂ enzyme activity by forming a stable hemiketal with the nucleophilic Ser²²⁸ residue of the catalytic triad (Clark et al., 1995; Street et al., 1993). These compounds do not inhibit 14-kDa secretory type IIA phospholipase A₂, CoA-independent transacylase nor lipoxygenases and cyclooxygenases (Bartoli et al., 1994; Riendeau et al., 1994; Tibes et al., 1997). They reduce arachidonic acid release and formation of eicosanoids and platelet-activating factor in platelets and leukocytes upon stimulation with lipopolysaccharide, Ca²⁺-ionophore or thrombin (Lo et al., 1997; Murakami et al., 1997b; Shamsuddin et al., 1997; Withnall et al., 1995). We have shown previously that in addition to this primary mode of action the trifluoromethylketones display antiphlogistic effects in vitro and in vivo: they (a) exhibited therapeutic benefit in animal models of acute, allergic and chronic inflammation (Tibes et al., 1997; Tibes et al., 1995), (b) prevented chemotaxis and Ca²⁺-ionophore-induced expression of the β₂-integrin Mac-1 (CD11b/CD18) in human neutrophils, and (c) suppressed lipopolysaccharide-stimulated protein synthesis and steady-state mRNA levels of interleukin-1β in human peripheral blood mononuclear cells (CD14⁺ monocytes) with IC₅₀ values of 10–20 μM (Amandi-Burgermeister et al., 1997). In the present study, we investigated by which subcellular mechanisms trifluoromethylketones interfere with interleukin-1β gene expression in monocytes. We show that these inhibitors of the cytosolic phospholipase A₂ enzyme activity prevent degradation of inhibitor-κBα and subsequent cytoplasmatic mobilisation, nuclear translocation and DNA-binding of nuclear factor-κB. Thereby, transcriptional activation of the interleukin-1β gene is abrogated.