Extranuclear Lipid Bodies, Elicited by CCR3-mediated Signaling Pathways, Are the Sites of Chemokine-enhanced Leukotriene C4 Production in Eosinophils and Basophils

Abstract

Eosinophils and basophils, when activated, become major sources of cysteinyl leukotrienes, eicosanoid mediators pertinent to allergic inflammation. We show that the C-C chemokines, eotaxin and RANTES (regulated upon activation normal T cell expressed and secreted), activate eosinophils and basophils for enhanced leukotriene C 4 (LTC 4 ) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles. Chemokineinduced lipid body formation and enhanced LTC 4 release were both mediated by CCR3 receptor G protein-linked downstream signaling involving activation of phosphoinositide 3-kinase, extracellular signal-regulated kinases 1 and 2, and p38 mitogen-activated protein kinases. Chemokine-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC 4 production; and as demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid, lipid bodies were the predominant sites of LTC 4 synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases both elicits the formation of lipid body domains and promotes LTC 4 formation at these specific extranuclear sites.

Mechanisms of leukocyte lipid body formation and function in inflammation

“…5-LO was shown to localize within the nuclear environment (nuclear membrane and euchromatin) of alveolar macrophages and basophilic leukemia cells, whereas 5-LO was found to be predominantly cytosolic in human neutrophils and resting peritoneal macrophages (Peters-Golden & Brock 2000, Bandeira-Melo & Weller 2003. In addition to the nuclear environment, we have demonstrated the compartmentalization of the key enzyme for leukotriene production, 5-LO, within human eosinophil and basophil lipid bodies (Bozza et al 1997, Bandeira-Melo et al 2001a). In addition, immuno-reactivity for 5-LO was also detected in lipid bodies from enucleated human eosinophils (Bozza et al 1997).…”

“…Direct assessment of intracellular sites for eicosanoid has been elusive, as those mediators are newly formed, non-storable and rapidly released upon cell stimulation. Recently, a new strategy to cross-link newly formed LTC 4 at its sites of synthesis within in vitro stimulated eosinophils was described, enabling the immunofluorescent localization of newly formed LTC 4 at its intracellular formation locale (Bandeira-Melo et al 2001a, 2002b. Lipid bodies were the predominant sites of LTC 4 synthesis in chemokine-stimulated human eosinophils.…”

“…Interestingly, the pretreatment of leukocytes with protein synthesis inhibitors acting on transcription (actinomycin D) and translation (cycloheximide) significantly, although partially, inhibited lipid body formation induced by PAF and fatty acids in vitro but not by eotaxin and RANTES, thus indicating that according to the stimulated conditions induction of lipid bodies depends on new protein synthesis and its likely that specific and transcriptionaly regulated early response genes are activated during the process of lipid body formation (Bozza et al 1996a, b, 1997, Bandeira-Melo et al 2001a. A role for peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor supergene family that function in ligand-activated transcription, in leukocyte lipid body formation has recently been suggested.…”

“…2). For instance, lipid bodies can be elicited in vivo and in vitro after stimulation of cells with either lipid agonists such as, cis-unsaturated fatty acids, PAF, LPS or PKC activators (Weller et al 1989, 1991Bozza et al 1996a, b, Pacheco et al 2002 or protein stimuli such as chemokines and interleukines , Bartemes et al 1999, Bandeira-Melo et al 2001a, 2002a.…”

“…Moreover, chemokines acting via CCR3 receptors, including RANTES, eotaxin, eotaxin-2, and eotaxin-3, can initiate intracellular signaling in eosinophils, but not neutrophils, that also culminate in de novo formation of lipid bodies (Bandeira- Melo et al 2001a, b). Differently from the signaling pathways described by PAF, eotaxin and RANTES activating CCR3 receptor signals through phosphoinositide 3-kinase (PI3K) and the ERK1/2 and p38 MAP kinases (Bandeira- Melo et al 2001a).…”