Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice.In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB 4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and ␤2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of ␤2-integrins in LTB 4 -stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of ␤2-integrindeficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a ␤2-integrindependent mechanism. (Blood. 2012; 120(7):1489-1498) IntroductionNeutrophils are one of the first cell types to reach sites of infection or acute inflammation. Recruitment involves an orchestrated sequence of events in which circulating neutrophils respond to chemotactic signals to become firmly adherent to activated endothelium, followed by transendothelial migration via either a paracellular or transcellular route. 1,2 Once at a site of inflammation, neutrophils contribute to ongoing leukocyte recruitment and tissue injury by releasing lipid mediators, proteases, reactive oxygen species (ROS), and other factors. [3][4][5] Whereas they are critical to immune defense, neutrophils can, also play a pathogenic role in chronic inflammatory diseases and therefore their recruitment is subject to numerous levels of control, not all of which are understood completely. 1 Delineating these regulatory pathways will provide insights into the mechanisms of tissue injury in inflammatory disorders and novel targets for drug development.Much of the experimental evidence implicating neutrophils in disease has been obtained through murine studies in which this lineage was selectively depleted using Abs that bind the neutrophil surface antigen Ly6G, such as RB6-8C5 (more typically termed anti-Gr-1). 6,7 However, the function of Ly6G remains unknown. Ly6G is a small protein of approximately 25 kDa that is tethered to the cell surface via a GPI linker. 7 In bone marrow (BM), peripheral blood, and wound exudates, the expression of Ly6G is limited to cells with granulocyte morphology. 7,8 Structurally, Ly6G belongs to the Ly6/urokinase pl...

The interaction between bacteria and endothelial cell plasma membrane is mediated by components of the bacterial wall outer membrane, the most important being LPS.3 LPS binds to CD14 (1-3) and Toll-like receptor 4 (TLR4) (1-4) expressed in the membrane. NF-B, the transcription factor activated by LPS-CD14-TLR4 signaling (5), results in the transcriptional induction of cytokines (interleukin-1 (IL-1), IL-6, IL-8), tissue factor, and adhesion molecules (E-and P-selectins, VCAM-1 (vascular cell adhesion molecule), and ICAM-1) (6). Cav-1, the structural protein of caveolae in endothelial cells and other cell types, regulates the formation of caveolae, the vesicle carriers involved in the transcytosis of albumin across the endothelial barrier (7). Studies showed that caveolae-mediated transcytosis contributes to the regulation of microvascular permeability (7) secondary to the activation of Src kinase (8). Cav-1-null mice, lacking caveolae (9), showed defective albumin transcytosis (10). In an experimental model of diabetes, increased Cav-1 expression in endothelial cells was associated with increased transcytosis of albumin (11). LPS was shown to induce the expression of Cav-1 in endothelial cells (12) and murine macrophages (13, 14); however, the mechanisms of the response and its consequences in regulating endothelial barrier function are not clear.NF-B is composed of dimers of five different proteins (p50, p52, p65/RelA, RelB, c-Rel) (15). These dimers exist in the cytoplasm in inactive forms bound to the inhibitory protein I-B (IB) (15). A variety of agonists activate IB kinases ␣ and ␤ (15), which in turn phosphorylate serines 32 and 36 of IB␣ and serines 19 and 23 of IB␤, respectively (15). Phosphorylation of IB␣ and IB␤ leads to the proteolytic degradation of IB and dissociation of NF-B, and NF-B translocates to the nucleus to induce gene transcription (15). The IB kinase complex consists of two catalytic IKK␣ and IKK␤, and a regulatory subunit, IKK␥ (or NF-B essential modulator (NEMO)) (16). NEMO interaction with IKK␣ and IKK␤ is required for IB kinase catalytic activity. Based on our observation that the intronic region of Cav-1 contains NF-B consensus sites, we addressed the possibility that LPS mediates Cav-1 expression by an NF-B-dependent mechanism. We surmised that this pathway thereby contributes to the mechanism of increased transendothelial albumin permeability seen with LPS. We demonstrate here that LPS activation of endothelial cells increased Cav-1 protein expression as well as caveolae number and that both were dependent on activation of NF-B. Moreover, inhibiting NF-B activation pharmacologically, knockdown of p65/RelA expression and knockdown of Cav-1 expression each interfered with the increase in transendothelial albumin permeability induced

Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.inflammation ͉ multiprotein complex ͉ 5-lipoxygenase ͉ 5-lipoxygenase-activating protein L eukotrienes (LTs) are lipid signaling molecules derived from arachidonic acid (AA) that initiate and amplify innate and adaptive immune responses by regulating the recruitment and activation of leukocytes in inflamed tissues (1-3). Cells employ multiple mechanisms to prevent the inappropriate onset of pro-inflammatory signaling while requiring tightly coupled processes to trigger the generation of pro-inflammatory signaling molecules. The interplay of these processes is epitomized by the synthesis of LTB 4 and LTC 4 . In unstimulated myeloid cells, including mast cells, LT formation is held in abeyance by the cytosolic compartmentalization of cytosolic phospholipase A 2 (cPLA 2 ) (4, 5) and the cytosolic/nucleoplasmic localization of 5-lipoxygenase (5-LO) (6). LT formation is initiated by translocation of cPLA 2 to the Golgi and ER/nuclear envelope to release AA (4, 5). In parallel, 5-LO targets to the inner and outer nuclear membranes to initiate LT synthesis by converting AA to 5-HPETE and LTA 4 , a process that requires the integral nuclear envelope protein 5-lipoxygenase-activating protein (FLAP) (7-9). Although we have shown that LTC 4 synthase and FLAP constitutively interact on the nuclear envelope (10), how or whether 5-LO interacts with these proteins on the nuclear envelope in response to cell signaling is unknown.Understanding how cells couple the reorganization of the LT biosynthetic enzymes to efficient AA utilization is central to understanding the signal transduction pathways that control the synthesis of bioactive lipids derived from AA. Scaffold/docking proteins can localize components of biochemical reactions at membrane interfaces; examples include protein kinase A anchoring proteins (11) and the integral membrane proteins caveolins 1-3 (12). The dependence of cellular LT synthesis on FLAP (7, 8) and its membrane localization (9) make it a conceptually appealing candidate for a 5-LO scaffold. However, no interaction of 5-LO with any membrane protein has been detected.A closely related question is: how do different combinations of extracellular signals lead to LT generation? For example, in mast cells, the engage...

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Leukotrienes are bioactive signaling molecules derived from arachidonic acid that initiate and amplify innate immunity. A single structure, the leukotriene synthetic complex, on the nuclear membrane of neutrophils integrates and transduces extracellular signals to generate the chemotactic lipid LTB4.

Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB4 synthesis and to respond to LTB4 within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase–/– and leukotriene A4 (LTA4) hydrolase–/– mice, we demonstrate that FLSs generate sufficient levels of LTB4 production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs—which comprise the predominant lineage populating the synovial lining—are competent to metabolize exogenous LTA4 into LTB4 ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB4 3-fold without inducing the expression of LTA4 hydrolase protein. Moreover, LTB4 (acting via LTB4 receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB4 in joints, placing LTB4 regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.

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