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Human pituitary adenomas are one of the most common intracranial neoplasms. Although most of these tumors are benign and can be treated medically or by transsphenoidal surgery, a subset of these tumors are fast-growing, aggressive, recur, and remain a therapeutic dilemma. Because antibodies against immune checkpoint receptors PD-1 and CLTA-4 are now routinely used for cancer treatment, we quantified the expression of mRNA coding for PD-1, CLTA-4, and their ligands, PD-L1, PD-L2, CD80, and CD86 in human pituitary adenomas and normal pituitary glands, with the ultimate goal of exploiting immune checkpoint therapy in aggressive pituitary adenomas. Aggressive pituitary adenomas demonstrated an increased expression of PD-L2, CD80, and CD86 in compared to that of normal human pituitary glands. Furthermore, aggressive pituitary tumors demonstrated significantly higher levels of CD80 and CD86 compared to non-aggressive tumors. Our results establish a rationale for studying a potential role for immune checkpoint inhibition therapy in the treatment of pituitary adenomas.

The imprinted delta like 1 homolog (DLK1) - thyroxine deiodinase type III (DIO3) locus regulates development and growth. Its imprinting regulation involves two differentially methylated regions (DMRs), intergenic-DMR (IG-DMR) and maternally expressed gene 3-DMR (Meg3-DMR). In mice, a maternal deletion of the IG-DMR leads to LOI in the locus, proving that the IG-DMR is a cis-acting imprinting control region of the locus. However, the Meg3-DMR overlaps with the promoter, exon 1 and intron 1 of the Meg3 gene. Because deletion of the Meg3-DMR inactivates the Meg3 gene, their roles in imprinting regulation of Meg3-DMR mice is unknown. Therefore, we generated two mouse models: Meg3Δ(1-4) and Meg3Δ(2-4), respectively targeting exons 1–4 and exons 2–4 of the Meg3 gene. A maternal deletion of Meg3Δ(1-4) caused embryonic death and LOI in both embryos and placentas, but did not affect methylation status of the IG-DMR. In contrast, mice carrying a maternal deletion of Meg3Δ(2-4) were born normally and did not have LOI. These data indicate that it is the Meg3-DMR, not the Meg3 gene, which regulates imprinting of the Dlk1-Dio3 locus.

The functions of long noncoding (lnc)RNAs, such as MEG3, are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with mouse double minute 2 homolog (Mdm2). To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy, a single-molecule localization microscopy technique, to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross-nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the association between FKBP1A and mTOR, MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.

Identifying secreted mediators that drive the cognitive benefits of exercise holds great promise for the treatment of cognitive decline in ageing or Alzheimer's disease (AD). Here, we show that irisin, the cleaved and circulating form of the exercise-induced membrane protein FNDC5, is sufficient to confer the benefits of exercise on cognitive function. Genetic deletion of Fndc5/irisin (global Fndc5 knock-out (KO) mice; F5KO) impairs cognitive function in exercise, ageing and AD. Diminished pattern separation in F5KO mice can be rescued by delivering irisin directly into the dentate gyrus, suggesting that irisin is the active moiety. In F5KO mice, adult-born neurons in the dentate gyrus are morphologically, transcriptionally and functionally abnormal. Importantly, elevation of circulating irisin levels by peripheral delivery of irisin via adeno-associated viral overexpression in the liver results in enrichment of central irisin and is sufficient to improve both the cognitive deficit and neuropathology in AD mouse models. Irisin is a crucial regulator of the cognitive benefits of exercise and is a potential therapeutic agent for treating cognitive disorders including AD.

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase–activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C(4) production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A(2) (cPLA(2)) and were linked to LTB(4) production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA(2) were clustered with 5-LO, and 77 (33, 114) molecules of cPLA(2) were associated with FLAP. These assemblies were tightly linked to LTB(4) formation. The activation-dependent close associations of cPLA(2), FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA(2) in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

The functions of long noncoding (lnc)RNAs such as MEG3 are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with Mdm2. To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule localization microscopy (SMLM) technique to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross-nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the association between FKBP1A and mTOR, MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.

The ability of the long noncoding RNA MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has previously been shown to bind to p53 in vitro, which led us to hypothesize that MEG3 functions by disrupting the interaction of p53 and its E3 ubiquitin ligase Mdm2. To test this hypothesis in vivo, we built a cross-nearest neighbor/Monte Carlo analytical method based on two color direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule localization microscopy (SMLM) technique. Our data support the interaction of MEG3 and p53. Surprisingly, this association had no effect on the binding of p53 and Mdm2, distinct from the most commonly proposed model for the mechanism of MEG3 action. Additionally, our mathematical approach to analyzing SMLM data has general applicability to assessing molecular interactions in a native cellular context.

The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate leukotriene C4. We identified the time-dependent reorganization of both 5-LO and FLAP into higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC analysis identified a subset of these clusters with a high degree of interaction between 5-LO and FLAP that specifically correlates with the time course of LTC4synthesis, strongly suggesting their role in the initiation of leukotriene biosynthesis.

Antibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin- independent neutrophil migration into inflamed lung. In peritoneum, the role of β2 integrins varied with stimulus, proving dispensable for neutrophil entry in E. coli peritonitis but contributory in IL-1-mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin- mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of β2 integrin dependence in neutrophil migration.KEY POINTSThe contribution of β2 integrins to neutrophil migration into inflamed tissues varies with site and stimulus.Ligation of Ly6G, a GPI-linked neutrophil surface protein, selectively attenuates β2 integrin-dependent neutrophil migration in vivo.Blockade correlates with disrupted interaction between Ly6G and β2 integrins and impaired integrin-mediated postadhesion strengthening.

Summary Increased consumption of fats and added sugars has been associated with an increase in metabolic syndromes. Here we show that mice chronically fed an energy-rich diet (ERD) with high fat and moderate sucrose have enhanced the absorption of a gastrointestinal fructose load, and this required expression of the arrestin domain protein Txnip in the intestinal epithelial cells. ERD feeding induced gene and protein expression of Glut5, and this required the expression of Txnip. Furthermore, Txnip interacted with Rab11a, a small GTPase that facilitates the apical localization of Glut5. We also demonstrate that ERD promoted Txnip/Glut5 complexes in the apical intestinal epithelial cell. Our findings demonstrate that ERD facilitates fructose absorption through a Txnip-dependent mechanism in the intestinal epithelial cell, suggesting that increased fructose absorption could potentially provide a mechanism for worsening of metabolic syndromes in the setting of a chronic ERD., Graphical Abstract, Highlights • Consumption of energy-rich diets (ERDs) promotes intestinal fructose absorption • Intestinal Txnip is required for increased fructose absorption by ERD • Txnip increases fructose transporter Glut5 protein and gene expression • Txnip forms a complex with Rab11a to facilitate the apical localization of Glut5, Human Metabolism; Molecular Biology

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration.

CD47 plays an important but incompletely understood role in innate and adaptive immune responses. CD47 associates in cis with T-cell LFA-1 integrins and regulates expression of high-affinity conformations of both LFA-1 and VLA-4., CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47−/− Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47−/− Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)–activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α–activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn2+ or Mg2+/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.

Metabolic studies suggest that the absorptive capacity of the small intestine for fructose is limited, though the molecular mechanisms controlling this process remain unknown. Here we demonstrate that thioredoxin-interacting protein (Txnip), which regulates glucose homeostasis in mammals, binds to fructose transporters and promotes fructose absorption by the small intestine. Deletion of Txnip in mice reduced fructose transport into the peripheral bloodstream and liver, as well as the severity of adverse metabolic outcomes resulting from long-term fructose consumption. We also demonstrate that fructose consumption induces expression of Txnip in the small intestine. Diabetic mice had increased expression of Txnip in the small intestine as well as enhanced fructose uptake and transport into the hepatic portal circulation. The deletion of Txnip in mice abolished the diabetes-induced increase in fructose absorption. Our results indicate that Txnip is a critical regulator of fructose metabolism and suggest that a diabetic state can promote fructose uptake. DOI: http://dx.doi.org/10.7554/eLife.18313.001, eLife digest Fructose is a type of sugar that is found naturally in fruits, and it is closely related to glucose. The amount of fructose in our diet has increased dramatically in the last few decades. Growing evidence suggests that excessive amounts of fructose contribute to several metabolic diseases, including fatty liver disease and diabetes. Fructose is absorbed in the small intestine via transport proteins called GLUT2 and GLUT5 and then travels to the liver where it can stimulate the cells to make fats. However, it is not clear how fructose uptake is regulated in the small intestine. Glucose is taken into cells by a transport protein that is closely related to GLUT2 and GLUT5. Another protein called thioredoxin-interacting protein (Txnip) interacts with the glucose transporter and regulates glucose uptake. Here, Dotimas et al. investigated whether Txnip also regulates the activities of GLUT2 and GLUT5 to control how cells absorb fructose. Initial experiments in cells showed that Txnip binds to both GLUT2 and GLUT5 and increases the amount of fructose taken up by both mouse and human cells. Cells from mutant mice that do not produce Txnip absorbed less fructose than normal cells did. Furthermore, the mutant mice had lower levels of fructose in the blood and less severe metabolic disease after consuming fructose regularly for six months. Mice with diabetes absorbed more fructose through the small intestine than normal mice, and the loss of Txnip from these mice abolished this effect. Together the findings of Dotimas et al. suggest that Txnip plays an important role in regulating fructose absorption and indicate that, at least in some circumstances, diabetes may lead to more fructose being absorbed in the small intestine. The next steps following on from this work are to understand the molecular details of how Txnip regulates fructose uptake and to determine if other forms of diabetes also show increased fructose uptake. DOI: http://dx.doi.org/10.7554/eLife.18313.002

Alzheimer's disease (AD) is characterized by memory loss, insidious cognitive decline, profound neurodegeneration, and the extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques and intracellular accumulation of tau in neurofibrillary tangles. Loss and dysfunction of synapses are believed to underlie the devastating cognitive decline in AD. A large amount of evidence suggests that oligomeric forms of Aβ associated with senile plaques are toxic to synapses, but the precise sub-synaptic localization of Aβ and which forms are synaptotoxic remain unknown. Here, we characterize the sub-synaptic localization of Aβ oligomers using three high-resolution imaging techniques, stochastic optical reconstruction microscopy, immunogold electron microscopy, and Forster resonance energy transfer in a plaque-bearing mouse model of AD. With all three techniques, we observe oligomeric Aβ inside synaptic terminals. Further, we tested a panel of Aβ antibodies using the relatively high-throughput array tomography technique to determine which forms are present in synapses. Our results show that different oligomeric Aβ species are present in synapses and highlight the potential of array tomography for rapid testing of aggregation state specific Aβ antibodies in brain tissue.

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 LTB4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and β2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of β2-integrins in LTB4-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of β2-integrin–deficient 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 β2-integrin–dependent mechanism.

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.

Leukotriene B4 (LTB4) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB4 by ω-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB4 by CYP4Fs to down-regulating functional responses of cells to LTB4 is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB4 in terminating the responses of PMN to LTB4 and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the ω-1 and ω-2 oxidation of LTB4 in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB4 to 19-OH LTB4 and to a lesser extent 18-OH LTB4. Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB4. CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I·C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB4 metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A.

The Golgi complex has a central role in the intracellular sorting of secretory proteins. Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation. Because this explanation is now appreciated to be incomplete, interest has developed in understanding tubules that connect the Golgi cisternae. Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.

The CD200:CD200R1 inhibitory signaling pathway has been implicated in playing a prominent role in limiting inflammation in a wide range of inflammatory diseases. CD200R1 signaling inhibits the expression of proinflammatory molecules including tumor necrosis factor, interferons, and inducible nitric oxide synthase in response to selected stimuli. Unsurprisingly, due to the regulatory role that CD200R1 plays in multiple inflammatory pathways, an increasing number of parasitic, bacterial, and viral pathogens exploit this pathway to suppress host defenses. A complete understanding of the pathways regulated by CD200R1 signaling and the diverse mechanisms that pathogens have evolved to manipulate the CD200:CD200R1 pathway can help identify clinical situations where targeting this interaction can be of therapeutic benefit. In this review, we compare CD200R1 to other pathogen-targeted inhibitory receptors and highlight how this signaling pathway is utilized by a diverse number of pathogens and, therefore, may represent a novel targeting strategy for the treatment of infectious diseases.

Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.

Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.

Cytochrome P450 4F3 (CYP4F3) catalyzes the inactivation of leukotriene B4 by ω-oxidation in human neutrophils. To understand the regulation of CYP4F3 expression, we analyzed the CYP4F3 gene and cloned a novel isoform (CYP4F3B) that is expressed in fetal and adult liver, but not in neutrophils. The CYP4F3 gene contains 14 exons and 13 introns. The cDNAs for CYP4F3A (the neutrophil isoform) and CYP4F3B have identical coding regions, except that they contain exons 4 and 3, respectively. Both exons code for amino acids 66–114 but share only 27% identity. When expressed in COS-7 cells, the K m of CYP4F3B was determined to be 26-fold higher than the K m of CYP4F3A using leukotriene B4 as a substrate. 5′-Rapid amplification of cDNA end studies reveal that the CYP4F3A and CYP4F3B transcripts have 5′-termini derived from different parts of the gene and are initiated from distinct transcription start sites located 519 and 71 base pairs (bp), respectively, from the ATG initiation codon. A consensus TATA box is located 27 bp upstream of the CYP4F3B transcription start site, and a TATA box-like sequence is located 23 bp upstream of the CYP4F3A transcription start site. The data indicate that the tissue-specific expression of functionally distinct CYP4F3 isoforms is regulated by alternative promoter usage and mutually exclusive exon splicing.

Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 microM ATP and greater than 50% with 100 microM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4.

Background: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. Objectives: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. Methods: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. Results: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. Conclusion: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs. © 2013 American Academy of Allergy, Asthma & Immunology.

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., Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase–activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA4. We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony–stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/-) mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1(-/-) peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam(2)CSK(4) and to HSV-1. CD200R1(-/-) macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/-) mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/-) mice and CD200R1(-/-) fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.

In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.

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.

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB(4) by omega-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB(4) by CYP4Fs to down-regulating functional responses of cells to LTB(4) is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB(4) in terminating the responses of PMN to LTB(4) and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the omega-1 and omega-2 oxidation of LTB(4) in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB(4) to 19-OH LTB(4) and to a lesser extent 18-OH LTB(4). Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB(4). CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I.C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB(4) metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A.

Cell signaling leading to the formation of leukotriene (LT)C 4 requires the localization of the four key biosynthetic enzymes on the outer nuclear membrane and endoplasmic reticulum. Whether any macromolecular organization of these proteins exists is unknown. By using fluorescence lifetime imaging microscopy and biochemical analysis, we demonstrate the presence of two distinct multimeric complexes that regulate the formation of LTs in RBL-2H3 cells. One complex consists of multimers of LTC 4 synthase and the 5-lipoxygenase activating protein (FLAP). The second complex consists of multimers of FLAP. Surprisingly, all LTC 4 synthase was found to be in association with FLAP. The results indicate that the formation of LTC 4 and LTB 4 may be determined by the compartmentalization of biosynthetic enzymes in discrete molecular complexes.

The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B4, a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3+ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123–155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468–872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation.

Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC(4) synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC(4) synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC(4) synthase. The active site of LTC(4) synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB(4) and LTC(4) occurs in different subcellular locations and suggests that LTC(4) must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane.