In inflamed venules, neutrophils rolling on E-selectin induce integrin ␣ L ␤ 2 -dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-␥ (FcR␥), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential Eselectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raftdependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, Eselectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce ␣ L ␤ 2 -dependent slow rolling. (Blood. 2010;116(3):485-494) IntroductionCirculating leukocytes enter inflamed tissues through sequential adhesive and signaling events. 1 Neutrophils first tether to and roll on P-and E-selectin expressed on activated endothelial cells. 2 They roll on P-selectin through interactions with P-selectin glycoprotein ligand-1 (PSGL-1) 3,4 and on E-selectin through interactions with PSGL-1, CD44, and E-selectin ligand-1. [5][6][7] Rolling neutrophils encounter immobilized chemokines that signal through G␣ i proteincoupled receptors. These signals activate integrins ␣ L ␤ 2 and ␣ M ␤ 2 to their high-affinity states, enabling interactions with intercellular adhesion molecule-1 (ICAM-1) that promote arrest, adhesion strengthening, intraluminal crawling, and transendothelial migration. 1 Importantly, E-selectin directly triggers signals in rolling neutrophils that cooperate with chemokine signals to maximize neutrophil recruitment during inflammation. 8 In autoperfused whole blood, neutrophils rolling on immobilized E-selectin activate ␣ L ␤ 2 to an intermediate-affinity state, which slows rolling on coimmobilized ICAM-1. 8 In vivo, neutrophils roll slowly on E-selectin expressed in tumor necrosis factor-␣ (TNF-␣)-stimulated postcapillary venules. 9 Injecting anti-␣ L ␤ 2 antibody increases rolling velocities, documenting the physiologic importance of integrins for slow rolling in vivo. 8,10 Integrin-dependent slow rolling prolongs transit times in inflamed venules 11 and enhances neutrophil recruitment. 8 In autoperfused whole blood, it was reported that integrinmediated slow rolling on E-selectin and ICAM-1 is eliminated in neutrophils lacking PSGL-1 but only marginally impaired in ne...

SummaryDuring acute inflammation, P-selectin is transiently mobilized from Weibel-Palade bodies to the surface of histamine-activated endothelial cells, where it mediates rolling adhesion of neutrophils under hydrodynamic flow. During chronic or allergic inflammation, sustained expression of P-selectin on the endothelial cell surface has been observed. We found that the cytokines interleukin 4 (IL-4) or oncostatin M (OSM) induced a five-to ninefold increase in P-selectin messenger RNA (mlLNA) in human umbilical vein endothelial cells (HUVEC) that persisted as long as 72 h. IL-4 elevated P-selectin mRNA by increasing its transcription rate rather than by prolonging its already long half-life. Stimulation of P-selectin transcription by IL-4 or OSM required new protein synthesis and tyrosine phosphorylation of cellular proteins. Tumor necrosis factor 0r IL-113, lipopolysaccharide, or IL-3 did not increase P-selectin mlLNA in HU-VEC, and did not augment the IL-4-induced increase in P-selectin transcripts. IL-4 or OSM increased P-selectin protein on the cell surface as well as in Weibel-Palade bodies. Under flow conditions, neutrophils rolled on P-selectin expressed by IL-4-treated H UVEC, and even more neutrophils rolled on P-selectin after IL-4-treated HUVEC were stimulated with histamine. These data demonstrate that IL-4 or OSM stimulates endothelial cells to synthesize more P-selectin over prolonged periods. The increased expression of P-selectin may facilitate the emigration of leukocytes into sites of chronic or allergic inflammation.

Fibrosis is a common disease process in which profibrotic cells disturb organ function by secreting disorganized extracellular matrix (ECM). Adipose tissue fibrosis occurs during obesity and is associated with metabolic dysfunction, but how profibrotic cells originate is still being elucidated. Here, we use a developmental model to investigate perivascular cells in white adipose tissue (WAT) and their potential to cause organ fibrosis. We show that a Nestin-Cre transgene targets perivascular cells (adventitial cells and pericyte-like cells) in WAT, and Nestin-GFP specifically labels pericyte-like cells. Activation of PDGFRα signaling in perivascular cells causes them to transition into ECM-synthesizing profibrotic cells. Before this transition occurs, PDGFRα signaling up-regulates mTOR signaling and ribosome biogenesis pathways and perturbs the expression of a network of epigenetically imprinted genes that have been implicated in cell growth and tissue homeostasis. Isolated Nestin-GFP+ cells differentiate into adipocytes ex vivo and form WAT when transplanted into recipient mice. However, PDGFRα signaling opposes adipogenesis and generates profibrotic cells instead, which leads to fibrotic WAT in transplant experiments. These results identify perivascular cells as fibro/adipogenic progenitors in WAT and show that PDGFRα targets progenitor cell plasticity as a profibrotic mechanism.

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P-and L-selectin but not thrombin. Heparin prevents mucin binding to P-and L-selectin and mucininduced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P-and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet Pselectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. IntroductionTrousseau syndrome is classically defined as a thrombotic event preceding or appearing concomitantly with a visceral malignancy. 1,2 The hallmarks are venous and arterial microthrombi with secondary microangiopathic hemolytic anemia. Notwithstanding its classic definition, Trousseau syndrome is sometimes used to describe any thrombotic complication associated with cancer. 2 Early studies of Trousseau syndrome emphasized the activation of fluid-phase coagulation. A cysteine protease in carcinoma extracts was reported to directly activate factor X. 3 Many tumors express tissue factor, 4 and tissue factor-bearing microparticles derived from platelets 5 or tumor cells 6,7 have been observed. However, heparin prevents recurrent thrombosis much more effectively than vitamin K antagonists such as warfarin. 1,[8][9][10][11][12][13] Although heparin might block tissue factor-triggered coagulation more effectively than warfarin, its clinical superiority implies that other mechanisms also contribute to Trousseau syndrome. The association of Trousseau syndrome with mucinous adenocarcinomas suggests a pathogenic role for tumor-secreted mucins. 1 Carcinoma cells express higher levels of mucins, 14 and elevated levels of these mucins and their fragments circulate in patients. 15,16 Mucins bearing highly sialylated Oglycans selectively resist clearance and circulate for long periods. 17 During inflammation or injury, interactions of selectins with their glycosylated ligands initiate adhesion of leukocytes to activated platelets and/or endothelial cells. 18 L-selectin is expressed on leukocytes. Thrombin and other agonists cause plat...

Basal and inducible expression of human P-selectin in transgenic mice differs from that of murine P-selectin, resulting in distinct functions.

In vitro studies suggest that bone marrow endothelial cells contribute to multilineage hematopoiesis, but this function has not been studied in vivo. We used a Cre/loxP-mediated recombination to produce mice that lacked the cytokine receptor subunit gp130 in hematopoietic and endothelial cells. Although normal at birth, the mice developed bone marrow dysfunction that was accompanied by splenomegaly caused by extramedullary hema- IntroductionThe interleukin-6 (IL-6) family of cytokines includes IL-6, IL-11, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotropin-1. All these cytokines bind to receptors that share the signaling subunit gp130. 1,2 Because many cells express these receptors, gp130-dependent signaling is thought to have numerous important functions. Indeed, mice lacking gp130 in all cells die of diverse morphologic abnormalities during development, 3 and postnatally induced deletion of gp130 in all cells causes defects in many systems. 4 Because gp130 is so widely expressed, clarifying gp130-dependent functions requires targeted deletion of gp130 in specific cell types. For example, myocyte-restricted deletion of gp130 in mice causes apoptosis that results in heart failure. 5 Global deletion of gp130 decreases hematopoietic progenitors in fetal liver, 3 and postnatally induced deletion of gp130 causes hematopoietic defects. 4 It is unknown whether these abnormalities result from loss of gp130 signaling in hematopoietic cells, other cells in the microenvironment, or both. Cultured hematopoietic cells respond directly to IL-6-related cytokines. [6][7][8][9][10] However, endothelial cells also express gp130, 11 and there is a close developmental relationship between hematopoietic and endothelial cells that may arise from hemangioblasts or specialized hemogenic endothelial cells in early fetal life. [12][13][14][15][16] Cultured endothelial cells derived from adult bone marrow or fetal yolk sac support multilineage hematopoiesis, 12,13,17,18 but this broad function has not been demonstrated in vivo. Stimulation of megakaryocyte progenitors with some chemokines, but not with IL-6 or IL-11, causes them to adhere to bone marrow endothelial cells, which enhances thrombopoiesis in thrombopoietin-deficient mice. 19 This study provides evidence that endothelial cells contribute to hematopoiesis in one lineage, but it does not address whether IL-6 family cytokines participate in cross-talk between hematopoietic and endothelial cells during hematopoiesis.Here, we used the Cre/loxP-mediated gene recombination to selectively delete gp130 in hematopoietic and endothelial cells of mice. Although hematopoiesis was normal throughout the fetal and neonatal periods, bone marrow dysfunction developed in adult mice. Intrinsic defects in hematopoietic stem and progenitor cells were not apparent even when anemia was severe. Transplantation experiments revealed that hematopoiesis required the expression of gp130 in the bone marrow microenvironment. These data provide evidence that gp130-depend...

The escalating epidemic of obesity has increased the incidence of obesity-induced complications to historically high levels. Adipose tissue is a dynamic energy depot, which stores energy and mobilizes it during nutrient deficiency. Excess nutrient intake resulting in adipose tissue expansion triggers lipid release and aberrant adipokine, cytokine and chemokine production, and signaling that ultimately lead to adipose tissue inflammation, a hallmark of obesity. This low-grade chronic inflammation is thought to link obesity to insulin resistance and the associated comorbidities of metabolic syndrome such as dyslipidemia and hypertension, which increase risk of type 2 diabetes and cardiovascular disease. In this review, we focus on and discuss members of the chemokine system for which there is clear evidence of participation in the development of obesity and obesity-induced pathologies.

Tumor necrosis factor-␣ (TNF-␣) or lipopolysaccharide (LPS) increases expression of the P-selectin gene in murine, but not in human, endothelial cells. These mediators augment expression of a reporter gene driven by the murine, but not the human, P-selectin promoter in transfected endothelial cells. The regions from ؊593 to ؊474 and from ؊229 to ؊13 in the murine P-selectin promoter are required for TNF-␣ or LPS to stimulate reporter gene expression. Within these regions, we identified two tandem B elements, a reverse-oriented B site and a variant activating transcription factor/cAMP response element (ATF/CRE), that participate in TNF-␣-or LPS-induced expression. The tandem B elements bound to NF-B heterodimers and p65 homodimers, the reverse-oriented B site bound to p65 homodimers, and the variant ATF/CRE bound to nuclear proteins that included activating transcription factor-2. Mutations in each individual element eliminated binding to nuclear proteins and decreased by 20 -60% the TNF-␣-or LPSinduced expression of a reporter gene driven by the murine P-selectin promoter in transfected endothelial cells. Simultaneous mutations of all elements further decreased, but did not abolish, induced expression. Cooverexpression of p50 and p65 enhanced murine P-selectin promoter activity in a B site-dependent manner. These data indicate that the B sites and the variant ATF/CRE are required for TNF-␣ or LPS to optimally induce expression of the murine P-selectin gene. The presence of these elements in the murine, but not the human, P-selectin gene may explain in part why TNF-␣ or LPS stimulates transcription of P-selectin in a species-specific manner.