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Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-1 (PAR1) can disrupt endothelial barrier integrity, whereas the bioactive lipid sphingosine 1-phosphate (

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immu-

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western lifestyle. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn's disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD.

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Attraction of neutrophils to sites of infection or tissue injury is an essential prerequisite for an efficient innate immune response. Herein, we provide novel evidence that the antimicrobial protein, neutrophil gelatinase associated lipocalin (24p3 or lipocalin-2, Lcn2) is a central regulator of this process. Lcn2 is produced by several cell types but high amounts are released by neutrophils. Using human and murine neutrophils, we found that the addition of recombinant Lcn2 significantly stimulated their migration, which was independent of IL-8/keratinocyte chemokine formation. Mechanistically, this could be traced back to Lcn2-mediated changes of Erk1/2 signaling. Accordingly, the i.p. injection of Lcn2 into C57BL/6 mice stimulated the mobilization of neutrophils while we found a significantly reduced neutrophil chemotactic activity of cells obtained from Lcn2 KO mice. This observation transmitted to a reduced accumulation of neutrophils in intradermal lesions infected with Salmonella typhimurium in Lcn2 KO mice as compared to WT mice. This was not only due to a reduced chemotaxis but also to an impaired cellular adhesion of neutrophils in the absence of Lcn2. We herein describe a novel role of Lcn2 as an important paracrine chemoattractant and an indispensable factor for neutrophil function in inflammation.Keywords: Chemotaxis r Lipocalin-2 (Lcn2) r polymorphonuclear neutrophils (PMNs) Supporting Information available online IntroductionTissue infiltration of leukocytes in response to inflammatory or infectious stimuli warrants previous adhesion of leukocytes to endothelial cells and subsequent migration across subendothelial basement membranes. Following cellular damage, epithelial cells produce IL-8 or its murine ortholog keratinocyte chemokine (KC), respectively, which in turn attracts neutrophil granulocytes (PMNs, polymorphonuclear neutrophils) to cross the epitheCorrespondence: Prof. Guenter Weiss e-mail: guenter.weiss@i-med.ac.at lial barrier to the affected site [1]. As part of their antiinfective armory, PMNs produce and release several antimicrobial peptides and proteolytic enzymes. One of these peptides is the 21 kDa protein neutrophil gelatinase associated lipocalin also called lipocalin-2 (Lcn2) [2]. Lcn2 is stored in so-called secondary granules together with lactoferrin, calprotectin (S100A8/A9), or Mac-1 (CD11b/CD18), which play essential roles for neutrophil effector functions and migration [3,4]. * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 3346-3357 Innate immunity 3347Lipocalins are a family of structurally related proteins characterized by eight β-strands that form a β-barrel defining a calyx [3,5]. Lcn2 is expressed and secreted by immune cells, hepatocytes and renal tubular cells, in which it is involved in different biological functions [3,[6][7][8][9][10][11].On the one hand Lcn2 acts as an antimicrobial protein, and this function is based on its ability to capture and deplete bacter...

Summary.  Coagulation and inflammation are intimately linked and cellular signaling by coagulation proteases through protease‐activated receptors (PARs) may affect pro‐ and anti‐inflammatory responses. Permeability of the endothelial cell barrier at the blood–tissue interface plays a key role in inflammatory disorders such as sepsis. We have recently shown that PAR1 signaling by activated protein C or low concentrations of thrombin can enhance endothelial barrier integrity. In the present study, we analyzed effects of coagulation factor Xa (FXa), which is known to activate both endothelial cell PAR1 and PAR2, on monolayer integrity using a transformed human umbilical vein endothelial cell (HUVEC) line in a dual‐chamber system. Preincubation with FXa potently reduced high‐dose thrombin‐mediated hyperpermeability and basal permeability. FXa was protective at concentrations of 5 nm or higher and proteolytic activity was required. Barrier protective FXa signaling was not affected by cleavage‐blocking anti‐PAR1 antibodies or by a PAR1 antagonist. Similarly, cleavage‐blocking anti‐PAR2 alone had no effect, but blocking both PAR1 and PAR2 inhibited barrier protection by FXa. Incubation of the cell layer with a PAR2‐specific agonist peptide reduced thrombin‐mediated hyperpermeability and basal permeability similar to FXa. In conclusion, not only PAR1, but also PAR2 can mediate barrier protection in endothelial cells and FXa can use either receptor to enhance barrier integrity. Although it is currently unknown whether PAR signaling by FXa has a physiological role, the results suggest a potential protective effect of FXa and other agonists of endothelial PAR2, which should be explored in models of local and systemic inflammation in vivo.

IntroductionRecombinant human activated protein C (APC) has powerful protective effects in systemic inflammation that led to its approval to treat patients with severe sepsis. 1 Protein C (PC) is physiologically activated on the endothelial cell surface by the key procoagulant enzyme thrombin and APC down-regulates thrombin formation in a negative feedback loop. 2 However, this anticoagulant effect of APC is unlikely to explain its benefit in systemic inflammation because other anticoagulants with comparable effect do not improve survival in septic patients 3,4 or in animal models. 5 More recently, APC signaling through protease activated receptor-1 (PAR1) emerged as an alternative mechanism for APC's beneficial effects. 6,7 In cultured endothelial cells PAR1 mediates protective effects of APC on gene expression, 6,8 survival, 9,10 and barrier integrity. 11 PAR1 has also been implicated in mouse models analyzing neuroprotective effects of APC in vivo. 9,12,13 PAR1 is a 7-transmembrane G-protein-coupled receptor that is enzymatically cleaved after Arg41 to expose a new extracellular N-terminus that acts as a tethered activating ligand. 14 PAR1 is the prototypical thrombin receptor and thrombin cleaves and activates PAR1 with high efficiency because it directly binds to PAR1 in an orientation that favors cleavage. 15 In contrast, APC needs to be recruited to a coreceptor, the endothelial protein C receptor (EPCR), to induce PAR1-dependent signaling 6 but APC remains less efficient than thrombin. 16 Systemic inflammation leads to generation of thrombin and in view of the relatively low efficiency of PAR1 activation by APC it has been argued that a role of APC-PAR1 signaling in sepsis is unlikely. 16,17 Here we demonstrate that APC can mediate significant PAR1 cleavage even in the presence of thrombin. In contrast to thrombincleaved PAR1, the APC-cleaved PAR1 accumulates on the endothelial cell surface. The findings support the concept that PAR1 can mediate protective APC signaling in conditions where thrombin is also present. Methods Reagents and assaysHuman thrombin was as described. 6,18 Human plasma-derived APC and PC were from Haematologic Technologies (Essex Junction, VT). Recombinant human wild-type APC and mutant APC S360A were a gift from Dr John Griffin (Scripps Research Institute, La Jolla, CA). 19 All experiments involving stimulation with APC included hirudin (Calbiochem, La Jolla, CA) unless indicated otherwise or if cells were coincubated with APC and thrombin. Control experiments demonstrated that hirudin alone had no effect in any of our assays. Brefeldin A was from Calbiochem. Monoclonal anti-PAR1 ATAP2, WEDE15, and SPAN11 were as described. 20,21 Monoclonal rat anti-EPCR RCR-92 (nonblocking) and RCR-252 (blocking) were provided by Dr Kenji Fukudome (Saga Medical School, Saga, Japan) and were used at 25 g/mL. 22 Amidolytic assays for APC activity were as described previously. 23Cell culture, permeability assay, and surface immunoassays EA.hy926 cells 24 and primary human umbilical vein endothelial ...