Objective-Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1␣ (Ccl3/MIP-1␣) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Methods and Results-Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1␣ induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (Ͼ70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes Key Words: leukocyte Ⅲ migration Ⅲ chemokines Ⅲ permeability Ⅲ basement membrane L eukocyte recruitment from the microvasculature to sites of inflammation is a key event in both innate and adaptive immunity. In this process, a diversity of adhesion molecules, proteases, and chemokines are involved regulating the sequential steps of leukocyte rolling, firm adherence, and transmigration. 1,2 Chemokines are small molecules (8 to 14 kDa) which can be classified into C, CC, CXC, and CX 3 C chemokines according to the arrangement of their N-terminal cysteine residues. Increased levels of chemokines and their respective receptors have been found in numerous pathological conditions. According to the current paradigm, chemokine receptors on circulating leukocytes are supposed to interact with chemokines presented on the venular endothelium. These interactions immediately activate leukocyte integrins which, in turn, facilitate firm adherence and transmigration of leukocytes. [3][4][5] In the past years, particularly CC chemokines have been extensively studied in various inflammatory pathologies. Concluding from these studies, CC chemokines such as monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1␣ (Ccl3/MIP-1␣) have been suggested to exclusively mediate the migration of monocytes and lymphocytes. [3][4][5] However, there is a growing body of evidence that Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ are also critically involved in the recruitment of neutrophils. 6,7 The underlying mechanisms, however, remain largely unclear.Recently, it has been reported that both Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ are able to induce the release of lipid mediators such as leukotriene-B 4 (LTB 4 ). 8 -10 The functional relevance of endogenously generated lipid mediators including prostaglandins, leukotrienes, and PAF for each single step of the recruitment process of neutrophils elicited by Ccl2/JE/ In addition to leukocyte migration, Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ have been implicated in the control of microvascular permeability. 11,12 Moreover, Ccl3/MIP-1␣ has recently been demonstrated to induce remodeling of the perivascular basement membrane, a process which might promote microvascular leakage during inflammatory conditions. 11 The contribution of neutrophils to these events, however, has not yet been studied.Therefore, the objective of the present study...

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Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.

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Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury.

Background-Urokinase-type plasminogen activator (uPA) has recently been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury. The underlying mechanisms remain largely unclear. Methods and Results-Using in vivo microscopy on the mouse cremaster muscle, I/R-elicited firm adherence and transmigration of neutrophils were found to be significantly diminished in uPA-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)-deficient mice. Interestingly, postischemic leukocyte responses were significantly reduced on blockade of the integrin CD11b/Mac-1, which also serves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a selectively reduced transmigration in wild-type animals. On I/R or stimulation with recombinant uPA, Ͼ90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence microscopy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. Conclusions-Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the subsequent paracellular transmigration of neutrophils during I/R. This process is regulated via CD11b/Mac-1, and does not require uPAR. Pharmacological blockade of uPA interferes with these events and effectively attenuates postischemic tissue injury. (Circulation. 2011;124:1848-1859.)Key Words: ischemia Ⅲ leukocytes Ⅲ plasminogen activators Ⅲ reperfusion Ⅲ urokinase I schemia-reperfusion (I/R) injury is considered to be the most common cause of organ dysfunction and failure after myocardial infarction, hemorrhagic shock, and transplantation. Leukocyte infiltration of postischemic tissue is a key event in the pathogenesis of I/R injury. In this multistep cascade, a diversity of adhesion molecules, chemoattractants, and proteases are involved, regulating intravascular rolling and firm adherence as well as transendothelial migration of leukocytes to the reperfused tissue. [1][2][3][4] Clinical Perspective on p 1859Urokinase-type plasminogen activator (uPA) is a serine protease that has been implicated in a variety of physiological and pathophysiological processes. In this context, uPA is known to activate extracellular matrix-degrading enzymes and, through interaction with the urokinase receptor (uPAR; CD87), uPA is thought to induce intracellular signaling pathways, ultimately regulating cell adhesion and migration. 5 Moreover, uPA mediates the conversion of plasminogen to plasmin, which, in addition to its fibrinolytic properties, is also able to degrade components of the ECM as well as to activate intracellular signaling mechanisms. 6 Plasminogen activators, such as recombinant uPA, are therapeutically used for the activation of the fibrinolytic system during thrombembolic events. 7 Interestingl...

Injury to parathyroid glands during thyroid and parathyroid surgery is common and postoperative hypoparathyroidism represents a serious complication. Parathyroid glands possess a unique autofluorescence in the near-infrared spectrum which could be used for their identification and protection at an early stage of the operation. In the present study parathyroid autofluorescence was visualized intraoperatively using a standard Storz laparoscopic near-infrared/indocyanine green (NIR/ICG) imaging system with minor modifications to the xenon light source (filtered to emit 690 nm to 790 nm light, less than 1% in the red and green above 470 nm and no blue light). During exposure to NIR light parathyroid tissue was expected to show autofluorescence at 820 nm, captured in the blue channel of the camera. Over a period of 5 years, we investigated 205 parathyroid glands from 117 patients. 179 (87.3%) glands were correctly identified by their autofluorescence. Surrounding structures such as thyroid, lymph nodes, muscle, or adipose tissue did not reveal substantial autofluorescence. We conclude that parathyroid glands can be identified by their unique autofluorescence at an early stage of the operation. This may help to preserve these fragile structures and their vascularization and lower the rate of postoperative hypocalcemia.

Key Points The density of the interstitial collagen network increases in inflamed tissue, providing physical guidance to infiltrating neutrophils. Neutrophil interstitial migration does not require the pericellular degradation of collagen fibers, but it is modulated by MMPs.