During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site. IntroductionChemokine-induced recruitment of circulating leukocytes is fundamental in the immune response to bacterial infections. The leukocyte recruitment cascade is initiated by endothelial cell activation and presentation of chemokines to rolling leukocytes, which, by activating leukocyte integrins, results in leukocyte adhesion to and diapedesis through the vessel wall. [1][2][3] Recently, an additional step in the leukocyte recruitment cascade was detected bridging adhesion and diapedesis, namely, Mac-1-mediated intraluminal crawling. [4][5][6][7] In these studies, neutrophils were observed to crawl on endothelium in all directions before transmigration through endothelial junctions; and if crawling were disabled, diapedesis was delayed and occurred preferentially through the transcellular pathway. 4,5 However, chemokine presence on endothelium is not enough to initiate leukocyte diapedesis; a chemotactic gradient over the vessel wall with a higher extravascular concentration is required. 8 Outside the vasculature, leukocytes chemotax along a chemical gradient in the extracellular matrix toward the chemokine source. 9,10 During infection, a multitude of chemotactic factors are present simultaneously, and the ability of leukocytes to prioritize between end-target (bacterial peptides) and intermediate (eg, macrophage inflammatory protein-2 [MIP-2]) chemotactic cues is crucial for leukocytes to find the site of in...
Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer’s disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood–brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-β peptide, implicated in Alzheimer’s disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-β elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood–brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer’s disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-0997-1) contains supplementary material, which is available to authorized users.
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