A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils

Abstract

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...

Glioblastoma-synthesized G-CSF and GM-CSF contribute to growth and immunosuppression: Potential therapeutic benefit from dapsone, fenofibrate, and ribavirin

“…Neutrophils move along an IL-8 gradient. 94,95 IL-8 also activates neutrophils, increasing surface expression and binding activity of MAC1 (a complement receptor). 96,97 In patients with dermatitis herpetiformis, serum IL-8 is elevated following the immune response to dietary gluten in the small bowel.…”

Intravascular Leukocyte Chemotaxis: The Rules of Attraction

“…Indeed, this chemotactic gradient is sequestered on endothelial HS and provides directional cues to crawling neutrophils, which follow this gradient to optimal transmigration sites close to the origin of the infection (Massena S. et al, 2010). However, whether haptotactic gradients can be established by all chemokines remains unclear, since different chemokines have diverse affinity to HS (Lindhal U., Kjellén L., 1991;Lindhal U., 2007).…”

“…It is generally believed that increased permeability supports chemokine influx into the vessels and one hypothesis is that the increase in vascular permeability during inflammation grants the paracellular transport of chemokines for rapid presentation to intravascular leukocytes, guiding them out to the afflicted area in the tissue. A recent study using intravital spinning-disk confocal microscopy in anesthetized mice revealed that chemokines added extravascularly became accumulated intra-luminally at endothelial cell junctions (Massena S. et al, 2010). High junctional sequestration of chemokines suggests that chemokines are transported either paracellularly into blood vessels or longitudinally on the apical endothelial cell membrane towards junctions after being transcytosed.…”

“…Chemokines and a variety of positively charged proteins bind to HS through specific and/or electrostatic interactions (Lindhal U., Kjellén L., 1991;Lindhal U., 2007). Indeed, interstitially released chemokines were shown to cross the endothelium and to be presented by luminal HSPGs to leukocytes both in vitro (Ihrcke N. S. et al, 1993;Parish C., 2005;Wang L. et al, 2005;Lindhal U., 2007) and in vivo (Massena S. et al, 2010). Binding of chemokines to endothelial HSPG may promote molecular encounters between rolling leukocytes and chemokines, and thereby further inducing leukocyte activation.…”

“…More recently, the existence of an intravascular gradient of chemokines (macrophage inflammatory protein-2, CXCL2 [MIP-2]; keratinocyte-derived chemokine, CXCL1 [KC]) originating from the infection or released by tissue leukocytes on endothelial cells has been described (Massena S. et al, 2010). Indeed, this chemotactic gradient is sequestered on endothelial HS and provides directional cues to crawling neutrophils, which follow this gradient to optimal transmigration sites close to the origin of the infection (Massena S. et al, 2010).…”