Artikel
Matrix metalloproteinases modulate ameboid-like migration of neutrophils through inflamed interstitial tissue
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Veröffentlicht: | 24. April 2015 |
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Gliederung
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Introduction: Recruitment of leukocytes to the site of inflammation is a crucial step in the development of inflammatory processes. The single steps of the leukocyte recruitment process, described as rolling, firm adherence and transendothelial migration have been studied in detail in the past decades, identifying a number of leukocyte- as well as endothelial-bound members of the immunoglobulin-superfamily to be involved. In contrast to that, the mechanisms underlying interstitial migration of leukocytes remained largely unclear. According to the current paradigm, leukocytes are suggested to locomote in an ameboid fashion being largely independent of pericellular proteolysis. However, our knowledge about interstitial migration of leukocytes largely relies on in vitro data which do not respect the complex architecture of the interstitial tissue as well as the dramatic phenotypic and functional changes leukocytes undergo during their transmigration. In particular, the role of proteases- especially serine proteases and matrix metalloproteinases for interstitial migration of leukocytes is poorly understood.
Material and methods: M. cremaster assay: Leukocyte recruitment was analyzed in the cremaster muscle of anesthetized C57BL/6 mice using in vivo microscopy. Interstitial migration of leukocytes was induced via visually controlled semiautomatic microinjection of CCL3 or PAF.
Peritonitis assay: Leukocyte recruitment to the peritoneal cavity was induced via intraperitoneal injection of CCL3 or PAF.
Phenotyping of transmigrated leukocytes was performed by immunohistochemistry. Remodeling processes within the perivenular basement membrane were analyzed by confocal laser scanning microscopy. Interstitial collagen expression was visualized using multiphoton in vivo microscopy. Surface expression of proteases on neutrophils upon transmigration was determined via flow cytometry.
Results: RLOT microscopy on the inflamed mouse cremaster muscle revealed that intravascular accumulation and transmigration of neutrophils was significantly reduced after blockade of serine proteases or of matrix metalloproteinases (MMPs). A novel in vivo chemotaxis assay showed that directional interstitial migration of neutrophils was induced via perivenular microinjection of inflammatory mediators. Blockade of actin polymerization almost completely abolished neutrophil interstitial migration directionality, velocity and distance. Multi-photon laser scanning in vivo microscopy demonstrated that the density of the interstitial collagen network increases in inflamed tissue thereby providing physical guidance to infiltrating neutrophils. Despite neutrophils locomoting through the interstitial tissue without pericellular collagen degradation, inhibition of MMPs, but not of serine proteases, significantly reduced their polarization and interstitial locomotion. In this context, blockade of MMPs was found to modulate expression of adhesion/signaling molecules on neutrophils.
Conclusion: In conclusion, our data indicate that Serine proteases are critically involved in extravasation of neutrophils, whilst these proteolytic enzymes are dispensable for the subsequent extravascular locomotion of these inflamatory cells. Interstitial migration of transmigrated neutrophils strictly relies on actin polymerisation and does not require the pericellular degradation of the extracellular matrix, though it is influenced by the activity of MMPs.
Figure 1 [Fig. 1]