Visualization of leukocyte transendothelial and interstitial migration using reflected light oblique transillumination in intravital video microscopy

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration

Intravascular Immune Surveillance by CXCR6(+) NKT Cells Patrolling Liver Sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance

Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator

Dissemination of the apicomplexan parasite, Toxoplasma gondii

The parasitic protist Toxoplasma gondii is a common pathogen of rodents and felines that also infects humans. The most severe clinical manifestations of toxoplasmosis in humans derive from the systemic dissemination of T. gondii, during which the parasite penetrates biological barriers and accesses protected host compartments such as the central nervous system. T. gondii dissemination is enabled by the intrinsic gliding motility of extracellular parasites, which allows for travel to new host cells and tissues, and also powers the invasion of diverse host cells including migratory leukocytes. Dissemination is further advanced when migrating infected leukocytes shuttle intracellular parasites to new locations as they traffic throughout the host. All T. gondii gliding motility and host cell invasion was long presumed to be powered by the work of a parasite actin-myosin motor. The possibility of alternative gliding and invasion mechanisms was suggested by the development of inducible Cre-Lox technology that facilitated inducible disruption of genes thought to encode critical components of the T. gondii invasion machinery, including the parasite actin gene ACT1. To determine whether ACT1-independent invasion was likely, inducible Δact1 parasites were examined for uniformity of ACT1 protein depletion. Individual parasites with residual ACT1 protein persisted long after inducible ACT1 excision. Suggesting the residual ACT1 content of these parasites was functionally relevant, the invasion of Δact1 parasites was highly sensitive to an actin polymerization inhibitor. Parasite invasive ability was also found to negatively correlate with the length of time parasites were subjected to ACT1 depletion. Although the existence of ACT1-independent invasion mechanisms cannot be formally excluded, they do not appear to comprise robust alternatives to actin-dependent gliding and invasion in T. gondii. As the most abundantly infected circulating leukocyte during murine toxoplasmosis, monocytes have been theorized to be poised to deliver intracellular T. gondii across the blood-brain barrier and into the central nervous system. However, in vivo evidence supporting this theory was scarce. In vitro models had demonstrated that infection could alter the motility of monocytes when interacting with endothelial vasculature. However, whether infected monocytes could efficiently traverse the specialized endothelium that comprises the blood-brain barrier had not been tested, nor had the ability of infected monocytes to migrate through the tissue environments where T. gondii is first encountered. Models of peripheral and blood-brain barrier endothelium were used to show that infection markedly inhibited monocyte transendothelial migration. In contrast, infected monocytes and macrophages migrated through three-dimensional matrices in vitro and collagen-rich tissues in vivo with enhanced efficiency. Enhanced tissue migration relied on host Rho/ROCK and formin signaling, and the secreted T. gondii kinase ROP17. In a murine model, infection with Δrop17 parasites that fail to enhance tissue migration resulted in delayed dissemination and prolonged mouse survival. These results implicate monocytes in advancing the tissue spread of T. gondii during in vivo dissemination

Endothelial C-type natriuretic peptide maintains vascular homeostasis

PMCID: PMC4151218Wellcome Trust (084449/Z/07/Z and 078496/Z/05/Z

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features

Plasminogen activator-coated nanobubbles targeting cell-bound β2-glycoprotein I as a novel thrombus-specific thrombolytic strategy

Beta2-glycoprotein I (β2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with anti-phospholipid syndrome (APS). β2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NBs) coated with recombinant tissue plasminogen activator (rtPA) and recombinant antibody specific for cell-bound β2-GPI. The therapeutic efficacy of targeted nanobubbles was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: 1) thrombosis developed in a rat model of APS; 2) ferric chloride-induced mesenteric thrombosis in rats, and 3) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted nanobubbles bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through β2-GPI expressed on activated cells. In vitro, rtPA-targeted NBs (rtPA-tNBs) induced greater lysis of platelet-rich blood clots than untargeted NBs. In a rat model of APS, administration of rtPA-tNBs caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNBs, but not untargeted NBs and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNBs, that target β2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NBs. Our findings suggest that targeting cell-bound β2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APSunrelated thrombotic conditions

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo