Kindlin-3 recruitment to the plasma membrane precedes high-affinity ß2-integrin and neutrophil arrest from rolling.

Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the ß2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates ß2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and ß2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ ß2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity ß2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ ß2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity ß2-integrin.

Bioengineered in vitro models of leukocyte-vascular interactions.

Leukocytes continuously circulate our body through the blood and lymphatic vessels. To survey invaders or abnormalities and defend our body against them, blood-circulating leukocytes migrate from the blood vessels into the interstitial tissue space (leukocyte extravasation) and exit the interstitial tissue space through draining lymphatic vessels (leukocyte intravasation). In the process of leukocyte trafficking, leukocytes recognize and respond to multiple biophysical and biochemical cues in these vascular microenvironments to determine adequate migration and adhesion pathways. As leukocyte trafficking is an essential part of the immune system and is involved in numerous immune diseases and related immunotherapies, researchers have attempted to identify the key biophysical and biochemical factors that might be responsible for leukocyte migration, adhesion, and trafficking. Although intravital live imaging of in vivo animal models has been remarkably advanced and utilized, bioengineered in vitro models that recapitulate complicated in vivo vascular structure and microenvironments are needed to better understand leukocyte trafficking since these in vitro models better allow for spatiotemporal analyses of leukocyte behaviors, decoupling of interdependent biological factors, better controlling of experimental parameters, reproducible experiments, and quantitative cellular analyses. This review discusses bioengineered in vitro model systems that are developed to study leukocyte interactions with complex microenvironments of blood and lymphatic vessels. This review focuses on the emerging concepts and methods in generating relevant biophysical and biochemical cues. Finally, the review concludes with expert perspectives on the future research directions for investigating leukocyte and vascular biology using the in vitro models.

Selective Recruitment of Monocyte Subsets by Endothelial N-Glycans.

Monocyte rolling, adhesion, and transmigration across the endothelium are mediated by specific interactions between surface adhesion molecules. This process is fundamental to innate immunity and to inflammatory disease, including atherosclerosis, where monocyte egress into the intimal space is central to formation of fatty plaques. Monocytes are a heterogeneous population of three distinct subsets of cells, all of which play different roles in atherosclerosis progression. However, it is not well understood how interactions between different monocyte subsets and the endothelium are regulated. Furthermore, it is appreciated that endothelial adhesion molecules are heavily N-glycosylated, but beyond regulating protein trafficking to the cell surface, whether and if so how these N-glycans contribute to monocyte recruitment is not known. This review discusses how changes in endothelial N-glycosylation may impact vascular and monocytic inflammation. It will also discuss how regulating N-glycoforms on the endothelial surface may allow for the recruitment of specific monocyte subsets to sites of inflammation, and how further understanding in this area may lead to the development of glyco-specific therapeutics in the treatment of cardiovascular disease.

Critical Role of the CXCL10/C-X-C Chemokine Receptor 3 Axis in Promoting Leukocyte Recruitment and Neuronal Injury during Traumatic Optic Neuropathy Induced by Optic Nerve Crush.

Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma. Loss of retinal ganglion cells (RGCs) is a key pathological process in TON, yet mechanisms responsible for RGC death remain unclear. In a mouse model of TON, real-time noninvasive imaging revealed a dramatic increase in leukocyte rolling and adhesion in veins near the optic nerve (ON) head at 9 hours after ON injury. Although RGC dysfunction and loss were not detected at 24 hours after injury, massive leukocyte infiltration was observed in the superficial retina. These cells were identified as T cells, microglia/monocytes, and neutrophils but not B cells. CXCL10 is a chemokine that recruits leukocytes after binding to its receptor C-X-C chemokine receptor (CXCR) 3. The levels of CXCL10 and CXCR3 were markedly elevated in TON, and up-regulation of CXCL10 was mediated by STAT1/3. Deleting CXCR3 in leukocytes significantly reduced leukocyte recruitment, and prevented RGC death at 7 days after ON injury. Treatment with CXCR3 antagonist attenuated TON-induced RGC dysfunction and cell loss. In vitro co-culture of primary RGCs with leukocytes resulted in increased RGC apoptosis, which was exaggerated in the presence of CXCL10. These results indicate that leukocyte recruitment in retinal vessels near the ON head is an early event in TON and the CXCL10/CXCR3 axis has a critical role in recruiting leukocytes and inducing RGC death.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Cell adhesion during bullet motion in capillaries.

A numerical analysis is presented of cell adhesion in capillaries whose diameter is comparable to or smaller than that of the cell. In contrast to a large number of previous efforts on leukocyte and tumor cell rolling, much is still unknown about cell motion in capillaries. The solid and fluid mechanics of a cell in flow was coupled with a slip bond model of ligand-receptor interactions. When the size of a capillary was reduced, the cell always transitioned to "bullet-like" motion, with a consequent decrease in the velocity of the cell. A state diagram was obtained for various values of capillary diameter and receptor density. We found that bullet motion enables firm adhesion of a cell to the capillary wall even for a weak ligand-receptor binding. We also quantified effects of various parameters, including the dissociation rate constant, the spring constant, and the reactive compliance on the characteristics of cell motion. Our results suggest that even under the interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin, which is mainly responsible for leukocyte rolling, a cell is able to show firm adhesion in a small capillary. These findings may help in understanding such phenomena as leukocyte plugging and cancer metastasis.

Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest: short communication.

RATIONALE: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. OBJECTIVE: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. METHODS AND RESULTS: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4(-/-) mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe(-/-) mice on high-fat diet. CONCLUSIONS: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice.

Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered ß2 integrin-dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti-P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.

Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells.

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.

Endoscopic time-lapse imaging of immune cells in infarcted mouse hearts.

RATIONALE: High-resolution imaging of the heart in vivo is challenging owing to the difficulty in accessing the heart and the tissue motion caused by the heartbeat. OBJECTIVE: Here, we describe a suction-assisted endoscope for visualizing fluorescently labeled cells and vessels in the beating heart tissue through a small incision made in the intercostal space. METHODS AND RESULTS: A suction tube with a diameter of 2 to 3 mm stabilizes the local tissue motion safely and effectively at a suction pressure of 50 mm Hg. Using a minimally invasive endoscope integrated into a confocal microscope, we performed fluorescence cellular imaging in both normal and diseased hearts in live mice for an hour per session repeatedly over a few weeks. Real-time imaging revealed the surprisingly rapid infiltration of CX3CR1(+) monocytes into the injured site within several minutes after acute myocardial infarction. CONCLUSIONS: The time-lapse analysis of flowing and rolling (patrolling) monocytes in the heart and the peripheral circulation provides evidence that the massively recruited monocytes come first from the vascular reservoir and later from the spleen. The imaging method requires minimal surgical preparation and can be implemented into standard intravital microscopes. Our results demonstrate the applicability of our imaging method for a wide range of cardiovascular research.

A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis.

BACKGROUND: Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. METHODS AND RESULTS: Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR(-/-) mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE(-/-) mice. CONCLUSIONS: Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

Involvement of SSAO/VAP-1 in oxygen-glucose deprivation-mediated damage using the endothelial hSSAO/VAP-1-expressing cells as experimental model of cerebral ischemia.

BACKGROUND: In the acute phase of ischemic stroke, endothelial cells are activated and induce the expression of adhesion molecules. Vascular adhesion protein 1 (VAP-1) is a proinflammatory protein that mediates leukocyte recruitment through its semicarbazide-sensitive amine oxidase (SSAO) activity (EC 1.4.3.21). Plasmatic SSAO activity predicts the appearance of parenchymal hemorrhages after tissue plasminogen activator treatment in ischemic stroke patients, and it is increased as well in hemorrhagic stroke patients. The aim of this study has been to elucidate the role of SSAO/VAP-1 present in endothelial cells during ischemic stroke conditions. METHODS: Based on the use of endothelial cells expressing, or not expressing, the human SSAO/VAP-1 protein, we have set up an easy ischemic model using oxygen-glucose deprivation (OGD) as an experimental approach to the stroke process. Different OGD and reoxygenation conditions have been analyzed. Western blotting has been used to analyze the activated apoptotic pathways. Several metalloproteinase inhibitors were also used to determine their role in the SSAO/VAP-1 release from the membrane of endothelial cells to the culture media, as a soluble form. Adhesion assays were also performed in order to assess the SSAO/VAP-1-dependent leukocyte adhesion to the endothelia under different OGD and reoxygenation conditions. RESULTS: Our results show that SSAO/VAP-1 expression increases the susceptibility of endothelial cells to OGD, and that its enzymatic activity, through specific substrate oxidation, increases vascular cell damage under these experimental conditions. Caspase-3 and caspase-8 are activated during the death process. In addition, OGD constitutes a stimulus for soluble SSAO/VAP-1 release, partly mediated by metalloproteinase-2-dependent shedding. Short-time OGD induces SSAO/VAP-1-dependent leukocyte binding on endothelial cells, which is partly dependent on its enzymatic activity. CONCLUSIONS: Our results show that SSAO/VAP-1 could participate in some of the processes occurring during stroke. Its expression in endothelial cells increases the OGD-associated cell damage. SSAO/VAP-1 mediates also part of the tissue damage during the reoxygenation process by oxidizing its known enzymatic substrate, methylamine. Also, OGD constitutes a stimulus for its soluble-form release, found elevated in many pathological conditions including stroke. OGD induces SSAO-dependent leukocyte-binding activity, which may have consequences in disease progression, since leukocyte infiltration has shown a determinant role in cerebral ischemia. For all the stroke-related processes in which SSAO/VAP-1 participates, it would be an interesting therapeutic target. Therefore, this model will be a very useful tool for the screening of new molecules as therapeutic agents for cerebral ischemia.

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLß2.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)ß(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)ß(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)ß(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)ß(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal ß(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the ß(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)ß(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)ß(2)-mediated arrest.

Ezrin-radixin-moesin-binding sequence of PSGL-1 glycoprotein regulates leukocyte rolling on selectins and activation of extracellular signal-regulated kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)ß(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)ß(2)-dependent leukocyte slow rolling.

αB-crystallin/HspB5 regulates endothelial-leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin.

αB-crystallin is a small heat shock protein, which has pro-angiogenic properties by increasing survival of endothelial cells and secretion of vascular endothelial growth factor A. Here we demonstrate an additional role of αB-crystallin in regulating vascular function, through enhancing tumor necrosis factor α (TNF-α) induced expression of endothelial adhesion molecules involved in leukocyte recruitment. Ectopic expression of αB-crystallin in endothelial cells increases the level of E-selectin expression in response to TNF-α, and enhances leukocyte-endothelial interaction in vitro. Conversely, TNF-α-induced expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin is markedly inhibited in endothelial cells isolated from αB-crystallin-deficient mice. This is associated with elevated levels of IκB in αB-crystallin deficient cells and incomplete degradation upon TNF-α stimulation. Consistent with this, endothelial adhesion molecule expression is reduced in inflamed vessels of αB-crystallin deficient mice, and leukocyte rolling velocity is increased. Our data identify αB-crystallin as a new regulator of leukocyte recruitment, by enhancing pro-inflammatory nuclear factor κ B-signaling and endothelial adhesion molecule expression during endothelial activation.

Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling.

We introduce quantitative dynamic footprinting microscopy to resolve neutrophil rolling on P-selectin. We observed that the footprint of a rolling neutrophil was fourfold larger than previously thought, and that P-selectin-PSGL-1 bonds were relaxed at the leading edge of the rolling cell, compressed under the cell center, and stretched at the trailing edge. Each rolling neutrophil formed three to four long tethers that extended up to 16 µm behind the rolling cell.

Rapid histamine-induced neutrophil recruitment is sphingosine kinase-1 dependent.

Leukocyte recruitment to sites of inflammation is critical for the development of acute allergic responses. Rapid P-selectin up-regulation by endothelial cells is a key promoter of leukocyte infiltration in response to mediators such as histamine. However, the mechanisms underpinning this process are still incompletely understood. We examined the role of the sphingosine kinase/sphingosine-1-phosphate (SK/S1P) pathway and showed that in human umbilical vein endothelial cells, histamine rapidly activates SK in an extracellular signal-regulated kinase (ERK) 1/2-dependent manner, concurrent with the induction of P-selectin expression. Histamine activated both SK-1 and SK-2 isoforms; inhibition of SK-1, but not SK-2, attenuated histamine-induced P-selectin up-regulation and neutrophil rolling in vitro. S1P receptor antagonists failed to prevent histamine-induced P-selectin expression, and exogenous S1P did not increase P-selectin expression, suggesting that S1P cell surface receptors are not involved in this process. Finally, the role of both SK-1 and SK-2 in histamine-induced leukocyte rolling in vivo was assessed using pharmacological and genetic methods. Consistent with the in vitro findings, mice pretreated with either sphingosine kinase inhibitor or fingolimod (FTY720) significantly attenuated histamine-induced leukocyte rolling in the cremaster muscle. Similarly, Sphk1(-/-) but not Sphk2(-/-) mice exhibited reduced histamine-induced leukocyte rolling. These findings demonstrate a key role for SK-1 in histamine-induced rapid P-selectin up-regulation and associated leukocyte rolling, and suggest that endothelial SK-1 is an important contributor to allergic inflammation.

Estradiol receptors agonists induced effects in rat intestinal microcirculation during sepsis.

The steroid hormone estradiol is suggested to play a protective role in intestinal injury during systemic inflammation (sepsis). Our aim was to determine the effects of specific estradiol receptor (ER-α and ER-ß) agonists on the intestinal microcirculation during experimental sepsis. Male and sham ovariectomized female rats were subjected to sham colon ascendens stent peritonitis (CASP), and they were compared to male and ovariectomized female rats underwent CASP and either estradiol receptor α (ER-α) agonist propyl pyrazole triol (PPT), estradiol receptor ß (ER-ß) agonist diarylpropiolnitrile (DPN), or vehicle treatment. Intravital microscopy was performed, which is sufficiently sensitive to measure changes in the functional capillary density (FCD) as well as the major steps in leukocyte recruitment (rolling and adhesion). The leukocyte extravasations were also quantified by using histological paraffin sections of formalin fixed intestine. We found that either DPN (ER-ß) or PPT (ER-α) significantly reduced (P<0.05) sepsis-induced leukocyte-endothelial interaction (rolling, adherent leukocytes and neutrophil extravasations) and improved the intestinal muscular FCD. [PPT: Female; Leukocyte rolling (n/min): V(3) 3.7±0.7 vs 0.8±0.2, Leukocyte adhesion(n/mm(2)): V(3) 131.3±22.6 vs 57.2±13.5, Neutrophil extravasations (n/10000 µm(2)): 3.1±0.7 vs 6 ±1. Male; Leukocyte adhesion (n/mm(2)): V(1) 154.8±19.2 vs 81.3±11.2, V(3) 115.5±23.1 vs 37.8±12]. [DPN: Female; neutrophil extravasations (n/10000 µm(2)) 3.8±0.6 vs 6 ±1. Male; Leukocyte adhesion (n/mm(2)) V(1) 154.8±19.2 vs 70±10.5, V(3) 115.5±23.1 vs 52.8±9.6].Those results suggest that the observed effects of estradiol receptors on different phases of leukocytes recruitment with the improvement of the functional capillary density could partially explain the previous demonstrated salutary effects of estradiol on the intestinal microcirculation during sepsis. The observed activity of this class of compounds could open up a new avenue of research into the potential treatment of sepsis.

Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte-endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice.

OBJECTIVE: P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to play a significant role in septic lung injury. However, the detailed role of PSGL-1 in the pulmonary leukocyte recruitment remains elusive. We have developed a method based on intravital fluorescence microscopy of the lung microcirculation to examine the role of PSGL-1 in the extravasation process of leukocytes in septic lung damage. METHODS: Male C57BL/6 mice were treated with a control antibody or an anti-PSGL-1 antibody prior to cecal ligation and puncture (CLP). Leukocyte-endothelium interactions and microvascular hemodynamics were studied in pulmonary arterioles, capillaries and venules 4 h after CLP. RESULTS: Immunoneutralization of PSGL-1 decreased CLP-induced leukocyte rolling in pulmonary arterioles and venules significantly. Inhibition of PSGL-1 had no effect on leukocyte adhesion in venules, whereas the number of adherent leukocytes in lung arterioles and the number of trapped leukocytes in capillaries were markedly decreased. Moreover, immunoneutralization of PSGL-1 improved microvascular perfusion in the lung of septic animals. CONCLUSIONS: Taken together, these results document that PSGL-1 mediates leukocyte rolling in arterioles and venules. However, inhibition of PSGL-1 only decreases leukocyte adhesion in arterioles, suggesting that leukocyte rolling is not a prerequisite for pulmonary venular adhesion of leukocytes in sepsis. In addition, our data show that capillary trapping of leukocytes is dependent on PSGL-1 function.

Cation-pi interaction regulates ligand-binding affinity and signaling of integrin alpha4beta7.

Integrin α(4)ß(7) mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α(4)ß(7) for ligand is dynamically regulated by three interlinked metal ion-binding sites in ß(7)-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in ß(7)-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α(4)ß(7), and only slightly affected rolling adhesion mediated by low affinity α(4)ß(7). Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α(4)ß(7)-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α(4)ß(7)-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.