Search

von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollate-induced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier.

Background/Aims This study was designed to investigate the role of endothelial cell-selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. Methods The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90min/30–360min). Results As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. Postischemic neutrophil transmigration was significantly attenuated in ESAM−/− mice after 2h of reperfusion, whereas it was completely restored after 6h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM−/− mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM−/− mice. Conclusions ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.

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,[for full text, please go to the a.m. URL], 132. Kongress der Deutschen Gesellschaft für Chirurgie

Summary. Background: The translocation of nanoparticles in the lung toward effector organs via the circulation is considered an important direct pathway for systemic effects of nanoparticles after inhalation. Recently, we have reported that a moderate dose of systemically administered nanosized carbon black particles exerted thrombogenic effects in hepatic microvessels of healthy mice. Objectives: This study addresses the questions of whether similar thrombogenic effects are also evoked upon inhalation of nanosized carbon particles (NCP) and whether NCP-induced hepatic platelet accumulation is associated with pulmonary or systemic inflammation. Methods: Two and 8 h after a 24-h exposure to either filtered air or to NCP, intravital fluorescence microscopy of the hepatic microcirculation was performed in C57Bl/6 mice. Parameters of pulmonary or systemic inflammatory response were determined in bronchoalveolar lavage and blood/plasma samples. Results: Inhalative exposure to NCP caused platelet accumulation in the hepatic microvasculature, whereas leukocyte recruitment and sinusoidal perfusion did not differ from controls. Fibrinogen deposition was detected by immunohistochemistry in both hepatic and cardiac microvessels from NCP-exposed mice. In contrast, inhalation of NCP affected neither the plasma levels of proinflammatory cytokines nor blood cell counts. Moreover, the bronchoalveolar lavage data indicate that no significant inflammatory response occurred in the lung. Conclusions: Thus, exposure to NCP exerts thrombogenic effects in the microcirculation of healthy mice independent of the route of administration (i.e. inhalation or systemic intra-arterial administration). The NCP-induced thrombogenic effects are not liver specific, are associated with neither a local nor a systemic inflammatory response, and seem to be independent of pulmonary inflammation.

Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.

Objective—Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1α (Ccl3/MIP-1α) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear.Methods and Results—Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1α induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B4(LTB4) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB4, but not Ccl2/JE/MCP-1 and Ccl3/MIP-1α, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/MCP-1- and Ccl3/MIP-1α-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice.Conclusions—Ccl2/JE/MCP-1 and Ccl3/MIP-1α mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage.

Background: The endothelial receptors that control leukocyte transmigration in the postischemic liver are not identified. This study was designed to investigate the role of endothelial cell selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. Methods and Results: The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90 min/30–360 min). As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. The postischemic neutrophil transmigration was significantly attenuated in ESAM−/− mice after 2 h of reperfusion, whereas it was completely restored after 6 h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM−/− mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30 min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM−/− mice. Conclusions: ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.

Ischemia-reperfusion (I/R) activates innate immunity involving Toll-like receptor (TLR) 2 and TLR-4 signaling. Leukocyte migration and vascular permeability contribute to postischemic tissue damage. We hypothesized that TLR-2 and TLR-4 directly mediate leukocyte migration and vascular permeability during I/R. We used in vivo microscopy on postischemic murine cremaster muscle to quantify leukocyte adhesion as well as transendothelial and interstitial migration in sham-operated wild-type mice and in wild-type, TLR-2(-/-), and TLR-4-mutant mice 30 and 120 min after I/R. Alterations in fluorescein isothiocyanate-dextran leakage across cremasteric venules were determined as a measure of endothelial permeability. I/R-induced leukocyte adhesion in TLR-2(-/-) and TLR-4-mutant mice was comparable to that in wild-type mice. The number of transmigrated leukocytes was increased upon I/R in wild-type mice as compared with the sham-operated group. In contrast, leukocyte transmigration was significantly attenuated in TLR-2(-/-) but not in TLR-4-mutant mice. Motility and polarization of interstitially migrating leukocytes did not significantly differ in TLR-2(-/-) and TLR-4-mutant mice from wild-type mice. Postischemic vascular leakage was significantly lower in both TLR-2(-/-) and TLR-4-mutant than in wild-type mice. We conclude that both TLR-2 signaling and TLR-4 signaling enhance postischemic vascular permeability and that TLR-2 has additional effects on the transendothelial migration of leukocytes at the postischemic vascular wall.

Endothelial barrier dysfunction is a hallmark of many severe pathologies, including sepsis or atherosclerosis. The cardiovascular hormone atrial natriuretic peptide (ANP) has increasingly been suggested to counteract endothelial leakage. Surprisingly, the precise in vivo relevance of these observations has never been evaluated. Thus, we aimed to clarify this issue and, moreover, to identify the permeability-controlling subcellular systems that are targeted by ANP. Histamine was used as important pro-inflammatory, permeability-increasing stimulus. Measurements of fluorescein isothiocyanate (FITC)-dextran extravasation from venules of the mouse cremaster muscle and rat hematocrit values were performed to judge changes of endothelial permeability in vivo. It is noteworthy that ANP strongly reduced the histamine-evoked endothelial barrier dysfunction in vivo. In vitro, ANP blocked the breakdown of transendothelial electrical resistance (TEER) induced by histamine. Moreover, as judged by immunocytochemistry and Western blot analysis, ANP inhibited changes of vascular endothelial (VE)-cadherin, beta-catenin, and p120(ctn) morphology; VE-cadherin and myosin light chain 2 (MLC2) phosphorylation; and F-actin stress fiber formation. These changes seem to be predominantly mediated by the natriuretic peptide receptor (NPR)-A, but not by NPR-C. In summary, we revealed ANP as a potent endothelial barrier protecting agent in vivo and identified adherens junctions and the contractile apparatus as subcellular systems targeted by ANP. Thus, our study highlights ANP as an interesting pharmacological compound opening new therapeutic options for preventing endothelial leakage.

Mechanisms mediating CD4+ T cell activation during hepatic ischemia-reperfusion (I/R) remain not fully understood. In this study, we tested the hypothesis that CD4+ T cells and Kupffer cells (KCs) interact during I/R via proinflammatory mediators causing reciprocal activation. In mice, accumulation of fluorescence-labeled CD4+ T cells was analyzed using intravital fluorescence microscopy in a sham-operated group, a group after warm lobar hepatic I/R (90/30–120min), and an additional I/R-group after depletion of KCs with GdCl3 (n = 6 each group). The I/R-induced accumulation of CD4+ T cells in sinusoids was significantly attenuated in mice undergoing depletion of KCs. To investigate whether KCs activate CD4+ T cells by releasing reactive oxygen species (ROS) and IL-6 as well as TNF-alpha, accumulation of CD4+ T cells was quantified in mice treated with the ROS scavenger glutathione, in IL-6-/- mice, and in wild-type mice after infusion of TNF-receptor-1-/- CD4+ T cells. Reduction of ROS and IL-6 as well as TNF-R1 deficiency attenuated postischemic CD4+ T cell accumulation (p < 0.05). Using flow cytometry we show in vitro that TNF-alpha and IL-6 are able to directly activate isolated CD4+ T cells and upregulate the expression of adhesion molecules on sinusoidal endothelial cells. Finally, we assessed whether CD4+ T cells might, in turn, trigger KC activation. As shown by an intravital microscopic analysis of the clearance kinetics of fluorescence-labeled latex beads, phagocytic activity of KCs was significantly depressed after I/R and this effect was enhanced in CD4-/- mice. In conclusion, this study demonstrates that KCs support the intrasinusoidal recruitment of CD4+ T cells during I/R. This effect is mediated by release of ROS and the cytokines TNF-alpha and IL-6, which together trigger activation of both CD4+ T cells and sinusoidal endothelial cells. In addition, CD4+ T cells seem to enhance the activation of KCs in the postischemic liver.

T cells are suggested to be involved in the induction of antigen-independent hepatic ischemia-reper-fusion (I/R) injury. In the present study, we analyzed the recruitment of T cell subpopulations in the postischemic liver in vivo and investigated by which pathway T cells mediate tissue injury. In a murine model of warm hepatic I/R (I: 90min, R: 30, 120 min), the recruitment of freshly isolated (magnetic immunosorting) and CFDA-SE-labeled syngeneic CD4+ and CD8+ T cells was analyzed using intra-vital microscopy (n = 6 each group). Hepatic I/R induced accumulation of CD4+ T cells in sinusoids (4.4 ± 0.7 vs. 1.6 ± 0.2/acinus in the sham-operated group) as well as transmigration into the perivas-cular space. In contrast, postischemic recruitment of CD8+ T cells was very low and did not differ from controls. To investigate the role of CD4+ cells during hepatic I/R, we performed an in vivo analysis of sinusoidal colocalization of CD4+ cells with platelets (n = 6). After I/R (90/30 min), 27 ± 3% of adherent CD4+ cells were colocalized with platelets, a fact pointing to interactions between both types of blood cells. In a third set of experiments (n = 6), we tested the hypothesis that CD4+ T cells mediate microvascular I/R injury by enhancing platelet recruitment. Indeed, the postischemic increase in the number of accumulated platelets was significantly lower in sinusoids of CD4-/- mice than in those of wild-type controls. Moreover, the postischemically impaired sinusoidal perfusion was ameliorated in CD4-/- mice (WT 30 ± 1; CD4-/- 18 ± 2% non-perfused sinusoids). In summary, these in vivo data indicate that hepatic I/R induces accumulation as well as transmigration of CD4+but not CD8+ T cells in sinusoids during early reperfusion. CD4+ T cells are colocalized with platelets in sinusoids, suggesting a reciprocal activation of both cell types either due to direct interactions or through activation of hepatic endothelium. CD4+ T cell deficiency is associated with an attenuation of postischemic platelet recruitment as well as with an improvement of postischemic tissue perfusion. Supported by DFG (FOR440/2)

T cells are suggested to participate in the manifestation of ischemia-reperfusion-induced inflammatory responses in the liver. The aim of this study was to investigate the recruitment of T cells in the postischemic hepatic microvasculature in vivo and to test the hypothesis that this recruitment is mediated by Kupffer cell activation. In C57B1/6 mice, ischemia of the left liver lobe was induced for 90 min. CD4+ and CD8+ T cells were isolated from spleens of syngenic mice by magnetic cell sorting and labeled ex vivo with CFDA-SE. After 30 min of reperfusion, either 1 × 107 CD4+ or 0.7 × 107CD8+ T cells were infused intraarterially (n = 5 each group). In control experiments, an identical number of either CD4+ or CD8+ T cells was infused into sham-operated animals. In an additional ischemia-reperfusion group, activation of Kupffer cells was blocked by gadolinium chloride (10 µg/kg, i.V., n = 5). The number of T cells accumulated in hepatic microvessels as well as of T cells transmigrated to the perivascular space was quantitatively analyzed by means of intravital video fluorescence microscopy. Hepatic ischemia-reperfusion induced an increase in the number of adherent CD4+ T cells in postsinusoidal venules and sinusoids after 30 min as well as 120 min of reperfusion as compared to the sham-operated group. The prolongation of reperfusion time from 30 min to 120 min significantly enhanced T cell transmigration. The blockade of Kupffer cells with gadolinium chloride completely attenuated both postischemic adherence and transmigration of CD4+ T cells. In contrast, the postischemic recruitment of CD8+ T cells was very low in all segments of the hepatic microvasculature and did not differ among experimental groups. Thus, normothermic hepatic I/R induces accumulation of CD4+, but not CD8+ T cells, in the hepatic microcirculation. This accumulation is triggered by Kupffer cells and occurs in sinusoids and to a lesser extent in postsinusoidal venules during early reperfusion. CD4+ T cell transmigration is enhanced as reperfusion time is prolonged up to 120 min. Accumulation of T cells in the postischemic liver may provide a potential mechanism underlying postischemic liver injury.