Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators

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

Atrial Natriuretic Peptide Protects against Histamine-Induced Endothelial Barrier Dysfunction in Vivo

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, β-catenin, and p120ctn 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

ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability

Endothelial cell–selective adhesion molecule (ESAM) is specifically expressed at endothelial tight junctions and on platelets. To test whether ESAM is involved in leukocyte extravasation, we have generated mice carrying a disrupted ESAM gene and analyzed them in three different inflammation models. We found that recruitment of lymphocytes into inflamed skin was unaffected by the gene disruption. However, the migration of neutrophils into chemically inflamed peritoneum was inhibited by 70% at 2 h after stimulation, recovering at later time points. Analyzing neutrophil extravasation directly by intravital microscopy in the cremaster muscle revealed that leukocyte extravasation was reduced (50%) in ESAM−/− mice without affecting leukocyte rolling and adhesion. Depletion of >98% of circulating platelets did not abolish the ESAM deficiency–related inhibitory effect on neutrophil extravasation, indicating that it is only ESAM at endothelial tight junctions that is relevant for the extravasation process. Knocking down ESAM expression in endothelial cells resulted in reduced levels of activated Rho, a GTPase implicated in the destabilization of tight junctions. Indeed, vascular permeability stimulated by vascular endothelial growth factor was reduced in ESAM−/− mice. Collectively, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall, possibly by influencing endothelial cell contacts

Flavopiridol Protects Against Inflammation by Attenuating Leukocyte-Endothelial Interaction via Inhibition of Cyclin-Dependent Kinase 9

Objective: The cyclin-dependent kinase (CDK) inhibitor flavopiridol is currently being tested in clinical trials as anticancer drug. Beyond its cell death–inducing action, we hypothesized that flavopiridol affects inflammatory processes. Therefore, we elucidated the action of flavopiridol on leukocyte–endothelial cell interaction and endothelial activation in vivo and in vitro and studied the underlying molecular mechanisms. Methods and Results: Flavopiridol suppressed concanavalin A–induced hepatitis and neutrophil infiltration into liver tissue. Flavopiridol also inhibited tumor necrosis factor-α–induced leukocyte– endothelial cell interaction in the mouse cremaster muscle. Endothelial cells were found to be the major target of flavopiridol, which blocked the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin), as well as NF-κB-dependent transcription. Flavopiridol did not affect inhibitor of κB (IκB) kinase, the degradation and phosphorylation of IκBα, nuclear translocation of p65, or nuclear factor-κB (NF-κB) DNA-binding activity. By performing a cellular kinome array and a kinase activity panel, we found LIM domain kinase-1 (LIMK1), casein kinase 2, c-Jun N-terminal kinase (JNK), protein kinase Cθ (PKCθ), CDK4, CDK6, CDK8, and CDK9 to be influenced by flavopiridol. Using specific inhibitors, as well as RNA interference (RNAi), we revealed that only CDK9 is responsible for the action of flavopiridol. Conclusion: Our study highlights flavopiridol as a promising antiinflammatory compound and inhibition of CDK9 as a novel approach for the treatment of inflammation-associated diseases

To Protect Fatty Livers from Ischemia Reperfusion Injury: Role of Ischemic Postconditioning

BACKGROUND The benefit of ischemic postconditioning (IPostC) might be the throttled inflow following cold ischemia. The current study investigated advantage and mechanisms of IPostC in healthy and fatty rat livers. METHODS Male SD rats received a high-fat diet to induce fatty livers. Isolated liver perfusion was performed after 24 h ischemia at 4°C as well as in vivo experiments after 90 min warm ischemia. The so-called follow-up perfusions served to investigate the hypothesis that medium from IPostC experiments is less harmful. Lactate dehydrogenase (LDH), transaminases, different cytokines, and gene expressions, respectively, were measured. RESULTS Fatty livers showed histologically mild inflammation and moderate to severe fat storage. IPostC reduced LDH and TXB2 in healthy and fatty livers and increased bile flow. LDH, TNF-\textgreeka, and IL-6 levels in serum decreased after warm ischemia + IPostC. The gene expressions of Tnf, IL-6, Ccl2, and Ripk3 were downregulated in vivo after IPostC. CONCLUSIONS IPostC showed protective effects after ischemia in situ and in vivo in healthy and fatty livers. Restricted cyclic inflow was an important mechanism and further suggested involvement of necroptosis. IPostC represents a promising and easy intervention to improve outcomes after transplantation

Optimized dispersion of nanoparticles for biological in vitro and in vivo studies

Background: The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies. Results: TiO(2) (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 10(5) kJ/m(3) was sufficient to disaggregate TiO(2) (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO(2) (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO(2) (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO(2) (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO(2) (rutile), ZnO, Ag, SiO(x), SWNT, MWNT, and diesel SRM2975 particulate matter. Conclusion: The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates

Ischemic Postconditioning (IPostC) Protects Fibrotic and Cirrhotic Rat Livers after Warm Ischemia

Background. Decreased organ function following liver resection is a major clinical issue. The practical method of ischemic postconditioning (IPostC) has been studied in heart diseases, but no data exist regarding fibrotic livers. Aims. We aimed to determine whether IPostC could protect healthy, fibrotic, and cirrhotic livers from ischemia reperfusion injury (IRI). Methods. Fibrosis was induced in male SD rats using bile duct ligation (BDL, 4 weeks), and cirrhosis was induced using thioacetamide (TAA, 18 weeks). Fibrosis and cirrhosis were histologically confirmed using HE and EvG staining. For healthy, fibrotic, and cirrhotic livers, isolated liver perfusion with 90 min of warm ischemia was performed in three groups (each with n=8): control, IPostC 8x20 sec, and IPostC 4x60 sec. additionally, healthy livers were investigated during a follow-up study. Lactate dehydrogenase (LDH) and thromboxane B-2 (TXB2) in the perfusate, as well as bile flow (healthy/TAA) and portal perfusion pressure, were measured. Results. LDH and TXB2 were reduced, and bile flow was increased by IPostC, mainly in total and in the late phase of reperfusion. The follow-up study showed that the perfusate derived from a postconditioned group had much less damaging potential than perfusate derived from the nonpostconditioned group. Conclusion. IPostC following warm ischemia protects healthy, fibrotic, and cirrhotic livers against IRI. Reduced efflux of TXB2 is one possible mechanism for this effect of IPostC and increases sinusoidal microcirculation. These findings may help to improve organ function and recovery of patients after liver resection

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

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

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo

Cortactin is required for endothelial barrier function and leukocyte recruitment in vivo