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The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca 2+ signals with Ca 2+ reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca 2+ transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca 2+ signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca 2+ channel Orai1 and Gα i -coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca 2+ signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca 2+ oscillations. Together, these findings establish Ca 2+ frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.

Microvascular dysfunction plays a fundamental role in the pathogenesis of salivary gland disorders. Restoring and preserving microvascular integrity might therefore represent a promising strategy for the treatment of these pathologies. The mechanisms underlying microvascular dysfunction in salivary glands, however, are still obscure, partly due to the unavailability of adequate in vivo models. Here, we present a novel experimental approach that allows comprehensive in vivo analyses of the salivary gland microvasculature in mice. For this purpose, we employed different microscopy techniques including multi-photon in vivo microscopy to quantitatively analyze interactions of distinct immune cell subsets in the submandibular gland microvasculature required for their infiltration into the surrounding parenchyma and their effects on microvascular function. Confocal microscopy and multi-channel flow cytometry in tissue sections/homogenates complemented these real-time analyses by determining the molecular phenotype of the participating cells. To this end, we identified key adhesion and signaling molecules that regulate the subset- and tissue-specific trafficking of leukocytes into inflamed glands and control the associated microvascular leakage. Hence, we established an experimental approach that allows in vivo analyses of microvascular processes in healthy and diseased salivary glands. This enables us to delineate distinct pathogenetic factors as novel therapeutic targets in salivary gland diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Uhl, Braun, Dominik, Luft, Canis and Reichel.)

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate β2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Immler, Lange-Sperandio, Steffen, Beck, Rohwedder, Roth, Napoli, Hupel, Pfister, Popper, Uhl, Mannell, Reichel, Vielhauer, Scherberich, Sperandio and Pruenster.)

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells "find" their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation.

Background and Purpose: Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation., Experimental Approach: Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine., Key Results: In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine., Conclusions and Implications: Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.

ABSTRACT Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg-1 body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg-1 body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]