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.

Objective-Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1␣ (Ccl3/MIP-1␣) 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-1␣ induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (Ͼ70% 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 Key Words: leukocyte Ⅲ migration Ⅲ chemokines Ⅲ permeability Ⅲ basement membrane L eukocyte recruitment from the microvasculature to sites of inflammation is a key event in both innate and adaptive immunity. In this process, a diversity of adhesion molecules, proteases, and chemokines are involved regulating the sequential steps of leukocyte rolling, firm adherence, and transmigration. 1,2 Chemokines are small molecules (8 to 14 kDa) which can be classified into C, CC, CXC, and CX 3 C chemokines according to the arrangement of their N-terminal cysteine residues. Increased levels of chemokines and their respective receptors have been found in numerous pathological conditions. According to the current paradigm, chemokine receptors on circulating leukocytes are supposed to interact with chemokines presented on the venular endothelium. These interactions immediately activate leukocyte integrins which, in turn, facilitate firm adherence and transmigration of leukocytes. [3][4][5] In the past years, particularly CC chemokines have been extensively studied in various inflammatory pathologies. Concluding from these studies, CC chemokines such as monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1␣ (Ccl3/MIP-1␣) have been suggested to exclusively mediate the migration of monocytes and lymphocytes. [3][4][5] However, there is a growing body of evidence that Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ are also critically involved in the recruitment of neutrophils. 6,7 The underlying mechanisms, however, remain largely unclear.Recently, it has been reported that both Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ are able to induce the release of lipid mediators such as leukotriene-B 4 (LTB 4 ). 8 -10 The functional relevance of endogenously generated lipid mediators including prostaglandins, leukotrienes, and PAF for each single step of the recruitment process of neutrophils elicited by Ccl2/JE/ In addition to leukocyte migration, Ccl2/JE/MCP-1 and Ccl3/MIP-1␣ have been implicated in the control of microvascular permeability. 11,12 Moreover, Ccl3/MIP-1␣ has recently been demonstrated to induce remodeling of the perivascular basement membrane, a process which might promote microvascular leakage during inflammatory conditions. 11 The contribution of neutrophils to these events, however, has not yet been studied.Therefore, the objective of the present study...

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Although different nanosized materials, including quantum dots (QDs), are intended to be used for biomedical applications, their interactions with microvessels and their inflammatory potential are largely unknown. In this in vivo study we report that leukocyte recruitment is modulated in the presence of quantum dots. We found that the surface chemistry of QDs strongly affects their localization in postcapillary venules, their uptake by perivascular macrophages, and their potential to modify steps of leukocyte recruitment.

In the present study, the role of gelatinases [matrix metalloproteinase-2 and -9 (MMP-2 and -9)] for leukocyte rolling, adherence, and transmigration was analyzed in the mouse cremaster muscle under different inflammatory conditions including ischemia-reperfusion (I/R) and stimulation with MIP-1alpha or platelet-activating factor (PAF). Using zymography, we detected a significant elevation of MMP-9 activity in response to the stimuli applied, and MMP-2 expression was not altered. However, treatment with a specific MMP-2/-9 inhibitor significantly abrogated elevated MMP-9 activity. As observed by intravital microscopy, all inflammatory conditions induced a significant increase in numbers of adherent and transmigrated leukocytes (>80% Ly-6G(+) neutrophils). Blockade of gelatinases significantly diminished I/R- and MIP-1alpha-induced leukocyte adherence and subsequent transmigration, and upon stimulation with PAF, gelatinase inhibition had no effect on leukocyte adherence but selectively reduced leukocyte transmigration. Concomitantly, we observed an increase in microvascular permeability after I/R and upon stimulation with MIP-1alpha or PAF, which was almost completely abolished in the inhibitor-treated groups. Using immunofluorescence staining and confocal microscopy, discontinuous expression of collagen IV, a major substrate of gelatinases within the perivascular basement membrane (BM), was detected in postcapillary venules. Analysis of intensity profiles demonstrated regions of low fluorescence intensity, whose size was enlarged significantly after I/R and upon stimulation with MIP-1alpha or PAF as compared with unstimulated controls. However, this enlargement was abolished significantly after inhibition of gelatinases, respectively. In conclusion, these data demonstrate that gelatinases strictly regulate microvascular permeability and BM remodeling during the early inflammatory response, whereas concomitant leukocyte recruitment is mediated by these proteases in a stimulus-specific manner.

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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-1α (MIP-1α/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 Cx3CR1gfp/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.