In Vivo Imaging of Lymphocyte Trafficking

Halin, Cornelia; Mora, J. Rodrigo; Sumen, Cenk; Andrian, Ulrich H. von

doi:10.1146/annurev.cellbio.21.122303.133159

Cited by 154 publications

(139 citation statements)

References 95 publications

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“…Moreover, Chen et al (5) showed that LN NK cells efficiently lysed tumor cells and suppressed B16 melanoma metastasis to draining LN. These findings suggest that NK cells actively patrol the LN in search of cognate target and transformed cells, perhaps exhibiting robust motility similar to other lymphocyte subsets, such as T and B cells (6,7). Therefore, it was surprising that NK cells isolated by DX5-positive selection were reported to move with slow velocity (2.75 Ϯ 0.17 m/min) when imaged in LN by two-photon microscopy (8).…”

mentioning

confidence: 99%

Natural killer cells actively patrol peripheral lymph nodes forming stable conjugates to eliminate MHC-mismatched targets

Garrod¹,

Wei²,

Parker

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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motility ͉ two-photon microscopy N atural killer (NK) cells are a specialized subset of lymphocytes capable of mediating effector functions without the need for prior sensitization. To avoid aberrant autoimmune attack, NK cells express a diverse repertoire of inhibitory receptors that suppress lytic activity. Polymorphic Ly49 molecules specific for self-MHC class I constitute one of the best characterized family of inhibitory receptors (1). Under normal conditions, persistent engagement of Ly49 by cognate selfligands results in recruitment of SHP-1 to the receptor's cytoplasmic immunoreceptor tyrosine-based inhibitory motif domain and subsequent dephosphorylation of activating signaling pathways (2). This inhibition of NK cell effector function is alleviated in the absence of self-molecules and is often the first step in acute rejection of allogeneic grafts. Although NK cellmediated clearance of circulating MHC-mismatched targets is well established (3), the ability of NK cells to migrate to and eliminate allogeneic target cells from peripheral lymph nodes (LN) under homeostatic conditions remains poorly defined.Recent evidence suggests that NK cells may exert important functional responses within LN. Specifically, enhanced recruitment of NK cells to antigen-stimulated LN facilitated the induction of a productive immune response (4). Moreover, Chen et al. (5) showed that LN NK cells efficiently lysed tumor cells and suppressed B16 melanoma metastasis to draining LN. These findings suggest that NK cells actively patrol the LN in search of cognate target and transformed cells, perhaps exhibiting robust motility similar to other lymphocyte subsets, such as T and B cells (6, 7). Therefore, it was surprising that NK cells isolated by DX5-positive selection were reported to move with slow velocity (2.75 Ϯ 0.17 m/min) when imaged in LN by two-photon microscopy (8).DX5 is a monoclonal antibody commonly used to identify and isolate murine NK cells. DX5 binds the ␣ 2 -integrin CD49b (9). Integrins are heterodimers comprised of ␣-and ␤-subunits and are of key importance in several complex biological processes, including cell adhesion and migration (10). Experimental evidence indicates that integrin engagement can result in the activation of key signaling events (11). Specifically, cross-linking of ␤ 1 -integrins expressed on human NK cells transduces intracellular signals, leading to the activation of the Ras-MAPK cascade, subsequent IFN-␥ production, and enhanced cytotoxicity (12). Therefore, CD49b cross-linking could induce signaling events that render positively selected NK cells immotile. To investigate this possibility, we used two-photon microscopy to visualize the in situ basal motility of unmanipulated NK cells within intact LN and examine their interaction dynamics with syngeneic and allogeneic B cells. Our results demonstrate that CD49b cross-linking strongly modulates motility, adhesion to collagen, and effector function, whereas unmanipulated NK cells are highly motile and preferentially form stable conjugate...

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mentioning

confidence: 99%

Natural killer cells actively patrol peripheral lymph nodes forming stable conjugates to eliminate MHC-mismatched targets

Garrod¹,

Wei²,

Parker

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Apart from using different guidance receptors, such as GPCRs and RTKs, cells also differ in whether, under physiological conditions, they migrate individually (5) or collectively, as part of a group (6). When migrating individually, each cell is expected to detect and respond to spatial cues directly and independently.…”

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confidence: 99%

Direct detection of guidance receptor activity during border cell migration

Janssens

Sung

Rørth

2010

Proc. Natl. Acad. Sci. U.S.A.

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Guidance receptor signaling is crucial for steering migrating cells. Despite this, we generally lack direct measurements of such signaling. Border cells in Drosophila migrate as a tightly associated group, but dynamically, with front and rear cells exchanging places. They use the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) as a guidance receptor, perceiving the attractant Pvf1. Here we determine the spatial distribution of PVR signaling by generating an antibody that specifically detects activated PVR in situ. PVR activity is very low in migrating border cells, due to strong activity of cellular phosphatases. Measurements of signal at the cell cortex show variability but a strong bias for both total active PVR and specific activity of PVR to be elevated at the front versus side of the leading cell, often with several-fold difference in signal levels. This polarized active PVR signal requires the E3 ubiquitin ligase Cbl and the recycling regulator Rab11, indicating a dependency on receptor trafficking. The endogenous ligand gradient contributes to shaping of signaling by increasing the specific activity of PVR toward the source in front cells. Surprisingly, signaling is also elevated at the back versus the side of rear cells. This distally polarized distribution of active PVR is ligand independent. Thus the actual guidance signal transmitted in border cells appears to integrate perceived ligand distribution with cell polarity or cell orientation with respect to the cluster. A general implication is that both group configuration and extrinsic cues can directly modulate guidance receptor signaling during collective cell migration.

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“…In contrast to CFDA-SE, rhodamine 6G, a fluorescent membrane-permeable dye accumulating in mitochondria, has been used in countless IVM experiments. Upon intravenous injection, the dye selectively labels leukocytes and platelets in vivo, and its unbound portion is rapidly cleared from the circulation [8,16].…”

Section: Discussionmentioning

confidence: 99%

“…Acridine orange presents with limitations due to its accumulation in parenchymal tissue and its potential light-induced phototoxic effect that alters hemodynamics and cell adhesion. The same holds true for rhodamine 6G, the widely applied dye for in vivo labelling of leukocytes for IVM studies [7,8]. In contrast, carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) has been rarely applied for IVM investigations [9][10][11] even though it is discussed as an ideal dye for flow cytometry-based proliferation studies [5,6,12].…”

Section: Biophotonicsmentioning

confidence: 99%

Comparative suitability of CFDA‐SE and rhodamine 6G for in vivo assessment of leukocyte‐endothelium interactions

Jbeily

Claus

Dahlke

et al. 2014

Journal of Biophotonics

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Intravital fluorescence microscopy (IVM) is a predestined tool for investigating the fate of leukocytes during the process of leukocyte recruitment. In the present study, the commonly used dye for this purpose, rhodamine 6G, and carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE) were compared for leukocytes labelling with respect to suitability for IVM studies. Their potential in labelling different leukocytes subpopulations as well as their fluorescence intensities were assessed by flow cytometry revealing distinct differences between both dyes. These differences had a profound impact on their application for in vivo imaging of leukocyte‐endothelium interactions. In summary, CFDA‐SE revealed superior in labelling leukocytes for in vivo microscopy with respect to image quality. In addition, we could show the efficiency of CFDA‐SE also under disease condition in an animal model of sepsis. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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In Vivo Imaging of Lymphocyte Trafficking

Cited by 154 publications

References 95 publications

Natural killer cells actively patrol peripheral lymph nodes forming stable conjugates to eliminate MHC-mismatched targets

Natural killer cells actively patrol peripheral lymph nodes forming stable conjugates to eliminate MHC-mismatched targets

Direct detection of guidance receptor activity during border cell migration

Comparative suitability of CFDA‐SE and rhodamine 6G for in vivo assessment of leukocyte‐endothelium interactions

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