Imaging receptor microdomains on leukocyte subsets in live mice

Chiang, Elaine Y.; Hidalgo, Andrés; Chang, Jungshan; Frenette, Paul S.

doi:10.1038/nmeth1018

Cited by 81 publications

(84 citation statements)

References 16 publications

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“…Multiphoton microscopy is extremely useful for studying distinct populations of leukocytes outside the vasculature, where dynamic events are sufficiently slow so that they can be observed using time-lapse systems, but it cannot be used to track leukocytes in the vasculature due to slow imaging speed. Recently, Frenette and colleagues (43) reported that high-speed, high-resolution multichannel fluorescence intravital videomicroscopy could be used to track different leukocyte subsets in response to a cytokine. We used similar spinning disk multichannel fluorescence intravital videomicroscopy (for the first time to our knowledge) to examine the very early dynamic events in blood vessels during CS.…”

Section: Discussionmentioning

confidence: 99%

Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement

Norman

Hulliger

Colarusso

et al. 2008

The Journal of Immunology

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Contact sensitivity (CS) is one of the primary in vivo models of T cell-mediated inflammation. The presence of CS-initiating CD4 T lymphocytes at the time of challenge is essential for transfer and full development of the late phase CS inflammatory response. From this observation investigators have speculated that early recruitment of CD4 T cells to the site of challenge must occur. Moreover, there must be rapid synthesis/release and disappearance of an important mediator during the first hours after hapten challenge. Using spinning disk confocal microscopy, we observed the very early effector events of the immune response. Simultaneous, real-time visualization of predominant neutrophil and extremely rare CD4 T cell trafficking in the challenged skin vasculature was noted (one rolling CD4 T cell for every 10–18 rolling and adherent neutrophils). We demonstrate that neutrophil adhesion during the early CS response was reduced in C5a receptor-deficient (C5aR−/−) mice or leukotriene B4 receptor antagonist-treated mice, whereas CD4 T cell recruitment was only inhibited in C5aR−/− mice. In line with these observations, leukocyte infiltration and the associated tissue damage were significantly reduced in C5aR−/− mice but not in leukotriene B4 receptor antagonist-treated wild-type mice 24 h after challenge. C5a receptor expression on T cells and not on tissue resident cells was important for the development of a CS response. Thus, by using spinning disk confocal microscopy we visualized the early events of an adaptive immune response and identified the rare but essential recruitment of CD4 T cells via the complement pathway.

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Section: Discussionmentioning

confidence: 99%

Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement

Norman

Hulliger

Colarusso

et al. 2008

The Journal of Immunology

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“…Specific leukocyte subset adhesion. To examine adhesion of specific leukocyte subsets, confocal intravital microscopy was used (35,36). To enable identification of specific leukocyte subsets, mice received the following Abs (eBioscience, San Diego, CA): anti-Gr-1-PE (0.8 mg) to label neutrophils, and anti-CD4-FITC (2 mg) and anti-CD8-allophycocyanin (2 mg) to label CD4 + and CD8 + cells, respectively, administered i.v.…”

Section: Histologymentioning

confidence: 99%

Endogenous Regulatory T Cells Adhere in Inflamed Dermal Vessels via ICAM-1: Association with Regulation of Effector Leukocyte Adhesion

Deane

Abeynaike

Norman

et al. 2012

The Journal of Immunology

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Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4+ T cells increased during CS, peaking 8–24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4+ T cells were Foxp3+ Tregs. CD4+ T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti–ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4+ T cell depletion caused an increase in adhesion of CD8+ T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1–deficient mice.

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“…29,30 To discriminate accurately between leukocyte subsets, we injected intravenously very low amounts of fluorescently conjugated leukocyte-specific antibodies at the time of imaging, a procedure that does not alter the dynamic behavior of intravascular leukocytes. 31 T lymphocytes were thus distinguished by CD4 labeling, and neutrophils by Gr-1 labeling ( Figure 1A; see also Supplemental Figure S2 at http://ajp.amjpathol.org). Under noninflammatory conditions, about one-third of passing neutrophils were found rolling on dermal microvessels (see Supplemental Figure S2 at http://ajp.amjpathol.org).…”

Section: In Vivo Imaging Of Inflammatory T-lymphocyte Recruitment In mentioning

confidence: 99%

Physiological Contribution of CD44 as a Ligand for E-Selectin during Inflammatory T-Cell Recruitment

Nácher

Blázquez

Shao

et al. 2011

The American Journal of Pathology

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Endothelial selectins guide the migration of inflammatory T cells to extralymphoid tissues. Whereas Pselectin glycoprotein ligand-1 (PSGL-1) functions as the exclusive ligand for P-selectin, it acts in coordination with additional glycoproteins to mediate Eselectin binding. CD44 can act as one such ligand in neutrophils, but its contribution in inflammatory T lymphocytes remains unexplored. We have used realtime in vivo imaging of the cremasteric and dermal microcirculations to explore the dynamics of leukocyte recruitment, as well as the physiological contribution of CD44 in a model of Th1-driven inflammation. CD4؉ T-cell rolling frequency and kinetics, as well as arrest, were dependent on endothelial selectins and were markedly altered under inflammatory conditions. CD44 extracted from Th1 cells bound to soluble E-selectin in vitro and cooperated with PSGL-1 by controlling rolling velocities and promoting firm arrest. Using several competitive recruitment assays in a delayed-type hypersensitivity model, we show that the combined absence of CD44 and PSGL-1 impairs inflammatory T-cell recruitment beyond that of PSGL-1 alone. Differential expression of leukocyte fucosyltransferases in these cells may account for the differential use of E-selectin ligands relative to neutrophils. Our results identify additional mechanisms by which CD44 modulates the inflammatory response. The trafficking of T lymphocytes is regulated by their repertoire of adhesion and chemotactic receptors. Naïve T cells recirculate through secondary lymphoid organs in search of their cognate antigen, a process facilitated by their specific binding to high endothelial venules through L-selectin, ␤2 integrins, and the chemokine receptor CCR7. Once activated, effector and memory T cells switch their adhesive and signaling repertoire to one that allows immune surveillance of tissues. In particular, skin and inflamed areas are dynamically infiltrated by activated T cells, a process that underlies a number of pathogenic inflammatory diseases.1 It is generally accepted that this infiltration is initiated by extravasation from the blood microvasculature through a multistep cascade similar to that undergone by neutrophils, including initial tethering and rolling events followed by firm arrest and diapedesis.2 Tethering and rolling are initiated by labile interactions mediated by endothelial P-and E-selectins (encoded by Selp and Sele genes, respectively) expressed constitutively in the skin microvasculature 3 and induced in other tissues during inflammation.2 Induction of ligands for endothelial selectins is therefore critical for the acquired migrating properties of inflammatory lymphocytes. 4 Two glycoproteins expressed on Th1 lymphocytes, Pselecting glycoprotein ligand-1 (PSGL-1; encoded by Selplg) and the sialomucin CD43, have been shown to

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Imaging receptor microdomains on leukocyte subsets in live mice

Cited by 81 publications

References 16 publications

Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement

Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement

Endogenous Regulatory T Cells Adhere in Inflamed Dermal Vessels via ICAM-1: Association with Regulation of Effector Leukocyte Adhesion

Physiological Contribution of CD44 as a Ligand for E-Selectin during Inflammatory T-Cell Recruitment

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