Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0309214 on October 21, 2009

Published online before print October 21, 2009
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(Journal of Leukocyte Biology. 2009;86:1285-1294.)
© 2009 Society for Leukocyte Biology

Dynamic alterations in chemokine gradients induce transendothelial shuttling of human T cells under physiologic shear conditions

Jack Y. Lee*, Catherine D. Buzney*, Mark C. Poznansky{dagger},1 and Robert Sackstein*,{ddagger},1,2

* Harvard Skin Disease Research Center and Department of Dermatology, Brigham & Women’s Hospital, Boston, Massachusetts, USA;
{dagger} Infectious Disease Medicine and Dana-Farber/Harvard Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA; and
{ddagger} Department of Medicine, Brigham & Women’s Hospital, and Department of Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA

2. Correspondence: Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, MA 02115. USA. E-mail: rsackstein{at}rics.bwh.harvard.edu

ABSTRACT

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies, yet the molecular effectors of this process are relatively uncharacterized. For extravasation, studies based predominantly on static transwell assays support a general model, whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However, this model of chemotaxis cannot readily reconcile how chemokines influence intravasation, as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus, to analyze how T cells integrate perivascular chemokine signals under physiologic flow, we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment, transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However, when exposed to abluminal CXCL12, transmigrated T cells underwent striking retro-TEM and re-entered the flow stream. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent, augmented by apical flow, blocked by antibodies to integrins, and reduced by AMD3100 in a dose-dependent manner. Moreover, CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream.

Key Words: chemotaxis • fugetaxis • intravasation • CXCL12 • transendothelial migration • shear stress • fluid shear


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