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

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 exposedto abluminal CXCL12, transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. 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.

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells

“…Because E-selectin and VCAM-1 are natively expressed by bone marrow microvascular endothelium (5), we analyzed TEM of hMSCs across HUVEC monolayers stimulated to express E-selectin and VCAM-1. Our data show that HCELL + hMSCs undergo VLA-4-mediated firm adherence and TEM through stimulated HUVEC in the absence of chemokine administration; in particular, we observed heightened firm adherence of HCELL + hMSCs on HUVEC under flow conditions known to dissipate chemokine gradients (21). To specifically evaluate whether CXCR4 signaling contributes to the observed hMSC TEM, we performed PCR to measure CXCR4 mRNA levels and Western blot analysis of CXCR4 protein, to- .…”

“…In cases where CXCR4 expression has been reported, the chemotactic response of hMSCs to CXCL12 is modest (17). Our prior work has shown that chemokine gradients established across an endothelial interface are rapidly dissipated by hemodynamic shear forces (21), suggesting that cells bearing modest expression of chemokine receptors would be at a disadvantage in responding to perivascular chemokines in vivo. Collectively, these prior studies and our results here indicate that although chemokine(s) could cooperate in activating hMSC VLA-4 binding to endothelium and subsequent TEM in vivo, engagement of hMSC HCELL with E-selectin natively expressed on primary endothelial cells can drive this process directly.…”

Leukocyte Migration into Inflamed Tissues

“…This leukocyte reverse TEM (rTEM) response has been reported for human monocyte, neutrophil, and T cell migration through cultured endothelial cells (Buckley et al, 2006;Lee et al, 2009;Randolph and Furie, 1996) and for neutrophils in zebra fish embryos (Mathias et al, 2006). Neutrophil rTEM, and a TEM mode that involves multiple oscillatory movements within endothelial cell junctions (termed ''hesitant'' TEM), have also been observed in stimulated mouse cremasteric venules in vivo (Woodfin et al, 2011).…”

Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature

“…These results do not allow us to dissect the mechanism(s) behind this long lasting effect, apart from proposing resistance to cleavage hence longer presence at the inflammatory site. We cannot exclude the involvement of other pathways in the striking effect of SAnxA1, persisting within the inflamed paw, on the 24-hour edema, including promotion of retrograde leukocyte transmigration 43,44 and/or removal of apoptotic leukocytes. 9 In any case, we observed major reductions in immune cell infiltrates in the 24-hours after -carrageenan.…”