Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Abstract

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing redfluorescent vasculature and yellowfluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steadystate and inflammation. (Blood. 2012; 120(11):2249-2258) IntroductionDendritic cells (DCs) are important in the initiation of adaptive immune responses. In peripheral tissues, such as the skin, DCs take up antigen, mature, and migrate via lymphatic vessels (LVs) to draining lymph nodes (dLNs), where they present antigen to resting T cells. Although this migration pattern has been known for more than 20 years, 1 DC migration into LVs is only now starting to be unraveled at the cellular level using live imaging technologies. [2][3][4] Before transmigrating across the lymphatic endothelium, DCs first need to squeeze through preformed, narrow pores present in the lymphatic basement membrane (BM). 3 DC entry into LVs is thought to occur at the level of primary lymphatic capillaries, which feature discontinuous cell junctions with "button"-like accumulations of cell adhesion molecules. 5 At the sites of such buttons, lymphatic endothelial cells (LECs) partially overlap and generate open flaps, through which leukocytes enter into lymphatics. 3,5 The most prominent mediator of DC migration into afferent lymphatics is CCL21, a chemokine constitutively expressed in LVs. 6,7 More recently, also other LEC-expressed molecules that mediate DC migration via LVs to dLNs have been identified. [8][9][10][11] Notably, ICAM-1 and VCAM-1, which are up-regulated in LVs during inflammation, 8,12 have been implicated in this process. 8 Intriguingly, experiments performed with pan-integrin knockout DCs revealed that DC migration to dLNs in steady-state was integrin-independent. 2 This ...

Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells

“…Our findings indicate that the dependency of BVs on Sema3A signals might be related to their maturation status and, potentially, to their anatomical location. A lack of VECad-Cre-mediated recombinatory events in the adult blood vessels can be excluded, because the VECad-Cre-driven reporter RFP remains highly expressed on adult endothelial cells in the ears (Nitschke et al, 2012). Taken together, our findings reveal that endothelial cell-derived Sema3A does not affect gross BV development or permeability, but specifically repels VEGF-A-induced tip cell filopodia and thus finetunes the angiogenic response during development.…”

Neutrophils Exhibit Differential Requirements for Homing Molecules in Their Lymphatic and Blood Trafficking into Draining Lymph Nodes

“…This discrepancy could be explained by differences in cell types and in requirement for adhesion molecules between steady-state and inflammation. It was shown that integrin ligands are induced on lymphatic endothelial cells only under inflammation, and that blockade of both Mac-1 and LFA-1 reduced DC crawling on activated lymphatic endothelium (35,36).…”

Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells

“…Constitutive VECad-Cre-driven recombinatory events might not be purely endothelial cell specific because VE-Cadherin is expressed in endothelial cells and leukocytes (Nitschke et al, 2012) in embryonic stages (Monvoisin et al, 2006). Thus, we performed experiments with inducible VECad-CreERT2; Sema3A flox/flox mice (Sem3A-iECKO) and applied tamoxifen at P1-P3.…”

“…Sema3A-loxed (Sema3a flox/flox ) and Sema3A lacZ knock-in (Sema3a +/lacZ ) mice (Taniguchi et al, 1997) were obtained from RIKEN, Japan. Vascularendothelial cadherin (VECad)-CreERT2 mice (Monvoisin et al, 2006) were provided by Dr Luisa Iruela-Arispe (UCLA, CA, USA) and VECad-Cre red fluorescent protein (RFP) mice (Nitschke et al, 2012) by Dr Cornelia Halin (ETH Zurich, Switzerland). To generate constitutive endothelial cellspecific Sema3A KO mice (Sema3A-cECKO), Sema3a flox/flox mice were crossed with constitutive VECad-Cre mice.…”