Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots

Abstract

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (TH1 cell) and type 1 cytotoxic T cell (TC1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide–binding proteins of the Gi type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration–promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

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Figure 1: Effector lymphocytes arrest in vivo on inflamed skin vessels independently of Gi protein signaling.
Figure 2: Effector human T lymphocytes arrest on TNF-activated HUVECs independently of chemokine Gi protein signaling.
Figure 3: Integrins of effector T cells are not conformationally activated but use constitutive PLC signaling for spontaneous adhesiveness.
Figure 4: Integrin outside-in Src signals trigger microvillar collapse and spreading of effector lymphocytes on isolated ligands independently of chemokine signals.
Figure 5: Transendothelial migration but not crawling of effector lymphocytes requires Gi protein signals.
Figure 6: CCR2 on effector lymphocytes is critical for their transendothelial migration through TNF-activated HUVECs and HDMECs.
Figure 7: Vesicle-stored CCL2 drives effector lymphocyte transendothelial migration.
Figure 8: CCL2 vesicles docked on actin fibers beneath the plasma membrane promote the transendothelial migration of effector lymphocytes.

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Acknowledgements

We thank R. Wedlich-Soeldner (Max-Planck Institute for Biochemistry) for Lifeact-mRFP; A. Bershadsky (Weizmann Institute) for α-tubulin–mCherry; B. Geiger (Weizmann Institute) for paxillin-CFP; F. Sanchez-Madrid (Universidad Autónoma de Madrid) for ICAM-1-EGFP; Y. Kloog (Tel-Aviv University) for eGFP-th; R. Pardi (Dibit-Scientific Institute San Raffaele) for Rab11-DsRed; G. Shakhar and M. Fernandez-Borja for discussions, and S. Schwarzbaum for editorial assistance. Supported by The Linda Jacobs Chair in Immune and Stem Cell Research (R.A.), the Israel Science Foundation (R.A.), the Minerva Foundation, the Flight Attendant Medical Research Institute Foundation (R.A.) and the Germany-Israel Science Foundation (R.A.).

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Authors

Contributions

Z.S. designed the in vitro models, did most of the experiments and wrote parts of the manuscript; S.J.C. designed and did major parts of Figures 1 and 5b, and Supplementary Figures 2 and 3; B.R.,V.K. and R.J. did multiphoton intravital imaging; V.G. and L.S.-B. did flow chamber studies; S.W.F. did flow chamber experiments, biochemistry and data organization; E.K. and V.S. did EM analysis; T.M. did chemokine-GFP cloning; S.M.N. did LFA-1 phosphorylation analysis; I.G. did TH1-TC1 analysis; O.H. did GPCR blocker synthesis; C.G.G., A.E., W.W. and A.B.-B. supervised specific parts of the research; and R.A. designed the study, supervised the work and wrote the manuscript.

Corresponding author

Correspondence to Ronen Alon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Methods (PDF 5527 kb)

Supplementary Video 1

Intravital microscopy of OT-1 effectors interacting with skin vessels in a sterile inflammed ear model. (MOV 3277 kb)

Supplementary Video 2

Crawling of a PTX-pretreated CD3 effector on a CFA inflamed skin vessel. (MOV 419 kb)

Supplementary Video 3

The major TEM route of effectors is through endothelial junctions. (MOV 2884 kb)

Supplementary Video 4

Transendothelial migration but not apical crawling of effector lymphocytes requires chemokine signals. (MOV 4131 kb)

Supplementary Video 5

Formation of subluminal leading edge by effector lymphocytes requires endothelial based chemokine signals. (MOV 8652 kb)

Supplementary Video 6

Effector lymphocytes can cross the endothelium through paracellular and transcellular TEM routes. (MOV 9032 kb)

Supplementary Video 7

CCL2 containing vesicles are not recycling endosomes. (MOV 3796 kb)

Supplementary Video 8

CCL2 vesicles are mobilized on microtubules and docked on actin fibers in inflamed endothelial cells. (MOV 4883 kb)

Supplementary Video 9

CCL2 vesicles interaction with endothelial cytoskeleton. (MOV 3861 kb)

Supplementary Video 10

Treatment of endothelial cells with microtubules disrupting agent, halt vesicle motility. (MOV 4394 kb)

Supplementary Video 11

Disruption of actin filaments impairs chemokine vesicle docking without altering rapid vesicle motility. (MOV 4967 kb)

Supplementary Video 12

The basolateral leading edge of effector lymphocytes consumes vesicle stored CCL2 during productive TEM. (MOV 6138 kb)

Supplementary Video 13

Sticking of PTX-pretreated CD3 effectors on a CFA inflamed skin vessel. (MOV 796 kb)

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Shulman, Z., Cohen, S., Roediger, B. et al. Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots. Nat Immunol 13, 67–76 (2012). https://doi.org/10.1038/ni.2173

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