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


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.


  1. 1

    Campbell, J.J. & Butcher, E.C. Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12, 336–341 (2000).

    CAS  Article  Google Scholar 

  2. 2

    Ley, K., Laudanna, C., Cybulsky, M.I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Thelen, M. & Stein, J.V. How chemokines invite leukocytes to dance. Nat. Immunol. 9, 953–959 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Shulman, Z. et al. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity 30, 384–396 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Schreiber, T.H., Shinder, V., Cain, D.W., Alon, R. & Sackstein, R. Shear flow-dependent integration of apical and subendothelial chemokines in T-cell transmigration: implications for locomotion and the multistep paradigm. Blood 109, 1381–1386 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Carman, C.V. et al. Transcellular diapedesis is initiated by invasive podosomes. Immunity 26, 784–797 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Ravkov, E.V., Myrick, C.M. & Altman, J.D. Immediate early effector functions of virus-specific CD8+CCR7+ memory cells in humans defined by HLA and CC chemokine ligand 19 tetramers. J. Immunol. 170, 2461–2468 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Shiow, L.R. et al. CD69 acts downstream of interferon-α/β to inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature 440, 540–544 (2006).

    CAS  Article  Google Scholar 

  9. 9

    von Andrian, U.H. & Mackay, C.R. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Masopust, D. et al. Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin. J. Immunol. 172, 4875–4882 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Bromley, S.K., Mempel, T.R. & Luster, A.D. Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat. Immunol. 9, 970–980 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Xie, H., Lim, Y.C., Luscinskas, F.W. & Lichtman, A.H. Acquisition of selectin binding and peripheral homing properties by CD4+ and CD8+ T cells. J. Exp. Med. 189, 1765–1776 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Siegelman, M.H., Stanescu, D. & Estess, P. The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion. J. Clin. Invest. 105, 683–691 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Warnock, R.A., Askari, S., Butcher, E.C. & von Andrian, U.H. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205–216 (1998).

    CAS  Article  Google Scholar 

  15. 15

    Shulman, Z. & Alon, R. Real-time in vitro assays for studying the role of chemokines in lymphocyte transendothelial migration under physiologic flow conditions. Methods Enzymol. 461, 311–332 (2009).

    CAS  Article  Google Scholar 

  16. 16

    McKinstry, K.K. et al. Rapid default transition of CD4 T cell effectors to functional memory cells. J. Exp. Med. 204, 2199–2211 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Lim, Y.C. et al. α4 β1-integrin activation is necessary for high-efficiency T-cell subset interactions with VCAM-1 under flow. Microcirculation 7, 201–214 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Constantin, G. et al. Chemokines trigger immediate β2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13, 759–769 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Beals, C.R., Edwards, A.C., Gottschalk, R.J., Kuijpers, T.W. & Staunton, D.E. CD18 activation epitopes induced by leukocyte activation. J. Immunol. 167, 6113–6122 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Bolomini-Vittori, M. et al. Regulation of conformer-specific activation of the integrin LFA-1 by a chemokine-triggered Rho signaling module. Nat. Immunol. 10, 185–194 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Fagerholm, S.C., Hilden, T.J., Nurmi, S.M. & Gahmberg, C.G. Specific integrin α and β chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms. J. Cell Biol. 171, 705–715 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Baker, R.G. & Koretzky, G.A. Regulation of T cell integrin function by adapter proteins. Immunol. Res. 42, 132–144 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Hogg, N., Patzak, I. & Willenbrock, F. The insider's guide to leukocyte integrin signalling and function. Nat. Rev. Immunol. 11, 416–426 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Atarashi, K., Hirata, T., Matsumoto, M., Kanemitsu, N. & Miyasaka, M. Rolling of Th1 cells via P-selectin glycoprotein ligand-1 stimulates LFA-1-mediated cell binding to ICAM-1. J. Immunol. 174, 1424–1432 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Cinamon, G., Shinder, V. & Alon, R. Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines. Nat. Immunol. 2, 515–522 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Woolf, E. et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat. Immunol. 8, 1076–1085 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Zhang, H.H. et al. CCR2 identifies a stable population of human effector memory CD4+ T cells equipped for rapid recall response. J. Immunol. 185, 6646–6663 (2010).

    CAS  Article  Google Scholar 

  28. 28

    Cinamon, G., Shinder, V., Shamri, R. & Alon, R. Chemoattractant signals and β2 integrin occupancy at apical endothelial contacts combine with shear stress signals to promote transendothelial neutrophil migration. J. Immunol. 173, 7282–7291 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Hillyer, P., Mordelet, E., Flynn, G. & Male, D. Chemokines, chemokine receptors and adhesion molecules on different human endothelia: discriminating the tissue-specific functions that affect leucocyte migration. Clin. Exp. Immunol. 134, 431–441 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Bao, X. et al. Endothelial heparan sulfate controls chemokine presentation in recruitment of lymphocytes and dendritic cells to lymph nodes. Immunity 33, 817–829 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Øynebråten, I., Bakke, O., Brandtzaeg, P., Johansen, F.E. & Haraldsen, G. Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments. Blood 104, 314–320 (2004).

    Article  Google Scholar 

  32. 32

    Øynebråten, I. et al. Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol. J. Immunol. 175, 5358–5369 (2005).

    Article  Google Scholar 

  33. 33

    Klausner, R.D., Donaldson, J.G. & Lippincott-Schwartz, J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116, 1071–1080 (1992).

    CAS  Article  Google Scholar 

  34. 34

    Mamdouh, Z., Mikhailov, A. & Muller, W.A. Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment. J. Exp. Med. 206, 2795–2808 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Middleton, J. et al. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385–395 (1997).

    CAS  Article  Google Scholar 

  36. 36

    Palframan, R.T. et al. Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J. Exp. Med. 194, 1361–1373 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Pruenster, M. et al. The Duffy antigen receptor for chemokines transports chemokines and supports their promigratory activity. Nat. Immunol. 10, 101–108 (2009).

    CAS  Article  Google Scholar 

  38. 38

    Guarda, G. et al. L-selectin-negative CCR7-effector and memory CD8+ T cells enter reactive lymph nodes and kill dendritic cells. Nat. Immunol. 8, 743–752 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Wolf, M., Albrecht, S. & Marki, C. Proteolytic processing of chemokines: implications in physiological and pathological conditions. Int. J. Biochem. Cell Biol. 40, 1185–1198 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Sauty, A. et al. The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells. J. Immunol. 162, 3549–3558 (1999).

    CAS  PubMed  Google Scholar 

  41. 41

    Park, J.J. & Loh, Y.P. How peptide hormone vesicles are transported to the secretion site for exocytosis. Mol. Endocrinol. 22, 2583–2595 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Rondaij, M.G. et al. Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells. Blood 112, 56–63 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Pober, J.S. & Sessa, W.C. Evolving functions of endothelial cells in inflammation. Nat. Rev. Immunol. 7, 803–815 (2007).

    CAS  Article  Google Scholar 

  44. 44

    Soehnlein, O., Lindbom, L. & Weber, C. Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood 114, 4613–4623 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Jamieson, T. et al. The chemokine receptor D6 limits the inflammatory response in vivo. Nat. Immunol. 6, 403–411 (2005).

    CAS  Article  Google Scholar 

  46. 46

    Proudfoot, A.E. The biological relevance of chemokine-proteoglycan interactions. Biochem. Soc. Trans. 34, 422–426 (2006).

    CAS  Article  Google Scholar 

  47. 47

    Grabovsky, V. et al. Subsecond induction of α4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192, 495–506 (2000).

    CAS  Article  Google Scholar 

  48. 48

    Proudfoot, A.E. et al. Amino-terminally modified RANTES analogues demonstrate differential effects on RANTES receptors. J. Biol. Chem. 274, 32478–32485 (1999).

    CAS  Article  Google Scholar 

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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.).

Author information




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).

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