Adhesion Molecules And Lymphocyte Homing. Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 30 & Feb 4, 2014

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1 Adhesion Molecules And Lymphocyte Homing Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 30 & Feb 4, 2014

2 Outline General introduction to lymphocyte trafficking homeostatic recirculation vs active recruitment. Homeostatic trafficking of lymphocytes across specialized high endothelial venules (HEVs) Structure/function of adhesion molecules involved in initial tethering/rolling (L-selectin/PNAd) Structure/function of chemokine/chemokine receptors that mediate activation (CCR7/CCL21) Structure/function of adhesion molecules required for firm arrest/transendothelial migration (LFA-1/ICAM-1 & α4β7 integrin/madcam-1) Leukocyte recruitment in acute and chronic inflammation.

3 Lymphocyte Extravasation in LN HEV Initial Attachment Rolling Firm Adhesion Transmigration 1 L-Selectin/PNAd-1 3 LFA-1/ICAM-1/2 2 Chemokines (CCL21)

4 The CC Chemokine SLC/6CKine/CCL21 Mediates Tissue-Specific Homing L-selectin CCR7 LFA-1 ss ss PNAd ss ICAM ss ss 1 LN HEV CCL21 2 3&4

5 Chemokines C CC CXC CXXXC N C C N C CC +++ COOH +++ N C CXC COOH N C CXXXC C C C CCL (ligand) CCL21 (SLC) CXCL (ligand) CXCL12 (SDF-1) CCR (chemokine receptor) CCR7 CXCR (chemokine receptor) CXCR4

6 Structure of CC Chemokines CCL21 NH 2 C C CC +++ COOH Basic (positively charged) amino acids in C-terminus required for binding to glycosaminoglycans (heparin sulfate) that make up glycocalyx on endothelial cells 3-dimensional structure at N- terminus required for binding to chemokine receptor α-helixβ Anti-parallel β-pleated sheet

7 (CXCL12) (CXCL13) (CCL19, CCL21)

8 Chemokines Stimulate Chemotaxis and Migration Transwell Migration Assays 5 μm pore Chemokine (CCL21)

9 Chemokines Stimulate Chemotaxis and Migration Transwell Migration Assays 5 μm pore Chemokine (CCL21)

10 Evidence that CCL21 is a Tissue Specific Homing Chemokine Tissue specific localization of CCL21 in HEV Capsule L-selectin CCR7 LFA-1 B cell area (follicle) PNAd ss ss ss ss ss ICAM T cell area (paracortex CCL21 HEV HEV CCL21 mrna Lymph Node

11 CCL21 (SLC) is Expressed by Lymph Node HEV In Situ Analysis (mrna) BCL/CXCL13 CCL21/SLC IHC Analysis (protein) PNAd CCL21 WT +/+ plt/plt Science 286:2098, 1999 J. Exp. Med. 191:61, 2000 BCL/CXCL13 binds CXCR5 (involved in B cell migration) SLC/CCL21 binds CCR7 (mediates B & T cell homing across HEV

12 In vivo Imaging of CCL21 Localization on PLN HEV by Intravital Microscopy (IVM) Femoral Femoral Artery Artery Injection Injection of of FITC-labeled FITC-labeled anti-ccl21 anti-ccl21 Ab) Ab) VCR PC Inguinal LN dapted from von Andrian et al, Curr. Op. Immunol. 2004

13 CCL21 is Expressed by Lymph Node HEV in Wild Type But Not plt/plt Mice WT plt/plt. Exp. Med. 191:61, 2000 Evidence that CCL21 and CCR7 receptor critical for T lymphocyte extravasation across HEV HEV express CCL21 [SLC] mrna and protein HEV of plt/plt mice lack CC21 [SLC] and CCL19 [ELC/MIP3β); can t support firm adhesion or homing Reconstitution of CCL21 in HEV of plt/plt mice restores firm adhesion T cells from CCR7 -/- suppressed in ability to enter LN, PP Chemokine desensitization of CCR7 (using high concentrations of CCL21) blocks arrest of rolling cells

14 Chemokine (CCL21) Chemokine (CCL21) Chemokine Receptor (CCR7) G αi GDP GTP G γ G αi GTP G γ G β G β PTX PI3K ERK Signaling Ca ++ Adhesion to HEV, EC, or ECM Migration Cytoskeletal Reorganization Gene Transcription Proliferation Differentiation

15 CCL21 Is Required for Lymphocyte Trafficking Across HEV Short-Term Homing Assay (PLN) 80 *p < PTX CCR7 Desens CCR7 CCL21 mab Cells / Unit Area 40 * * * HEV Chen et al., Nature Immunology, 2006 PTX : CCR7 Desens: CCL21 mab :

17 Dynamic Regulation of Leukocyte-Endothelial Adhesion HEV Adapted from Immunol. Rev Tethering/Rolling L-selectin/PNAd α4β7/madcam-1 Activation CCL21/CCR7 Sticking LFA-1/ICAM-1/2 Transmigration HOMING

18 LAD Leukocyte Adhesion Deficiency TIME MAGAZINE 1992 LAD patient leukocytes do not express LFA-1 (leukocyte-function associated antigen-1) cannot make β2 integrin chain

19 Integrin Family αlβ2 α4β1 α4β7 Nature VOL August 1990

20 LFA-1 is Localized on Planar Membranes of Lymphocytes TK1 cells (low mag) LFA-1 Butcher and von Andrian Cell 199

21 Ig Superfamily Nature VOL August 1990

22 LFA-1 is Required for Lymphocyte Homing Across HEV of Lymphoid Organs Treat + LFA-1 blocking Ab TRITClabeled cells Cells / Unit Area Isotype Control LFA-1 Blocking Ab 1 h Quantify redlabeled cells in organs 50 * * * PLN MLN PP SPL Pancreas Chen et al., Nature Immunol. 2006

23 LFA-1 Binds to Domain 1 (D1) of ICAM-1 Dimers D1 LFA-1 D1 D1-D1 dimer D2 D3 ICAM-1 D2 D4 D5 D3 D4 D5 ICAM-1 D4-D4 dimer D4-D4 dimer Y Yang, Molecular Cell 14: , April 23, 2004

24 CCL21 and ICAM-1 Engagement are both Required for Maximal LFA-1 Affinity Lymphocyte Inside-out Signaling LFA-1 CCR7 Microclusters formed Actin Folded (<5 nm) Low affinity CCL21 Extended (25 nm) Intermediate affinity Extended High affinity ICAM-1 Outside-in Signaling Alon et al., Nature Immunol Endothelium Actin

25 Dynamic Regulation of Leukocyte-Endothelial Adhesion HEV Adapted from Immunol. Rev Tethering/Rolling L-selectin/PNAd α4β7/madcam-1 Activation CCL21/CCR7 Sticking LFA-1/ICAM-1/2 Transmigration LFA-1/ICAM-1/2 HOMING

26 ICAM-1-Enriched Microvilli-Like Projections Form Cup- Like Structure that Guides Transendothelial Migration LFA-1 ICAM-1 Adherent Leukocyte Cup-like Structure (forms in min.) LFA-1 ICAM-1 Endothelial Cell Carman and Springer, J. Cell Biol Shaw et al., J. Exp. Med Yang et al., Blood 2005

27 Lymphocyte-Endothelial Adhesion in Peyer s Patches L-selectin α4β7 integrin CCR7 α4β7 integrin LFA-1 MAdCAM-1 ss ss ss ss ss ss ss ss ss ICAM-1/2 MAdCAM-1 1 CCL21 2 3&4 HEV

28 Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) MAdCAM-1 staining of PP HEV with MECA-367 mab MAdCAM-1 structure Briskin, Butcher et al. Nature 363:461, 1993

29 MAdCAM-1 GLDTSL Ig Domain 1 Ig Domain 2 DE antenna Function blocking mab DATK32, HP1/2 I domain MIDAS Mg ++ β 7 XXXX β propeller α 4 Mucin Domain Ca ++ Lectin L-selectin Ig Domain 3 Glycocalyx 10 nm HEV Function blocking mab MEL-14, DREG-56 Lymphocyte Microvillous Membrane

30 Lymphocyte-Endothelial Adhesion in Lymph Nodes L-selectin CCR7 LFA-1 PNAd ss ss ss ss ss ICAM-1/2 1 CCL21 2 3&4 HEV

31 Homing Receptors Direct Lymphocyte Trafficking Naïve Central Memory L-selectin hi α4β7 + LFA-1 + CCR7 + Lymphoid Tissues (LN, PP) Inflamed Tissues (Autoimmune pathology) Effector Effector Memory L-selectin neg α4β7 hi LFA-1 hi CCR7 neg CLA + CCR5/CXCR3 + Extralymphoid Sites Infection, Inflammation Tumor Tissues

32 Current View of Lymphocyte Trafficking in PLN HEVs Girard JP et al., Nat Rev Immunol. 12:762-73, 2012

33 DCs Maintain Homeostatic Trafficking in HEVs Girard JP et al., Nat Rev Immunol. 12:762, 2012 Moussion C and Girard JP, Nature 13;479, 2011

35 Lymphocyte Recirculation and Recruitment Recruitment Trafficking to extralymphoid sites (HEV-like): Low basal level Active recruitment induced by infection, injury, acute & chronic inflammation Recirculation Efficient trafficking to lymphoid organs: PLN (HEV) Spleen (no HEV) Peyer s Patches (HEV)

36 Dynamic Regulation of Endothelial Adhesion at Sites of Infection/ Injury/ Inflammation Role of cell adhesion receptors in inflammation. An inflammatory response to foregin antigens of bacteria begins with the attraction of leukocytes that release biologically active mediators (A). These cytokines or bacterial products stimulate the expression of adhesion molecules such as ICAM-1, E-selectin, P-selectin, and VCAM-1 on the surface of cuboidal endothelial cells (B). White blood cells then adhere to the activated endothelial cells, migrate through the endothelium and enter areas of infection or tissue damage (D). ( ) Up-regulated adhesion receptors on the surfaces of cells; Endo, endothelium. Control Inflammation ICAM-1 Chen, et al. Nature Immunol. 2006

37 Local Cytokines Increase Lymphocyte Recruitment at Inflammatory Sites TNF-α LTαβ IL-1β IL-6 IFN-γ Sites of Infection, Local Tissue Damage Transcription/ Expression of Chemokines & Vascular Adhesion Molecules ICAM-1 VCAM-1 E/P-selectin CXCL9/10 CCL5

38 mrna Expression in Human Umbilical Vein Endothelial Cells (HUVEC) Following TNF-α Treatment Hrs: VCAM-1 Terminal Ig domains VCAM Lectin, EGF Repeat E-selectin E-selectin

39 TNF-α TNFR Endothelium TRADD TRAF2 RIP MEKK3 P Cytoplasm IKK P IκB P IκB NF-κB Nuclear Translocation Nucleus κbbe VCAM-1 ICAM-1 E-selectin

40 Taming the Flames Within FEBRUARY 17, 2004

41 Venules Lined by High Endothelium in Synovium of Rheumatoid Arthritis Patients RA Jalkanen, Butcher et al. Science Vol. 233:556, 1986

42 Chronic Inflammatory Diseases Associated With HEV-Like Vasculature Disease Affected organ Plump CAMs endothelium on endothelium Crohn s disease Gut + MAdCAM-1/PNAd/ICAM-1 Ulcerative colitis Gut + MAdCAM-1/PNAd/ICAM-1 Diabetes mellitus Pancreas + MAdCAM-1/PNAd/ICAM-1 Grave s disease Thyroid + PNAd/ICAM-1 Hashimoto s thyroiditis Thyroid + PNAd/ICAM-1 Rheumatoid arthritis Synovium + PNAd/ICAM-1 Cutaneous lesions Skin + PNAd/ICAM-1 Sjogren's syndrome Lacrimal/salivary glands + PNAd/MAdCAM-1/ICAM-1 Adapted from J.P. Girard and T.A. Springer, Immunol. Today 16: ; S.D. Rosen, Annual Rev. Immunol. 22:129, 2004.

43 Mechanisms of Neutrophil Recruitment to Inflamed Tissues PMN Mucin-like CAM E/P-Selectin CXCR2 ss ss ss ss ss LFA-1 ICAM-1 1 IL-8/MIP2 KC 2 3&4 Endothelium

44 Intravital Microscopy

45 Ischemia-Reperfusion Injury Control: Ischemia: Reperfusion: Cells move through venules at high velocity (~2400 μm/sec); few adherent cells. Reduced blood flow; distinct cells visible. Increased number of rolling (~40 μm/sec) and adherent cells; adhesion is L-selectin and P- selectin dependent.

46 Process of Metastasis of Malignant Cells Involves Similar Adhesive Mechanisms As Lymphocyte Trafficking R. Hynes, Cell 68: , 1992