1. Homing and Inflammation How do cells migrate from blood into tissue? –the four step model –role of selectins, chemokines and integrins What controls recruitment of appropriate cell types (neutrophils, monocytes, lymphocytes) to the site of inflammation? How do cells exit from tissue into circulation? Are homing defects involved in human disease? Micro 204. Molecular and Cellular Immunology Lecturer: Jason Cyster jason.cyster@ucsf.edu

3. Lymphocyte Homing Constitutive trafficking of naive T and B lymphocytes to secondary lymphoid organs in lymph nodes, Peyer’s patches, tonsil this requires active migration across blood vessels Entry into secondary lymphoid organs is highly selective for lymphocytes Egress from lymphoid organs involves distinct molecular mechanisms from entry

4. Lymphocyte homing and recirculation Adapted from Anderson, artnscience.us (PP)

5. Inflammation involves local release of cytokines and chemokines by tissue cells in response to pathogen products or damage cytokines cause increase in vascular permeability leading to local swelling, increased entry of antibody, complement, etc. cytokines cause increased expression of adhesion molecules on vascular endothelium and these work together with chemokines to recruit cells - neutrophils, monocytes, NK cells and, later, effector lymphocytes

6. The cascade (multistep) model of leukocyte extravasation

7. Selectins are calcium-dependent (C-type) lectins (carbohydrate binding proteins) L-selectin - entry to LNs, PPs –on lymphocytes (neutrophils) –binds specialized sulfated mucins (‘peripheral node addressins’ or PNAd) made by high endothelial venules (HEV) P-selectin - early role in entry to site of inflammation –in Weibel-Palade bodies in endothelial cells and  -granules of platelets –translocates to membrane in response to thrombin, histamine, C5a, etc –binds PSGL-1, a tyrosine sulfated mucin present on neutrophils, some effector T cells E-selectin - delayed role in entry to site of inflammation –cytokine inducible on endothelial cells (especially cutaneous) –binds carbohydrate ligand (sialyl-Le x ) on neutrophil and effector T cell glycoproteins /glycolipids

8. Selectin ligands

9. Lymphocytes traverse high endothelial venules (HEVs) to enter lymph nodes LN section stained with: B220 (pan B cell marker) PNAd (‘peripheral node address’ – sulfated glycans recognized by mAb MECA79) T cell area (paracortex) HEV Follicle or B zone

10. Key property of selectins is fast binding kinetics The rapid kon and koff of selectin–carbohydrate ligand interaction allows flowing leukocytes to tether and roll along endothelial cells under shear flow Rolling slows down flowing leukocytes and places them in proximity to endothelial cells where chemokines are transported and expressed

11. Yang et al. 2000, JEM 190, 1769 Leukocyte rolling immediately after mild trauma depends on P-selectin - PSGL1 interactions wild-typePSGL1-deficient - both movies are of cremaster muscle venules within 30 minutes of surgery (selectins induced by inflammatory trauma) PSGL-1 = P-selectin glycoprotein ligand-1

12. The cascade (multistep) model of leukocyte extravasation

13. GG  NH2 S-S >40, structurally related basic proteins of ~10kD Four families: C, CC, CXC, CX3C chemokine receptor Chemokines Lymphoid chemokines – help direct the homeostatic trafficking of cells into and through peripheral lymphoid tissues (e.g. CCR7-CCL21 and CCL19; CXCR5-CXCL13) Inflammatory chemokines – help recruit cells to sites of inflammation (e.g. CXCR2 / IL-8; CCR2 / MCP1; CCR5 / MIP1  )

14. Chemokine (IL8)-mediated firm adhesion of rolling neutrophils

15. Lymphocyte adhesion to HEV depends on chemokines normal lymph node CCL21/CCL19-deficient lymph node Stein et al., (2000) J. Exp. Med. 191, 61

16. The cascade (multistep) model of leukocyte extravasation

17. Questions / Experiments for group discussion 1.What mechanisms might allow chemokines to act locally on endothelium? 2.Does the chemokine have to be expressed by the endothelial cell? 3.Could other classes of chemoattractants work at this step?

18. The Integrin Family integrins are dimers of  and  chains Most bind extracellular matrix proteins (e.g. fibronectin, laminin) but some have transmembrane ligands

19. Integrins involved in leukocyte attachment to endothelium Integrin Ligands  L  2 ICAM-1, ICAM-2 (ICAM-3) (CD11a/CD18, LFA1)  4  1 VCAM-1, MADCAM-1, FN  MAdCAM-1, VCAM-1, FN ICAM1 and VCAM1 are constitutively expressed on lymph node HEV and are upregulated on inflammed endothelium MAdCAM-1 is expressed by HEV in mucosal lymphoid tissues

20. Integrins can be in resting (low affinity for ligand) and active states intracellular signals cause ‘inside-out’ signaling in the integrin, converting it from an inactive to an active state chemokine signaling can cause ‘inside-out’ signaling and activate integrins ligand binding can cause ‘outside-in’ signaling that also promotes formation of the active state Integrin Activation switchblade-like activation

21. Inside-out signaling occurs by separation of the integrin cytoplasmic domains Kim, Carman & Springer 2003 Science 301, 1720

22. Conformational change of integrin during activation

23. Girard JP, Moussion C, Förster R. Nat Rev Immunol. 2012 Multistep Model of Lymphocyte Transmigration into LNs

24. Diapedesis / transendothelial migration The forward migration of leukocytes across endothelium Integrins may be involved in migration from point of attachment to endothelial cell junctions Additional molecules involved – e.g. monocyte transmigration involves Junctional Adhesion Molecules (JAMs) and PECAM-1 (CD31) Signaling occurs into the endothelial cell May be promoted by shear stress (via mechanoreceptors?)

25. Questions / Experiments for group discussion 1.What mechanisms might allow chemokines to act locally on endothelium? 2.Does the chemokine have to be expressed by the endothelial cell? 3.Could other classes of chemoattractants work at this step? 4.Do leukocytes only cross endothelium at cell-cell junctions?

27. Questions / Experiments for group discussion 1.What mechanisms might allow chemokines to act locally on endothelium? 2.Does the chemokine have to be expressed by the endothelial cell? 3.Could other classes of chemoattractants work at this step? 4.Do leukocytes only cross endothelium at cell-cell junctions? 5.What mechanism might underly tissue-specific homing of lymphocytes? And other cell types?

28. Homing specificity provided by the selectin/chemokine/integrin code

29. Tissue selective chemokine and adhesion molecule expression in the organization of the immune system Kunkel & Butcher (2002) Immunity 16, 1

30. Multistep cascade of lymphocyte migration to site of infection/inflammation: same logic, different ‘area code’ Lipid Mediators (Prostaglandins & leukotrienes) can function as chemoattractants Activated components of complement (C5a, C3a) also act as chemoattractants

31. Leukocyte adhesion deficiency (LAD) type I: –defects in  2 integrin -> defective neutrophil migration to inflammed skin, peritoneum; lymphocytes less affected due to continued use of  –LAD patients have recurrent bacterial infections Experiment: You have a mutant mouse with symptoms of LAD but its integrins are intact. What other classes of molecules might you test as being potentially defective?

32. How do cells exit from lymphoid tissues? MEDULLARY SINUSES Diagram courtesy of Ted Yednock

33. Lymphoid Organ S1PR 1 wt S1PR 1 ko Sphingosine-1-phosphate receptor-1 (S1PR1) is required intrinsically in lymphocytes for: - T cell egress from the thymus - T and B cell egress from peripheral lymphoid organs 20 60 100 140 180 Blood Spleen Peripheral LNs Mesenteric LNs Peyer’s Patches Lymph wt ko CD4 T % of co-transferred wt control Matloubian, Lo, Cinamon, Cyster Nature 2004 1 day after i.v. transfer HEV Efferent lymphatic

34. Sphingosine 1-phosphate (S1P) Abundant in plasma (~1uM) and lymph (~0.1uM) Made intracellulary by all cell types during sphingolipid degradation but only secreted by some cell types Ligand for a family of G-protein coupled receptors (S1PR1-5) S1P receptors have roles in blood vessel and heart development Sphingosine S1P Ethanolamine + Hexadecanal Sph kinases Lipid phosphatases S1P lyase S1PR1 (Edg1) S1PR2S1PR4S1PR5S1PR3

35. blood thymus, spleen S1PR 1 S1P S1P lyase radiation resistant cells RBC lymph lymph node, Peyer’s patch S1PR 1 S1P S1P lyase radiation resistant cells Lymphocytes express S1PR1 and exit lymphoid organs in response to S1P S1P is abundant in blood and lymph RBC major source of blood S1P Lymphatic endothelial cells are source of lymph S1P Extracellular S1P is kept low inside lymphoid tissue S1PR1 is a point of egress control Lymphatic Endothelial cells

36. Lymph Node Egress Pathway? Cortex

37. T cell accumulation within cortical sinusoids requires S1PR1 LYVE-1 is a lymphatic marker Note: the sinsuses are packed with cells Pham et al. 2008 Immunity 28, 122

38. T cells extend processes into cortical sinuses during egress decision-making Grigorova et al. 2009 Nature Immunol. 10, 58

39. Multi-step model of T cell egress 1.Cortical sinus probing 1.S1PR1 dependent entry 3. Capture in a region of flow and exit from lymph node

40. Grigorova et al. 2009 Nature Immunol. 10, 58

42. Innate Immune stimuli cause a generalized lymph node egress shutdown 6 h IFN  Non-specific Ag-specific Infection (e.g. dsRNA) 12 h Lymph Node Lymphocytes

43. Model to account for IFN  -mediated block in egress CD69 Egress promoting signal S1P 1 S1P IFN  receptor CD69 IFN  etc. Innate Immune Stimuli Internalize Egress Blocked

44. CD69 gates migration of activated lymphocytes into cortical sinuses and egress from LN Grigorova et al., 2014 PNAS 107, 20447

45. S1PR 1 Prolonged retention of antigen- activated lymphocytes Antigen activated T cells downregulate S1PR1 mRNA and are retained Recovery of S1PR1 after ~4 cell divisions associated with egress >Retention may help ensure appropriate clonal expansion and receipt of instruction signals before exiting Activated B cells downregulate S1PR1 A subset of plasma cells upregulate S1PR1 and exit spleen, LN to travel to BM Naïve Activated Effector

46. Translational Impact of Homing Research  blocking antibody (Natalizumab/Tysabri®) in use for treatment of Multiple Sclerosis and Crohn’s disease  blocking antibody (Vedolizumab/Entyvio®) approved May 2014 for ulcerative colitis and Crohn’s disease CCR9 antagonist in phase III trial for Crohn’s disease FTY720 (Fingolimod) first oral treatment for relapsing/remitting multiple sclerosis; in trial for ulcerative colitis Inhibitors of prostaglandin and leukotriene synthesis have mutliple anti-inflammatory effects, including antagonizing cell recruitment CXCR4 antagonists used to mobilize HSC from bone marrow Led to advanced understanding of causes of Leukocyte Adhesion Deficiency (LAD)

47. Recommended reading: Girard JP, Moussion C, Förster R. HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes. Nat Rev Immunol. 2012. 12:762-73 Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu Rev Immunol. 2012. 30:69-94. Sperandio M, Gleissner CA, Ley K. Glycosylation in immune cell trafficking. Immunol Rev. 2009 230:97-113 Muller WA. Mechanisms of transendothelial migration of leukocytes. Circ Res. 2009 105:223- 30 Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol. 2008 20:533-40 Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, Sarnacki S, Cumano A, Lauvau G, Geissmann F. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007 317:666-70. Shamri R, Grabovsky V, Gauguet JM, Feigelson S, Manevich E, Kolanus W, Robinson MK, Staunton DE, von Andrian UH, Alon R. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol. 2005 6:497-506. von Andrian, U. H. and Mempel, T. R. 2003. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3, 867 Lowe JB. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr Opin Cell Biol. 2003 15:531-8 Carman CV, Springer TA. Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr Opin Cell Biol. 2003 Kunkel & Butcher (2002) Chemokines and the tissue-specific migration of lymphocytes. Immunity 16, 1