Homing and Inflammation

1. Homing and Inflammation How do cells exit 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?

2. 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

3. 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

4. The cascade (multistep) model of leukocyte extravasation

5. 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) Can be shed upon lymphocyte activation P-selectin - early role in entry to site of inflammation in Weibel-Palade bodies in endothelial cells and a-granules of platelets translocates to membrane in response to thrombin, histamine, C5a, etc binds PSGL-1, a tyrosine sulfated mucin - 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-Lex) on neutrophil glycoproteins /glycolipids and cutaneous leukocyte antigen (CLA) on effector T cells The initial step is rolling, which is mediated by selectins and their carbohydrate ligands and, additionally,a4 integrins. 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.The initial step is rolling, which is mediated by selectins and their carbohydrate ligands and, additionally,a4 integrins. 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.

6. Carbohydrate selectin ligands. Sialyl Lewis X (sLex) binds all three selectins with modest affinity (indicated in boxes as Kd or IC50) (14�16). Binding of P-selectin is improved by core 2 and adjacent Y-SO4 (left) (15). Binding to L-selectin is enhanced by GlcNAc-6-sulfation (right) (16). The precise requirements for E-selectin binding beyond sLex are not known. Binding affinities of selectins to natural glycoprotein ligands like GlyCAM-1 are much higher, with apparent Kd in the nM range, suggesting that other determinants are involved or ligand clustering occurs. GlcNAc-6-O-sulfation is catalyzed by GlcNAc-6-O-sulfotransferases. Tyrosine O-sulfation occurs in the trans-Golgi network and is catalysed by two known tyrosyl protein sulfotransferases (Tpst1 and 2) which transfer a sulfuryl group from the sulfate donor 3�-phosphoadenosine 5�-phosphosulfate (PAPS) to the hydroxyl group of peptidyl-tyrosine. Immunological Reviews 2009 (Klaus Ley) Vol. 230: 97�113 Carbohydrate selectin ligands. Sialyl Lewis X (sLex) binds all three selectins with modest affinity (indicated in boxes as Kd or IC50) (14�16). Binding of P-selectin is improved by core 2 and adjacent Y-SO4 (left) (15). Binding to L-selectin is enhanced by GlcNAc-6-sulfation (right) (16). The precise requirements for E-selectin binding beyond sLex are not known. Binding affinities of selectins to natural glycoprotein ligands like GlyCAM-1 are much higher, with apparent Kd in the nM range, suggesting that other determinants are involved or ligand clustering occurs. GlcNAc-6-O-sulfation is catalyzed by GlcNAc-6-O-sulfotransferases. Tyrosine O-sulfation occurs in the trans-Golgi network and is catalysed by two known tyrosyl protein sulfotransferases (Tpst1 and 2) which transfer a sulfuryl group from the sulfate donor 3�-phosphoadenosine 5�-phosphosulfate (PAPS) to the hydroxyl group of peptidyl-tyrosine. Immunological Reviews 2009 (Klaus Ley) Vol. 230: 97�113

7. Selectin ligands are complex carbohydrates

8. 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 a4 integrins can also support rolling interactionsa4 integrins can also support rolling interactions

9. Glyco-Sulfotransferase deficiency leads to faster L-selectin mediated rolling and reduced ability to undergo the rolling-to-sticking transition

10. 

11. The cascade (multistep) model of leukocyte extravasation

12. Cyster (1999) Science 286, 2098Cyster (1999) Science 286, 2098

13. Chemokines in Inflammation The large number of chemokines and chemokine receptors allows for a significant amount of homing specificity to be imparted by these molecules Examples: (PARTIAL LIST) Cell type Chemokine Receptors Ligands Neutrophils CXCR1, CXCR2 IL-8, GCP-2, Gro-a Eosinophils CCR1, CCR3 Eotaxin, MIP-1a, MCP-3 Monocytes CCR1, CCR2, CCR5 MCP-1, 2, 3, 5, RANTES, MIP-1a Na�ve T CCR7, CXCR4 SLC, SDF-1 Na�ve B CXCR5, CXCR4, CCR7 BLC, SDF-1, SLC Th1 effector CCR2, CCR5, CXCR3 MIP-1a, MCP1, RANTES, IP10 Th2 effector CCR3, CCR4, CCR8 Eotaxin, MDC, TARC, I309 CD8 effector CCR2, CCR5, CXCR3 MCP1, MIP1a, RANTES, IP10 Immature DC CCR1,2,3,4,5,6 MCP-1, 2, 3, 5, RANTES, MIP-1a (see Zlotnik and Yoshie, Immunity 12, 121 (2000) for standardized chemokine nomenclature) Other chemoattractants: - monocytes, neutrophils are attracted by C5a, fMLP, PAF - Th2 cells, eosinophils, basophils express CRTH2, a receptor for prostaglandin D2

14. Chemokine code required for T and B cell entry to lymph nodes and Peyer�s patches Also, can be transported across the EC by non-signaling chemokine receptors (e.g. duffy transports IL-8) or possibly by the chemokine receptors themselvesAlso, can be transported across the EC by non-signaling chemokine receptors (e.g. duffy transports IL-8) or possibly by the chemokine receptors themselves

15. The cascade (multistep) model of leukocyte extravasation

16. A and b pairing forms 24 heterodimersA and b pairing forms 24 heterodimers

18. A switchblade-like model for integrin activation

19. Integrin Activation

20. Leukocyte adhesion deficiency (LAD) type I: defects in b2 integrin -> defective neutrophil migration to inflammed skin, peritoneum; lymphocytes less affected due to continued use of a4b1, a4b7 LAD patients have recurrent bacterial infections Other types of LAD involve defects in expression of glycosyltransferases needed to make selectin ligands and defects in intracellular signaling molecules needed for chemokine-mediated integrin activation LAD II is a defect in a the gene encoding a multispanning transmembrane protein that transports GDP-fucose from its cytosolic site of synthesis to the lumen of the Golgi. In LAD II patients (and FX-null mice) neutrophils are devoid of E- and P-selectin activites.LAD II is a defect in a the gene encoding a multispanning transmembrane protein that transports GDP-fucose from its cytosolic site of synthesis to the lumen of the Golgi. In LAD II patients (and FX-null mice) neutrophils are devoid of E- and P-selectin activites.

21. Inside-out signaling occurs by separation of the integrin cytoplasmic domains Bidirectional Transmembrane Signaling by Cytoplasmic Domain Separation in Integrins Minsoo Kim,* Christopher V. Carman,* Timothy A. Springer - Science, Vol 301, Issue 5640, 1720-1725 Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 ({alpha}L�2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein�fused and yellow fluorescent protein�fused {alpha}L and �2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the {alpha} and � cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane. (The cytoskeletal protein, Talin, is involved in promoting a spatial separation of the cytoplasmic domains).Bidirectional Transmembrane Signaling by Cytoplasmic Domain Separation in Integrins Minsoo Kim,* Christopher V. Carman,* Timothy A. Springer - Science, Vol 301, Issue 5640, 1720-1725 Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 ({alpha}L�2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein�fused and yellow fluorescent protein�fused {alpha}L and �2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the {alpha} and � cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane. (The cytoskeletal protein, Talin, is involved in promoting a spatial separation of the cytoplasmic domains).

23. Diapedesis / transendothelial migration �The forward migration of leukocytes through endothelial junctions� Integrins may be involved in migration of cells from point of attachment to EC junctions monocyte transmigration regulated by several cell adhesion molecules e.g. JAMs, PECAM1 (CD31), CD99 Shear stress (via mechanoreceptors?) may be involved in promoting diapedesis Some cells may migrate through the body of the endothelial cell after diapedesis further molecular steps govern migration across the subendothelial basal lamina and through interstitial tissues

24. Integrins function during transmigration Additional molecules are likely to be involved Monocyte transmigration involves Junctional Adhesion Molecules (JAMs) and PECAM-1 (CD31) Signaling occurs into the Endothelial Cell Figure 1. Monocyte locomotion on vascular endothelium: directional transport to the site of transmigration (TEM) and an 'educational' process for essential leukocyte and endothelial cell polarization. Nature Immunology 5, 351 - 353 (2004) I and II, arrested leukocytes require beta2 integrin ligands (ICAMs) to reach endothelial junctions. III, integrins and their respective endothelial ligands at the leukocyte-endothelial interface must rearrange specialized cytoskeletal networks for initiation of TEM8. IV, integrin blockade prevents both directional locomotion and formation of the TEM leukocyte-endothelial synapse. JAMs, junctional adhesion molecules; VE, vascular endothelial; PECAM, platelet-endothelial cell adhesion molecule.Figure 1. Monocyte locomotion on vascular endothelium: directional transport to the site of transmigration (TEM) and an 'educational' process for essential leukocyte and endothelial cell polarization. Nature Immunology 5, 351 - 353 (2004) I and II, arrested leukocytes require beta2 integrin ligands (ICAMs) to reach endothelial junctions. III, integrins and their respective endothelial ligands at the leukocyte-endothelial interface must rearrange specialized cytoskeletal networks for initiation of TEM8. IV, integrin blockade prevents both directional locomotion and formation of the TEM leukocyte-endothelial synapse. JAMs, junctional adhesion molecules; VE, vascular endothelial; PECAM, platelet-endothelial cell adhesion molecule.

25. proposed that dynamic podosomes-like protrusions serve to stochastically �probe� or �palpate� the endothelial surface as a means to efficiently identify locations of relatively low endothelial resistance, at which, protusions are able to progressively extend thereby driving trans-cellular pore formation Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol. 2008 20:533-40 proposed that dynamic podosomes-like protrusions serve to stochastically �probe� or �palpate� the endothelial surface as a means to efficiently identify locations of relatively low endothelial resistance, at which, protusions are able to progressively extend thereby driving trans-cellular pore formation Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol. 2008 20:533-40

27. Matrix metalloproteases - mediators of extracellular proteolysis Once in the tissue, cells have to digest extracellular matrix to move protease release induced by activation e.g. IL8 induces gelatinase release from neutrophils Types of metalloproteases 1. collagenases 2. gelatinases 3. stromelysins 4. membrane-type metalloproteases

28. Important: the chemokine receptor is on the lymphocyte and the chemokine is made by the tissue cells Important: the chemokine receptor is on the lymphocyte and the chemokine is made by the tissue cells

29. Tissue selective chemokine and adhesion molecule expression in the systemic organization of the immune system One thing we�ll come back to is that effector T cells are �imprinted� in the draining lymphoid organ to acquire homing properties that direct them to the appropriate type of tissueOne thing we�ll come back to is that effector T cells are �imprinted� in the draining lymphoid organ to acquire homing properties that direct them to the appropriate type of tissue

30. Drugs that inhibit lymphocyte egress from lymphoid organs (e.g. FTY720) are being tested as immunosuppressants e.g. in transplant and MS patientsDrugs that inhibit lymphocyte egress from lymphoid organs (e.g. FTY720) are being tested as immunosuppressants e.g. in transplant and MS patients

31. 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 (S1P1-5, formerly known as EDG receptors) S1P receptors have roles in blood vessel and heart development

32. Lymphocytes express S1P1 and exit lymphoid organs in response to S1P

33. Nature 427, 355 (2004) Nature 427, 355 (2004)

34. Go over anatomy basics: cortex is composed of T cell rich area and B cell follicles; medulla is a macrophage and lymphatics sinus rich area and historically has been thought of as the site of egress; lymph flows in via afferents and travels via the subcapsular space to the medullary sinuses Lmb3 - Theofilopoulos, D. Kono DOCK3 - Y. Fukui; J. Stein Go over anatomy basics: cortex is composed of T cell rich area and B cell follicles; medulla is a macrophage and lymphatics sinus rich area and historically has been thought of as the site of egress; lymph flows in via afferents and travels via the subcapsular space to the medullary sinuses Lmb3 - Theofilopoulos, D. Kono DOCK3 - Y. Fukui; J. Stein

35. T cell accumulation within cortical sinusoids requires S1P1 Note that structures appear packed full of cells Trung Pham et al., Immunity 2008 Note that structures appear packed full of cells Trung Pham et al., Immunity 2008

37. Proposed model for S1P1 and CCR7 action during cortical sinus �entry decision-making�

38. Innate Immune stimuli cause a generalized lymph node egress shutdown 1. Hall, J. G. & Morris, B. The immediate effect of antigens on the cell output of a lymph node. Br. J. Exp. Pathol. 46, 450-454 (1965). 2. Degre, M. Influence of polyinosinic: polycytidylic acid on the circulating white blood cells in mice. Proc Soc Exp Biol Med 142, 1087-1091 (1973). 3. Korngold, R., Blank, K. J. & Murasko, D. M. Effect of interferon on thoracic duct lymphocyte output: induction with either poly I:poly C or vaccinia virus. J Immunol 130, 2236-2240 (1983). 4. Kalaaji, A. N., Abernethy, N. J., McCullough, K. & Hay, J. B. Recombinant bovine interferon-alpha I 1 inhibits the migration of lymphocytes from lymph nodes but not into lymph nodes. Reg Immunol 1, 56-61 (1988). 5. Young, A. J., Seabrook, T. J., Marston, W. L., Dudler, L. & Hay, J.1. Hall, J. G. & Morris, B. The immediate effect of antigens on the cell output of a lymph node. Br. J. Exp. Pathol. 46, 450-454 (1965). 2. Degre, M. Influence of polyinosinic: polycytidylic acid on the circulating white blood cells in mice. Proc Soc Exp Biol Med 142, 1087-1091 (1973). 3. Korngold, R., Blank, K. J. & Murasko, D. M. Effect of interferon on thoracic duct lymphocyte output: induction with either poly I:poly C or vaccinia virus. J Immunol 130, 2236-2240 (1983). 4. Kalaaji, A. N., Abernethy, N. J., McCullough, K. & Hay, J. B. Recombinant bovine interferon-alpha I 1 inhibits the migration of lymphocytes from lymph nodes but not into lymph nodes. Reg Immunol 1, 56-61 (1988). 5. Young, A. J., Seabrook, T. J., Marston, W. L., Dudler, L. & Hay, J.

39. Model to account for IFNa/b-mediated block in egress

40. Prolonged retention of antigen-activated lymphocytes Antigen activated T cells downregulate S1P1 mRNA and are retained Recovery of S1P1 after ~4 cell divisions associated with egress Retention may help ensure appropriate clonal expansion and receipt of instruction signals before exiting

41. Recommended reading: 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 Cyster, JG. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu Rev Immunol. 2005;23:127-59 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