Homing and Inflammation!

Transcription

1 Homing and Inflammation! Micro 204. Molecular and Cellular Immunology! Lecturer: Jason Cyster! 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?!

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 Lymphocytes traverse HEVs to enter lymph nodes Follicle or B zone HEV T cell area (paracortex) LN section stained with: B220 PNAd

4 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

5 The cascade (multistep) model of leukocyte extravasation! 1) Tethering and Rolling Blood Flow 2) Triggering/Activation of Integrins 3) Firm Adhesion 4) Diapedesis Endothelial Cells Selectins Chemokines Integrins

6 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 α-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!

7 Selectin ligands

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!

9 The cascade (multistep) model of leukocyte extravasation! 1) Tethering and Rolling Blood Flow 2) Triggering/Activation of Integrins 3) Firm Adhesion 4) Diapedesis Endothelial Cells Selectins Chemokines Integrins

10 Chemokines! >40, structurally related basic proteins of ~10kD! Four families: C, CC, CXC, CX3C chemokine! NH2 S-S receptor! Gα β γ 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 / MIP1α)

11 The Integrin Family! integrins are dimers of α and β chains! Most bind extracellular matrix proteins (e.g. fibronectin, laminin) but some have transmembrane ligands!

12 Integrins involved in leukocyte attachment to endothelium! Integrin! Ligands! αlβ2!! ICAM-1, ICAM-2 (ICAM-3)! (CD11a/CD18, LFA1)! α4β1!! VCAM-1, MADCAM-1, FN! α4β7! 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!

13 Integrin Activation 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! switchblade-like activation

14 Inside-out signaling occurs by separation of the integrin cytoplasmic domains! Kim, Carman and Springer (2003). Science 301, 1720

15 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?

16 Multistep Model of Lymphocyte Transmigration into Lymph Nodes ligand

17 Diapedesis / transendothelial migration! The forward migration of leukocytes through endothelial junctions or the body of the endothelial cell Integrins may be involved in migration of cells 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?)

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20 Homing specificity provided by the selectin/chemokine/integrin code!

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

22 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

23 Leukocyte adhesion deficiency (LAD) type I:! defects in β2 integrin -> defective neutrophil migration to inflammed skin, peritoneum; lymphocytes less affected due to continued use of α4β1, α4β7! 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?

24 How do cells exit from lymphoid tissues?! MEDULLARY SINUSES! Diagram courtesy of Ted Yednock

25 S1PR1 is required intrinsically in lymphocytes for:! - T cell egress from the thymus! - T and B cell egress from peripheral lymphoid organs! Lymphoid Organ! S1PR 1 wt S1PR 1 ko % of co-transferred wt control! 180! 140! 100! 60! 20! CD4 T! wt! ko! Blood! Spleen! Peripheral! LNs! Mesenteric! LNs! Peyer s! Patches! Lymph! Matloubian, Lo, Cinamon, Cyster Nature 2004

26 Sphingosine 1-phosphate (S1P)! Sph kinases! Sphingosine! Lipid phosphatases! S1P! S1P lyase! Ethanolamine! +! Hexadecanal!! S1PR1 S1PR2 S1PR3 S1PR4 S1PR5 (Edg1) 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!

27 Lymphocytes express S1PR1 and exit lymphoid organs in response to S1P! thymus, spleen lymph node, Peyer s patch S1PR 1 S1PR 1 S1P lyase S1P lyase radiation resistant cells blood S1P RBC Lymphatic radiation resistant Endothelial cells cells lymph 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!

28 Lymph Node Egress Pathway?! Cortex

29 T cell accumulation within cortical sinusoids requires S1P 1! LYVE-1 is a lymphatic marker Note: the sinsuses are packed with cells

30 Multi-step model of T cell egress 1. Cortical sinus probing 2. S1PR1 dependent entry 3. Capture in a region of flow and exit from lymph node

31 Innate Immune stimuli cause a generalized lymph node egress shutdown Lymph Node! Infection! (e.g. dsrna)! 6 h IFNα/β! 12 h Non-specific! Ag-specific! Lymphocytes!

32 Model to account for IFNα/β-mediated block in egress Innate Immune Stimuli! IFNα/β etc.! S1P! IFNα/β! receptor! CD69! S1P 1! Internalize! CD69! Egress promoting signal! Egress Blocked!

33 Prolonged retention of antigenactivated lymphocytes S1PR 1 Activated 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 Naïve Effector Activated B cells downregulate S1PR1 A subset of plasma cells upregulate S1PR1 and exit spleen, LN to travel to BM

34 Translational Impact of Homing Research α4β1 blocking antibody (Natalizumab/Tysabri ) in use for treatment of Multiple Sclerosis and Crohn s disease FTY720 (Fingolimod) first oral treatment for relapsing/remitting MS Inhibitors of prostaglandin and leukotriene synthesis have mutliple anti-inflammatory effects, including antagonizing cell recruitment CXCR4 antagonists used to mobilize HSC from bone marrow CCR9 antagonist in phase III trial for Crohn s disease Led to advanced understanding of causes of Leukocyte Adhesion Deficiency (LAD)

35 Experiments for group discussion -> You have a mouse that has reduced numbers of lymphocytes in its LNs. How do you test if this is due to defective homing into the LN? How does the experiment distinguish an entry defect from a retention defect/accelerated egress?

36 Recommended reading:! Girard JP, Moussion C, Förster R. HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes. Nat Rev Immunol :762-73! Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu Rev Immunol :69-94.! Sperandio M, Gleissner CA, Ley K. Glycosylation in immune cell trafficking. Immunol Rev :97-113! Muller WA. Mechanisms of transendothelial migration of leukocytes. Circ Res :223-30! Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol :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 : ! 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 : ! von Andrian, U. H. and Mempel, T. R 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 :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!!