Leukocyte Migration and Inflammation 1-26-2018 Janusz Kabarowski janusz@uab.edu 6-2082

INFLAMMATION IS A PRIMARY RESPONSE TO INFECTION OR TISSUE INJURY. The primary objective of inflammation is to localize and eradicate the infectious agent responsible and to facilitate subsequent tissue repair mechanisms. Photomicrographs show leukocyte infiltration in response to inflammation in skin tissue.

ADHESION MOLECULES General Properties: 1. 2. Cell to Cell Contact Functions in Immune Responses: Cell to Cell Contact Homing for tissue development Recruitment of phagocytic cells (neutrophils and monocyte-derived macrophages) Homing of naïve/effector/memory lymphocytes

Adhesion Molecule Biology Techniques - How they do it 1. Intra-vital microscopy 2. Flow Chamber Systems

Timothy A. Springer, Harvard Medical School Intravital Microscopy - the mouse mesentery preparation. In vivo microscopy of postcapillary venules Timothy A. Springer, Harvard Medical School

Timothy A. Springer, Harvard Medical School Parallel Plate Flow Chamber - visualization and measurement of interactions between flowing leukocytes and adhesion molecules coated directly on a flow chamber slide, or expressed on a cell monolayer (e.g. endothelial cells). A vacuum holds the top and bottom of the parallel plate flow chamber together. Medium then flows through the chamber along the flow channel. A syringe pump is also used to circulate fresh medium throughout the system at a specific rate. Flow rate determines the shear stress, which is measured in dynes/cm2. 1 dyne is the force required to accelerate a mass of 1 gram at a rate of 1cm per second squared. The flow chamber is utilized at various flow rates to produce varying degrees of shear stress (from low shear stress of ~0.7 dynes/cm2 up to high shear stress of ~7 dynes/cm2). Timothy A. Springer, Harvard Medical School

Timothy A. Springer, Harvard Medical School Recording leukocyte adhesion to endothelial monolayers under conditions mimicking normal blood flow Timothy A. Springer, Harvard Medical School

The Big Picture - leukocyte adhesion cascade Endothelial Cells Rolling Activation Blood Flow --> Firm Adhesion Neutrophil Diapedesis The leukocyte adhesion cascade is a series of adhesion and activation events that culminate in the extravasation (diapedesis) of leukocytes across the endothelium at sites of infection/inflammation where they can ultimately exert effects aimed at eradicating the infectious or injurious agent (phagocytosis, release of bacteriacidal molecules by neutrophils). Basement membrane

Physical Factors Influencing Leukocyte Migration 1. 2. 3. Blood Flow Physical Barriers Ability to Detect Infection

MOLECULES CONTROLING LEUKOCYTE MIGRATION 1. 2. 3. 4. SELECTINS - Family of C-type Lectins INTEGRINS - Family of Heterodimeric Receptors; b2-integrins IMMUNOGLOBULIN (Ig) SUPERGENE FAMILY Composed of Ig Domains --> CAMs SIGNALING MOLECULES (Receptors/Ligands) Chemoattractant and Chemokine Receptors

Step 1: Rolling

Selectins & Carbohydrate (CHO) Ligands Step 1: Rolling Selectins & Carbohydrate (CHO) Ligands * Endothelium Leukocyte P-Selectin L-Selectin Proteins with specific carbohydrate groups on their surface (glycoproteins) act as ligands for Selectins. P-Selectin and E-Selectin are expressed at low levels on normal endothelium, but their expression is induced on the surface of endothelial cells in response to inflammatory mediators. P-Selectin Glycoprotein Ligand-1 (PSGL-1) on leukocytes binds P-Selectin, a major adhesion molecule expressed on the surface of activated endothelial cells involved in the capture and subsequent rolling of leukocytes at endothelium adjacent to sites of infection/inflammation. E-Selectin

Sialyl Lewis X (sLeX) is a Carbohydrate determinant on Selectin Ligands - e.g. P-Selectin Glycoprotein Ligand-1 (PSGL-1) The carbohydrate sLeX is a tera-saccharide comprising N-Acetylneuraminic acid (Neu5Ac), Galactose (Gal), N-Acetylglucosamine (GlcNAc) and Fucose (Fuc).

The affinity of Selectin-CHO binding is sufficiently low so that the binding between endothelial cells and the leukocyte is transient, resulting in “rolling” of the leukocyte over the endothelial surface as Selectin-CHO interactions are sequentially made and broken under the influence of blood flow.

Video Acknowledgment: Dr. Tim Springer, Harvard University

Video Acknowledgment: Dr. Tim Springer, Harvard University

X LAD II - Leukocyte Adhesion Deficiency Type 2 Incomplete glycosylation of sLex: no binding to selectins Many cases of LAD II are due to a metabolic disorder in the production of GDP-L-Fucose which is critical for the generation of Sialyl Lewis X (sLeX) carbohydrate groups on Selectin ligands. X GDP-L-Fucose

Incomplete sLeX glycosylation prevents selectin-dependent rolling.

Step 2: Activation

Chemotactic Receptors Step 2: Activation Chemotactic Receptors & Ligands Endothelium Leukocyte fMLP IL-8 C5a MCP1 Infection Integrin Molecules that act as ligands for chemotactic receptors expressed on leukocytes are produced during infection. Some are bacterial products, such as fMLP (N-Formyl Methionyl Leucyl Phenylalanine), others are produced by endothelial cells and/or resident tissue macrophages (IL-8: Interleukin-8, MCP1: Monocyte Chemotactic Protein-1). C5a is a proteolytic fragment of Complement 5 produced in response to inflammation.

Chemotactic Receptors C5a PAF LTB4 LPC MCP1 fMLP IL-8 Plasma membrane Diverse molecules act as chemokines. For example, N-formyl Methionyl Leucyl Phenylalanine (fMLP) is a peptide produced by bacteria, MCP1 (Monocyte Chemotactic Protein-1) and IL-8 (Interleukin-8) are produced by endothelial cells, C5a (Complement 5 anaphylatoxin) is a proteolytic fragment of C5 with potent chemotactic activity towards neutrophils and capable of stimulating histamine release from mast cells. PAF (Platelet Activating Factor) and LPC (Lysophosphatidylcholine) are chemotactic lipids produced by injured cells and activated macrophages.   Heterotrimeric G protein Complex

Integrin Molecules (2 Integrins) Ligand Binding α β I Domain Ca++, Mg++ b-Subunit (CD18) a-Subunit (CD11a-c) Beta-2 integrins include Leukocyte Function-associated Antigen-1: LFA-1 (CD11a/CD18), MAC-1 (CD11b/CD18) and p150/95 (CD11c/CD18). The “I domain” of the alpha subunit is a divalent cation binding site that is important for the activation of integrins to a high affinity binding state. Leukocyte

Activation of Integrins by Chemokine Receptors Activated (high affinity) Low affinity β α β α Chemokines activate chemotactic receptors of the G protein-coupled receptor (GPCR) family which signal via heterotrimeric G proteins. Signaling responses include the release of intracellular calcium (Ca++) which contributes to the activation of integrins through the divalent cation binding “I domain” of the alpha subunit. Gq/11 GTP G13 Gi

Endothelium Immunoglobulin Supergene Family VCAM PECAM ICAM-1 Ig Domain ICAM-2 In addition to extracellular matrix components such as fibronectin, integrins bind cell adhesion molecules (CAMs) of the Immunoglobulin Supergene Family. ICAM-1 (Intercellular Cell Adhesion Molecule-1), ICAM-2 (Intercellular Cell Adhesion Molecule-2), VCAM (Vascular Cell Adhesion Molecule), PECAM (Platelet Endothelial Cell Adhesion Molecule).

Step 3: Firm Adhesion

Infection Inflammatory Mediators

Infection Activation and firm adhesion Chemokines Inflammatory Mediators Infection

Firm adhesion of Leukocytes in response to Chemokine addition Video Acknowledgment: Dr. Tim Springer, Harvard University

Video Acknowledgment: Dr. Tim Springer, Harvard University

LAD I - Leukocyte Adhesion Deficiency Type 1 Missing functional CD18 : no expression of CD11a-c

Normal human Neutrophils Roll, are Activated, and undergo Firm Arrest. Healthy neutrophils roll a cell diameter or two on platelets, are activated, and arrest through integrins LFA-1 and Mac-1 Normal human Neutrophils Roll, are Activated, and undergo Firm Arrest. This is a succession of clips from different time points . First neutrophils accumulate on the substrate, and then the flow rate and hence the wall shear stress is increased. The healthy neutrophils remain firmly adherent at the highest shears. Diacovo, T. G., Roth, S. J., Buccola, J. M., Bainton, D. F., and Springer, T. A. (1996). Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the 2-integrin CD11b/CD18, Blood 88, 146-157. Weber, C., and Springer, T. A. (1997). Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to IIb3 and stimulated by platelet-activating factor, J Clin Invest 100, 2085-2093. Video Acknowledgment: Dr. Tim Springer, Harvard University

2 Integrin-deficient Neutrophils from LAD1 patients roll and become activated, but lack integrins LFA-1 and MAC-1 so do not arrest. This is a succession of clips from different time points . First neutrophils accumulate on the substrate, and then the flow rate and hence the wall shear stress is increased. The LAD neutrophils roll at the higher shear stresses, because they lack  integrins and do not develop firm adhesion to the substrate. Diacovo, T. G., Roth, S. J., Buccola, J. M., Bainton, D. F., and Springer, T. A. (1996). Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the 2-integrin CD11b/CD18, Blood 88, 146-157. Weber, C., and Springer, T. A. (1997). Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to IIb3 and stimulated by platelet-activating factor, J Clin Invest 100, 2085-2093. Video Acknowledgment: Dr. Tim Springer, Harvard University

Step 4: Diapedesis

Step 4: Diapedesis PECAM:PECAM Integrin:ICAM JAM-1:Integrin The transmigrating leukocyte extravasates between neighboring endothelial cells without disrupting the integrity of the endothelial barrier. This is accomplished by homotypic binding between PECAM on the leukocyte and PECAM expressed on the endothelial cell at tight junctions. Activated integrins such as LFA-1 (CD11a/CD18) on the surface of the leukocyte can similarly “compete” for binding to Junctional Adhesion Molecule-1 (JAM-1) expressed at tight junctions between endothelial cells and thus facilitate leukocyte extravasation into the adjacent inflamed tissue. JAM-1:Integrin JAM-1:JAM-1

Video Acknowledgment: Dr. Tim Springer, Harvard University

Endothelial Cells Blood Flow Leukocyte Rolling Selectin-mediated 1 Activation Cytokine-mediated 2 Firm Adhesion Diapedesis Integrin-mediated 3 4

Phagocytosis: Getting to the Target The cellular actin cytoskeleton is associated with the intracellular portion of integrins. Activation of integrins results in signaling events that induce modification of the actin cytoskeleton (such as focal adhesion complex and stress fiber formation) that provides traction for diapedesis. Metalloproteinases Integrin-dependent Pulling (Traction) Cytoskeleton and Molecular Motors

LYMPHOCYTE TRAFFICKING AND IMMUNE SURVEILLANCE Photomicrograph of a High Endothelial Venule (HEV) through which lymphocytes enter lymph nodes.

Peripheral Lymph Nodes, Peyer’s Patch, Spleen B or T cells regions LYMPHOCYTE RECIRCULATION Bone Marrow Thymus B Cells T Cells T Cells Peripheral Lymph Nodes, Peyer’s Patch, Spleen B or T cells regions + Antigen Germinal Center T cells Zone Naïve B and T lymphocytes produced in the bone marrow and thymus respectively recirculate between the blood and secondary lymphoid organs in which antigens are presented. Continual recirculation between the blood and lymph nodes (green arrows) is vital for efficient antigen surveillance by naïve T lymphocytes. Having encountered its cognate antigen through its antigen receptor, a naïve T lymphocyte becomes activated, undergoes clonal proliferative expansion, and differentiates into an antigen-specific effector T cell capable of homing to sites of inflammation throughout the body (rather than recirculating through lymph nodes). Entry of these effector T lymphocytes into inflamed sites in which the antigen is present results in the activation of their effector functions (cytokine production, modulation of other inflammatory cell-types). Plasma B Cells EffectorT cells Bone Marrow, Mucosa, etc Sites of Infection/Inflammation

LYMPHOCYTE TRAFFICKING AND IMMUNE SURVEILLANCE - naïve T Lymphocytes recirculate through Lymph Nodes via High Endothelial Venules (HEVs) T Blood Thoracic duct T Efferent lymphatic B T T HEV L-selectin - PNAd CCR7 - SLC Specific adhesion molecules and chemokines control naïve T lymphocyte entry into lymph nodes through HEVs. L-Selectin expressed on the surface of naïve T lymphocytes binds Peripheral Node Addresin (PNAd) constitutively expressed on the surface of HEVs. The chemotactic receptor, CCR7, on naïve T lymphocytes is activated by Secondary Lymphoid tissue Chemokine (SLC) produced by endothelial cells lining the HEV. Antigen Afferent lymphatic

Naïve Lymphocyte Homing in Lymph Nodes Step 1: Rolling L-selectin PNAd PNAd - peripheral node addresin Step 2: Activation CCR7 SLC GAG SLC - secondary lymphoid tissue chemokine GAG - glycosaminoglycan Step 3: Firm Adhesion ICAM-1 LFA-1 Peripheral Node Addressin (PNAd) is the major L-Selectin binding ligand mediating entry of naïve T lymphocytes into lymph nodes. Secondary Lymphoid Tissue Chemokine (SLC) is an important chemokine mediating integrin activation on the surface of naïve T lymphocytes entering lymph nodes. Note that SLC is present on the surface of HEV endothelial cells bound to Glycosaminoglycan (GAG) long-chain carbohydrates. HEV

L-selectin and the Hyaluronan Receptor, CD44, control Naïve T Lymphocyte homing to Lymph Nodes and entry into “T cell areas” Effector T cells (CD44highL-selectinlow) Inflamed Tissues Lymph Nodes CD44 expression L-Selectin is an adhesion receptor for Peripheral Node Addresin (PNAd) constitutively expressed on the surface of endothelial cells lining HEVs. High levels of surface L-Selectin expression by naïve T lymphocytes therefore allows them to initiate rolling on the endothelium lining HEVs. Differential expression of L-Selectin by naïve versus effector T lymphocytes contributes to their different homing characteristics: L-Selectinhigh naïve T lymphocytes preferentially home to lymph nodes, while L-Selectinlow effector T lymphocytes preferentially home to sites of inflammation. Naïve T cells (CD44highL-selectinhigh) L-selectin expression

Naïve Lymphocytes Home into Lymph Nodes through High Endothelial Venules (HEVs) Video Acknowledgment: Dr. Tim Springer, Harvard University

Migration of Naïve Lymphocytes to T cell areas of Lymph Nodes involves interaction of Lymphocyte CD44 with Hyaluronan CD44 is a receptor for Hyaluronan (a long chain carbohydrate). In lymph nodes, collagen fiber tracts decorated with Hyaluronan extend from HEVs to the T cell areas. Clark, R. A., Alon, R., and Springer, T. A. (1996). CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma, J Cell Biol 134, 1075-1087. While high L-Selectin expression by naïve T lymphocytes contributes to their preferential homing to lymph nodes via HEVs, once in the lymph node, CD44 expressed on their surface directs their movement towards T cell areas where they may encounter their cognate antigen. Video Acknowledgment: Dr. Tim Springer, Harvard University

Naïve Lymphocyte Homing in Peyer’s Patches (Mucosa-associated Lymphoid Tissue of the small intestine) Step 1: Rolling L-selectin MAdCAM-1 MAdCAM-1 - mucosal addresin CAM-1 Step 2: Activation CCR7 SLC GAG SLC - secondary lymphoid tissue chemokine GAG - glycosaminoglycan Step 3: Firm Adhesion ICAM-1 LFA-1 Homing of naïve T lymphocytes to Peyer’s Patches involves adhesion molecules and chemotactic receptors analagous to those mediating homing to peripheral lymph nodes. Note that interaction of L-Selectin on naïve T lymphocytes with MAdCAM-1, rather than PNAd, expressed by the endothelium in Peyer’s Patches mediates their initial rolling prior to activation, firm adhesion and diapedesis.

Lipids are important chemoattractants too. Lipids control neutrophil recruitment during inflammation. Leukotriene B4 (LTB4) activation of BLT-1 receptor mediates the initial wave of neutrophil infiltration into inflamed arthritic joints, initiating a cytokine-chemokine cascade which amplifies and sustains neutrophil recruitment. Lipids control lymphocyte trafficking. Sphingosine-1-phosphate (S-1-P) gradients acting through lymphocyte S-1-P receptors control lymphocyte trafficking through lymph nodes.

Leukotriene B4 (LTB4) Sphingosine-1-phosphate (S-1-P) OH P O NH2 OH O Neutrophil recruitment Lymphocyte trafficking to inflamed tissue. through lymph nodes.

A Lipid-Cytokine-Chemokine cascade choreographs neutrophil recruitment into the inflamed arthritic joint. Lipid Cytokine Chemokine CCR1 BLT1 Neutrophil CXCR2 MIP-1 MIP-1 RANTES KC MIP-2 LIX LTB4 IL-1 Synovial cells Immune complexes Mast cells Macrophages Neutrophils

Arthritis - neutrophil recruitment into inflamed joints: Neutrophil recruitment into inflamed tissue is controlled by temporally distinct cascades of chemoattractants. Arthritis - neutrophil recruitment into inflamed joints: Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis. Chou, RC, et al, Immunity, vol 33:p266-278, 2010. (2) Neutrophil recruitment into sites of “sterile inflammation” (tissue injury/necrosis): Intravascular Danger Signals Guide Neutrophils to Sites of Sterile Inflammation. McDonald, B, et al, Science, vol 330:p362-366, 2010.

Sphingosine-1-phosphate (S-1-P) and S-1-P receptor expression control egress of T lymphocytes from lymph nodes. LOW S-1-P T T Efferent lymphatic T B HIGH S-1-P Afferent lymphatic High endothelial venule (HEV) Antigen

Blood Lymph node Lymph S-1-P T T T

S-1-P Receptor mediated lymphocyte egress from lymph nodes. S1PR1-mediated lymphocyte egress from lymph nodes. The level of sphingosine-1-phosphate (S1P) in lymphoid tissues is normally relatively low compared with the lymph, thereby forming a S1P gradient (shaded green). S1PR1 expressed on T cells is responsive to the S1P gradient and promotes T-cell egress from the lymphoid organ through the endothelial barrier into lymph. Upon activation of the T cell in the lymphoid organ on encountering an antigen-expressing dendritic cell (DC) or by type I interferon stimulation, S1PR1 expression is decreased. Mechanisms include direct protein:protein interaction with CD69, which is induced upon type I interferon stimulation, and through down-regulation of the transcription factor KLF2, which is a direct activator of the S1PR1 gene. Effector T cells eventually re-express S1PR1 and thereby egress from the lymph node to the lymph and into the peripheral tissues. If the levels of S1P are increased in lymphoid tissues, by inhibition of S1P lyase or possibly by inflammation, or in the presence of synthetic S1PR1 ligands such as FTY720, T-cell egress might be blocked by several possible mechanisms: dissipation of the S1P gradient, down-modulation of S1PR1 on T cells by ligand-induced internalization and S1PR1-mediated closure of egress ports on the endothelium by enhancement of junctional contacts. Juan-Rivera, et al, Nature Rev Immunol, 8:753-763, 2008. The alliance of sphingosine-1-phosphate and its receptors in Immunity.

Targeting S-1-P receptors to treat autoimmune disease ? Fingolimod (aka FTY720) is phosphorylated by sphingosine kinase-2 in vivo to form an S-1-P receptor agonist. Fingolimod has shown efficacy in multiple sclerosis patients. (Brinkmann, V, Br J Pharmacol, 158:1173-1182, 2009) FTY720 FTY720-phosphate Downregulation of S-1-P receptors (lymphocytes and CNS) Reduction of pathogenic T cells in CNS Sphk-2

Suggested Reading: Books: Review Articles: Research Article: Kuby, Chapter 13 - “Leukocyte activation & Migration” Review Articles: Leukocyte adhesion - Von Andrian and MacKay, NEW ENGLAND JOURNAL OF MEDICINE, vol 343:p1020, 2000. Lymphocyte trafficking - Von Andrian and Memper, NATURE REVIEWS IMMUNOLOGY, vol 3:p867, 2003. Chemokines: Sirens of neutrophil Recruitment - but is it just one song ? McDonald and Kubes, IMMUNITY, vol 33:p148, 2010. The alliance of sphingosine-1-phosphate and its receptors in Immunity - Juan-Rivera, et al, NATURE REV IMMUNOL, vol 8:p753-763, 2008. Research Article: Intravascular danger signals guide neutrophils to sites of sterile inflammation - McDonald, B, et al, SCIENCE, vol 330:p362, 2010.