Lymphocyte Migration Ppt
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Lymphocyte Migration ©Dr. Colin R.A. Hewitt crah1@le.ac.uk Differences between armed, effector T cells and naïve T cells - Naïve CD4 cells CD44 LFA-1 CD45RA CD2 CD45RO VLA-4 Activated L-selectin Naïve Associates with TcR and CD4 - phosphatase activity reduces threshold of T cell signalling + + + + + - - - ++ ++ ++ - + ++ Homing to lymph node Adhesion molecules Homing Differential to inflamed splicing of vascular CD45 mRNA in naïve & armed endothelium T cells Patterns of lymphocyte trafficking Naïve T cells Thymus Bone Marrow Naïve T cell Lymph node HEV High endothelial venules Post capillary venules in 2º lymphoid tissue HIGH ENDOTHELIAL VENULES. Specialised to allow lymphocytes and nothing else into the lymph node Post capillary venules in other tissues are lined by simple squamous epithelium HEV Role of endothelial cells in trafficking and recirculation Endothelial are involved in: Vasomotor tone, vascular permeability, regulation of coagulation, immune modulation and lymphocyte extravasation High endothelial venules Post-capillary venules Constitutively present in secondary lymphoid tissue Present in non-lymphoid tissues Need to allow egress of naïve cells from the circulation Molecules expressed by endothelial cells regulate trafficking and recirculation through lymphoid and non-lymphoid tissues The multi-step paradigm of leukocyte migration: Step 1: Tethering & rolling Cytokine activated endothelial cells express adhesion molecules Leukcocytes ‘marginate’ from the peripheral pool to the marginal pool Cells normally roll past resting endothelial cells Tethering Rolling 4000 microns/sec 40 microns/sec Tethering and rolling are mediated by SELECTINS and ADDRESSINS Selectins & addressins SELECTINS Leucocytes inc. Naive T cells: L SELECTIN Endothelial cells: P SELECTIN & E SELECTIN P selectin: Weibel-Palade bodies. E selectin: TNF & IL-1 induced A common core with different extracellular C type lectin domains that bind carbohydrates in a Ca2+ dependent manner. Each selectin binds to specific carbohydrates and is able to transduces signals into the cell VASCULAR ADDRESSINS On high endothelial venules in lymphoid tissue: Carbohydrates that “decorate” CD34 and GlyCAM-1 Sialyl LewisX molecules Peripheral Node addressins (PNAd) Mucosal endothelium: MAdCAM-1 Guides lymphocyte entry into lymphoid tissues Steps 2 & 3: Activation & arrest Cytokines from epithelium activate expression of Intracellular adhesion molecules (ICAMs) Rolling Neutrophil Selectin is shed Cell activation is activated changes INTEGRIN by integrin to (adhesion molecule) chemokines high affinity has low affinity for format ICAM Activation 1-3 seconds G-protein-linked seven transmembrane spanning receptors For granulocyte activation: Chemokines Platelet activating factor C5a In T cells: ?? Activation Inhibit G protein with pertussis toxin Occupancy of large numbers of surface receptors Rolling phenotype only - no stable adhesion Ligand of lymphocyte toxin-sensitive receptor not yet identified Steps 2 & 3: Activation & arrest Cytokines from epithelium activate expression of Intracellular adhesion molecules (ICAMs) Rolling Neutrophil Selectin is shed Cell activation is activated changes INTEGRIN by integrin to (adhesion molecule) chemokines high affinity has low affinity for format ICAM Arrest INTEGRIN Ig FAMILY LIGAND L2 (LFA-1) ICAM-1 Integrin Activation of lymphocyte increases affinity of integrin (Mn2+ in vitro) Ig family ligand Integrins Two chain molecules - that bind to Ig superfamily molecules and extracellular matrix components “Inside out” signalling Activation of lymphocyte Remove cytoplasmic tail of integrin -chain Activation of lymphocyte Activation of the extracellular high affinity integrin binding site is dependent upon activation of the lymphocyte, & the cytoplasmic domain of the integrin i.e. signals from “inside” the cell have an effect “outside” “Outside in” signalling Ligation of lymphocyte integrin by ligand Activation of lymphocyte High affinity interaction of integrins with their ligands may alter the behaviour of the cell i.e. signals from “outside” the cell have an effect “inside” Step 4: Migration and diapedesis Firm adhesion causes the leukocyte to flatten and migrate between the endothelial cells Leukocyte migrates towards site of infection by detecting and following a gradient of chemokine. Leukocytes migrate readily to the chemokine RANTES made by epithelilal cells that have encountered microorganisms Arrest is reversible if diapeisis does not occur ~10 Minutes Diapedesis PECAM expressed at intercellular junctions of endothelial cells and on the lymphocyte Metalloproteases digest the basement membrane Migration Signals similar to those important in step 2 are involved i.e. chemokines Extracellular matrix provides traction for moving cells Simultaneous occupancy of large numbers of surface receptors the cell will stay still. Chemotactic gradient Differential receptor occupancy between the trailing and leading edges of the cells. Operates at low levels of receptor expression Recirculation Non-lymphoid cells HEV Pass through the blood vessels in the lymph node and continue arteriovenous circulation Naïve lymphoid cells HEV Adhere to and squeeze between High Endothelial Venules (HEV), then percolate through the lymph node and exit via the efferent lymphatic vessel Inflammation Normal oesophagus Normal palatine tonsils Normal skin Candida infection Streptococcal infection Staphylococcal infection Role of endothelial cells in trafficking and recirculation Post-capillary venules High endothelial venules Constitutively present in secondary lymphoid tissue Need to allow egress of naïve cells from the circulation Present in non-lymphoid tissues Injury and inflammation alters morphology to resemble HEV Need to allow egress of memory cells to sites of infection Inflammation or injury induces changes in endothelial cells t = seconds Weibel-Palade bodies with pre-formed adhesion molecules Injury or irritation generates thrombin histamine, Leukotrienes etc Adhesion molecule expression t = hours IkB phosphorylated & degraded. NF-kB translocates to nucleus TNF & IL-1 released due to inflammation in tissue Adhesion molecule expression Memory and naïve T cells CD44 LFA-1 CD45RA CD2 CD45RO VLA-4 Activated L-selectin Naïve Associates with TcR and CD4 - phosphatase activity reduces threshold of T cell signalling + + + + + - - - ++ ++ ++ - + ++ Naïve cells need to access lymphoid tissue to become stimulated Memory cells need to access sites of inflammation Integrins facilitate the access of leukocytes to sites of inflammation Activated effector memory cell with L selectin shed from surface L2 (LFA-1) Peripheral vascular endothelium INFLAMMATION ICAM-1 41(VLA-4) VCAM-1 Activated vascular endothelium TNF- Trafficking, homing and adhesion Trafficking: Non-random movement of cells from tissues, blood or lymph. Includes migration to and from sites of lymphocyte maturation as well as homing. Adhesion: Binding of cells to other cells or Homing: extracellular matrix Tendency of lymphocytes activated in a particular region of the body to preferentially return to the same region Includes localisation of cells in distinct regions of lymphoid tissue. Evidence that lymphocytes exhibit specialised trafficking patterns 3H-labelled lymphocytes from mesenteric lymph nodes 3H-labelled Remove tissues, section and autoradiograph A lymphocytes from skin Section through A Discovery of the T cell gut-homing mechanism Murine Lymphoma TK-1 Lymph node HEV Peyer’s patch HEV Inhibition of binding using a panel of monoclonal antibodies identified the lymphocyte molecule that mediated binding to Peyer’s Patch HEV: the integrin 47. A similar approach was used to identify the endothelial ligand of 47: the mucosal addressin: MAdCAM-1 Skin-homing T cells Cutaneous T cell lymphomas Extensive infiltration of epidermis with T cells Cells home to the skin and express the cutaneous lymphocyte associated antigen (CLA) Apply contact sensitiser Induce delayed-type Sample T cells by hypersensitivity raising a suction blister Cells in the suction blister express CLA - the skin homing receptor E-selectin is the ligand of CLA Why is lymphocyte homing necessary? Tendency of lymphocytes activated in a particular region of the body to preferentially return to the same region. Gut pathogen e.g. rotavirus Gut Anti-rotavirus T cells will never be needed in the skin Anti-rotavirus T cells will be needed in the gut Anti-rotavirus T cells activated Response resolves, lymphocytes nonrandomly redistributed Quantitative aspects of lymphocyte migration Traffic between lymphoid/non-lymphoid tissues involves~ 5 x 1011 cells per day Only ~2% (1 x 1010) of these cells are in the blood at any one time Lymphocytes only stay in the blood for ~30 minutes Circulating blood pool of lymphocytes is exchanged 48 times a day However…… Less than 10% of blood lymphocytes migrate into lymph nodes, tonsils & Peyer’s patches. ~90% of lymphocytes leave the blood to enter organs such as the liver, lung spleen and bone marrow. Traffic is 5 times faster than traffic through lymphoid tissue Summary Naïve cells entering Peripheral Lymph Nodes Contact - Rolling - Arrest - Diapedesis T cells Endothelial cells L-selectin PNAd (CD34, Gly-CAM) L2 (LFA-1) ICAM-1 Naïve cells entering Peyer’s Patches Contact - Rolling - Arrest - Diapedesis T cells L-selectin 47 L2 (LFA-1) Endothelial cells MAdCAM carbohydrate MAdCAM-1 ICAM-1 Memory cells entering Inflamed tissue T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis 41 (VLA-4) VCAM-1 L2 (LFA-1) ICAM-1 Memory cells homing to Peyer’s Patches T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis 47 L2 (LFA-1) MAdCAM-1 ICAM-1 Memory cells homing to Skin T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis CLA E-selectin 41 (VLA-4) VCAM-1 L2 (LFA-1) ICAM-1
