(from Kerfoot & Kubes, J Immunol 2002; 169: 1000) Anti-P-selectin alone blocks rolling, not other selectins. Expression is seen in brain and spinal cord in EAE. Levels are low, but increased.

(from Kerfoot & Kubes, J Immunol 2002; 169: 1000) P-selectin blocking before assay inhibits both rolling and adhesion at peak disease (4d)  4 blockade inhibits adhesion more than rolling Since rolling precedes adhesion, then P-selectin seems more important that  4 integrins

Both memory and effector CD4 + T cells express CD45RO - how to distinguish between the two? Ans. CCR7 CCR7- CD45RO+ T cells are effector-memory T cells - make cytokines, perforin, granzymes These cells don’t enter lymph nodes ’Combination code' for entry of T cells to normal lymph nodes is L-selectin binding, followed by CCR7 signaling through SLC. This code is expressed by naive T cells and CCR7+ memory T cells, but not by certain cells that do not recirculate, and memory- effector T cells Other effector-memory T cells: CD8+ CD11b+ cells

DC enter LN via afferent lymph -> subcapsular sinus -> T cell areas SLC and ELC are involved in this migration Maturing DC express chemokines that can attract T cells Antigen regulates microenviromental homing Integrin activation Chemokine receptor expression CCR7 low CXCR5 + activated Th2 T cells may be favoured for T:B interaction - move towards and into B follicles for GC reaction Adhesion signaling also regulates microenviromental homing

L-selectin signaling Anti-L-selectin antibody induces Ca ++ fluxes and tyrosine phosphorylation in PMN's Other selectin signaling Interaction between PMN's or monocytes with HUVEC's induced Ca ++ fluxes in HUVEC's - blocked by antibodies against E-selectin or VCAM-1. Antibodies against P- or E-selectin induced rearrangement of EC microfilaments or actin stress fibers. ICAM-1 signaling Predominantly expressed on inflamed endothelia or on APC’s High affinity integrins bind ICAM-1 ICAM-1 induces endothelial cells to make TNF Induces astrocytes (brain), synovial cells (joints) and endothelial cells to make IL-1 and IL-6 Induces cell cycle arrest in B cells - MAPK, blocks NF  B,

Integrins  /ß heterodimers 17  ’s, 8 ß’s, making 22 integrin receptors Bind to specific receptors (ICAM’s), or to ECM (fibronectin, collagen) LFA-1 (CD11a/CD18, or  L:ß2) Mac-1 (CD11b/CD18, or  M:ß2) VLA-4 (a4:ß1) Many integrins recognize RGD motifs (arg-gly-asp) in ECM Integrin ligand-binding activity regulated by intracellular signaling (‘inside-out signaling’) Integrins associate with cytoskeleton (eg. Talin) when cells are activated.

Integrin signaling FAK (focal adhesion kinase) Integrin-associated tyrosine kinase, involved in integrin signaling for cytoskeletal reorganisation. GTP-binding proteins (Rho, Ras and Rac), MAPKinase. There are also FAK- and Ras-independent pathways Outcome of integrin signaling: prevention of apoptosis (stabilisation of Bcl-2 expression) cell cycle arrest/progression local regulation of adhesion, contractility and cell movement Shape change Stress fibers, and actin modulation of growth factor signaling

Ras PI-3-K Rac Rho PIP-5-K PIP2 Profilin Gelsolin Vinculin Rho-K Myosin phosphatase Focal adhesions Stress fibers Integrin signaling 101 FAK   (from Kumar, CC Oncogene 17:1365) Ras TCR/CD3 MAPK Survival Proliferation

Kim et al made constructs of  L and ß2 (  L/ß2 integrin = LFA-1) coupled to mCFP and mYFP respectively and used FRET to measure interaction between the two chains. Results show conformational changes in integrin cytoplasmic domains associated with activation in living cells. Activation of conformational change by PMA (‘inside-out’ signaling) resulted in physical separation of cytoplasmic regions. Ligand binding (sICAM-1) also induced spatial separation of cytoplasmic domains Not all activation stimuli did this (eg. Mn ++ ) Enforced proximity of cytoplasmic regions prevented activation GFKKR motif in  -chain transmembrane/cytoplasmic region critical for spatial separation Talin binding to LFA-1 induces spatial separation Chemokine signaling (SDF-1/CXCL12 to CXCR4) (‘inside-out’) also induced spatial separation

Kim, M., Carman, C.V., Springer, T.A Science 301: