(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, granzymesThese 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 areasSLC and ELC are involved in this migration

Maturing DC express chemokines that can attract T cells

Antigen regulates microenviromental homingIntegrin activationChemokine receptor expression

CCR7low 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 signalingAnti-L-selectin antibody induces Ca++ fluxes and tyrosine phosphorylation in PMN's

Other selectin signalingInteraction 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-1ICAM-1 induces endothelial cells to make TNFInduces astrocytes (brain), synovial cells (joints) and endothelial cells to make IL-1 and IL-6Induces cell cycle arrest in B cells - MAPK, blocks NFB,

Integrins

/ß heterodimers17 ’s, 8 ß’s, making 22 integrin receptorsBind 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 signalingFAK (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 changeStress fibers, and actin

•modulation of growth factor signaling

Ras

PI-3-K

Rac

Rho PIP-5-K

PIP2

ProfilinGelsolinVinculin

Rho-K

Myosin phosphatase

Focal adhesionsStress fibers

Integrin signaling 101

FAK

(from Kumar, CC. 1998. Oncogene 17:1365)

Ras

TCR/CD3

MAPK

SurvivalProliferation

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. 2003. Science 301:1720-1725

Kim, M., Carman, C.V., Springer, T.A. 2003. Science 301:1720-1725