(from Kerfoot & Kubes, J Immunol 2002; 169: 1000) Anti-P-selectin alone blocks rolling, not other...
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Transcript of (from Kerfoot & Kubes, J Immunol 2002; 169: 1000) Anti-P-selectin alone blocks rolling, not other...
(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