Integrin activation

The ability of rolling leukocytes to arrest on target endothelial sites depends on rapid activation of their integrins. Studying LFA-1 activation as a paradigm for leukocyte integrins, we find that rapid LFA-1 activation by endothelial-displayed chemokines involves local GPCR signals which trigger conformational rearrangements of the integrin within millisecond contacts (Scheme 1). These spatially confined events involve the cytoskeletal regulatory GTPases RhoA and Rap-1 and require proper integrin activation and anchorage to the cortical actin cytoskeleton via cytoskeletal linkers such as talin and Kindlin-3. An inherited integrin activation deficiency identified by us, termed LAD-III, is linked to deficiency in Kindlin-3, a hematopoietic member of the Kindlin adaptor family. A possible crosstalk between chemokine stimulated Rap-1 and RhoA GTPases and these integrin cytoskeletal linkers and their associated effectors is currently under investigation in both normal and malignant lymphocytes.

Scheme 1 A proposed model for rapid integrin activation by endothelial-immobilized chemokines. Flow chamber analysis is shown in movies 3 and 4. Bidirectional activation switches the integrin (LFA-1 in this demonstration) from an inactive bent state to an extended state within 0.1 sec (step 1). This critical event primes the integrin to bind its endothelial ligand and undergo a further conformational shift (step 2, depicted by the red star). This activation of the integrin headpiece is stabilized by further separation of the integrin subunit tails (step 3). Associations of the integrin subunit tails with talin and additional adaptors provide critical mechanical stabilization of the nascent adhesive bond.

The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in T lymphocytes and in LFA-1 dependent adhesion strengthening to ICAM-1. In a follow up study, in collaboration with C. Laudanna (Verona) we showed that a specific RhoA 23/40 effector region is vital for the earliest LFA-1 dependent arrests of lymphocytes on HEVs. Blocking the RhoA 23/40 region in human T lymphocytes in vitro impaired the subsecond CXCL12-triggered LFA-1 mediated T cell arrest on ICAM-1 by preventing the induction of an extended LFA-1 conformational state. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine induced inside-out LFA-1 extension prior to ligand binding, but is not required for a variety of non chemokine signals that strengthen LFA-1-ICAM-1 bonds via outside in conformational changes, such as TCR signals.