The Force-dependent Mechanism Of Integrin α₄β₇-MAdCAM-1 Interaction

There are a large number of lymphocytes migrate to the inflammation site of intestinal mucosa due to the overactive immune disorder in inflammatory bowel disease（IBD）,hence leading to the intestinal mucosal damage.This process includes the interaction of integrinα₄β₇with mucosal vascular addressin cell adhesion molecule-1（MAdCAM-1）,which mediates the adhesion of circulating lymphocytes to the surface of high endothelial venules（HEVs）.Lymphocyte adhesion is a multistep cascade process including tethering,rolling,firm adhesion and crawling,Integrinα₄β₇ is involved not only in firm adhesion as well as in rolling adhesion process.Targeting integrinα₄β₇ or its ligand MAdCAM-1 can reduce the severity of inflammation in IBD,so as to achieve the therapeutic effect.In addition,the interaction between integrinα₄β₇ and its ligand occurs in the blood flow environment,therefore this interaction is regulated by intrinsic chemical factors,as well as blood flow force.However,the question how does flow shear stress modulate integrinα₄β₇-MAdCAM-1 interaction is not studied.Here,we perform the kinetic measurements in order to explore the force-dependent mechanism of the interaction between integrinα₄β₇ and MAdCAM-1.In this study,parallel plate flow chamber was used to simulate human physiological environment.Expressing integrinα₄β₇ RPMI 8226 cells were driven to adhere to MAdCAM-1coated substrate under various wall shear stresses.At the same time,divalent metal ions Ca²⁺,Ca²⁺+Mg²⁺,Mg²⁺or Mn²⁺were added to solution in order to activate integrinα₄β₇ to different active states.Under these conditions,the changes of adhesion behavior RPMI 8226 cells were quantified and compared.The lifetime of the interaction between integrinα₄β₇-MAdCAM-1and the rolling velocity of cells were measured.The results showed that in the Ca²⁺or Mg²⁺solution environment,the mean rolling velocity of cells first decreased and then increased with the increasing of the force,which behaved a phenomenon of flow-enhanced adhesion.In the meanwhile,the lifetime of tether adhesion and the mean stop time of rolling adhesion also showed similar phenomenon that first increased and then decreased with increasing fluid shear stress.The inverse tendency of rolling velocity against bond lifetime indicates that the phenomenon of force-dependent flow-enhanced adhesion mediated by integrinα₄β₇-MAdCAM-1 was regulated by catch bond mechanism.Furthermore,the interaction between integrinα₄β₇ and MAdCAM-1 was further scrutized by atomic force microscopy.Frequency of adhesion and lifetime were measured during the interaction beteeen MAdCAM-1 and integeinα₄β₇ in different active states in the presence of various ions.The results of AFM,consistent with the results of PPFC,showed that lifetime of integrinα₄β₇-MAdCAM-1 increased first and then decreased with the increase of force.This biphase curve was regulated by the conversion of catch-slip bond.In addition,the results showed that the process was also regulated by ions.Integrinα₄β₇ may be activated to low affinity state under Ca²⁺ion conditions,while integrinα₄β₇ was activated to a medium affinity and high affinity state respectively under conditions of Mg²⁺ions and Mn²⁺ions.Therefore,the interaction between integrinα₄β₇ and MAdCAM-1 is a force-dependent and metal-ion-dependent process.In conclusion,the flow-enhanced phenomenon mediated by integrinα₄β₇-MAdCAM-1 is regulated by catch bond mechanism in this research.The interaction of integrinα₄β₇-MAdCAM-1 plays a key role in inflammatory bowel disease.Therefore,the perform of this study would deepen the understanding of lymphocyte homing,inflammatory bowel disease and other physiological and pathological processes,and provide a new perspective for the intervention of immune response and the development of new anti-inflammatory therapy.