CD40 receptor and its ligand CD40 L are the members of TNFR and TNF family,respectively.CD40 is expressed on antigen-presenting cells(APC)as a monomer,and would be dimerized upon its oligomerization through binding to...CD40 receptor and its ligand CD40 L are the members of TNFR and TNF family,respectively.CD40 is expressed on antigen-presenting cells(APC)as a monomer,and would be dimerized upon its oligomerization through binding to its ligand CD40 L.Such dimer formation was shown to be essential for some CD40 signaling events,including phosphoinositide-3 kinase(PI-3 K)activation,the subsequent B7-2 upregulation and the production of IL-8.CD40 L is primarily expressed on activated T cells and activated platelets,and acts as an important costimulatory molecule especially on T follicular helper cells.It is believed that CD40-CD40 L interaction deeply involved in many immune events for the host defense against pathogens and cancer.CD40-CD40 L interaction mediates intracellular signaling along different pathways,such as the canonical and noncanonical nuclear factorκB pathway,mitogen-activated protein kinases,phosphatidylinositol-3 kinase(PI3K)and the phospholipase Cy pathway,is necessary for successful adaptive immune responses mainly relevant to development of CD8+CTLs,and is deeply involved in cross-talks among T cell,B cell,tumor cell,platelet and etc.However,interaction of CD40 with platelet CD40 L would occur in hemodynamics environments,and the regulation of force on this interaction remain unclear.Besides polymerization of CD40,the hemodynamic environment could be believed to regulate interaction between CD40 and its ligand.To reveal the mechanical regulation mechanism of interaction between CD40 L and CD40,atomic force microscope(AFM)was used in the adhesion frequency assay for measuring two-dimensional(2D)kinetics of CD40 L with CD40(the monomer and its dimer)at zero-force and force-clamp assay for measuring single-bond lifetimes of CD40 L/CD40 complex in a range of forces at single-molecule level.AFM cantilever probes were functionalized by incubation with CD40 L in buffer,and soaked in phosphate-buffered saline(PBS)containing 1%bovine serum albumin(BSA)to block nonspecific binding.Polystyrene dishes were coated with CD40(the monomer and its dimer)and then filled with PBS containing 1%BSA.Our data showed that the adhesion frequency of CD40 L with CD40 monomer and its dimer was 0.10 and0. 15,respectively,showing a higher affinity of CD40 L to CD40 dimer rather than its monomer;the rupture force for CD40 L dissociating from CD40 monomer and its dimer was 23 and 31pN,respectively,demonstrating a higher mechanical strength of complex of CD40 L with CD40 dimer instead of monomer;and increasing tension will prolong first and then shorten the bond lifetime of CD40 L-CD40 complex with force threshold of about 1OpN,and dimerization of CD40 had significantly increased enhanced the bond lifetime of CD40 L-CD40 complex,showing a dimerization-enhanced affinity of CD40 to CD40 L and'Catch-Slip'bond mechanism of CD40 L-CD40 interaction.These results suggest that,through upregulating the binding affinity with CD40 L,the polymerization of CD40 stabilizes the linkers and thereby communication between cell and cell,so does the force being small than the force threshold Both CD40 polymerization-and'Catch bond'-induced enhancement of affinity of CD40 with CD40 L may be necessary for stable cross talks between two cells.This finding may be useful for understanding CD40/CD40 L-induced events importantly in physiological or pathological processes at molecular and cellular level.展开更多
As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that...As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that mediates platelet aggregation and leukocyte immune response.Unlike soluble CD40 L,the interaction between transmembrane platelet CD40 L and its receptors occurs within the cell junction surface,usually,in a physiological and pathological high blood flow shear stress environment.This two-dimensional reaction kinetics should be a mechano-chemical coupling process.In addition to its classical receptor CD40,CD40 L also binds to receptorα5β1,CD40 L can bind to the resting state of integrinα5β1,but the mechanical regulation mechanism of integrinα5β1 activation under fluid shear stress remains unclear.We assume that the force can promote CD40 L-inducedα5β1 activation.To check this hypothesis,we performed flow chamber experiment to investigate interaction of CD40 L andα5β1.In experiments,the bottom of the flow chamber is functionalized by a suitable concentration of CD40 L,and the fiber spheres of 6μm diameter was coated withα5β1.The selection of CD40 L concentration was based on the observation that as many tether events ofα5β1-coated spheres as possible were observed rather than stable adhesion events of these spheres.Theα5β1-coated sphere suspension was poured over the CD40 L-coated substrates in the flow chamber under different shear rates.A high-speed camera was used to observe and record tether events of fiber spheres at a rate of 100 frames per second.According to our affinity state transition model for integrin,the data were analyzed to obtain the rate of integrin activation and its mechanical regulation characteristics.Our results demonstrated that the interaction betweenα5β1 and CD40 L is biphasic force-dependent,showing mechano-chemical regulation mechanism of'Catch-slip bond'transition.The affinity jumping model was well fitted with the data obtained from flow chamber experiment at various wall shear stresses.We found that,CD40 L ligation-induced jumping ofα5β1 affinity state from low to medium(or high)one will occur within 0.5-1.0 second,resulting in prolonging of bond lifetimes.And,frequency distribution of the tether events number with tether lifetime under each force,exhibits obvious doublet peaks,one within 0.5-1 s and second within 1.5-2.5 s,indicating theα5β1 affinity state transform from low to high one.The probability distribution of the tether lifetime under different shear forces are not linear,and exists a turning point,which shows that the rate ofα5β1 dissociation from CD40 L is fast first,and then become slow,showing a force-induced conformation transformation of the integrinα5β1 from low affinity state to high affinity one.Our findings suggest that,the continuous force stimulation will quickly cause the affinity state change of integrinα5β1. The dissociation rate of theα5β1/CD40 L complex decreases first and then increases with wall shear stress,exhibiting a'Catch-slip bond'transformation of interaction betweenα5β1-CD40 L.This mechanical regulation mechanism exists in interaction of CD40 L not only toα5β1 at low affinity state but also to one at high affinity state.Our results should be useful in understanding the mechanical regulation mechanism of a5β1-CD40 L interaction-mediated cellular immune response and inflammatory processes.展开更多
基金supported by the National Natural Science Foundation of China ( 116272109, 11432006)
文摘CD40 receptor and its ligand CD40 L are the members of TNFR and TNF family,respectively.CD40 is expressed on antigen-presenting cells(APC)as a monomer,and would be dimerized upon its oligomerization through binding to its ligand CD40 L.Such dimer formation was shown to be essential for some CD40 signaling events,including phosphoinositide-3 kinase(PI-3 K)activation,the subsequent B7-2 upregulation and the production of IL-8.CD40 L is primarily expressed on activated T cells and activated platelets,and acts as an important costimulatory molecule especially on T follicular helper cells.It is believed that CD40-CD40 L interaction deeply involved in many immune events for the host defense against pathogens and cancer.CD40-CD40 L interaction mediates intracellular signaling along different pathways,such as the canonical and noncanonical nuclear factorκB pathway,mitogen-activated protein kinases,phosphatidylinositol-3 kinase(PI3K)and the phospholipase Cy pathway,is necessary for successful adaptive immune responses mainly relevant to development of CD8+CTLs,and is deeply involved in cross-talks among T cell,B cell,tumor cell,platelet and etc.However,interaction of CD40 with platelet CD40 L would occur in hemodynamics environments,and the regulation of force on this interaction remain unclear.Besides polymerization of CD40,the hemodynamic environment could be believed to regulate interaction between CD40 and its ligand.To reveal the mechanical regulation mechanism of interaction between CD40 L and CD40,atomic force microscope(AFM)was used in the adhesion frequency assay for measuring two-dimensional(2D)kinetics of CD40 L with CD40(the monomer and its dimer)at zero-force and force-clamp assay for measuring single-bond lifetimes of CD40 L/CD40 complex in a range of forces at single-molecule level.AFM cantilever probes were functionalized by incubation with CD40 L in buffer,and soaked in phosphate-buffered saline(PBS)containing 1%bovine serum albumin(BSA)to block nonspecific binding.Polystyrene dishes were coated with CD40(the monomer and its dimer)and then filled with PBS containing 1%BSA.Our data showed that the adhesion frequency of CD40 L with CD40 monomer and its dimer was 0.10 and0. 15,respectively,showing a higher affinity of CD40 L to CD40 dimer rather than its monomer;the rupture force for CD40 L dissociating from CD40 monomer and its dimer was 23 and 31pN,respectively,demonstrating a higher mechanical strength of complex of CD40 L with CD40 dimer instead of monomer;and increasing tension will prolong first and then shorten the bond lifetime of CD40 L-CD40 complex with force threshold of about 1OpN,and dimerization of CD40 had significantly increased enhanced the bond lifetime of CD40 L-CD40 complex,showing a dimerization-enhanced affinity of CD40 to CD40 L and'Catch-Slip'bond mechanism of CD40 L-CD40 interaction.These results suggest that,through upregulating the binding affinity with CD40 L,the polymerization of CD40 stabilizes the linkers and thereby communication between cell and cell,so does the force being small than the force threshold Both CD40 polymerization-and'Catch bond'-induced enhancement of affinity of CD40 with CD40 L may be necessary for stable cross talks between two cells.This finding may be useful for understanding CD40/CD40 L-induced events importantly in physiological or pathological processes at molecular and cellular level.
基金supported by the National Natural Science Foundation of China ( 116272109, 11432006)
文摘As a key regulator of immune response,CD40 L is usually associated with chronic disease-related inflammation,autoimmune diseases and malignant diseases.Receptor recognition of platelet CD40 L is the initial event that mediates platelet aggregation and leukocyte immune response.Unlike soluble CD40 L,the interaction between transmembrane platelet CD40 L and its receptors occurs within the cell junction surface,usually,in a physiological and pathological high blood flow shear stress environment.This two-dimensional reaction kinetics should be a mechano-chemical coupling process.In addition to its classical receptor CD40,CD40 L also binds to receptorα5β1,CD40 L can bind to the resting state of integrinα5β1,but the mechanical regulation mechanism of integrinα5β1 activation under fluid shear stress remains unclear.We assume that the force can promote CD40 L-inducedα5β1 activation.To check this hypothesis,we performed flow chamber experiment to investigate interaction of CD40 L andα5β1.In experiments,the bottom of the flow chamber is functionalized by a suitable concentration of CD40 L,and the fiber spheres of 6μm diameter was coated withα5β1.The selection of CD40 L concentration was based on the observation that as many tether events ofα5β1-coated spheres as possible were observed rather than stable adhesion events of these spheres.Theα5β1-coated sphere suspension was poured over the CD40 L-coated substrates in the flow chamber under different shear rates.A high-speed camera was used to observe and record tether events of fiber spheres at a rate of 100 frames per second.According to our affinity state transition model for integrin,the data were analyzed to obtain the rate of integrin activation and its mechanical regulation characteristics.Our results demonstrated that the interaction betweenα5β1 and CD40 L is biphasic force-dependent,showing mechano-chemical regulation mechanism of'Catch-slip bond'transition.The affinity jumping model was well fitted with the data obtained from flow chamber experiment at various wall shear stresses.We found that,CD40 L ligation-induced jumping ofα5β1 affinity state from low to medium(or high)one will occur within 0.5-1.0 second,resulting in prolonging of bond lifetimes.And,frequency distribution of the tether events number with tether lifetime under each force,exhibits obvious doublet peaks,one within 0.5-1 s and second within 1.5-2.5 s,indicating theα5β1 affinity state transform from low to high one.The probability distribution of the tether lifetime under different shear forces are not linear,and exists a turning point,which shows that the rate ofα5β1 dissociation from CD40 L is fast first,and then become slow,showing a force-induced conformation transformation of the integrinα5β1 from low affinity state to high affinity one.Our findings suggest that,the continuous force stimulation will quickly cause the affinity state change of integrinα5β1. The dissociation rate of theα5β1/CD40 L complex decreases first and then increases with wall shear stress,exhibiting a'Catch-slip bond'transformation of interaction betweenα5β1-CD40 L.This mechanical regulation mechanism exists in interaction of CD40 L not only toα5β1 at low affinity state but also to one at high affinity state.Our results should be useful in understanding the mechanical regulation mechanism of a5β1-CD40 L interaction-mediated cellular immune response and inflammatory processes.
