Chunzhang Cao

Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration.

Migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. Here, we show that efficient macrophage migration in inflammatory environment depends on Mac‐1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue‐type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI‐1 enhances binding of the integrin–protease–inhibitor complex to the endocytic receptor LRP (lipoprotein receptor‐related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac‐1, tPA, PAI‐1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI‐1‐deficient mice can be restored by wild‐type but not by a mutant PAI‐1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac‐1‐mediated adhesion, whereas PAI‐1 and LRP facilitate its transition to cell retraction. Our results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).

Bisphosphonates cause osteonecrosis of the jaw-like disease in mice.

Bisphosphonate-associated osteonecrosis of the jaw (BONJ) is a morbid bone disease linked to long-term bisphosphonate use. Despite its broad health impact, mechanistic study is lacking. In this study, we have established a mouse model of BONJ-like disease based on the equivalent clinical regimen in myeloma patients, a group associated with high risk of BONJ. We demonstrate that the murine BONJ-like disease recapitulates major clinical and radiographical manifestations of the human disease, including characteristic features of osseous sclerosis, sequestra, avascular, and radiopaque alveolar bone in the jaw that persists beyond a normal course of wound healing following tooth extraction. We find that long-term administration of bisphosphonates results in an increase in the size and number of osteoclasts and the formation of giant osteoclast-like cells within the alveolar bone. We show that the development of necrotic bone and impaired soft tissue healing in our mouse model is dependent on long-term use of high-dose bisphosphonates, immunosuppressive and chemotherapy drugs, as well as mechanical trauma. Most importantly, we demonstrate that bisphosphonate is the major cause of BONJ-like disease in mice, mediated in part by its ability to suppress osseous angiogenesis and bone remodeling. The availability of this novel mouse model of BONJ-like disease will help elucidate the pathophysiology of BONJ and ultimately develop novel approaches for prevention and treatment of human BONJ.

The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti-inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the gamma-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase-1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Structure of the F-spondin domain of mindin, an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.

Molecular Basis for the Interaction of Low Density Lipoprotein Receptor-related Protein 1 (LRP1) with Integrin αMβ2 IDENTIFICATION OF BINDING SITES WITHIN αMβ2 FOR LRP1

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β2 integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes αMβ2 but not its homologous receptor αLβ2. The interaction between these two cellular receptors in macrophages is significantly enhanced upon αMβ2 activation by LPS and is mediated by multiple regions in both LRP1 and αMβ2. Specifically, we find that both the heavy and light chains of LRP1 are involved in αMβ2 binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For αMβ2, we find that the αM-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the αLβ2 receptor with that of αM confers the αLβ2 receptor with the ability to interact with LRP1. Furthermore, we show that residues 160EQLKKSKTL170 within the αM-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of αMβ2, our finding suggests that binding of LRP1 to αMβ2 could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits αMβ2-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β(2) integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes α(M)β(2) but not its homologous receptor α(L)β(2). The interaction between these two cellular receptors in macrophages is significantly enhanced upon α(M)β(2) activation by LPS and is mediated by multiple regions in both LRP1 and α(M)β(2). Specifically, we find that both the heavy and light chains of LRP1 are involved in α(M)β(2) binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For α(M)β(2), we find that the α(M)-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the α(L)β(2) receptor with that of α(M) confers the α(L)β(2) receptor with the ability to interact with LRP1. Furthermore, we show that residues (160)EQLKKSKTL(170) within the α(M)-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of α(M)β(2), our finding suggests that binding of LRP1 to α(M)β(2) could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits α(M)β(2)-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.

Molecular basis for the interaction of the LDL receptor-related protein 1 (LRP1) with the integrin alpambeta2: identification of binding sites within alphambeta2 for LRP1

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β2 integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes αMβ2 but not its homologous receptor αLβ2. The interaction between these two cellular receptors in macrophages is significantly enhanced upon αMβ2 activation by LPS and is mediated by multiple regions in both LRP1 and αMβ2. Specifically, we find that both the heavy and light chains of LRP1 are involved in αMβ2 binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For αMβ2, we find that the αM-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the αLβ2 receptor with that of αM confers the αLβ2 receptor with the ability to interact with LRP1. Furthermore, we show that residues 160EQLKKSKTL170 within the αM-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of αMβ2, our finding suggests that binding of LRP1 to αMβ2 could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits αMβ2-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β(2) integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes α(M)β(2) but not its homologous receptor α(L)β(2). The interaction between these two cellular receptors in macrophages is significantly enhanced upon α(M)β(2) activation by LPS and is mediated by multiple regions in both LRP1 and α(M)β(2). Specifically, we find that both the heavy and light chains of LRP1 are involved in α(M)β(2) binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For α(M)β(2), we find that the α(M)-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the α(L)β(2) receptor with that of α(M) confers the α(L)β(2) receptor with the ability to interact with LRP1. Furthermore, we show that residues (160)EQLKKSKTL(170) within the α(M)-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of α(M)β(2), our finding suggests that binding of LRP1 to α(M)β(2) could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits α(M)β(2)-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes

While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its βN-domains, and this interaction is inhibited by RAP and (β15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing βN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.

Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments.

Summary.  Background: The interaction of the fibrin βN‐domain with VE‐cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the β15–42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (β15–66)2 fragment, corresponding to the full‐length βN‐domain and mimicking its dimeric arrangement in fibrin, bound to VE‐cadherin. Objective: To test our hypothesis that dimerization of β15–42‐containing fragments increases their affinity for VE‐cadherin and ability to inhibit transendothelial migration of leukocytes. Methods: Interaction of β15–42‐containing fragments with VE‐cadherin was characterized by ELISA and surface plasmon resonance. The inhibitory effect of such fragments was tested in vitro with a leukocyte transendothelial migration assay and in vivo with mouse models of peritonitis and myocardial ischemia–reperfusion injury. Results: First, we prepared the monomeric β15–42 and β15–64 fragments and their dimeric forms, (β15–44)2 and (β15–66)2, and studied their interaction with the fibrin‐binding domain of VE‐cadherin, VE‐cad(3). The experiments revealed that both dimeric fragments bound to VE‐cad(3) with high affinity, whereas the affinities of β15–42 and β15–64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo, and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, (β15–44)2 significantly reduced myocardial injury induced by ischemia–reperfusion. Conclusion: The results confirm our hypotheses and indicate that (β15‐66)2 and (β15‐44)2, which exhibited much higher affinity for VE‐cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

Activated protein C accelerates venous thrombus resolution through heme oxygenase-1 induction.

Thrombus resolution is a complex process that involves thrombosis, leukocyte‐mediated thrombolysis, and the final resolution of inflammation. Activated protein C (APC) is an anticoagulant that also possesses immunoregulatory activities.

Anti-VLDL receptor monoclonal antibodies inhibit fibrin-VLDL receptor interaction and reduce fibrin-dependent leukocyte transmigration

Our previous studies revealed that the interaction of fibrin with the very low density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation, and localised the fibrin-binding site to CR-domains 2-4 of this receptor. In the present study, we tested interaction of three anti-VLDLR monoclonal antibodies, mAb 1H10, 1H5, and 5F3, with recombinant fragments of VLDLR containing various combinations of its CR-domains and found that the epitopes for mAb 1H10 and mAb 1H5 overlap with the fibrin-binding site of VLDLR. Based on these findings, we hypothesised that mAb 1H10 and mAb 1H5 should inhibit fibrin-VLDLR interaction and modulate leukocyte transmigration. To test this hypothesis, we first demonstrated that these monoclonal antibodies both have high affinity to the fibrin-binding fragments of the VLDL receptor and efficiently inhibit interaction between the VLDLR-binding fragment of fibrin and the fibrin-binding fragments of VLDLR. Next, in the in vitro experiments using leukocyte transendothelial migration assay we found that both monoclonal antibodies efficiently inhibit leukocyte transmigration induced by fibrin mimetic NDSK-II. Finally, in vivo experiments using mouse model of peritonitis revealed that mAb 1H10 and mAb 1H5 both significantly reduce infiltration of leukocytes into the peritoneum. Furthermore, our experiments using mouse model of myocardial ischemia-reperfusion injury revealed that both monoclonal antibodies significantly reduce myocardial injury induced by ischaemia-reperfusion. Thus, the results obtained indicate that monoclonal antibodies 1H10 and 1H5 are novel specific inhibitors of fibrin-VLDLR-dependent leukocyte transmigration pathway. They may represent potential therapeutics for treatment of fibrin-dependent inflammation including myocardial ischaemia-reperfusion injury.