Fibrin-VLDL Receptor-Dependent Pathway Promotes Leukocyte Transmigration by Inhibiting Src Kinase Fyn and is a Target for Fibrin ß15-42 Peptide.

According to the current view, binding of fibrin degradation product E1 fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and fibrin-derived ß15-42 peptide reduces leukocyte transmigration by competing with E1 for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of ß15-42 to VE-cadherin raised a question about its ability to inhibit E1-VE-cadherin interaction. Further, our previous study revealed that fibrin promotes leukocyte transmigration through the very-low-density lipoprotein (VLDL) receptor (VLDLR)-dependent pathway and suggested a possible link between the inhibitory properties of ß15-42 and this pathway. To test such a link and the proposed inhibitory mechanisms for ß15-42, we performed in vitro experiments using surface plasmon resonance, enzyme-linked immunosorbent assay, and leukocyte transendothelial migration assay, and in vivo studies with wild-type and VLDLR-deficient mice using mouse model of peritonitis. The experiments revealed that ß15-42 cannot inhibit E1-VE-cadherin interaction at the concentrations used in the previous in vivo studies leaving the proposed Fyn-dependent signaling mechanism as a viable explanation for the inhibitory effect of ß15-42. While testing this mechanism, we confirmed that Fyn plays a critical role in controlling fibrin-induced transendothelial migration of leukocytes and found that signaling through the VLDLR-dependent pathway results in inhibition of Fyn, thereby increasing leukocyte transmigration. Furthermore, our in vivo experiments revealed that ß15-42 inhibits this pathway, thereby preventing inhibition of Fyn and reducing leukocyte transmigration. Thus, this study clarifies the molecular mechanism underlying the VLDLR-dependent pathway of leukocyte transmigration and reveals that this pathway is a target for ß15-42.

Microglial-mediated PDGF-CC activation increases cerebrovascular permeability during ischemic stroke.

Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1-/-) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1-/- mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b+ cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1-/- mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.

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.

Mac-1 Regulates IL-13 Activity in Macrophages by Directly Interacting with IL-13Rα1.

Mac-1 exhibits a unique inhibitory activity toward IL-13-induced JAK/STAT activation and thereby regulates macrophage to foam cell transformation. However, the underlying molecular mechanism is unknown. In this study, we report the identification of IL-13Rα1, a component of the IL-13 receptor (IL-13R), as a novel ligand of integrin Mac-1, using a co-evolution-based algorithm. Biochemical analyses demonstrated that recombinant IL-13Rα1 binds Mac-1 in a purified system and supports Mac-1-mediated cell adhesion. Co-immunoprecipitation experiments revealed that endogenous Mac-1 forms a complex with IL-13Rα1 in solution, and confocal fluorescence microscopy demonstrated that these two receptors co-localize with each other on the surface of macrophages. Moreover, we found that genetic inactivation of Mac-1 promotes IL-13-induced JAK/STAT activation in macrophages, resulting in enhanced polarization along the alternative activation pathway. Importantly, we observed that Mac-1(-/-) macrophages exhibit increased expression of foam cell differentiation markers including 15-lipoxygenase and lectin-type oxidized LDL receptor-1 both in vitro and in vivo. Indeed, we found that Mac-1(-/-)LDLR(-/-) mice develop significantly more foam cells than control LDLR(-/-) mice, using an in vivo model of foam cell formation. Together, our data establish for the first time a molecular mechanism by which Mac-1 regulates the signaling activity of IL-13 in macrophages. This newly identified IL-13Rα1/Mac-1-dependent pathway may offer novel targets for therapeutic intervention in the future.

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 (ß15-66)(2) fragment, which mimics a pair of fibrin ßN-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 (ß15-66)(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 ßN-domains, and this interaction is inhibited by RAP and (ß15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing ßN-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.

Molecular basis for the interaction of low density lipoprotein receptor-related protein 1 (LRP1) with integrin alphaMbeta2: 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 ß(2) integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes α(M)ß(2) but not its homologous receptor α(L)ß(2). The interaction between these two cellular receptors in macrophages is significantly enhanced upon α(M)ß(2) activation by LPS and is mediated by multiple regions in both LRP1 and α(M)ß(2). Specifically, we find that both the heavy and light chains of LRP1 are involved in α(M)ß(2) binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For α(M)ß(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)ß(2) receptor with that of α(M) confers the α(L)ß(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)ß(2), our finding suggests that binding of LRP1 to α(M)ß(2) could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits α(M)ß(2)-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.

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-A resolution. The structure revealed an eight-stranded antiparallel beta-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.

Cytoskeletal protein radixin activates integrin alpha(M)beta(2) by binding to its cytoplasmic tail.

Talin binding of integrins, via its band 4.1, ezrin, radixin, and moesin (FERM)-homologous domain, directly activates the integrin receptor. However, it is not known whether other FERM-containing proteins also possess such an integrin activating capability. We report here that radixin, one of the original FERM-domain proteins, binds to the membrane-proximal region of the integrin beta(2) but not alpha(M) cytoplasmic tail. Importantly, we show that radixin binding significantly enhances the adhesive activity of integrin alpha(M)beta(2). Given the distinct biological activities of radixin and talin, radixin may represent a novel talin-independent pathway for integrin activation under specific settings.

Mac-1 promotes FcgammaRIIA-dependent cell spreading and migration on immune complexes.

The integrin Mac-1 plays a critical role in Fc receptor (FcR)-mediated antibody-dependent cellular cytotoxicity (ADCC). However, the mechanism by which Mac-1 facilitates the functions of FcgammaRIIA, a major FcR expressed on human leukocytes, is not fully understood. We report here that Mac-1 sustains cell adhesion, enhances cell spreading, and accelerates cell migration on preformed immune complexes (ICs) by directly interacting with FcgammaRIIA but not with the IC substrate. Coupling Mac-1 to FcgammaRIIA allows FcgammaRIIA to reside in the leading front of actin polymerization at the filopodial extension and thus could potentially enhance FcgammaRIIA-mediated cell spreading and migration. The direct interaction between Mac-1 and FcgammaRIIA is demonstrated by co-immunoprecipitation, by cell surface co-localization, and by solid-phase binding assays using recombinant alpha(M)I-domain and soluble FcgammaRIIA. Further mutational analysis identifies the E(253)-R(261) sequence within the alpha(M)I-domain as part of the FcgammaRIIA binding interface within Mac-1. Altogether, these results demonstrate that FcgammaRIIA recognizes Mac-1 via the alpha(M)I-domain but not the lectin domain, a distinct feature from other FcRs, and that Mac-1 binding confers FcgammaRIIA with the ability to prolong cell adhesion as well as to spread and migrate on the ICs, leading to effective cell killing by ADCC.

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).

Defective osteogenesis of the stromal stem cells predisposes CD18-null mice to osteoporosis.

Osteogenesis by the bone marrow stromal stem cells (BMSSCs) supports continuous bone formation and the homeostasis of the bone marrow microenvironment. The mechanism that controls the proliferation and differentiation of BMSSCs is not fully understood. Here, we report that CD18, a surface protein present primarily on hematopoietic cells, but not on differentiated mesenchymal cells, is expressed by the stromal stem cells and plays a critical role in the osteogenic process. Constitutive expression of CD18 on BMSSCs using a retroviral promoter significantly enhances bone formation in vivo, whereas genetic inactivation of CD18 in mice leads to defective osteogenesis due to decreased expression of the osteogenic master regulator Runx2/Cbfa1. The defective osteogenesis of the CD18-null BMSSCs can be restored by expressing full-length, but not cytoplasmic domain-truncated, CD18. Radiographic analyses with dual-energy x-ray absorptiometry and 3D microcomputed tomography show that mice lacking CD18 have decreased bone mineral density and exhibit certain features of osteoporosis. Altogether, this work demonstrates that CD18 functions critically in the osteogenesis of BMSSCs, and thus lack of CD18 expression in the leukocyte adhesion deficiency patients may predispose them to osteoporosis.

A specific role of integrin Mac-1 in accelerated macrophage efflux to the lymphatics.

In response to injury, monocytes migrate to the site of inflammation, where they differentiate into macrophages and participate in various biologic processes. However, their fate during the resolution of acute inflammation is not fully understood. Here, we show that inflammatory macrophages do not die locally by apoptosis; rather, they migrate across the peritoneal mesothelium to the lymphatics, through which they further migrate to the lymph nodes and to the blood circulation. Macrophage efflux is enhanced considerably on cell activation, and such accelerated macrophage migration is dependent specifically on integrin Mac-1, and can be blocked by addition of its antagonist. Thus, genetic inactivation of Mac-1 in mice inhibits the accelerated macrophage efflux from the inflammatory site to the lymphatics, but it does not compromise the accumulation of blood monocytes into the inflammatory site. Together, our study demonstrates that Mac-1 is involved specifically in the efflux of activated macrophages to the lymphatics, suggesting that Mac-1 may play an important role in the removal of local inflammatory macrophages and in their subsequent migration to the lymph nodes, a process that is critical to the development of the adaptive immunity.