NF-κB directs dynamic super enhancer formation in inflammation and atherogenesis.

Proinflammatory stimuli elicit rapid transcriptional responses via transduced signals to master regulatory transcription factors. To explore the role of chromatin-dependent signal transduction in the atherogenic inflammatory response, we characterized the dynamics, structure, and function of regulatory elements in the activated endothelial cell epigenome. Stimulation with tumor necrosis factor alpha prompted a dramatic and rapid global redistribution of chromatin activators to massive de novo clustered enhancer domains. Inflammatory super enhancers formed by nuclear factor-kappa B accumulate at the expense of immediately decommissioned, basal endothelial super enhancers, despite persistent histone hyperacetylation. Mass action of enhancer factor redistribution causes momentous swings in transcriptional initiation and elongation. A chemical genetic approach reveals a requirement for BET bromodomains in communicating enhancer remodeling to RNA Polymerase II and orchestrating the transition to the inflammatory cell state, demonstrated in activated endothelium and macrophages. BET bromodomain inhibition abrogates super enhancer-mediated inflammatory transcription, atherogenic endothelial responses, and atherosclerosis in vivo.

Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas.

Antibodies that bind CD47 on tumor cells and prevent interaction with SIRPα on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPα interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab')2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fcγ receptor interactions. In contrast, F(ab')2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fcγ receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G.

Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1ß-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.

Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies.

The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article reviews basic mechanisms in the recruitment of leukocytes to tissues and then selectively reviews new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. Investigators have also begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise.

Emerging mechanisms of neutrophil recruitment across endothelium.

Neutrophils are the all-terrain vehicle of the innate immune system because of their ability to gain entry into tissues and organs, and thus, play an essential role in host defense. Exactly how this marvel of nature works is still incompletely understood. In the past 2-3 years, new players and processes have been identified in the endothelial-leukocyte adhesion cascade. Novel signaling pathways have been discovered in both the endothelium and the neutrophils that regulate various steps in the recruitment process. This review focuses on these emerging pathways and the mechanisms that regulate neutrophil recruitment across endothelium.

Novel role of CD47 in rat microvascular endothelium: signaling and regulation of T-cell transendothelial migration.

OBJECTIVE: Although endothelial CD47, a member of the immunoglobulin superfamily, has been implicated in leukocyte diapedesis, its capacity for intracellular signaling and physical localization during this process has not been addressed in detail. This study examined endothelial CD47 spatiotemporal behavior and signaling pathways involved in regulating T-cell transendothelial migration. APPROACH AND RESULTS: By biochemical methods, transmigration assays, and live-cell microscopy techniques, we show that endothelial CD47 engagement results in intracellular calcium mobilization, increased permeability, and activation of Src and AKT1/phosphoinositide 3-kinase in brain microvascular endothelial cells. These signaling pathways converge to induce cytoskeleton remodeling and vascular endothelial cadherin phosphorylation, which are necessary steps during T-cell transendothelial migration. In addition, during T-cell migration, transmigratory cups and podo-prints enriched in CD47 appear on the surface of the endothelium, indicating that the spatial distribution of CD47 changes after its engagement. Consistent with previous findings of intercellular adhesion molecule 1, blockade of CD47 results in decreased T-cell transmigration across microvascular endothelium. The overlapping effect of intercellular adhesion molecule 1 and CD47 suggests their involvement in different steps of the diapedesis process. CONCLUSIONS: These data reveal a novel role for CD47-mediated signaling in the control of the molecular network governing endothelial-dependent T-cell diapedesis.

Difference in Th1 and Th17 lymphocyte adhesion to endothelium.

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.

Endothelial CD47 promotes vascular endothelial-cadherin tyrosine phosphorylation and participates in T cell recruitment at sites of inflammation in vivo.

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47⁻/⁻ mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α-induced inflammation. Reconstitution of CD47⁻/⁻ mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47⁻/⁻ endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(αi)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(αi), had no inhibitory effect, whereas G(αi) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(αi). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.

IL-17 and TNF-α sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation.

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.

Foxp3+-inducible regulatory T cells suppress endothelial activation and leukocyte recruitment.

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg, as well as their direct influence at the site of inflammation, remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3(+) adaptive/inducible Treg (iTreg), but not naturally occurring Treg, efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial Ag presentation or immobilized anti-CD3ε, Foxp3(+) iTreg suppressed TNF-α- and IL-1ß-mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGF-ß-induced activin receptor-like kinase 5 signaling in endothelial cells. In addition, Foxp3(+) iTreg adhered to inflamed endothelium in vivo, and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3(+) iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.

p120-Catenin prevents neutrophil transmigration independently of RhoA inhibition by impairing Src dependent VE-cadherin phosphorylation.

Leukocyte transendothelial migration (TEM) is regulated by several signaling pathways including Src family kinases (SFK) and the small RhoGTPases. Previous studies have shown that vascular endothelial-cadherin (VE-cad) forms a complex with ß-,γ-, and p120-catenins and this complex disassociates to form a transient gap during leukocyte TEM. Additionally, p120-catenin (p120-1A) overexpression in human umbilical vein endothelial cells (HUVEC) stabilizes VE-cad surface expression, prevents tyrosine phosphorylation of VE-cad, and inhibits leukocyte TEM. Based on reports showing that p120 overexpression in fibroblasts or epithelial cells inhibits RhoA and activates Rac and Cdc42 GTPases, and on other reports showing that RhoA activation in endothelial cells is necessary for leukocyte TEM, we reasoned that p120 overexpression inhibited TEM through inhibition of RhoA. To test this idea, we overexpressed a mutant p120 isoform, p120-4A, which does not interact with RhoA. p120-4A colocalized with VE-cad in HUVEC junctions and enhanced VE-cad surface expression, similar to overexpression of p120-1A. Interestingly, overexpression of either p120-4A or p120-1A dramatically blocked TEM, and overexpression of p120-1A in HUVEC did not affect RhoA basal activity or activation of RhoA and Rac induced by thrombin or ICAM-1 crosslinking. In contrast, biochemical studies revealed that overexpression of p120-1A reduced activated pY416-Src association with VE-cad. In summary, p120 overexpression inhibits neutrophil TEM independently of an effect on RhoA or Rac and instead blocks TEM by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex.

SIRPα - CD47 axis regulates dendritic cell-T cell interactions and TCR activation during T cell priming in spleen.

The SIRPα-CD47 axis plays an important role in T cell recruitment to sites of immune reaction and inflammation but its role in T cell antigen priming is incompletely understood. Employing OTII TCR transgenic mice bred to Cd47-/- (Cd47KO) or SKI mice, a knock-in transgenic animal expressing non-signaling cytoplasmic-truncated SIRPα, we investigated how the SIRPα-CD47 axis contributes to antigen priming. Here we show that adoptive transfer of Cd47KO or SKI Ova-specific CD4+ T cells (OTII) into Cd47KO and SKI recipients, followed by Ova immunization, elicited reduced T cell division and proliferation indices, increased apoptosis, and reduced expansion compared to transfer into WT mice. We confirmed prior reports that splenic T cell zone, CD4+ conventional dendritic cells (cDCs) and CD4+ T cell numbers were reduced in Cd47KO and SKI mice. We report that in vitro derived DCs from Cd47KO and SKI mice exhibited impaired migration in vivo and exhibited reduced CD11c+ DC proximity to OTII T cells in T cell zones after Ag immunization, which correlates with reduced TCR activation in transferred OTII T cells. These findings suggest that reduced numbers of CD4+ cDCs and their impaired migration contributes to reduced T cell-DC proximity in splenic T cell zone and reduced T cell TCR activation, cell division and proliferation, and indirectly increased T cell apoptosis.

CD47 antibody blockade suppresses microglia-dependent phagocytosis and monocyte transition to macrophages, impairing recovery in EAE.

Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model of multiple sclerosis. During the early phase of EAE, infiltrating monocytes and monocyte-derived macrophages contribute to T cell recruitment, especially CD4+ T cells, into the CNS, resulting in neuronal demyelination; however, in later stages, they promote remyelination and recovery by removal of myelin debris by phagocytosis. Signal regulatory protein α and CD47 are abundantly expressed in the CNS, and deletion of either molecule is protective in myelin oligodendrocyte glycoprotein-induced EAE because of failed effector T cell expansion and trafficking. Here we report that treatment with the function blocking CD47 Ab Miap410 substantially reduced the infiltration of pathogenic immune cells but impaired recovery from paresis. The underlying mechanism was by blocking the emergence of CD11chiMHCIIhi microglia at peak disease that expressed receptors for phagocytosis, scavenging, and lipid catabolism, which mediated clearance of myelin debris and the transition of monocytes to macrophages in the CNS. In the recovery phase of EAE, Miap410 Ab-treated mice had worsening paresis with sustained inflammation and limited remyelination as compared with control Ab-treated mice. In summary, Ab blockade of CD47 impaired resolution of CNS inflammation, thus worsening EAE.

Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo.

Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.

Endothelial STING controls T cell transmigration in an IFNI-dependent manner.

The stimulator of IFN genes (STING) protein senses cyclic dinucleotides released in response to double-stranded DNA and functions as an adaptor molecule for type I IFN (IFNI) signaling by activating IFNI-stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared with control EC, whereas T cell adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell-expressed molecules ICAM1 and VCAM1, compared with WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC, and genetic loss or pharmacologic antagonisms of IFNI receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC-STING during T cell TEM that is dependent on the ISG CXCL10 and on IFNI/IFNAR signaling.

Endothelial CD47 interaction with SIRPgamma is required for human T-cell transendothelial migration under shear flow conditions in vitro.

Leukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPalpha and SIRPgamma. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3(+) T cells express SIRPgamma but not SIRPalpha, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPgamma. We tested, therefore, whether CD47-SIRPgamma interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPgamma in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-alpha-activated murine CD47(-/-) endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPgamma. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPgamma, play an important role in T-cell transendothelial migration.

p120-Catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation.

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endothelial cell borders and forms a complex with alpha-, beta-, gamma-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendothelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endothelial cells transduced with an adenovirus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leukocyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions.

Expanding the utility of beta-galactosidase complementation: piece by piece.

The ability to image and quantify multiple biomarkers in disease necessitates the development of split reporter fragment platforms. We have divided the beta-galactosidase enzyme into unique, independent polypeptides that are able to reassemble and complement enzymatic activity in bacteria and in mammalian cells. We created two sets of complementing pairs that individually have no enzymatic activity. However, when brought into close geometric proximity, the complementing pairs associated resulting in detectable enzymatic activity. We then constructed a stable ligand complex composed of reporter fragment, linker, and targeting moiety. The targeting moiety, in this case a ligand, allowed cell surface receptor targeting in vitro. Further, we were able to simultaneously visualize two cell surface receptors implicated in cancer development, epidermal growth factor receptor and transferrin receptor, using complementing pairs of the ligand-reporter fragment complex.

Eosinophils improve cardiac function after myocardial infarction.

Clinical studies reveal changes in blood eosinophil counts and eosinophil cationic proteins that may serve as risk factors for human coronary heart diseases. Here we report an increase of blood or heart eosinophil counts in humans and mice after myocardial infarction (MI), mostly in the infarct region. Genetic or inducible depletion of eosinophils exacerbates cardiac dysfunction, cell death, and fibrosis post-MI, with concurrent acute increase of heart and chronic increase of splenic neutrophils and monocytes. Mechanistic studies reveal roles of eosinophil IL4 and cationic protein mEar1 in blocking H2O2- and hypoxia-induced mouse and human cardiomyocyte death, TGF-ß-induced cardiac fibroblast Smad2/3 activation, and TNF-α-induced neutrophil adhesion on the heart endothelial cell monolayer. In vitro-cultured eosinophils from WT mice or recombinant mEar1 protein, but not eosinophils from IL4-deficient mice, effectively correct exacerbated cardiac dysfunctions in eosinophil-deficient ∆dblGATA mice. This study establishes a cardioprotective role of eosinophils in post-MI hearts.