Amha Atakilit

Integrin α9β1 Directly Binds to Vascular Endothelial Growth Factor (VEGF)-A and Contributes to VEGF-A-induced Angiogenesis

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin α9β1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from α9β1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike α3β1or αvβ3 integrins, α9β1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because α9β1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that α9β1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin α9β1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.

Expression of integrin β6 enhances invasive behavior in oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the αvβ6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the β6 subunit into poorly invasive SCC9 cells to establish the SCC9β6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of β6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to αvβ6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-α5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of β6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9β6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-αvβ6 antibodies. The expression of β6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9β6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-αvβ6 antibodies. These results demonstrate that expression of β6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin αvβ6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.

IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.

A recipe for intestinal lgA Our guts are teeming with microbes, some friendly and others not. Plasma cells in the gut secrete immunoglobulin A (IgA), which helps to keep the peace with resident commensal bacteria and fights pathogens. B cell isotype switching to IgA occurs in lymphoid tissues called Peyers patches. Reboldi et al. studied the cellular processes that guide B cells toward making IgA in mice. B cells took an unexpected journey from Peyers patches follicles to the intestinal mucosa to interact with specialized IgA-triggering dendritic cells. The B cells then migrated back to the follicles to become IgA-producing B cells. Science, this issue p. 10.1126/science.aaf4822 Multiple cellular and molecular cues in the intestine instruct B cells to switch to producing immunoglobulin A. INTRODUCTION Secretory immunoglobulin A (IgA) is made by intestinal plasma cells and has roles both in protection from gut pathogens and in maintaining homeostasis of intestinal commensals. Peyer’s patches (PPs)—the major organized lymphoid tissues of the small intestine, numbering 100 to 200 in humans and 6 to 12 in mice—are the dominant source of IgA-producing cells. A number of molecular factors have been identified that promote B cell switching from IgM to IgA, the best defined being transforming growth factor–β (TGFβ). TGFβ is made in a latent form and must be activated before it can induce TGFβ receptor (TGFβR) signaling. In this study, we explore the requirements for B cell IgA switching in PPs, concentrating on the location where it takes place and the key cell types involved. RATIONALE Mice deficient in the chemokine receptor CCR6 had been reported to mount poor IgA responses, but the mechanism responsible was unclear. The CCR6 ligand, CCL20, is abundant in the subepithelial dome (SED) of the PP, and one thought was that CCR6 was required for positioning dendritic cells (DCs) in the SED. However, CCR6 was known to be expressed by B cells and to be up-regulated following B cell activation. In this study, we have pursued the hypothesis that CCR6 is required within B cells to promote migration events and cellular interactions in the SED necessary for PP IgA responses. RESULTS Using bone marrow (BM) chimera and cell transfer approaches, we find that CCR6 expression in PP B cells is necessary for their efficient switching to IgA and for production of intestinal IgA against cholera toxin and commensal bacteria. Loss- and gain-of-function approaches establish that intrinsic CCR6 expression is necessary and sufficient for B cells to access the SED. CCR6 is up-regulated on pre–germinal center (GC) B cells in a CD40-dependent manner, and a transfer model indicates a more prominent role for CCR6 in T cell–dependent than in T cell–independent IgA responses. PP pre-GC B cells are shown to express IgA germline transcripts and activation-induced cytidine deaminase (AID), consistent with IgA switching initiating in this compartment. Using intravital two-photon microscopy, we find that B cells within the SED undergo prolonged interactions with DCs. Using BM chimera experiments and blocking reagents, we establish that SED DCs are dependent on the cytokine lymphotoxin-α1β2 (LTα1β2). RORγt+ innate lymphoid cells (ILCs) are identified as a necessary source of this cytokine. Deficiency in LTβR-dependent DCs or RORγt-dependent ILCs results in reduced IgA+ B cell frequencies in PPs. Reciprocally, transgenic overexpression of LTα1β2 increases SED DCs and IgA switching. We then examined how the SED DCs augment IgA switching and found that they abundantly expressed αvβ8, an integrin that has an established role in converting TGFβ from its latent to its active state. Experiments with Itgb8f/f Cd11c-Cre mice and with an αvβ8 blocking antibody established that DC αvβ8 expression was necessary for PP IgA switching. In vitro experiments provided further evidence that DC αvβ8 could directly activate TGFβ during DC–B cell interactions and showed that LTβR and retinoic acid signaling promote αvβ8 expression on DCs. CONCLUSION Our study defines a role for the PP SED as a niche that supports events necessary for IgA switching, in particular the induction of TGFβ activation, and it provides an example of a DC–B cell interaction acting to guide B cell fate. By defining a network of interactions required for IgA switching, this study identifies approaches that could be used to augment IgA responses while also defining sites for defects that could underlie IgA deficiency, the most common immune deficiency syndrome in humans. B cell and dendritic cell distribution in mouse Peyer’s patch. Image is a cross-sectional view of a single PP dome and the neighboring villous epithelium of the small intestine. The 7-µm frozen section was stained to detect naïve and pre-GC B cells (IgD, blue) that occupy the follicle and SED; dendritic cells (CD11c, green) that are abundant in the SED, the interfollicular T zone, and the intestinal lamina propria; T cells (CD8, red) that are present in the interfollicular T zone, the lamina propria, and in the epithelium; and nuclei (DAPI, gray). Red staining also occurred nonspecifically in association with the epithelium, and this was most prominent for the follicle-associated epithelium that overlies the SED. The follicle-associated epithelium is the site of intestinal antigen delivery into the PP. The dark (IgD-negative) oval-shaped structure within the follicle is a GC. Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer’s patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-β receptor (LTβR)–dependent maintenance of DCs. PP DCs augment IgA production by integrin αvβ8-mediated activation of transforming growth factor–β (TGFβ). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC–B cell interactions needed for TGFβ activation and induction of mucosal IgA responses.

Integrin Dependence of Brain Natriuretic Peptide Gene Promoter Activation by Mechanical Strain

Expression of the brain natriuretic peptide (BNP) gene in cultured neonatal rat ventricular myocytes is activated by mechanical strain in vitro. We explored the role of cell-matrix contacts in initiating the strain-dependent increment in human BNP (hBNP) promoter activity. Coating the culture surface with fibronectin effected a dose-dependent increase in basal hBNP luciferase activity and amplification of the response to strain. Preincubation of myocytes with an RGD peptide (GRGDSP) or with soluble fibronectin, each of which would be predicted to compete for cell-matrix interactions, resulted in a dose-dependent reduction in strain-dependent hBNP promoter activity. A functionally inert RGE peptide (GRGESP) was without effect. Using fluorescence-activated cell sorting, we demonstrated the presence of β1, β3, and αvβ5 integrins in myocytes as well as non-myocytes and α1 only in non-myocytes in our cultures. Inclusion of antibodies directed against β1, β3, or αvβ5, but not α1, α2, or cadherin, was effective in blocking the BNP promoter response to mechanical strain. These same antibodies (anti-β3, -β1, and -αvβ5) had a similar inhibitory effect on strain-stimulated ERK, p38 MAPK, and, to a lesser extent, JNK activities in these cells. Cotransfection with chimeric integrin receptors capable of acting as dominant-negative inhibitors of integrin function demonstrated suppression of strain-dependent BNP promoter activity when vectors encoding β1 or β3, but not β5, α5, or a carboxyl-terminal deletion mutant of β3(β3B), were employed. These studies underscore the importance of cell-matrix interactions in controlling cardiac gene expression and suggest a potentially important role for these interactions in signaling responses to mechanical stimuli within the myocardium.

Absence of Integrin αvβ3 Enhances Vascular Leak in Mice by Inhibiting Endothelial Cortical Actin Formation

RATIONALE Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms. OBJECTIVES We hypothesized that integrin αvβ3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI. METHODS β3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of β3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvβ3 antibodies were evaluated for sphingosine-1 phosphate (S1P)–mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization. MEASUREMENTS AND MAIN RESULTS β3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvβ3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvβ3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvβ3 localization after S1P was decreased by αvβ3 antibodies, suggesting that ligation of the αvβ3 with its extracellular matrix ligands is required to stabilize cortical αvβ3 focal adhesions. CONCLUSIONS Our studies identify a novel mechanism by which αvβ3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvβ3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.

Differential enzymatic activity of common haplotypic versions of the human acidic Mammalian chitinase protein.

Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.

Role of the Cytoplasmic Domain of the β-Subunit of Integrin αvβ6 in Infection by Foot-and-Mouth Disease Virus

ABSTRACT Field isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, αvβ6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the αvβ6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the β6 cytoplasmic domain on infection. Our results show that although loss of the β6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in αvβ6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.

Integrin α9β1 in airway smooth muscle suppresses exaggerated airway narrowing

Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).

Effective treatment of mouse sepsis with an inhibitory antibody targeting integrin αvβ5.

Objective:Integrin &agr;v&bgr;5 has been identified as a regulator of vascular leak and endothelial permeability. We hypothesized that targeting &agr;v&bgr;5 could represent a viable treatment strategy for sepsis. Design:Integrin &bgr;5 subunit knockout and wild-type 129/svJae mice and wild-type mice treated with &agr;v&bgr;5 blocking or control antibodies were tested in models of intraperitoneal lipopolysaccharide and cecal ligation and puncture. Human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers were treated with &agr;v&bgr;5 antibodies to assess for effects on lipopolysaccharide-induced changes in transendothelial resistance and on patterns of cytoskeletal reorganization. Setting:Laboratory-based research. Subjects:Mice and endothelial cell monolayers. Interventions, Measurements, and Main Results:Measurements taken after intraperitoneal lipopolysaccharide and/or cecal ligation and puncture included mortality, vascular leak, hematocrit, quantification of a panel of serum cytokines/chemokines, and assessment of thioglyccolate-induced leukocyte migration. &bgr;5 knockout mice had decreased mortality after intraperitoneal lipopolysaccharide and cecal ligation and puncture and decreased vascular leak, as measured by extravasation of an I125-labeled intravascular tracer. Treating clinically ill mice with &agr;v&bgr;5 antibodies, up to 20 hrs after intraperitoneal lipopolysaccharide and cecal ligation and puncture, also resulted in decreased mortality. &agr;v&bgr;5 antibodies attenuated lipopolysaccharide-induced transendothelial resistance changes and cytoskeletal stress fiber formation in both human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers. &agr;v&bgr;5 antibodies had no effect on cytokine/chemokine serum levels after cecal ligation and puncture. &bgr;5 knockout mice and wild-type controls did not exhibit differences in thioglyccolate-induced leukocyte migration. Conclusions:Our studies suggest that &agr;v&bgr;5 is an important regulator of the vascular endothelial leak response in sepsis and that &agr;v&bgr;5 blockade may provide a novel approach to treating this devastating disease syndrome.