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Background & Aims: The reason why small intestinal cancer is rarer than colorectal cancer is not clear. We hypothesized that intraepithelial lymphocytes (IELs), which are enriched in the small intestine, are the closest immune cells to epithelial cells, exclude tumor cells via cell-to-cell contact., Methods: We developed DPE-green fluorescent protein (DPE-GFP) × adenomatous polyposis coli; multiple intestinal neoplasia (APC min ) mice, which is a T-cell-reporter mouse with spontaneous intestinal tumors. We visualized the dynamics of IELs in the intestinal tumor microenvironment and the interaction between IELs and epithelial cells, and the roles of cell-to-cell contact in anti-intestinal tumor immunity using a novel in vivo live-imaging system and a novel in vitro co-culture system., Results: In the small intestinal tumor microenvironment, T-cell movement was restricted around blood vessels and the frequency of interaction between IELs and epithelial cells was reduced. Genetic deletion of CD103 decreased the frequency of interaction between IELs and epithelial cells, and increased the number of small intestinal tumors. In the co-culture system, wild-type IELs expanded and infiltrated to intestinal tumor organoids from APC min mice and reduced the viability of them, which was cell-to-cell contact and CD103 dependent., Conclusions: The abundance of IELs in the small intestine may contribute to a low number of tumors, although this system may not work in the colon because of the sparseness of IELs. Strategies to increase the number of IELs in the colon or enhance cell-to-cell contact between IELs and epithelial cells may be effective for the prevention of intestinal tumors in patients with a high cancer risk., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Thyroid cancer is one of the common malignancies of the endocrine system and the number of thyroid cancer cases is increasing constantly. Significant work has focused on the molecular mechanisms of thyroid cancer, but many mechanisms remain undiscovered. In this study, we employed a comprehensive analysis of whole-transcriptome resequencing derived from paired papillary thyroid cancer (PTC) and normal thyroid tissues. We performed a massive parallel whole-transcriptome resequencing of matched PTC and normal thyroid tissues in 19 patients and found that integrin subunit alpha 7 (ITGA7) was downregulated in thyroid tumor tissues, but the function of ITGA7 in this cancer is still unclear. We also discovered that ITGA7 gene in thyroid cancer tissues was downregulated compared to paired adjacent non-tumor tissues by real-time quantitative polymerase chain reaction. After transfection with small interfering RNA to knock down ITGA7, the abilities of colony formation, proliferation, migration, and invasion were enhanced in PTC cell lines (TPC1 and KTC-1). Meanwhile, ITGA7 knockdown decreased apoptotic cell death in thyroid cells but promoted the expressions of N-cadherin and vimentin and decreased E-cadherin expression by epithelial-to-mesenchymal transition, which may induce invasion and migration. In conclusion, these results indicated that ITGA7 is involved in the progress of PTC and might act as a tumor suppressor gene., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDβ2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDβ2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement., Competing Interests: The authors have declared that no competing interests exist.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMβ2, β1, α2, αIIbβ3, and α5 migrated to the cathode, whereas cells expressing β3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6β4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

Wound reepithelialization is an evolutionarily conserved process in which skin cells migrate as sheets to heal the breach and is critical to prevent infection but impaired in chronic wounds. Integrin heterodimers mediate attachment between epithelia and underlying extracellular matrix and also act in large signaling complexes. The complexity of the mammalian wound environment and evident redundancy among integrins has impeded determination of their specific contributions to reepithelialization. Taking advantage of the genetic tools and smaller number of integrins in Drosophila, we undertook a systematic in vivo analysis of integrin requirements in the reepithelialization of skin wounds in the larva. We identify αPS2-βPS and αPS3-βPS as the crucial integrin dimers and talin as the only integrin adhesion component required for reepithelialization. The integrins rapidly accumulate in a JNK-dependent manner in a few rows of cells surrounding a wound. Intriguingly, the integrins localize to the distal margin in these cells, instead of the frontal or lamellipodial distribution expected for proteins providing traction and recruit nonmuscle myosin II to the same location. These findings indicate that signaling roles of integrins may be important for epithelial polarization around wounds and lay the groundwork for using Drosophila to better understand integrin contributions to reepithelialization.

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α 3 , α 5 , α 6 , α 9 , α V , and β 1 subunits, but not the α 1 , α 2 , α 4 , α 7 , and α 8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α 5 β 1 , α 9 β 1 , or α V β 1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α 6 β 1 heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α 5 β 1 , α 9 β 1 , and α V β 1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α 3 (presumed) and α 6 subunits., (© 2018 International Federation for Cell Biology.)

Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Murine contact hypersensitivity (CHS) is a dendritic cell (DC)-dependent T-cell-mediated inflammation with CD8+ T cells as effectors and CD4+ T cells as regulators (Treg cells) that models human allergic contact dermatitis. The integrin αE(CD103) is expressed by some T-cell and DC subsets and has been implicated in epithelial lymphocyte localization, but its role in immune regulation remains enigmatic. We have identified a function for CD103 in the development of cutaneous allergic immune responses. CHS responses, but not irritant contact dermatitis, were significantly augmented in CD103-deficient mice in hapten-challenged skin. Phenotype and function of skin DCs during sensitization were normal, whereas adoptive transfer experiments revealed that the elevated CHS response in CD103-deficient mice is transferred by primed T cells and is independent of resident cells in recipient mice. While T-cell counts were elevated in challenged skin of CD103-deficient mice, the FoxP3 expression level of CD4+CD25+ Treg cells was significantly reduced, indicating impaired functionality. Indeed, Treg cells from CD103-deficient mice were not able to suppress CHS reactions during the elicitation phase. Further, CD103 on FoxP3+ Treg cells was involved in Treg retention to inflamed skin. These findings indicate an unexpected dichotomous functional role for CD103 on Treg cells by modulating FoxP3 expression.

α9β1 is the most recent addition to the integrin family of membrane receptors and consequently remains the one that is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the α9 gene. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to seeding HCECs increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

There is compelling evidence that autoreactive CD8(+)T cells play a central role in precipitating the development of autoimmune diabetes in non-obese diabetic (NOD) mice, but the underlying mechanisms remain unclear. Given that ITGAE (CD103) recognizes an islet-restricted ligand (E-cadherin), we postulated that its expression is required for initiation of disease. We herein use a mouse model of autoimmune diabetes (NOD/ShiLt mice) to test this hypothesis. We demonstrate that ITGAE is expressed by a discrete subset of CD8(+)T cells that infiltrate pancreatic islets before the development of diabetes. Moreover, we demonstrate that development of diabetes in Itgae-deficient NOD mice is significantly delayed at early but not late time points, indicating that ITGAE is preferentially involved in early diabetes development. To rule out a potential contribution by closely linked loci to this delay, we treated WT NOD mice beginning at 2 weeks of age through 5 weeks of age with a depleting anti-ITGAE mAb and found a decreased incidence of diabetes following anti-ITGAE mAb treatment compared with mice that received isotype control mAbs or non-depleting mAbs to ITGAE. Moreover, a histological examination of the pancreas of treated mice revealed that NOD mice treated with a depleting mAb were resistant to immune destruction. These results indicate that ITGAE(+) cells play a key role in the development of autoimmune diabetes and are consistent with the hypothesis that ITGAE(+)CD8(+)T effectors initiate the disease process., (© 2015 Society for Endocrinology.)

Despite extensive research, the pathogenesis of cigarette smoking (CS)-associated emphysema remains incompletely understood, thereby impeding development of novel therapeutics, diagnostics, and biomarkers. Here, we report a novel paradigm potentially involved in the development of epithelial death and tissue loss in CS-associated emphysema. After prolonged exposure of CS, CCN1 cleavage was detected both in vitro and in vivo. Full-length CCN1 (flCCN1) was secreted in an exosome-shuttled manner, and secreted plasmin converted flCCN1 to cleaved CCN1 (cCCN1) in extracellular matrix. Interestingly, exosome-shuttled flCCN1 facilitated the interleukin (IL)-8 and vascular endothelial growth factor (VEGF) release in response to cigarette smoke extract (CSE). Therefore, flCCN1 potentially promoted CS-induced inflammation via IL-8-mediated neutrophil recruitment and also maintained the lung homeostasis via VEGF secretion. Interestingly, cCCN1 abolished these functions. Furthermore, cCCN1 promoted protease and matrix metalloproteinase (MMP)-1 production after CSE. These effects were mainly mediated by the COOH-terminal fragments of CCN1 after cleavage. Both the decrease of VEGF and the elevation of MMPs favor the development of emphysema. cCCN1, therefore, likely contributes to the epithelial cell damage after CS. Additionally, CSE and cCCN1 both stimulated integrin-α7 expressions in lung epithelial cells. The integrin-α7 appeared to be the binding receptors of cCCN1 and, subsequently, mediated its cellular function by promoting MMP1. Consistent with our observation on the functional roles of cCCN1 in vitro, elevated cCCN1 level was found in the bronchoalveolar lavage fluid from mice with emphysematous changes after 6 mo CS exposure. Taken together, we hypothesize that cCCN1 promoted the epithelial cell death and tissue loss after prolonged CS exposure., (Copyright © 2014 the American Physiological Society.)

In the kidney, the α8 integrin chain (itga8) is expressed in mesenchymal cells and is upregulated in fibrotic disease. We hypothesized that itga8 mediates a profibrotic phenotype of renal cells by promoting extracellular matrix and cytokine expression. Genetic itga8 deficiency caused complex changes in matrix expression patterns in mesangial and smooth-muscle cells, with the only concordant effect in both cell types being a reduction of collagen III expression. Silencing of itga8 with siRNA led to a decline of matrix turnover with repression of matrix metalloproteinases and reduction of matrix production. In contrast, de novo expression of itga8 in tubular epithelial cells resulted in reduced collagen synthesis. Overexpression of itga8 in fibroblasts did not change the expression of matrix molecules or regulators of matrix turnover. Thus, the influence of itga8 on the expression of matrix components was not uniform and celltype dependent. Itga8 seems unlikely to exert overall profibrotic effects in renal cells.

Background: Previous studies have established that posthemorrhagic shock mesenteric lymph (PHSML) contains proinflammatory mediators, while the cellular basis of PHSML is less well characterized in acute models of injury. CD103 dendritic cells (DCs) have been identified in the mesenteric lymph (ML) in models of chronic intestinal inflammation, suggesting an important role in the gut response to injury. We have previously demonstrated the ability of vagal nerve stimulation (VNS) to prevent gut barrier failure after trauma/hemorrhagic shock (T/HS); however, the ability of VNS to alter ML DCs is unknown. We hypothesized that the CD103 MHC-II DC population would change in PHSML and that VNS would prevent injury-induced changes in this population in PHSML., Methods: Male Sprague-Dawley rats were randomly assigned to trauma/sham shock or T/HS. T/HS was induced by midline laparotomy and 60 minutes of HS (blood pressure, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals underwent cervical VNS after the HS phase. Gut tissue was harvested at 2 hours after injury for histologic analysis. ML was collected during the pre-HS, HS, and post-HS phase. For flow cytometric analysis, ML cells were subjected to staining with CD103 and MHC-II antibodies, and this cell population was compared in the pre-HS and post-HS phase from the same animal. The CD4Foxp3 cell (T reg) population in the ML node (MLN) was also tested to determine effects of CD103 DC modulation in the ML., Results: VNS reduced histologic gut injury and ML flow seen after injury. The CD103 MHC-II DC population in the PHSML was significantly decreased compared with pre-HS and was associated with decreased T reg expression in the MLN. VNS prevented the injury-induced decrease in the CD103 MHC-II+ DC population in the ML and restored the T reg population in the MLN., Conclusion: These findings suggest that VNS mediates the inflammatory responses in ML DCs and MLN T reg cells by affecting the set point of T/HS responsiveness.

Integrin α10β1 is the most abundant collagen-binding integrin in cartilaginous tissues and its expression pattern is distinct from that of other collagen-binding integrins. In vitro and in vivo studies have identified integrin α10β1 as a unique phenotypic marker for chondrocyte differentiation and a crucial mediator of cell-matrix interactions required for proper cartilage development. This chapter describes the structure of the integrin subunit α10, the tissue distribution of the integrin 10β1 and updates available information regarding its regulation and ligand binding. We also summarize current information on the functional roles of α10β1 in chondrogenesis of mesenchymal stem cells and in skeletal growth.

The extracellular matrix components osteopontin and tenascin-C are ligands of α9 integrin, and both play roles in corneal wound fibrosis and neovascularization. It has been shown that loss of osteopontin impairs closure of incisional wounds in the mouse cornea. Detailed analyses suggest that the loss of osteopontin reduces macrophage invasion and myofibroblast differentiation in the healing stroma in association with suppression of fibrogenic gene expression in response to injury. Cultured ocular fibroblasts derived from knockout mice showed an impairment of activation of p38 MAPK and Smad3 upon exposure to transforming growth factor β1. The loss of tenascin-C delays stromal healing in association with suppression of fibrogenic gene expression and macrophage invasion. With regard to neovascularization, the loss of either osteopontin or tenascin-C suppressed the growth of new blood vessels from the limbal region toward the central cornea in response to corneal cauterization in mice. Gene expression analysis further showed that lack of osteopontin or tenascin-C resulted in inhibition of angiogenic and proinflammatory gene expression. In conclusion, osteopontin or tenascin-C, α9 integrin ligands, play an important role in stromal healing (or fibrosis) and neovascularization in mouse cornea., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The metastasis suppressor KAI1/CD82 has been implicated in various cellular processes; however, its function in development is not fully understood. Here, we generated and characterized mutants of Tsp66E and Tsp74F, which are Drosophila homologues of KAI1/CD82 and Tspan11, respectively. These mutants exhibited egg elongation defects along with disturbed integrin localization and actin polarity. Moreover, the defects were enhanced by mutation of inflated, an αPS2 integrin gene. Mutant ovaries had elevated αPS2 integrin levels and reduced endocytic trafficking. These results suggest that Drosophila KAI1/CD82 affects the polarized localization and the level of integrin, which may contribute to epithelial cell polarity., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The Foxp3(+)CD4(+) regulatory T-cell (Treg)-deficient Scurfy (Sf) mice rapidly develop severe inflammation in the skin and lungs with expanded Th subsets bearing increased expression of various chemokine/chemoattractant/retention receptor genes (CRG). Nine different double mutants were generated to elucidate their roles in the skin and lung inflammation. The expanded Th2 response and the increased expression of several CRG for the skin and lung inflammation were inhibited in Sf.Il2(-/-) mice as previously described using microarray analysis. Herein in a reciprocal approach, we demonstrated that Sf.Il4(-/-) and Sf.Stat6(-/-) mice, despite lacking Th2 cytokines IL-4, IL-5, and IL-13, as well as the IL-4/STAT6-dependent CRG expression, the inflammation in the skin and lungs remained. The effect of the other Th1 cytokine IFN-γ was studied in Sf.Ifng(-/-) mice in which the multi-organ inflammation (MOI) was delayed but fully developed afterward with enhanced CRG expression except for the IFN-γ-dependent Cxcr3 in CD4(+) T-cells. Similarly, a transient delay of MOI was observed for Sf.Itgae(-/-) mice but their Th subsets and the critical CRG expansion remained. Ltb4r1(-/-), Alox5(-/-), Cx3cr1(gfp/gfp), or Il10(-/-) mutant genes also failed to effectively block inflammation in the skin and lungs in Sf mice. Our study has identified a novel function of IL-2 as a powerful Th1 cytokine that induces a panel of CRG in Th subsets required for skin and lung inflammation in Sf mice. The CRG panel induced by IL-2 but not by IL-4 or IFN-γ explains the apparent "organ-specific" display of the skin and lung inflammation in Sf mice., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dendritic cells (DC) play important roles in both tolerance and immunity to β cells in type 1 diabetes. How and why DC can have diverse and opposing functions in islets remains elusive. To answer these questions, islet DC subsets and their specialized functions were characterized. Under both homeostatic and inflammatory conditions, there were two main tissue-resident DC subsets in islets, defined as CD11b(lo/-)CD103(+)CX3CR1(-) (CD103(+) DC), the majority of which were derived from fms-like tyrosine kinase 3-dependent pre-DC, and CD11b(+)CD103(-)CX3CR1(+) (CD11b(+) DC), the majority of which were derived from monocytes. CD103(+) DC were the major migratory DC and cross-presented islet-derived Ag in the pancreatic draining lymph node, although this DC subset displayed limited phagocytic activity. CD11b(+) DC were numerically the predominant subset (60-80%) but poorly migrated to the draining lymph node. Although CD11b(+) DC had greater phagocytic activity, they poorly presented Ag to T cells. CD11b(+) DC increased in numbers and percentage during T cell-mediated insulitis, suggesting that this subset might be involved in the pathogenesis of diabetes. These data elucidate the phenotype and function of homeostatic and inflammatory islet DC, suggesting differential roles in islet immunity.

Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, α9β1 integrin is one of the least studied. α9, together with α4, comprise a more recent evolutionary sub-family of integrins that is only found in vertebrates. Since α9 was thought to have similar functions as α4, due to many shared ligands, it was a rather overlooked integrin until recently, when its importance for survival after birth was highlighted upon investigation of the α9 knockout mouse. α9β1 is expressed on a wide variety of cell types, interacts with many ligands for example fibronectin, tenascin-C and ADAM12, and has been shown to have important functions in processes such as cell adhesion and migration, lung development, lymphatic and venous valve development, and in wound healing. This has sparked an interest to investigate α9β1-mediated signaling and its regulation. This review gives an overview of the recent progress in α9β1-mediated biological and pathological processes, and discusses its potential as a target for cancer diagnosis and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The formation of a tubular organ, such as the heart, requires the communication of positional and polarity signals between migratory cells. Key to this process is the establishment of a new luminal domain on the cell surface, generally from the apical domain of a migratory cell. This domain will also acquire basal properties, as it will produce a luminal extracellular matrix. Integrin receptors are the primary means of cell adhesion and adhesion signaling with the extracellular matrix. Here we characterise the requirement of Integrins in a genetic model of vasculogenesis, the formation of the heart in Drosophila., Results: As with vertebrates, the Drosophila heart arises from lateral mesoderm that migrates medially to meet their contralateral partners, to then assemble a midline vessel. During migration, Integrins are among the first proteins restricted to the presumptive luminal domain of cardioblasts. Integrins are required for normal levels of leading edge membrane motility. Apical accumulation of Integrins is enhanced by Robo, and reciprocally, apicalisation of luminal factors like Slit and Robo requires Integrin function. Integrins may provide a template for the formation of a lumen by stabilising lumen factors like Robo. Subsequent to migration, Integrin is required for normal cardioblast alignment and lumen formation. This phenotype is most readily modified by other mutations that affect adhesion, such as Talin and extracellular matrix ligands., Conclusion: Our findings reveal an instructive role for Integrins in communicating polarising information to cells during migration, and during transition to an epithelial tube structure.

Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin., Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry., Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions., Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases., (© 2011 John Wiley & Sons A/S.)

Background: Peripheral nerve injury causes serious problems in orthopedic and plastic surgeries. Cell adhesion molecules such as integrin alpha7 provoke cell binding and signaling pathways within myofibers. Expression profiles of integrin alpha7 signaling pathways and the molecule's microscopic structure were assessed to investigate the long-term dynamic changes in denervated rat skeletal muscle., Methods: A denervated rat skeletal muscle model was established by severing the sciatic nerve for 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 20 weeks, and 26 weeks. Molecular expressions were investigated by mRNA and Western blot. The structural alterations were detected by immunohistochemistry, scanning electron microscopy, and transmission electron microscopy., Results: The denervated muscle atrophy presented the following dynamic molecular alterations: an initial increase around postdenervation in week (PIW) 8 and then a subsequent decay of integrin alpha7, integrin downstream signaling pathway (Ras or Raf or, ERK1/2), Akt, cleaved caspase-3, fast myosin heavy chain (MHC), beta actin, and RhoA. We demonstrated that the expressions of multiple signaling molecules were highly upregulated at PIW 8 (p<0.01). Scanning electron microscopy findings of the surface textures of myofibers showed more severe damage at PIW 8 and subsequently became smoother. Inner structures of myofibers separated with discontinuity on transmission electron microscopy examinations., Conclusion: Our novel finding showed that time-series alterations of integrin alpha7 signaling molecules and surface microstructures in the long-term denervated rat skeletal muscle are biphasic and coherently dynamic. Persisted p-Akt elevation suggested that denervated muscle may regenerate if reinnervation or other treatment was performed.

Development of diabetic nephropathy is accompanied by changes in integrin-mediated cell-matrix interactions. The α8-integrin chain is specifically expressed in mesangial cells of the glomerulus. During experimental hypertension, α8-integrin plays a protective role in the glomerulus. We hypothesized that α8-integrin is involved in maintaining the integrity of the glomerulus in diabetic nephropathy. Experimental streptozotocin (STZ) diabetes led to an increased expression and glomerular deposition of α8-integrin. To test the functional role of α8-integrin, STZ diabetes was induced in mice with a homozygous (α8-/-) or heterozygous (α8+/-) deletion of the α8-integrin gene and in wild-type litters (α8+/+). Blood glucose and mean arterial blood pressure were not different in α8-/- and α8+/+ mice after 6 wk of diabetes. However, diabetic α8-/- mice developed significantly higher albuminuria and more glomerulosclerosis than diabetic α8+/+ mice. Moreover, in diabetic α8-/- mice, the number of glomerular cells staining positive for the podocyte markers WT-1 and vimentin were reduced more prominently than in diabetic α8+/+. The filtration barrier protein nephrin was downregulated in diabetic glomeruli with the strongest reduction observed in α8-/- mice. Taken together, α8-/- mice developed more severe glomerular lesions and podocyte damage after onset of STZ diabetes than α8+/+ mice, indicating that α8-integrin is protective for the structure and function of the glomerulus and maintains podocyte integrity during the development of diabetic nephropathy.

Background: Integrins are heterodimeric cell adhesion molecules, with 18 α (ITGA) and eight β (ITGB) subunits forming 24 heterodimers classified into five families. Certain integrins, especially the α(4)/α(9) (ITGA4/ITGA9) family, interact with members of the ADAM (a disintegrin and metalloprotease) family. ADAM2 is among the better characterized and also of interest because of its role in sperm function. Having shown that ITGA9 on mouse eggs participates in mouse sperm-egg interactions, we sought to characterize ITGA4/ITGA9-ADAM2 interactions., Methodology/principal Findings: An anti-β(1)/ITGB1 function-blocking antibody that reduces sperm-egg binding significantly inhibited ADAM2 binding to mouse eggs. Analysis of integrin subunit expression indicates that mouse eggs could express at least ten different integrins, five in the RGD-binding family, two in the laminin-binding family, two in the collagen-binding family, and ITGA9-ITGB1. Adhesion assays to characterize ADAM2 interactions with ITGA4/ITGA9 family members produced the surprising result that RPMI 8866 cell adhesion to ADAM2 was inhibited by an anti-ITGA9 antibody, noteworthy because ITGA9 has only been reported to dimerize with ITGB1, and RPMI 8866 cells lack detectable ITGB1. Antibody and siRNA studies demonstrate that ITGB7 is the β subunit contributing to RPMI 8866 adhesion to ADAM2., Conclusions/significance: These data indicate that a novel integrin α-β combination, ITGA9-ITGB7 (α(9)β(7)), in RPMI 8866 cells functions as a binding partner for ADAM2. ITGA9 had previously only been reported to dimerize with ITGB1. Although ITGA9-ITGB7 is unlikely to be a widely expressed integrin and appears to be the result of "compensatory dimerization" occurring in the context of little/no ITGB1 expression, the data indicate that ITGA9-ITGB7 functions as an ADAM binding partner in certain cellular contexts, with implications for mammalian fertilization and integrin function.

The expression and function of integrin-type extracellular matrix receptors, VLA-4 and VLA-5, and laminin receptor VLA-6 on the surface of CD3(+)CD4(+) and CD3(+)CD8(+) defined T cell populations was evaluated in the blood of Duchenne muscular dystrophy (DMD) patients and healthy individuals. Both the number of CD4(+) and CD8(+) T cell subsets expressing VLA-4 or VLA-5 and the fibronectin-driven T cell migration was significantly higher in DMD patients. These data indicate that interactions of VLA-4 and/or VLA-5 with fibronectin may drive T lymphocytes to specific niches within muscle, contributing to tissue damage and fibrosis in DMD patients., (Copyright 2010 Elsevier B.V. All rights reserved.)

A recent study shows that one of the two known Caenorhabditis elegans homologs of the mammalian integrin alpha subunit plays an essential role in the clearance of apoptotic cells - efferocytosis - during nematode development. These new findings reveal that integrins have evolutionarily conserved functions in efferocytosis in metazoans.

Integrin alpha11beta1 is expressed by ectomesenchymally- and mesodermally-derived fibroblasts and is the major collagen receptor on embryonic fibroblasts. We have previously characterized a 3kb human alpha11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3kb alpha11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb alpha11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression. Not much is known about integrin alpha11 regulation by TGF-beta family members and the potential role of alpha11 in TGF-beta1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-beta1 induces alpha11 transcription in the fibrosarcoma cell line HT1080 as well as in primary fibroblasts. Co-transfection of an expression plasmid encoding constitutively active ALK5 together with alpha11 promoter-luciferase reporter constructs demonstrated that TGF-beta1 responsive elements are located within the 3kb alpha11 promoter. Serial deletions located TGF-beta1 responsiveness to the proximal promoter (nt -176/+25) as well as to the region extending to nt -330. Transfection and expression of the inhibitory Smad7 in the cells attenuated the TGF-beta1-dependent alpha11 induction both at the RNA and the protein level. Mutation and deletion analyses identified a Smad-binding element, SBE2 (nt -182/-176), as an important Smad3-binding site in this part of the promoter. Further analyses suggested that the Sp1-binding site SBS1 (nt -140/-134) takes part in the responsiveness to TGF-beta1 in a Smad2-dependent manner. In summary, our data confirm that 3kb of the alpha11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-beta1 responsiveness which appears to rely on both a Smad-binding element at nt -182/-176 and a Sp1-binding site at nt -140/-134. Our data furthermore indicate that additional elements needed for TGF-beta1 responsiveness are located upstream in the -2962/-330 promoter region., (Copyright 2009 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC, CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.

Scurfy (Sf) mice lack CD4(+)Foxp3(+) regulatory T cells and develop fatal multiorgan inflammation (MOI) mediated by CD4(+) T cells. Introducing Il2(-/-) gene into Sf mice (Sf.Il2(-/-)) inhibited inflammation in skin and lung. As a major integrin receptor for the organs, we compared CD103 expression on the CD4(+) T cells of B6, Il2(-/-), Sf, and Sf.Il2(-/-) mice. CD103(+)CD4(+) T cells, but not CD8(+) T cells or CD11c(+) dendritic cells, were significantly up-regulated only in Sf mice, indicating Il2(-/-) dominantly and specifically inhibited CD103 up-regulation in Sf CD4(+) T cells. In addition, CD4(+)Foxp3(+) regulatory T cell CD103 expression was not reduced in Il2(-/-) mice. Introducing CD103(-/-) into Sf mice inhibited inflammation in skin and lung as compared with age-matched Sf mice, but they died at approximately 7 wk old with inflammation developed in skin, lungs, and colon, demonstrating fatal MOI induced by CD103-independent mechanism. Transfer of Sf CD4(+) T cells induced MOI more rapidly than CD103(-)CD4(+) T cells, indicating the presence of CD103-dependent mechanism for inflammation. In vitro stimulation with anti-CD3 plus anti-CD28 beads confirmed that CD103 induction in the CD4(+)Foxp3(-) T cells in Il2(-/-) and Sf.Il2(-/-) is defective and cannot be restored by rIL-2 or rIL-15. The data indicate that IL-2 is required for optimal CD103 induction on CD4(+) T cells in Sf mice and this effect contributes to inflammation in an organ-specific manner. IL-2 also has additional roles because the protection of skin and lung inflammation in Sf.Il2(-/-), but not Sf.CD103(-/-) mice is lifelong and Sf.Il2(-/-) mice have longer lifespan than Sf.CD103(-/-) mice.

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.

Recent studies suggest a beneficial role for blocking CD103 signaling in preventing islet allograft rejection and thus Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, antibody blockade approaches generally raise anti-microbial safety issues, necessitating additional studies to address the possible adverse effects of antibody therapy. Here we report that CD103 had no significant impact on the development of primary and memory CD8(+) or CD4(+) responses after acute lymphocytic choriomeningitis virus (LCMV) infection. In addition, CD103 was found to be dispensable for T1D progression in a rapid, CD8-mediated virally-induced T1D model (the rat insulin promoter [RIP]-LCMV), suggesting that its previous efficacy in the NOD mouse model may not be related to its effect on the generation, memory conversion and/or effector function of CD8(+) or CD4(+) T cells. While the data does not preclude a role for CD103 in T1D in its entirety, the current study does provide much evidence to suggest that CD103 blockade may prove to be a safe intervention for autoimmunity and allo-transplantation. While in cases of rapid microbial (CD8)-driven T1D CD103 antibody blockade may not limit disease progression or severity, in mucosally-driven cases of T1D anti-CD103 antibody treatment may provide a new and safe therapeutic avenue.

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.

The purpose of this study was to examine the change in integrin expression in adult human retinal pigment epithelium (RPE) after culturing and to characterize the role of integrins in RPE adhesion to aged submacular human Bruch's membrane. Expression of alpha integrin subunits 1 through 6 in adult RPE cells, cultured or uncultured, was examined by reverse transcription/real-time polymerase chain reaction (PCR) and Western blotting. RPE was cultured on bovine corneal endothelial cell-secreted extracellular matrix (BCE-ECM). The role of alpha integrin subunits in RPE attachment was examined by immunofluorescent localization of these subunits at sites of focal adhesions in cultured adult RPE attached to laminin or collagen-I-coated culture dishes. Additionally, the effect of function-blocking antibodies to alpha integrin subunits on RPE attachment to laminin, collagen I, and aged submacular human Bruch's membrane was determined. Cultured adult RPE had increased expression of alpha1-5 integrin subunits by PCR compared to uncultured RPE. Western blots showed that alpha2, 3, and 5 subunit levels were low or absent in uncultured adult RPE. Cultured adult RPE had a substantially higher expression of these integrins. Alpha 1-3 subunits co-localized with phosphorylated focal adhesion kinase (FAK) at focal adhesions in RPE cells spread on laminin. Only alpha2 and alpha3 co-localized with phosphorylated FAK in focal adhesions of RPE on collagen I. Using function blocking antibodies, blocking alpha1 subunit singly or in combination with alpha2 and/or alpha3 significantly decreased RPE adhesion to laminin. Blocking alpha1 and alpha2 or blocking alpha1, alpha2, and alpha3 subunits significantly decreased RPE adhesion to collagen I. Compared to controls, significantly fewer RPE cells were able to spread on aged submacular human Bruch's membrane when alpha1-6 integrin subunits were blocked. These results indicate that alpha 1-5 subunits that are upregulated by culturing on BCE-ECM are necessary for RPE attachment to aged submacular human Bruch's membrane. Relative lack of these integrin subunits in uncultured adult RPE may be responsible for poor resurfacing of aged submacular human Bruch's membrane by these cells.

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.

Integrin alpha11 (ITGA11/alpha11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal alpha11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human alpha11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WT(shIGF2)) fibroblasts resulted in a decreased growth rate of A549+WT(shIGF2), compared with A549+WT tumors. The results indicate that alpha11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma-stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth.

We sought to understand the epithelial response to the T-cell mediated inflammatory process in kidney allograft rejection. Using microarrays, we studied transcriptome changes of kidney parenchymal cells and their relationship to the development of pathologic lesions such as tubulitis in mouse kidney allografts and isografts. Inflammatory infiltrate in allografts developed by day 5, but tubulitis first appeared at day 7 and was severe by day 21. Using microarrays, we selected 70 solute carrier transcripts with high renal parenchymal expression and known epithelial function. Transcript expression was reduced early in isografts and allografts, followed by progressive loss in allografts and recovery in isografts. The expression pattern of day 21 allografts developed progressively from the time of engraftment and was established before histologic lesions. These changes are probably functionally significant: selected proteins showed decreased immunostaining at days 7 and 21. Allospecific loss of transcripts was dependent on T cells but independent of perforin, granzymes A/B, CD103, or B cells. Weighted sum decomposition revealed multiple components of the epithelial response with allospecific changes from day 1. We conclude that loss of renal transcripts indicates an early stage of T-cell mediated alloimmune injury that later progresses to pathologic lesions such as tubulitis.

Eosinophilic inflammation is a common feature of numerous eosinophil-associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin-mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten-induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a beta7 -integrin addressin cell adhesion molecule-1-independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+ eosinophils express the intercellular adhesion molecule-1 (ICAM-1) counter-receptor integrins alphaL, alphaM, and beta2. Using ICAM-1-deficient mice and anti-ICAM-1 neutralizing antibodies, we show that hapten-induced colonic eosinophilic inflammation is critically dependent on ICAM-1. These studies demonstrate that beta2 -integrin/ICAM-1-dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.

CD18 hypomorph mice expressing reduced levels of the common beta2 integrin chain develop aggravated Lyme carditis, compared to that developed by wild-type (WT) mice, upon infection with the spirochete Borrelia burgdorferi. The enhancement of Lyme carditis in these mice is characterized by increased macrophage infiltration, correlating with augmented expression of the monocyte/macrophage chemoattractant protein 1 (MCP-1). The lack of CD18 results in the deficiency of all beta2 integrins, i.e., CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1/CR3), CD11c/CD18 (p150,95/CR4), and CD11d/CD18. To determine the roles of the various beta2 integrins in controlling the development of aggravated Lyme carditis, disease induction was analyzed in CD11a-/-, CD11b-/-, and CD11c-/- mice. CD11a-/- and CD11c-/- mice, but not CD11b-/- mice, developed aggravated Lyme carditis after exposure to B. burgdorferi. Similarly to CD18 hypomorph mice, CD11c-/- mice expressed higher levels of MCP-1, compared to both WT and CD11a-/- mice, as determined by in vitro analysis of MCP-1 secretion by bone marrow-derived dendritic cells and in vivo analysis of MCP-1 mRNA expression in B. burgdorferi-infected hearts. On the other hand, CD11a deficiency was associated with heightened heart B. burgdorferi burden relative to that of WT mice. Overall, our results suggest that the increased severity of Lyme carditis in CD18 hypomorph mice is caused by deficiency in CD11a or CD11c, possibly via different mechanisms.

Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.

Endogenous regulatory T cells (T(reg)) play a central role in the control of excessive or misdirected immune responses against self or foreign Ags. To date, virtually no data are available on the nature of the molecules and signals involved in the trafficking and retention of T(reg) in tissues where regulation is required. Here, we show that expression of alpha(E)beta(7) integrin is necessary for the homing of T(reg) at site of Leishmania major infection. The vast majority of T(reg) present in the dermis at steady-state conditions or during L. major infection express the alpha(E) chain (CD103) of alpha(E)beta(7). Genetically susceptible BALB/c mice that lack CD103 become resistant to infection, a phenotype that is associated with a poor capacity of T(reg) to be retained in the infected site. Such susceptible phenotype can be restored when T(reg) from wild-type mice were transferred in CD103(-/-) mice. The central role of CD103 in T(reg) retention was further demonstrated by usage of blocking Abs against CD103 and the transfer of T(reg) purified from CD103(-/-) mice. Our results strongly suggest that this molecule is induced and maintained on T(reg) following or just prior to their arrival in tissues. Furthermore, the expression of CD103 and the subsequent retention of T(reg) in tissues is highly regulated by their exposure to Leishmania Ag and the level of activation of the APCs they encounter. Thus, CD103, by controlling T(reg) retention, can contribute to the outcome of chronic infection by Leishmania.

Integrin alpha10beta1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the alpha10 integrin during development, we generated mice carrying a constitutive deletion of the alpha10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin alpha10beta1 plays a specific role in growth plate morphogenesis and function.

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Two activation-dependent Abs to the integrin alphaL-subunit were used to study conformational rearrangement of alphaLbeta2 on the cell surface. Activation lowered the concentration of Ca2+ required for maximal expression of each epitope. Each Ab requires the Ca2+-binding loop in the integrin genu and nearby species-specific residues in the thigh domain. Key thigh residues are shielded from Ab in the bent integrin conformation by the alpha-subunit calf-1 domain and the nearby bent beta leg, suggesting that extension at the genu is required for epitope exposure. Activating stimuli and alpha/beta I-like small molecule antagonists demonstrate that exposure of epitopes in the integrin alpha- and beta-subunit legs is coordinate during integrin activation. A coordinating residue donated by the calf-1 domain is as important as Ca2+ for mAb binding. Together with inspection of the alphaV structure, this result suggests that the genu/calf-1 interface is maintained in integrin activation, and that extension occurs by a rearrangement at the thigh/genu interface.

Infiltration of CD8(+)TCRalphabeta(+) T-effector populations (CD8 effectors) into graft epithelial compartments has long been recognized as a key lesion in progression of clinical renal allograft rejection. While the afferent phase of allograft immunity is increasingly well-defined, the efferent pathways by which donor-reactive CD8-effector populations access and ultimately destroy the graft renal tubules (rejection per se) have received remarkably little attention. This is an important gap in our knowledge of transplantation immunology, because epithelial compartments comprise the functional elements of most commonly transplanted organs including not only kidney, but also liver, lung, pancreas, and intestine. Furthermore, there is increasing evidence that attack of graft epithelial elements by CD8-effector populations not only causes short-term graft dysfunction but is also a major contributor to development of chronic allograft nephropathy and late graft loss, which now represent the salient clinical problems. Recent studies of the T-cell integrin, alpha(E)beta(7) (CD103), have provided insight into the mechanisms that promote interaction of CD8 effectors with graft epithelial compartments. The purpose of this communication is to review the known properties of the CD103 molecule and its postulated role in the efferent phase of renal allograft rejection.

The beta(2) integrin CD11a is involved in T cell-APC interactions, but the roles of CD11b, CD11c, and CD11d in such interactions have not been examined. To evaluate the roles of each CD11/CD18 integrin in T cell-APC interactions, we tested the ability of splenocytes of CD11-knockout (KO) mice to respond to staphylococcal enterotoxins (SEs), a commonly used superantigen. The defect in T cell proliferation with SEA was more severe in splenocytes from mice deficient in CD18, CD11b, or CD11d than in CD11a-deficient splenocytes, with a normal response in CD11c-deficient splenocytes. Mixing experiments showed that the defect of both CD11b-KO and CD11d-KO splenocytes was, unexpectedly, in T cells rather than in APC. Cytometric analysis failed to detect CD11b or CD11d on resting or activated T cells or on thymocytes of wild-type adult mice, nor did Abs directed to these integrins block responses in culture, suggesting that T cells educated in CD11b-KO or CD11d-KO mice were phenotypically altered. Consistent with this hypothesis, T cells from CD11b-KO and CD11d-KO splenocytes exhibited reduced intensity of CD3 and CD28 expression and decreased ratios of CD4/CD8 cells, and CD4(+) T cells were reduced among CD11b-KO and CD11d-KO thymocytes. CD11b and CD11d were coexpressed on a subset of early wild-type fetal thymocytes. We postulate that transient thymocyte expression of both CD11b and CD11d is nonredundantly required for normal thymocyte and T cell development, leading to phenotypic changes in T cells that result in the reduced response to SE stimulation.

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.

Background: Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, migration, and proliferation. The alpha8 integrin chain is expressed in the glomerulus exclusively by mesangial cells. The contribution of alpha8 to mesangial cell function, however, has not yet been studied., Methods: Mesangial cells from wild-type and alpha8-deficient mice were isolated and characterized. Integrin expression was assessed by real-time polymerase chain reaction (PCR), Western blot, or fluorescence-activated cell sorter (FACS) analysis. Cell adhesion was determined by conventional attachment assay and a centrifugal assay for cell adhesion. Cell migration was determined by a fluorescence-based transmigration assay and a chemotaxis assay. Proliferation rates were determined by BrdU and [3H]-thymidine assays., Results: On the alpha8 ligands fibronectin and vitronectin, but not on collagens, attachment of alpha8-deficient mesangial cells was reduced compared to wild-type cells. In contrast, alpha8-deficient mesangial cells migrated more easily and displayed an increased proliferative response on fibronectin or vitronectin, but not on collagens, compared to wild-type cells. These effects were not due to an up-regulation of the fibronectin or vitronectin receptors alpha5 or alphav in alpha8-deficient mesangial cells, as the cell surface expression of integrins alpha5 and alphav was comparable in wild-type and alpha8-deficient mesangial cells., Conclusion: These findings confirm a role for alpha8 integrin in the regulation of the mesangial cell phenotype. alpha8 integrin seems to promote adhesion, but inhibit migration and proliferation of mesangial cells. Thus, the data support the hypothesis that alpha8 integrin could play an important role for maintaining tissue integrity in the glomerulus during glomerular injury.