CCR6 expression in dendritic, T, and B cells suggests that this β-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6–/– mice have underdeveloped Peyer’s patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6–/– mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene–induced contact hypersensitivity (CHS) studies, CCR6–/– mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6–/– mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4+ T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6–/– mice as a model to study pathologies in these tissues.
Rosa Varona, Ricardo Villares, Laura Carramolino, Íñigo Goya, Ángel Zaballos, Julio Gutiérrez, Miguel Torres, Carlos Martínez-A., Gabriel Márquez
Approximately 40% of growth hormone–secreting pituitary adenomas have somatic mutations in the GNAS1 gene (the so-called gsp oncogene). These mutations at codon 201 or codon 227 constitutively activate the α subunit of the adenylate cyclase–stimulating G protein Gs. GNAS1 is subject to a complex pattern of genomic imprinting, its various promoters directing the production of maternally, paternally, and biallelically derived gene products. Transcripts encoding Gsα are biallelically derived in most human tissues. Despite this, we show here that in 21 out of 22 gsp-positive somatotroph adenomas, the mutation had occurred on the maternal allele. To investigate the reason for this allelic bias, we also analyzed GNAS1 imprinting in the normal adult pituitary and found that Gsα is monoallelically expressed from the maternal allele in this tissue. We further show that this monoallelic expression of Gsα is frequently relaxed in somatotroph tumors, both in those that have gsp mutations and in those that do not. These findings imply a possible role for loss of Gsα imprinting during pituitary somatotroph tumorigenesis and also suggest that Gsα imprinting is regulated separately from that of the other GNAS1 products, NESP55 and XLαs, imprinting of which is retained in these tumors.
Bruce E. Hayward, Anne Barlier, Márta Korbonits, Ashley B. Grossman, Philippe Jacquet, Alain Enjalbert, David T. Bonthron
Although hyperhomocysteinemia (HHcy) is a well-known risk factor for the development of cardiovascular disease, the underlying molecular mechanisms are not fully elucidated. Here we show that induction of HHcy in apoE-null mice by a diet enriched in methionine but depleted in folate and vitamins B6 and B12 increased atherosclerotic lesion area and complexity, and enhanced expression of receptor for advanced glycation end products (RAGE), VCAM-1, tissue factor, and MMP-9 in the vasculature. These homocysteine-mediated (HC-mediated) effects were significantly suppressed, in parallel with decreased levels of plasma HC, upon dietary supplementation with folate and vitamins B6/B12. These findings implicate HHcy in atherosclerotic plaque progression and stability, and they suggest that dietary enrichment in vitamins essential for the metabolism of HC may impart protective effects in the vasculature.
Marion A. Hofmann, Evanthia Lalla, Yan Lu, Michelle Ryu Gleason, Bonnie M. Wolf, Nozomu Tanji, Luis J. Ferran Jr., Brigitte Kohl, Vijay Rao, Walter Kisiel, David M. Stern, Ann Marie Schmidt
O2 deprivation can produce many devastating clinical conditions such as myocardial infarct and stroke. The molecular mechanisms underlying the inherent tissue susceptibility or tolerance to O2 lack are, however, not well defined. Since the fruit fly, Drosophila melanogaster, is extraordinarily tolerant to O2 deprivation, we have performed a genetic screen in the Drosophila to search for loss-of-function mutants that are sensitive to low O2. Here we report on the genetic and molecular characterization of one of the genes identified from this screen, named hypnos-2. This gene encodes a Drosophila pre-mRNA adenosine deaminase (dADAR) and is expressed almost exclusively in the adult central nervous system. Disruption of the dADAR gene results in totally unedited sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl-α) channels, a very prolonged recovery from anoxic stupor, a vulnerability to heat shock and increased O2 demands, and neuronal degeneration in aged flies. These data clearly demonstrate that, through the editing of ion channels as targets, dADAR, for which there are mammalian homologues, is essential for adaptation to altered environmental stresses such as O2 deprivation and for the prevention of premature neuronal degeneration.
Enbo Ma, Xiang-Qun Gu, Xiaohui Wu, Tian Xu, Gabriel G. Haddad
We describe here the immunologic characterization of a new mouse strain, SAMP1/Yit, which spontaneously develops a chronic intestinal inflammation localized to the terminal ileum. The resulting ileitis bears a remarkable resemblance to human Crohn’s disease. This strain of mice develops discontinuous, transmural inflammatory lesions in the terminal ileum with 100% penetrance by 30 weeks of age. The intestinal inflammation is characterized by massive infiltration of activated CD4+ and CD8α+TCRαβ+ T cells into the lamina propria and is accompanied by a dramatic decrease in the intraepithelial lymphocyte CD8α+TCRγδ+/CD8α+TCRαβ+ ratio. The results of adoptive transfer experiments strongly suggest that CD4+ T cells that produce a Th1-like profile of cytokines, e.g., IFN-γ and TNF, mediate the intestinal inflammation found in SAMP1/Yit mice. In addition, pretreatment of adoptive transfer recipients with a neutralizing anti-TNF antibody prevents the development of intestinal inflammation, suggesting that TNF plays an important role in the pathogenesis of intestinal inflammation in this model. To our knowledge, these data provide the first direct evidence that Th1-producing T cells mediate intestinal inflammation in a spontaneous animal model of human Crohn’s disease.
Michele M. Kosiewicz, Cynthia C. Nast, Anasuya Krishnan, Jesus Rivera-Nieves, Christopher A. Moskaluk, Satoshi Matsumoto, Kosuke Kozaiwa, Fabio Cominelli
While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell–expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.
Caiying Guo, Haisong Ju, Debbie Leung, Hamid Massaeli, Mingda Shi, Marlene Rabinovitch
Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.
Florian Sennlaub, Yves Courtois, Olivier Goureau
Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain receptors (DDRs) are a novel class of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinase (MMP) production, but little is known about their expression and function in the vasculature. We posited a critical role for the DDRs controlling smooth muscle cell migration and proliferation and thus repair following arterial injury. Smooth muscle cells were isolated from the aortas of mice with a targeted deletion of the DDR1 gene (DDR1-null) and studied in culture using models that mimic critical steps in neointimal thickening. Our studies suggest that DDR1 plays an important role in regulating attachment to collagen, chemotaxis, proliferation, and MMP production in smooth muscle cells. Following mechanical injury to the carotid arteries, cross-sectional area of the neointima was significantly lower in DDR1-null mice than in wild-type mice. There was also a significant decrease in collagen deposition in the injured arteries of the DDR1-null mice. Our results support the hypothesis that DDR1 plays an important role as a collagen receptor, mediating intimal thickening after vascular injury.
Guangpei Hou, Wolfgang Vogel, Michelle P. Bendeck
Autoantibodies to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus–infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab’s. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 ≥ GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [3H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti–AMPA-receptor Ab’s can be produced as the result of a virus infection, in part through molecular mimicry. These Ab’s may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.
Elena Koustova, Yoshitatsu Sei, Linda Fossom, Mei-Ling Wei, Peter N.R. Usherwood, N. Bradley Keele, Michael A. Rogawski, Anthony S. Basile
Hemangioma, the most common tumor of infancy, is a benign vascular neoplasm of unknown etiology. We show, for the first time to our knowledge, that endothelial cells from proliferating hemangioma are clonal, and we demonstrate that these hemangioma-derived cells differ from normal endothelial cells in their rates of proliferation and migration in vitro. Furthermore, migration of hemangioma endothelial cells is stimulated by the angiogenesis inhibitor endostatin, unlike the inhibition seen with normal endothelial cells. We conclude that hemangiomas constitute clonal expansions of endothelial cells. This is consistent with the possibility that these tumors are caused by somatic mutations in one or more genes regulating endothelial cell proliferation.
Eileen Boye, Ying Yu, Gretchen Paranya, John B. Mulliken, Bjorn R. Olsen, Joyce Bischoff
Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a –70T to C transition in a putative GATA-1 consensus binding element, (b) a –76G to A transition, (c) a –86C to A transversion in three unrelated patients, and (d) a –90C to A transversion in a putative CP2 binding motif. Also, a –224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 ± 1, 54 ± 3, 43 ± 6, and 8 ± 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the –70C mutation altered GATA1 binding, whereas the adjacent –76A mutation did not. Similarly, the –90C mutation altered CP2 binding, whereas the –86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.
Constanza Solis, Gerardo I. Aizencang, Kenneth H. Astrin, David F. Bishop, Robert J. Desnick
Stephen F. Porcella, Tom G. Schwan
Christopher D. Bayliss, Dawn Field, E. Richard Moxon