Albert Tousson

Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{{\cdot}-}}}\end{equation*}\end{document}) and hydrogen peroxide (H2O2) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{{\cdot}-}}}\end{equation*}\end{document} production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (⋅NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ⋅NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{{\cdot}-}}}\end{equation*}\end{document} levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming ⋅NO via O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{{\cdot}-}}}\end{equation*}\end{document}-dependent mechanisms.

The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane‐spanning domains (MSDs), two nucleotide‐binding domains (NBD) and a regulatory (R) domain. The NBDs and R‐domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj‐2) is a co‐chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj‐2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and ΔF508 CFTR can be isolated in complexes with Hdj‐2 and Hsc70. The ΔF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between ΔF508 CFTR and Hdj‐2/Hsp70 were ∼2‐fold higher than those with CFTR. The earliest stage at which Hdj‐2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj‐2 and nascent CFTR was greatly reduced after expression of the R‐domain. In experiments with purified components, Hdj‐2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj‐2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj‐2/Hsc70 is the formation of an intramolecular NBD1–R‐domain complex. Whether this step is defective in the biogenesis of ΔF508 CFTR will be discussed.

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Aminoglycoside suppression of a premature stop mutation in a Cftr–/– mouse carrying a human CFTR-G542X transgene

Abstract. Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since ~5% of all mutant CF alleles are stop mutations, it can be calculated that ~10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr–/–) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr–/–hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

CXC Chemokine Receptor 4 Expression and Function in Human Astroglioma Cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-γ-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-α, IL-1β, and IFN-γ. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-α and IL-1β. The peak of CXCR4 expression in response to TNF-α and IL-1β was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-α and IL-1β (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1α (SDF-1α), resulted in an elevation in intracellular Ca2+ concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1α treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-γ-inducible protein-10. SDF-1α-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.

Ca2+-Activated Cl– Channels: A Newly Emerging Anion Transport Family

Abstract. A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca2+-sensitive Cl– channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca2+-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca2+-mediated Cl– conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

CII-DC-AdTRAIL cell gene therapy inhibits infiltration of CII-reactive T cells and CII-induced arthritis

Previously, we described an APC-adenovirus (APC-Ad) FasL cell gene therapy method which could be used to deplete autoreactive T cells in vivo. FasL was toxic, however, and controlled regulation of FasL was not achieved. Here we describe an improved approach to delivering TNF-related apoptosis-inducing ligand (TRAIL) in vivo in which collagen II-induced (CII-induced) arthritis-susceptible (CIA-susceptible) DBA/1j mice were treated with CII-pulsed DCs that had been transfected with a novel Ad system. The Ad was engineered to exhibit inducible TRAIL under the control of the doxycycline-inducible (DOX-inducible) tetracycline response element (TRE). Four groups of mice were treated with CII-DC-AdTRAIL+DOX, CII-DC-AdTRAIL (no DOX), CII-DC-AdGFP+DOX, or DC-AdTRAIL+DOX (no CII), beginning 2 weeks after priming with CII in CFA. The incidence of arthritis and infiltration of T cells in the joint was significantly decreased in CII-DC-AdTRAIL+DOX-treated mice. The in vitro splenic T cell proliferative response and induction of IFN-gamma to bovine CII stimulation were also significantly reduced in mice treated with CII-DC-AdTRAIL+DOX. AdTRAIL+DOX was not toxic to DCs or mice but could induce activated T cells to undergo apoptosis in the spleen. Our results suggest that CII-DC-AdTRAIL+DOX cell gene therapy is a safe and effective method for inhibiting the development of CIA.

CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.

Molecular cloning of pituitary tumor transforming gene 1 from ovarian tumors and its expression in tumors

Pituitary tumor transforming gene 1 (PTTG1) recently cloned from human testis is a potent oncogene and is highly expressed in all the tumors analyzed to date. However, primary structure of PTTG1 and the cell types that express PTTG1 in tumors remained undescribed. We have used the reverse transcriptase-polymerase chain reaction technique to clone PTTG1 from ovarian tumors. Nucleotide sequencing of the PTTG1 cDNAs from various ovarian tumors showed identity with that of the human testis PTTG1. To determine the cell types that express PTTG1 in normal and tumor tissues, we performed in situ hybridization using digoxigenin-labeled cRNA as a probe. Our studies revealed a high level of expression of PTTG1 mRNA in both seminomatous and non-seminomatous testicular tumors; epithelial, sex-cord and stromal cell, and germ cell tumors of the ovary; and invasive ductal, ductal in situ and infiltrating ductal carcinoma of the breast. In normal tissues, expression of PTTG1 mRNA was very low or undetectable except in testis, where PTTG1 mRNA was found to be localized to spermatocytes and spermatids. Tumors that expressed high levels of PTTG1 mRNA also exhibited high levels of expression of basic fibroblast growth factor (bFGF), suggesting a correlation between PTTG1 and bFGF expression, and further suggesting that the PTTG1 protein may be involved in tumor angiogenesis and mitogenesis.