Issue published December 15, 1999 Previous issue | Next issue
- Volume 104, Issue 12
Go to section:
-
Research Articles
×
Abstract
Insulin receptor substrate–1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing β islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1–deficient β-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1–null islets and β-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways.
Authors
Rohit N. Kulkarni, Jonathon N. Winnay, Molly Daniels, Jens C. Brüning, Sarah N. Flier, Douglas Hanahan, C. Ronald Kahn
×Abstract
Antigen-specific T helper cells present in peripheral blood at very low frequencies are capable of rapid clonal expansion during antigenic challenge. The exquisite specificity of this response provides for activation and expansion of a very select cohort of T cells, a feature we have used to directly identify and quantify human epitope-specific T helper cells from peripheral blood. Soluble tetramerized class II MHC molecules, loaded with an immunodominant peptide from hemagglutinin (HA) and labeled with fluorescent dyes, were constructed and used to directly identify antigen-specific T cells from influenza-immune individuals. After 7 days of proliferation in response to stimulation by HA peptide or whole influenza vaccine, cells staining positive with the HA tetramer had undergone between 6 and 9 divisions and were CD3+, CD4+, CD25+, and CD8–, characteristic of activated T helper cells responding to antigen. The HA epitope-specific component of the complex response to whole influenza vaccine represented a major subset of proliferating T helper cells. Soluble class II tetramers allow a direct approach for the analysis of immunodominant antigenic specificities. The identification of antigen-specific T helper cells in the peripheral blood provides a means for tracking the immune response against infectious agents and in autoimmune disease.
Authors
Erik J. Novak, Andrew W. Liu, Gerald T. Nepom, William W. Kwok
×Abstract
Chronic interstitial nephritis frequently accompanies renal diseases of different etiologies. Far less common is the entity of primary interstitial nephritis wherein the glomerular and vascular structures of the kidney are not the primary focus of the disease process. Using in situ hybridization and the polymerase chain reaction, we detected DNA from the Epstein-Barr Virus (EBV) exclusively in renal tissue of patients with the idiopathic variety of chronic interstitial nephritis. The EBV genome, but not that of cytomegalovirus or adenovirus, was detected primarily in renal proximal tubule cells. Furthermore, the CD21 antigen, which serves as the receptor for EBV in B lymphocytes, was detected by immunocytochemistry primarily on proximal tubule cells and was markedly upregulated in the EBV-infected tissue. Western blot analysis of primary cultures of normal proximal tubule cells identified a 140-kDa protein, confirming the expression of the CD21 antigen. Colocalization experiments using proximal and distal tubule markers confirmed that EBV DNA and the CD21 antigen are found primarily in proximal tubule cells. EBV infection of renal proximal tubular cells may participate in evoking a cellular immune response that results in a damaged renal interstitium.
Authors
Joanne L. Becker, Frederick Miller, Gerard J. Nuovo, Christine Josepovitz, William H. Schubach, Edward P. Nord
×Abstract
Certain mutations in genes for sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). We have developed a transgenic rabbit model for HCM caused by a common point mutation in the β-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human β-MyHC cDNAs were cloned 3′ to a 7-kb murine β-MyHC promoter. We injected purified transgenes into fertilized zygotes to generate two lines each of the wild-type and mutant transgenic rabbits. Expression of transgene mRNA and protein were confirmed by Northern blotting and 2-dimensional gel electrophoresis followed by immunoblotting, respectively. Animals carrying the mutant transgene showed substantial myocyte disarray and a 3-fold increase in interstitial collagen expression in their myocardia. Mean septal thicknesses were comparable between rabbits carrying the wild type transgene and their nontransgenic littermates (NLMs) but were significantly increased in the mutant transgenic animals. Posterior wall thickness and left ventricular mass were also increased, but dimensions and systolic function were normal. Premature death was more common in mutant than in wild-type transgenic rabbits or in NLMs. Thus, cardiac expression of β-MyHC-Q403 in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to β-MyHC-Q403.
Authors
Ali J. Marian, Yun Wu, Do-Sun Lim, Meghan McCluggage, Keith Youker, Qun-tao Yu, Ramon Brugada, Francesco DeMayo, Miguel Quinones, Robert Roberts
×Abstract
The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a–/– mice, which lack AT1A receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT1) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT1 receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT1 signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT1 receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.
Authors
Chandra Nataraj, Michael I. Oliverio, Roslyn B. Mannon, Peter J. Mannon, Laurent P. Audoly, Carmen S. Amuchastegui, Phillip Ruiz, Oliver Smithies, Thomas M. Coffman
×Abstract
Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H–/– mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and β-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H–/– mice, but insulin-stimulated glucose uptake was abolished. G4H–/– mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.
Authors
E. Dale Abel, Helen C. Kaulbach, Rong Tian, James C.A. Hopkins, John Duffy, Thomas Doetschman, Timo Minnemann, Mary-Ellen Boers, Ed Hadro, Corinna Oberste-Berghaus, William Quist, Bradford B. Lowell, Joanne S. Ingwall, Barbara B. Kahn
×Abstract
Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1+ CD8+ cells play a critical role in this costimulation blockade–resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.
Authors
Joel Trambley, Adam W. Bingaman, Angello Lin, Eric T. Elwood, Seung-Yeun Waitze, Jongwon Ha, Megan M. Durham, Matthias Corbascio, Shannon R. Cowan, Thomas C. Pearson, Christian P. Larsen
×Abstract
Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR α-subunit (Hα1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Hα1-205–induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis.
Authors
Sin-Hyeog Im, Dora Barchan, Sara Fuchs, Miriam C. Souroujon
×Abstract
ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y1 receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y1-null mice were generated to define the role of the P2Y1 receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y1. These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y1-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y1. P2Y1-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y1 receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.
Authors
Catherine Léon, Béatrice Hechler, Monique Freund, Anita Eckly, Catherine Vial, Philippe Ohlmann, Andrée Dierich, Marianne LeMeur, Jean-Pierre Cazenave, Christian Gachet
×Abstract
CD34+ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34+ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34+ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34+ cells and CD34+KIT+ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34+KIT+ cells, even by RT-PCR. Conditioned media from CD34+ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34+ cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34+ cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.
Authors
Marcin Majka, Tomasz Rozmyslowicz, Benhur Lee, Samuel L. Murphy, Zbigniew Pietrzkowski, Glen N. Gaulton, Leslie Silberstein, Mariusz Z. Ratajczak
×Abstract
SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase– (PI-3K–) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell–derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP–/– cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.
Authors
Chang H. Kim, Giao Hangoc, Scott Cooper, Cheryl D. Helgason, Sandie Yew, R. Keith Humphries, Gerald Krystal, Hal E. Broxmeyer
×Abstract
Epidermal TNF expression increases in response to cutaneous permeability barrier disruption and wound healing. TNF signaling is mediated by acid and neutral sphingomyelinases (A- and N-SMase), which generate ceramide, an important regulator of proliferation, differentiation, and apoptosis. In the epidermis, ceramide is known to be an integral part of the extracellular stratum corneum (SC) lipid bilayers that constitute the permeability barrier of the skin. We show here that topical application of TNF after experimental injury to the SC of hairless mice (hr–/–) enhances barrier repair. In TNF receptor p55–deficient (TNF-R55–deficient) mice (hr+/+), cutaneous barrier repair was delayed compared with wild-type (hr+/+) or TNF-R75–deficient (hr+/+) animals. After barrier disruption in hairless (hr–/–) and wild-type (hr+/+), but not in TNF-R55–deficient (hr+/+) mice, the enzymatic activities of both A-SMase and N-SMase were significantly enhanced. Stimulation of SMase activities was accompanied by an increase in C24-ceramide levels. Most A-SMase activity in hairless mice (hr–/–) was found in the outer epidermal cell layers and colocalized in the lamellar bodies with A-SMase and sphingomyelin. Reduction of epidermal A-SMase activity by the inhibitor imipramine resulted in delayed permeability barrier repair after SC injury. Together, these results suggest that TNF-R55 signaling pathways contribute to cutaneous permeability barrier repair through SMase-mediated generation of ceramide.
Authors
Jens-Michael Jensen, Stefan Schütze, Michael Förl, Martin Krönke, Ehrhardt Proksch
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)