Expression of oxidant and antioxidant enzymes in human lung and interstitial lung diseases

86 Pages·2002·0.9 MB·English

by  Lakari, Essi

EXPRESSION OF OXIDANT ESSI
LAKARI
AND ANTIOXIDANT ENZYMES
IN HUMAN LUNG AND Department of Internal Medicine,
University Hospital of Oulu
INTERSTITIAL LUNG
Department of Pathology,
DISEASES University of Oulu
OULU 2002
ESSI LAKARI
EXPRESSION OF OXIDANT AND
ANTIOXIDANT ENZYMES IN
HUMAN LUNG AND INTERSTITIAL
LUNG DISEASES
Academic Dissertation to be presented with the assent of
the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium 1 of the University Hospital
of Oulu, on April 19th, 2002, at 12 noon.
OULUN YLIOPISTO, OULU 2002
Copyright © 2002
University of Oulu, 2002
Reviewed by
Docent Sisko Anttila
Docent Tarja Laitinen
ISBN 951-42-6662-5 (URL: http://herkules.oulu.fi/isbn9514266625/)
ALSO AVAILABLE IN PRINTED FORMAT
Acta Univ. Oul. D 670, 2002
ISBN 951-42-6661-7
ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)
OULU UNIVERSITY PRESS
OULU 2002

Lakari, Essi, Expression of oxidant and antioxidant enzymes in human lung and
interstitial lung diseases
Department of Internal Medicine, University Hospital of Oulu, P.O.Box 20, FIN-90029 OYS,
Finland, Department of Pathology, University of Oulu, P.O.Box 5000, FIN-90014 University of
Oulu, Finland
Oulu, Finland
2002
Abstract
Antioxidants function as blockers of radical processes and eliminate harmful reactive oxygen species
(ROS) produced during normal cellular metabolism. A complex antioxidant defence system has
evolved to protect the cellular homeostasis. This system includes antioxidant enzymes (AOEs), such
as superoxide dismutases (SODs), which are intracellular MnSOD and CuZnSOD and extracellular
ECSOD, H O scavenging enzymes catalase and glutathione peroxidase, and hemeoxygenase-1
2 2
(HO-1), an important enzyme in heme metabolism, which has also been suggested to have antioxidant
capacities. ROS play an important role in the pathogenesis of interstitial lung diseases. These diseases
represent a group of disorders with different etiology, histopathology, treatment and prognosis.
Sarcoidosis, extrinsic allergic alveolitis and two different forms of idiopathic pulmonary fibrosis,
usual interstitial pneumonia (UIP) and desquamative interstitial pneumonia (DIP) were included in
this study.
The purpose of this research was to evaluate the expressions of inducible nitric oxide synthase (i-
NOS), endothelial nitric oxide synthase (e-NOS) and xanthine oxidase (XAO), oxidant generating
enzymes commonly associated with tissue injury, and, on the other hand, the expressions of AOEs
suggested to be involved in the defence of lung tissue against oxidant stress. The methods included
immunohistochemistry on lung biopsies (n=48) and Western blotting, Northern blotting or reverse
polymerase chain reaction (RT-PCR) on human inflammatory cells and cells obtained from
bronchoalveolar lavage. I-NOS was intensively expressed in inflammatory, but not in fibrotic lesions,
similar e-NOS expression was found in control lung and in all interstitial lung diseases, while XAO
was mainly negative. MnSOD and HO-1 were highly expressed in the granulomas of sarcoidosis. In
contrast the expressions of MnSOD and HO-1 in late fibrotic lesions of UIP were low or undetectable
by immunohistochemistry. CuZnSOD and catalase showed similar immunoreactivity in healthy and
diseased lung. A cell specific expression and regulation of various enzymes may play an important
role during acute inflammatory diseases and also in the progression of lung fibrogenesis.
Keywords: antioxidant enzyme, oxidant, interstitial pneumonia, sarcoidosis, allergic alve-
olitis

Acknowledgements
This study was carried out at the Departments of Internal Medicine and Pathology of the
University of Oulu, during the years 1997-2001. I wish to express my sincerest gratitude
to Professor Antero Kesäniemi, Head of the Department of Internal Medicine, and
Professor Veli-Pekka Lehto, former Head of the Department of Pathology, and Professor
Frej Stenbäck, for proving excellent research facilities at my disposal.
I also wish to express my gratitude to all those who participated in this project. My
supervisor Professor Vuokko Kinnula, who introduced me to the fascinating world of
oxygen radicals, deserves my deepest gratitude for all the help, guidance and
encouragement she has given me during these years. With her enormous amounts of
integrity, determination and dedication she has gained my greatest admiration and
uppermost respect. Thank you.
My second supervisor, Docent Paavo Pääkkö has generously shared with me his vast
knowledge of pulmonary pathology, has been an endless source of advice and
encouragement, and also has an extraordinary capacity to see a silver lining even in a
darkest cloud, for all of which I’m eternally grateful.
Docent Ylermi Soini, who in the course of time eventually became an important
collaborant and my unofficial third supervisor, has greatly impressed me not only with his
scientific achievements, but also with the efficacy and tranquillity he makes them.
I also want to thank my co-authors, Professor Pirjo Koistinen for several useful
discussions, Professor Kari Raivio for kindly giving me the opportunity to visit his
laboratory at Children's Hospital in Helsinki to learn new techniques, and Marjaana Säily,
M.D., Ph.D., for mastering the RT-PCR method, and also for her unfailing optimism
during this project. My very special thanks are due to Petra Pietarinen-Runtti, M.D.,
Ph.D., for her skillful and always prompt, yet friendly, cooperation.
I’m very grateful to Docent Sisko Anttila M.D., Ph.D., and Docent Tarja Laitinen
M.D., Ph.D., for their constructive criticism and useful comments during the preparation
of this manuscript.
Professor James D. Crapo is gratefully acknowledged for kindly providing the
polyclonal antibodies for MnSOD, CuZnSOD, ECSOD and catalase, and Dr. Y.-S. Ho for
providing the human MnSOD, CuZnSOD, CAT and rat GPx cDNAs.
I'd also like to thank the other members of the pulmonary research team, especially
Riitta Kaarteenaho-Wiik, M.D., Ph.D., Airi Puhakka, M.D., Piritta Pylkäs, Med. Cand.,
Katriina Kahlos, M.D., Ph.D., Leena Tiitto, M.D., and Lauri Lammi, M.D., Ph.D. for
sharing these years of scientific work with me.
I’d like to thank all the pulmonary specialists at the Pulmonary division of Oulu, for
always patiently and kindly advicing me during my clinical work periods. Dr. Eeva-Maija
Ruotsalainen, M.D., deserves a very special thank you for skillfully performing most of
the bronchoscopies and collecting the BAL-samples used in this study. I'd also like to
thank all colleagues who have worked with me as pulmonary fellows, especially Doctors
Shirley Johnson, Marjo Juvonen, Tapio Kuha, Elsa Kurkela, Esko Kurttila, and Sakari
Saviaro, not only for being such pleasures to work with, but also for their valued
friendship.
Throughout this study, I have been very fortunate to enjoy skillful technical assistance:
Ms. Raija Sirviö has provided invaluable help with Western blotting, Mr. Manu Tuovinen,
master of immunohistochemistry and whose excellent taste of humor has enlightened
countless otherwise grey days, Ms. Mirja Vahera who first introduced me to the
techniques in immunohistochemistry, Ms. Ritva Löfman for her help during my weeks in
Children's Hospital in Helsinki, Ms. Satu Koljonen for her expertise with the RT-PCR
method, Ms. Päivi Koukkula for her help with the BAL-samples and Mr. Hannu
Wäänänen for his help with the photographs and figures. I have also vastly enjoyed the
working atmosphere at the Department of Pathology and want to warmly thank everyone
for extremely useful discussions of various problems with laboratory practice,
manuscripts – and gardening.
I owe warm thanks to Ms. Seija Leskinen, Mr. Mika Kihlström, and Ms. Liisa Kärki
for their never-failing patience with me during the numerous hours I have spent in their
laboratory preparing pictures for my articles and posters. I’m also very grateful to Ms.
Anne Salovaara, Ms. Terttu Niemelä, Ms. Irene Tuomela-Törmänen and Ms. Hilkka
Penttinen for all their help with various technical and practical problems.
At last, but far from least, I'd like to express my gratitude to all my friends and family
members for being there for me at the time I most needed you. Anne, Arja, Marja-Leena,
Sari, Sherry, and Tanja deserve a very special thank you for a friendship through thick,
thicker and thickest, as well as my brothers Jari and Ossi, sisters-in-law Laura and Eva-
Marie, nephews Ville and Joona, niece Noomi, and the energetic twins, my dear little
goddaughter Annika and her brother Jyrki, for providing countless successful diversions
from the scientific and clinical world, and also for their invaluable help with a million and
one practical matters.
This thesis is dedicated with love and gratitude to my mother and father for a lifetime
of love, encouragement, and unflinching faith in me. And to the loving memory of my
grandfather, Benjam Karjalainen, for possessing the greatest kindness and wisdom of
heart I have ever known.
This work was financially supported by Oulu University Hospital, Emil Aaltonen
Foundation, Finnish Tuberculosis Association Foundation, Paulo Foundation, Finnish
Medical Society Duodecim, AstraZeneca, and University of Oulu, which are all gratefully
acknowledged.
Oulu, March 2002 Essi Lakari
Abbreviations
ADP Adenosine diphosphate
AEC Aminoethyl carbazole
AIP Acute interstitial pneumonia
AMP Adenosine monophosphate
AOE Antioxidant enzyme
ARDS Adult respiratory distress syndrome
α-SMA α-smooth muscle actin
ATP Adenosine triphosphate
BAL Bronchoalveolar lavage
BIP Bronchiolitis obtiterans with interstitial pneumonia
BOOP Bronchiolitis obliterans with organizing pneumonia
BPD Bronchopulmonary dysplasia
BSO Buthionine sulfoximine
CAT Catalase
cDNA Complementary deoxyribonucleic acid
CDNB 1-chloro-2,4-dinitrobenzene
cGPx Cellular glutathione peroxidase
CO Carbon monoxide
CO Carbon dioxide
2
CuZnSOD Copper zinc superoxide dismutase
EDTA Ethylenediamine tetra-acetic acid
eGpx Extracellular glutathione peroxidase
eNOS Endothelial nitric oxide synthase
ELF Epithelial lining fluid
ET-1 Endothelin-1
Da Dalton
DIP Desquamative intersitial pneumonia
ECSOD Extracellular superoxide dismutase
FBS Fetal bovine serum
fMLP Formyl-methionyl-leucyl-phenylalanine
G6PD Glucose 6-phophate dehydrogenase
GIP Giant cell interstitial pneumonia
γ-GCS Gamma-glutamylcysteine synthetase
γ-GT Gamma-glutamyl transpeptidase
GPx Glutathione peroxidase
GR Glutathione reductase
GSH Glutathione
GSSG Oxidized glutathione
HL-60 Human leukemia cell line 60
HO Hydrogen peroxide
2 2
HO-1 Hemeoxygenase-1
HRCT High resolution computed tomography
HSP Heat shock protein
IFN-γ Interferon gamma
IGF-1 Insulin like growth factor-1
IL Interleukin
iNOS Inducible nitric oxide synthase
kb Kilobase
kD Kilodalton
K Michaelis constant
m
LIP Lymphoid interstitial pneumonia
LPS Lipopolysaccharide
MnSOD Manganese superoxide dismutase
MPO Myeloperoxidase
mRNA Messenger ribonucleic acid
MRP-1 Multi-drug resistance protein
NAC N-acetylcysteine
NADP Nicotinamide adenine dinucleotide phosphate
NADPH Reduced nicotinamide adenine dinucleotide phosphate
NF-κB Nuclear factor-kappaB
NO Nitric oxide
NO Nitrogen dioxide
2
NSIP Non specific interstitial pneumonia
O Molecular oxygen
2
O- Superoxide anion
2
O Ozone
3
OH. Hydroxyl radical
OONO- Peroxynitrite
PBS Phosphate buffered saline
PDGF Platelet derived growth factor
PEG-SOD Polyethylene glycol conjugated SOD
RBILD Respiratory bronchilitis with interstitial lung disease
ROS Reactive oxygen species
RT Room temperature
RT-PCR Reverse transcription polymerase chain reaction
SnPP Tin protoporphyrin
SOD Superoxide dismutase