Table Of ContentM E T H O D S I N M O L E C U L A R M E D I C I N ETM
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Interleukin Protocols
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
John M. Walker, SERIES EDITOR
69. Gene Therapy Protocols, 2nd ed., edited 51. Angiotensin Protocols, edited by Donna
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M E T H O D S I N M O L E C U L A R M E D I C I N ETM
Interleukin
Protocols
Edited by
Luke A. J. O'Neill
Department of Biochemistry and Biotechnology Institute,
Trinity College, Dublin, Ireland
and
Andrew Bowie
Department of Pharmacology, Conway Institute of Biomolecular
and Biomedical Research, University College Dublin, Dublin, Ireland
Humana Press Totowa, New Jersey
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Interleukin protocols / edited by Luke A. J. O'Neill and Andrew Bowie.
p. cm. -- (Methods in molecular medicine ; 60)
Includes bibliographical references and index.
ISBN 0-89603-738-X (alk. paper)
1. Interleukins--Laboratory manuals. I. O'Neill, Luke A. J. II. Bowie, Andrew. III.
Series.
QR185.8.I56 I587 2001
616.07'9--dc21
2001016960
Preface
Interleukins are a family of proteins that regulate the maturation, differ-
entiation, or activation of cells involved in immunity and inflammation, and
belong to a broader family termed cytokines. Collectively these proteins are
the key orchestrators of host defense and the response to tissue injury. There
are currently 23 different interleukins (numbered from IL-1 to IL-23), although
the full extent of the interleukin family will only become clear upon analysis
of the human genome sequence. Most important, interleukins are central to
the pathogenesis of a wide range of diseases that involve an immune compo-
nent, including such conditions as rheumatoid arthritis, multiple sclerosis,
ulcerative colitis, psoriasis, and asthma. Interleukins have also been impli-
cated in other conditions, including cancer, migraine, myocardial infarction,
and depression.
In essence, when cells are activated by interleukins, a program of gene
expression is initiated in the target cell that alters the cell’s phenotype, leading
to enhanced immune reactivity, inflammation, and/or proliferation. Interleukins
are therefore at the core of the cellular basis for many diseases. They are the
subject of intense investigation by biomedical researchers and the targeting or
use of interleukins in the clinic is proceeding apace. Approaches such as tar-
geting IL-4 in asthma or IL-1 in joint disease are being pursued, and it is likely
that in the next 5–10 years a number of new therapies based on either inhibit-
ing or administering interleukins will be available. In addition, the assaying of
interleukins has a role in the diagnosis and prognosis of disease, and polymor-
phisms in interleukin genes may well be found to predispose individuals to
disease.
The basis for these many advances in interleukin research lies in the use
of a range of methodologies for their study. In Interleukin Protocolswe have
brought together a critical mass of chapters covering the major techniques
currently available to researchers in this area. The book is divided into five
sections. Parts I and II concern a range of methods for assaying interleukin
protein and mRNA. The ELISA is the mainstay of assaying interleukin pro-
tein production and the chapters here cover the basic methodologies, where to
purchase reagents and also recent developments in the use of ELISA. The use
v
vi Preface
of FACS as a method of assaying interleukins intracellularly has been an
important advance and is also covered. The ability to measure interleukin
mRNA is another important technique in interleukin research and several chap-
ters describe quantitative methods that mainly rely on RT-PCR and RNAse
protection. Part II gives examples of how to measure specific interleukins in
order to illustrate the approaches that can be used for investigators interested
in a particular interleukin.
Part III covers the assays of interleukins in specific pathologies, including
breast cancer, depression, psoriasis, Grave's disease, migraine, and myocardial
infarction. Part IV is related to Part III in that it also concerns pathologies, but
has as its focus the assaying of interleukins in different biological fluids rel-
evant to disease. These include peritoneal fluids, sputum from asthma patients,
synovial fluid from arthritic joints, and cerebrospinal fluid from patients with
meningitis. More important, this section covers the difficulties associated
with the measuring of interleukins in such fluids. Finally, Part V concerns
newer methods in the study of interleukin signal transduction, analysis of poly-
morphisms in interleukin genes, and the use of cDNA arrays, areas that will
surely expand greatly in the next years as the feasibility of assaying the con-
sequences of interleukin action in disease becomes more apparent.
Interleukin Protocols will therefore be of interest to a wide range of
investigators, from molecular and cell biologists to immunologists to clinical
investigators. The discovery of interleukins and the analysis of their role in
disease represent major advances in molecular medicine. The methods
described will help researchers continue to advance, ultimately leading to bet-
ter diagnosis, prognosis, and treatment of many diseases where there
remains an unmet medical need.
Luke A. J. O'Neill
Andrew Bowie
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
PART I: GENERAL METHODS FOR ASSAYING INTERLEUKIN PROTEIN
AND MRNA................................................................................................. 1
1 ELISAs and Interleukin Research
Catherine Greene................................................................................... 3
2 ELISPOT Technique for Assaying Interleukins
Almuth Christine Hauer and Mona Bajaj-Elliott...............................17
3 Measurement of Interleukins by ELISPOT Assay with Particular
Application to Dual-Color Analysis (Stardust Assay)
Yoshihiro Okamoto and Mikio Nishida.............................................29
4 Assaying Interleukins Intracellularly by FACS
Liam O'Mahony and Maurice O’Donoghue.......................................41
5 Basic RT-PCR for Measurement of Cytokine Expression
Mona Bajaj-Elliott and Almuth Christine Hauer...............................49
6 Quantitative PCR for Measurement of Cytokine Expression
Ellen M. Nilsen, Finn-Eirik Johansen, and Per Brandtzaeg...........59
7 Single Cell RT-PCR for the Detection of Cytokine mRNA
Moshe Ligumsky, Hovav Nechushtan, and Ehud Razin.................77
8 Quantitation of Cytokine mRNA by Flash-Type Bioluminescence
Jeffrey K. Actor....................................................................................83
9 Measurement of Interleukin mRNA by Northern Blotting Using
a Nonradioactive Detection Method
Ginette Tardif, John A. Di Battista, Jean-Pierre Pelletier,
and Johanne Martel-Pelletier.........................................................99
10 Quantification of Mouse IL-6 and TNF-(cid:95) mRNA Using Xplore Assays
Scott W. Van Arsdell, Kevin P. Murphy, Csaba Pazmany,
Diane Erickson, and Mark D. Moody...........................................111
vii
viii Contents
11 Measurement of Cytokine and Chemokine mRNA Using Nonisotopic
Multiprobe RNase Protection Assay
Zheng-Ming Wang and Roman Dziarski.........................................125
12 Isolation of Total and Bioactive Interleukins
by Immunoaffinity-ReceptorAffinity Chromatography
Terry M. Phillips and Benjamin F. Dickens....................................145
13 Detection of Cytokines by Immunohistochemistry
Mahmoud Huleihel.............................................................................157
14 Whole Blood Assays and the Influence of Circadian Rhythmicity
on Human Cytokine Measurement
Nikolai Petrovsky...............................................................................163
PART II: ILLUSTRATION OF METHODOLOGIES USING INDIVIDUAL INTERLEUKINS..........175
15 Assaying Interleukin-8
Paul N. Moynagh................................................................................177
16 Measurement of Interleukin-2 by Bioassay
Lyanne Weston..................................................................................185
17 Assaying Interleukin-15
Amos Douvdevani, Michal Weiler, and Eli Lewis..........................193
PART III: ASSAYING INTERLEUKINS IN PARTICULAR PATHOLOGIES...........................205
18 Assaying Interleukin-6 in Breast Cancer
Judith Harmey, Amanda Haverty, Deirdre Foley,
and David Bouchier-Hayes...........................................................207
19 Cytokines, Stress, and Depressive Illness
Hymie Anisman, Shawn Hayley, Arun V. Ravindran, Zul Merali,
and Jenna Griffiths........................................................................217
20 Measurement of Interleukins in Cutaneous Disorders: Competitive
RT-PCR for Determination of IL-10 mRNA Expression in Psoriasis
Khusru Asadullah, Antje Haeussler-Quade, Wolf Dietrich Doecke,
Wolfram Sterry, and Hans-Dieter Volk.........................................239
21 Interleukins in Graves’ Disease
Jan Komorowski................................................................................251
22 Evaluation of Cytokines in Migraine Patients by ELISA
Irene Munno, Mariarosaria Marinaro, Giuseppe Lacedra,
Antonia Bassi, and Vincenzo Centonze.....................................263
23 Assaying Interleukins in Plasma in the Course of Acute Myocardial
Infarction
Radek Pudil........................................................................................273
Contents ix
PART IV: ASSAYING INTERLEUKINS IN PARTICULAR BIOLOGICAL FLUIDS.................283
24 Measurement of Cytokines in Peritoneal Fluids
Reijo Punnonen and Juha Punnonen.............................................285
25 Measurement of Cytokines in Seminal Plasma
Marek Glezerman, Eitan Lunenfeld and Mahmoud Huleihel........293
26 Measurement of Cytokines in Induced Sputum: Application
to the Investigation of Asthma and COPD
Peter G. Gibson and Jodie L. Simpson..........................................305
27 Cytokines in Blister Fluids
Franco Ameglio and Luciano D’Auria.............................................313
28 Measurement of Cytokines in Synovial Fluid
Fabrizio De Benedetti........................................................................327
29 Measurement of Cytokines in Cerebrospinal Fluid
Toshiyuki Yokoyama.........................................................................337
30 Detection of Cytokines in Tears
Archana Thakur and Mark D. P. Willcox.........................................345
PART V: NEWER TECHNIQUES:INTERLEUKIN SIGNAL TRANSDUCTION,
RECOMBINANT INTERLEUKIN PRODUCTION, INTERLEUKIN GENE POLYMORPHISMS,
ANDTHE USEOFCDNA ARRAYS...............................................................355
31 Measurement of Phosphoinositide 3-Kinase Activation
by Interleukins
Stephen G. Ward................................................................................357
32 Analysis of Interleukin-2 Signaling Using Affinity Precipitations
and Polyacrylamide Gel Electrophoresis
Paul Brennan and Verónica Athié-Morales....................................369
33 Applications for Green Fluorescent Protein in Cell Signaling
Paul Brennan and Emmanuelle Astoul...........................................385
34 Use of Pichia pastoris for Production of Recombinant
Cytokines
Kevin P. Murphy, Csaba Pazmany, and Mark D. Moody...............393
35 Analysis of Single Nucleotide Polymorphisms in the Interleukin-1B
Gene Using 5' Nuclease Assays
Emad M. El-Omar...............................................................................409
36 Measurement of Cytokine and Chemokine Gene Expression
Patterns Using cDNA Array
Zheng-Ming Wang and Roman Dziarski.........................................419
Index............................................................................................................439
