Macrophages: A Practical Approach

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Macrophages

The Practical Approach Series Related Practical Approach Series Titles Cytokine Cell Biology Cytokine Molecular Biology Animal Cell Culture 3/e Flow Cytometry 3/e Immunoassay Monoclonal Antibodies Cytoskeleton: signalling and cell regulation Lymphocytes 2/e Apoptosis Cell Growth, Differentiation, and Senescence Immunodiagnostics Growth Factors and Receptors Cell Separation Complement MHC 1 MHC 2 Affinity Separations Immunochemistry 1 Immunochemistry 2 Antibody Engineering Platelets Basic Cell Culture Please see the Practical Approach series website at http://www.oup.co.uk/pas for full contents lists of all Practical Approach titles.

Macrophages A Practical Approach Edited by

Donna M. Paulnock Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, USA

OXPORD UNIVERSITY PRESS

OXFORD UNIVERSITY PRESS

Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw with associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York © Oxford University Press, 2000 The moral rights of the author have been asserted Database right Oxford University Press (maker) First published 2000 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available Library of Congress Cataloguing in Publication Data 1 3 5 7 9 1 08 6 4 2 ISBN 0 19 963689 3 (Hbk.) ISBN 0 19 963688 5 (Pbk.) Typeset in Swift by Footnote Graphics, Warminster, Wilts Printed in Great Britain on acid-free paper by The Bath Press, Bath

Preface

Macrophages have long been recognized as a critical component of innate and acquired immune responses. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of cellular receptors responsible for microbe recognition has focused renewed scientific attention on macrophages, and has highlighted the need for an up-to-date summary of laboratory techniques effective for the isolation, identification, and functional analysis of these cells. The information in this book aims to provide investigators with a concise compilation of experimental techniques appropriate for studying diverse aspects of macrophage biology. Each chapter provides an overview of a relevant experimental topic as well as detailed and specific protocols for related laboratory procedures. I anticipate that this information will be of value to new investigators in this field and also will provide established investigators with readily accessible and indepth methodologies for their laboratories.

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Contents

Preface page v List of protocols xi Abbreviations xv 1 Isolation of macrophages from tissues, fluids, and Immune response sites 1 Mary Ellen Handel-Fernandez and Diana M. Lopez 1 Introduction T. 2 The heterogeneity of macrophages 1 3 Isolation of free mononuclear phagocytes 2 Murine peritoneal cells 2 Blood monocytes 6 Alveolar macrophages 7 4 Macrophages in haematopoietic tissues 10 5 Fixed tissue macrophages 14 Mechanical and enzymatic digestion of tissue 15 6 Macrophages in immune response sites 25 Macrophages in infection 25 Tumour-associated macrophages 26 References 28 2 Purification of macrophages 31 Sandra Gessani, Laura Fantuzzi, Patrizia Puddu, and Filippo Belardelli 1 Introduction 31 2 Purification of macrophages by adherence-based methods 32 Adhesion properties of macrophages 32 Macrophage adhesion molecules 33 Effect of adherence on macrophage gene expression 33 Effect of adherence on macrophage functional activities 35 3 Technical approaches for macrophage purification by adherence-based methods 35 Adherence to uncoated plastic or glass surfaces 36

vii

CONTENTS

4

5

6

7

8

Adherence to gelatin-coated surfaces 36 Adherence to microexudate-coated surfaces 38 Adherence to collagen matrices 39 Methods for detachment of adherent macrophages 40 Mechanical detachment 40 Recovery of adherent cells by EDTA treatment 41 Recovery of adherent macrophages by lignocaine treatment 42 General considerations on the adherence-based methods 43 Effects of different surfaces on macrophage morphology and functions 43 Effects of different detachment procedures on macrophage physiology 44 Physical methods of macrophage purification 45 Purification of macrophages by isopycnic gradient centrifugation 46 Isolation of whole mononuclear cells from peripheral blood by the Ficoll-Hypaque gradient 46 Purification of monocytes by Percoll gradient 48 Importance of the control of experimental conditions in the preparation of a gradient 49 Counterflow centrifugal elutriation 50 Additional issues to be considered in monocytes/macrophages purification 54 Macrophage heterogeneity 54 Comparison of the efficacy of different purification techniques with respect to the source of macrophages 55 Non-adherent versus adherent culture of macrophages 56 Problems caused by LPS contamination during the course of macrophage purification and culture 56 Conclusions 56 Acknowledgements 57 References 57

3 Characterization of macrophage antigens and receptors by immunochemistry and fluorescent analysis: expression, endocytosis, and phagocytosis 61 Leanne Peiser, Peter j. Gough, Elizabeth Darley, and Siamon Gordon 1 Introduction 61 2 Immunochemical labelling of monocytes and macrophages 62 Introduction 62 Practical considerations for immunofluorescent staining of macrophage populations in vitro 64 Preparation of in vitro macrophage cultures for immunochemical staining 65 3 Detection techniques for fluorescent analysis of macrophages 70 Flow cytometry 70 Fluorescent microscopy 71 4 Immunohistochemical staining of macrophages in mouse tissues 71 Preparation of tissue 72 Immunochemical staining of tissue 76 5 Fluorescent analysis of macrophage endocytic function 78 Introduction to the endocytic pathway 78 Practical considerations for testing macrophage endocytic function 85 Practical considerations for testing macrophage phagocytic function 86 viii

CONTENTS 6 Conclusion 89 Acknowledgements 90 References 90

4 Analysis of antigen processing and presentation 93 P. M. Kaye 1 Introduction 93 2 Preliminary considerations in study design 93 A definition of antigen processing and presentation 93 T cell choice restricts functional interpretation 94 'In vivo veritas' 95 The dendritic cell issue 95 Pathogens are not simple antigens 96 3 Analysis of class I and II antigen processing 96 Pathways of processing 96 Modified protocols for use with pathogens 105 Cell biology of antigen processing 110 4 Analysing antigen presenting function of macrophages 110 Correlative studies of cell phenotype 110 Functional assays of co-stimulation 111 References 112

5 Macrophage secretory products 115 Paola Allavena, Giancarlo Bianchi, Walter Luini, Andrea Doni, Pietro Transidico, Silvano Sozzani, and Alberto Mantovam 1 2 3 4 5 6

Introduction 115 Cytokines and chemokines: chemotaxis 116 Leukocyte transmigration 118 Reverse transmigration 121 Soluble cytokine receptors 122 Cross-linking of soluble receptors 124 References 125

6 Analysis of macrophage lytic functions 127 Maria Carla Bosco, Tiziana Musso, Luca Carta, and Luigi Varesio 1 Introduction 127 Biological perspective 127 Technical notes 129 2 Macrophage-mediated anti-tumour activity 130 Morphological tumour cell counting assay 130 Macrophage-mediated cytolysis: release of radioisotopes 133 Macrophage-mediated cytostasis of tumour cells: incorporation of radioactive labels 140 3 Target cell sensitivity 141

ix

CONTENTS 4 Microbicidal activity 142 Anti-Leishmania activity of monocytes/macrophages 144 Anti-fungal activity 347 References 353

7 Analysis of macrophage activity In vivo 157 Nico van Rooijen and Esther van Kesteren-Hendrikx 1 Introduction 357 2 Previous methods for blocking of phagocytosis 357 Silica, carrageenan, and dextran sulfate 357 Gadolinium chloride 158 Anti-macrophage antibodies and receptor antagonists 359 Competition 159 3 The liposome-mediated macrophage suicide technique 360 Principles 360 Liposomes 361 Liposome-encapsulated clodronate 162 Injection of liposomes and access to tissue macrophages 365 Selectivity of the approach with respect to macrophages 166 Duration of macrophage depletion 167 4 Practical applications of the technique 368 Improved efficacy of carrier-mediated gene transfer 168 Improved survival of human cells in immunodeficient (SCID) mice 169 Suppression of inflammatory reactions 170 Improved graft survival and functioning 170 References 171

8 Analysis of gene expression In mononuclear phagocytes 173 Joyce E. S. Doan, Thomas A. Hamilton, and Donna M. Paulnock 1 Introduction 173 2 Detection and quantification of specific RNA levels 174 Basic principles 174 Preparation of total cellular RNA 174 Nuclear run-on analysis 177 Quantification of specific mRNAs 183 3 Gene transfer 185 Basic principles 385 Experimental strategies 385 Transient transfection 186 Stable transfection 192 4 Measurement of protein-nucleic acid interactions 393 Basic principles 393 Sources and characteristics of binding factors 394 Measurements of DNA and RNA binding proteins 195 References 201

A1 List of suppliers 203 Index 209 X

Protocol list

Isolation of free mononuclear phagocytes

Harvesting of resident peritoneal macrophages 3 Elicitation of peritoneal macrophages using thioglycollate 5 Peritoneal macrophage elicitation using Bio-Gel polyaciylamide beads 5 Isolation of human blood monocytes 6 Isolation of human alveolar macrophages from tumour lung biopsies 8 Isolation of murine alveolar macrophages by lung lavage 8 Isolation of alveolar macrophages from whole murine lung 9 Macrophages In haematopoietic tissues

Isolation of resident bone marrow macrophages 11 Isolation of murine bone marrow-derived macrophages 12 Fixed tissue macrophages

Isolation of murine splenic macrophages 14 Isolation of human splenic macrophages 16 Isolation of murine Kupffer cells 18 Isolation of human Kupffer cells from liver wedge biopsies 19 Isolation of osteoclasts from murine bone 20 Isolation of rat microglial cells 21 Isolation of human microglial cells 22 Isolation and purification of lamina propria macrophages from human small intestine 23 Isolation of murine Langerhans cells 24 Macrophages In Immune response sites

Isolation of granuloma macrophages from the livers of infected mice 25 Isolation of macrophages from solid tumours (mechanical dissociation) 27 Isolation of tumour-associated macrophages (enzymatic method) 27 Technical approaches for macrophage purification by adherence-based methods

Purification Purification Purification Purification

of macrophages by adherence of macrophages by adherence of macrophages by adherence of macrophages by adherence

to uncoated plastic or glass surfaces 36 to gelatin-coated surfaces 37 to microexudate-coated surfaces 38 to collagen-coated surfaces 39

Methods for detachment of adherent macrophages

Mechanical detachment of macrophages 40

xi

PROTOCOL LIST

Detachment of macrophages by EDTA treatment 41 Detachment of macrophages by treatment with lignocaine 42 Physical methods of macrophage purification

Isolation of mononuclear cells by Ficoll-Hypaque gradient separation 47 One-step continuous Percoll gradient separation of monocytes 48 Purification of macrophages by counterflow centrifugal elutriation (CCE) 52 Immunochemical labelling of monocytes and macrophages

Preparation of isolated macrophages for immunochemical staining 66 Detaching cultured macrophages from plastic and glass surfaces using EDTA/Lidocaine 67 Preparation of 4% paraformaldehyde 68 Indirect immunofluorescent staining of cultured macrophages 68 Immunohlstochemical staining of macrophages In mouse tissues

Preparation of fresh tissue for immunohistochemistry 72 Preparation of periodate-lysine paraformaldehyde for perfusion of mouse tissue 73 Perfusion of mouse tissue 74 Immunochemical staining of mouse tissue 77 Fluorescent analysis of macrophage endocytlc function

Fluorescent labelling of proteins or particles 83 Quantitation of macrophage endocytic function 84 Phagocytic uptake of bacteria by macrophages 89 Analysis of class I and II antigen processing

Preparation of bone marrow macrophages 97 Induction of MHC class II antigens on bone marrow macrophages 99 Pulsing macrophages with soluble antigens for analysing class II-restricted processing 101 Assessment of the cellular characteristics of class II-restricted antigen processing 103 Loading soluble antigens into macrophages by osmotic lysis of pinosomes 104 Stimulation of class I-restricted responses by BMM0 105 Pulsing macrophages with particulate antigens in suspension 106 Enumeration of microbe uptake by macrophages using direct staining 108 Evaluating antigen transfer between APC populations 109 Analysing antigen presenting function of macrophages

Assay of co-stimulatory function of macrophages 111 Cytoklnes and chemokines: chemotaxis

Assessment of macrophage migration using a Boyden chamber 117 Leukocyte transmigration

Assessment of transendothelial migration by radioisotopic detection 119 Reverse transmigration

Assessment of leukocyte reverse transmigration in vitro 121 Cross-linking of soluble receptors

Identification of soluble cytokine receptors using radiolabelled ligands 124 Macrophage-mediated anti-tumour activity

Measurement of macrophage anti-tumour activity by the tumour cell counting assay 131

xii

PROTOCOL LIST Measurement of macrophage-mediated cytolysis by the 51Cr release assay 133 Assessment of macrophage-mediated cytotoxicity using [3H]TdR and [125I]dUrd release assays 135 Measurement of macrophage cytotoxicity using the111indium release assay 138 Assessment of macrophage-mediated cytostasis of tumour cells based on target cell incorporation of radioactive label 140 Microblcidal activity Assessment of macrophage anti-Leishmania cytolytic activity 145 Detection of phagocytosis of C. albicans by macrophages 148 Assessment of macrophage-mediated intracellular Candida killing using a colony counting technique 150 Assessment of macrophage-mediated extracellular Candida killing using a colorimetric assay 151 The llposome-medlated macrophage suicide technique Preparation of multilamellar clodronate-liposomes 162 Spectrophotometric determination of the amount liposome-encapsulated clodronate 164 Detection and quantification of specific RNA levels Preparation of total cellular RNA from cultured macrophages 175 Detection of nuclear RNA by nuclear run-on assay 178 Analysis of specific gene expression by RT-PCR 182 Gene transfer Purification of supercoiled plasmid DNA 187 DEAE dextran-mediated transfection of macrophages 189 Transfection of macrophages using lipophilic reagents 190 Transfection of macrophages by electroporation 191 Measurement of protein-nucleic acid interactions Preparation of nuclear extracts from macrophages 196 Preparation of oligonucleotide probes for EMSA 198 Electrophoretic mobility shift assay (EMSA) 199

xiii


Abbreviations

Ab antibody ADCC antibody-dependent cellular cytotoxicity APC antigen-presenting cell BMM0 bone marrow macrophages CCE counterflow centrifugal elutriation CPU colony-forming units DC dendritic cells DEPC diethyl pyrocarbonate DMEM Dulbecco's modified Eagle medium DTT dithiothreitol EC endothelial cells ' • ECM extracellular matrix EDTA ethylenediamine tetraacetic acid EMSA electrophoretic mobility shift assay FBS fetal bovine serum FTTC fluorescein isothiocyanate FN fibronectin GBSS Gey's balanced salt solution GM-CSF granulocyte-macrophage colony-stimulating factor HBSS Hank's balanced salt solution Hepes N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid IL interleukin LPS lipopolysaccharide MEM minimum essential medium MHC major histocompatibility complex MOI multiplicity of infection NO nitric oxide PBMC peripheral blood mononuclear cells PBS phosphate-buffered saline PDGF platelet-derived growth factor RNI reactive nitrogen intermediates ROI reactive oxygen intermediates RT room temperature XV

ABBREVIATIONS RT-PCR SER SOD TCR TNF VIA

reverse transcription-polymerase chain reaction sialoadhesin superoxide dismutase T cell receptor tumour necrosis factor very late activation antigen

Chapter 1 Isolation of macrophages from tissues, fluids, and immune response sites Mary Ellen Handel-Fernandez and Diana M. Lopez Department of Microbiology and Immunology, PO Box 016960 (R138), 1600 N.W. 10th Avenue, Miami, FLA 33101, USA

1 Introduction This chapter describes strategies for the isolation of macrophages from specific tissue sites under normal or pathological conditions. Macrophages and/or macrophage-like cells can be found in a variety of organs in the body; however, in many cases, only insufficient numbers can be obtained by practical methods and experimentation with such cell populations is limited to histological techniques. The protocols described below are those which consistently produce moderate to high yields of viable cells with consideration to time and practicality.

2 The heterogeneity of macrophages A significant aspect of macrophage function is their role in innate and specific immunity. Tissue macrophages stand guard against foreign invaders and are able to instantly defend as well as send signals for recruitment and present antigen to other immunological cells. A critical reason macrophages are so effective as a first line of defence is that they are distributed throughout the body in various organs, tissues, and fluids (1). It is estimated that the macrophage compartment of a healthy adult mouse consists of approximately 108 cells (2). In adult animals, mononuclear phagocytes arise in the bone marrow from myeloid stem cells and migrate to peripheral blood and various tissues. Macrophages display great diversity of phenotype and function resulting from their ability to adapt to the local environment (3). Table 1 lists some of the larger macrophage populations found in different organs. It is the exposure to particular tissues, cell types, and physiological states that leads tissue macrophages to vary maturationally, functionally, and metabolically as evidenced by their differential response to stimulation and their range of distinguishing markers (4). For example, although phagocytosis is a hallmark of

1

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ Table 1 Major sources of resident mononuclear phagocytes in tissues and fluids Tissue/location

Cell type

Peritoneal cavity

Peritoneal macrophage

Lung

Alveolar macrophage

Peripheral blood

Monocyte

Liver

Kupffer

Bone

Osteoclast

CNS

Microglial cell

Skin

Langerhans cells

Spleen

Fixed tissue macrophage

Thymus


Bone marrow

Monoblast, promonocyte, monocyte, macrophage

Lamina propria


macrophage activity, skin-associated macrophages, Langerhans cells, are poorly phagocytic (5). In addition, cytokine production, receptor expression, and perioxidatic activity are highly variable between macrophage subtypes (5-7). The diversity in macrophage phenotype and function has compounded the difficulty in the interpretation of the ever expanding volume of data concerning these cells. Since spatially distinct macrophages do not respond uniformly, assumptions about one population based on the evidence of another can be misleading. In truth, even macrophages within a single tissue do not behave similarly. For example, splenic red pulp macrophages express the antigens F4/80 and sialoadhesin dim, white pulp macrophages do not express F4/80 nor sialoadhesin, and macrophages from the marginal zone express F4/80 dim and sialoadhesin (8). Comparisons between rodents and humans is also a problem since much work in rodents is performed using peritoneal macrophages, while human studies are very often done using peripheral blood monocytes. Aside from the environmental diversity established under homeostatic conditions, other tiers of heterogeneity exist. Differentiation from promonoblast to monocyte to immature macrophage to mature macrophage leads to both the loss and acquisition of functions and/or phenotypes (9, 10). Pathological conditions and inflammatory events influence macrophage response and activation state. For example, a Gram-negative bacterial infection may lead to recruitment of fully mature, fully activated cells (11, 12). On the other hand, it has been reported that tumour-associated macrophages are more immature and in some cases unresponsive (13).

3 Isolation of free mononuclear phagocytes 3.1 Murine peritoneal cells Many researchers using rodent models rely on peritoneal macrophages due to the ease and large quantity of cells that can be obtained. Resident peritoneal 2

ISOLATION OF MACROPHAGES FROM TISSUES, FLUIDS, AND IMMUNE RESPONSE SITES

macrophages are free rather than fixed tissue macrophages, therefore their extraction from the peritoneal cavity does not require any special dissociative cocktails. A few million cells can be harvested from the peritoneal cavity of one mouse. When larger numbers are necessary, sterile inflammatory agents can be used to increase the cell number two- to fourfold. Such agents include protease peptone, casein, thioglycollate, and Bio-Gel polyacrylamide beads (14-16). There is an initial transient recruitment of polymorphonuclear leukocytes, then by day four, the majority of elicited cells are macrophages. These agents recruit a blood monocyte-derived cell population which is considered to be less mature than resident peritoneal macrophages (16). In addition, by their mere presence the eliciting agents may affect certain macrophage functions. A comparison of resident versus elicited peritoneal macrophages showed that resident macrophages secrete fivefold greater amounts of haemolytically active C4 (14). Stein and Gordon (6) reported that the capacity of peritoneal macrophage populations to release high levels of TNF depended on the process of recruitment as well as the subsequent stimuli. TNF can be released at high levels by LPS-stimulated, thioglycollate-elicited macrophages, but only in small amounts by LPS-stimulated resident or Bio-Gel polyacrylamide beads-recruited macrophages. These three cell populations release similar amounts of TNF when stimulation is phagocytosisdependent. Elicited macrophages spread more rapidly in culture than resident macrophages and are more responsive to growth factors in culture. These cells are poorly cytocidal on their own and require IFN-y to prime them for enhanced cytotoxic activity (5). Another important consideration is that macrophages can ingest thioglycollate products, whereas Bio-Gel polyacrylamide beads are too large to phagocytose (5). Therefore, choosing which peritoneal cell population to use depends on the stimulus with which the macrophages will be activated and the purpose for which the macrophage population will be used. The methods presented in this chapter (Protocols 1-3) (6, 15, 17) are relatively quick and do not require excessive manipulation which could compromise the expression of surface markers. Protocol I describes the harvesting of resident peritoneal macrophages from pathogen-free mice.

Harvesting of resident peritoneal macrophages Equipment and reagents • 5 ml syringe and 18-gauge needle (one per mouse) • Table-top centrifuge • Sterile scissors and forceps • Dissecting board

• Pathogen-free mice • 70%ethanol • RPMI 1640 containing 5% endotoxin-free FCS (Gibco BRL)3

3

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

Method 1

Sacrifice the mice by CO2 asphyxiation or cervical dislocation.

2

Place the mice abdomens up and wet them completely with 70% ethanol.

3

Make a transverse cut in the inguinal area and pull back the skin to expose the peritoneal wall. Soak the peritoneal wall with 70% ethanol.

4

Lift the peritoneal wall away from the cavity with sterile forceps and inject approx. 4 ml of cold RPMI containing 5% endotoxin-free FCS using a 5 ml syringe with an 18gauge needle (bevelled end of needle facing up). Remove the needle, massage the peritoneum, and insert the needle back into peritoneal cavity, drawing the fluid back into the syringe. Avoid puncturing the intestines when the needle is inserted. It may help to push on the syringe plunger to allow the medium to pass through the needle while the needle penetrates peritoneum.

5

Remove the needle from the syringe and dispense the fluid into 50 ml polypropylene tubes.b

6

Repeat this procedure two more times. Approx. 10 ml of fluid should be recovered.

7

Wash the cells three times by centrifugation at 300 g for 10 min at 4°C and resuspend them in RPMI containing 5% FBS 1640,

a

For all protocols PCS is heat-inactivated at 56°C for 30 min before it is first used unless otherwise stated. b Some protocols suggest keeping cells on ice while harvesting. However, in our experience, this may promote clumping of cells.

Protocol 1 yields approximately 3 x 106 cells containing 50-70% macrophages per mouse (16). Macrophages can be obtained at a purity of greater than 90% by adherence to plastic (see Chapter 2). Older male mice tend to have more fat in the peritoneum, which may clog the needle (16) and so female mice are therefore recommended for procedures involving peritoneal macrophages. Normally very few contaminating red blood cells are harvested with this method so an excessive number of red blood cells in the cell pellet may be a sign of infection. To prevent contamination by red blood cells, when using a number of mice, it may be advantageous to collect the cells into separate 50 ml tubes, and pool them after the cells have been observed under a microscope. Protocol 2 describes the elidtation of macrophages by thioglycollate. The use of aged thioglycollate will substantially augment the yield of cells. This is apparently due to increased glycation products (18). Thioglycollate elicits 8-12 X 106 cells approximately 70% of which are macrophages (16). Under the light microscope, these cells are clearly distinguishable from lymphocytes. In many cases, phagocytosed thioglycollate products can be seen in the lysosomes of the cells. This procedure is one of the easiest, least expensive methods of obtaining mouse macrophages. 4


Elicitation of peritoneal macrophages using thioglycollate Equipment and reagents • 23-gauge needles and 5 ml syringes • Pathogen-free mice • 70% ethanol • Brewer's thioglycollate medium. To prepare this weigh out 30 g of dehydrated thioglycollate medium and suspend in 1 litre of distilled water in a 2 litre Erhlenmeyer flask. Heat the thioglycollate solution to boil over a flame. Carefully

swirl the solution, dissolving the medium completely. The colour will change from brown to red. Take the solution off the flame after it begins to boil. Aliquot the thioglycollate into 100 ml or 250 ml bottles, and autoclave at 15 lb/in2, 121°C for 15 min, slow exhaust. Store in the dark at room temperature for one to two months before use.3

Method 1 Clean the abdomen of each mouse with 70% ethanol. 2 Draw 2-3 ml of thioglycollate medium into a 5 ml syringe, attach a 23-gauge needle and inject the solution i.p. A large gauge needle is recommended because of the viscosity of the medium. For mice younger than two months, use 1.0-1.5 ml medium. 3 To isolate the macrophage population, wait four days to harvest the cells. 4 After four days, harvest the macrophages as described in Protocol 1, steps 1-5. Cells extracted at times earlier than four days will contain a significantly greater number of gramilocytes. 5 Protocol 3 describes the use of Bio-Gel polyacrylamide beads for macrophage elicitation. Recruitment of macrophages by Bio-Gel polyacrylamide beads is a useful method, especially for procedures involving phagocytosis. Approx. 10 x 106 cells can be obtained from a single mouse and can be further purified by adherence (see ref. 14 and Chapter 2). a

3% thioglycollate is a clear brown solution. Cloudiness is a sign of contamination and media with this quality should be discarded.

Peritoneal macrophage elicitation using Bio-Gel polyacrylamide beads Equipment and reagents • 18-gauge needle, 5 ml syringe • Table-top centrifuge • 75 um sterile mesh screens (PGC Scientific) • 50 ml polypropylene tube

• Pathogen-free mice • Bio-Gel P-100 (fine) beads (Bio-Rad Laboratories) • 70% ethanol • RPMI 1640 with 10%FCS(Gibco BRL)

5


Method 1 Wash the sterile Bio-Gel beads in H2O by centrifugation for 5 min at 300 g. Repeat the procedure two times. 2 Resuspend the beads to give a 2% (v/v) suspension. 3 Divide the 2% (v/v) into 1 ml aliquots and autoclave at 15 lb/in2 for 20 min. 4 Clean the abdomen of the mouse with 70% ethanol and inject 1 ml of the 2% Bio-Gel suspension i.p. 5 Recover the macrophages as described in Protocol 1, steps 1-5 at four days after injection. 6 Remove the beads and other large particles from the cells by straining through the 0.75 um sterile mesh screen into a 50 ml polypropylene tube. 7 Wash the cells with RPMI 1640 with 10%FCSthree times by centrifugation at 300 g, for 10 min at 4 °C. Resuspend the cells in the same media.

3,2 Blood monocytes Monocytes are released into the blood within 21/2, days after their formation in the bone marrow and have a life span in the circulating blood of approximately 24 hours (9). Many emigrate into the tissues to mature into macrophages. There are approximately 2.7 x 105 monocytes/ml of blood. These cells account for 10-20% of all peripheral blood, mononuclcar tells (19). They are the most accessible mo no nuclear phagocytes to study in humans. The isolation of monocytes from blood involves the collection of the buffy coat, the white cell-enriched layer between the plasma and the erythrocytes (20). Different cell populations can be isolated by density gradient centrifugation with, for example, Percoll or sucrose (see Chapter 2). Since there are certain levels of lymphocyte contamination associated with isolation by these methods, cells can subsequently be further purified by adherence.

Isolation of human blood monocytes Equipment and reagents • 50 ml polypropylene tube • 10mlEDTA(K 3 )Vacutainers(Beckton Dickinson) • Table-top centrifuge • Hepes-buffered saline (HBS): 0.8% (w/v) NaCl, 10 mM Hepes-NaOH pH 7.4 (filter sterilized)

6

• OptiPrep (Accurate Chemical Co.) • Solution A: HBS, 10 mM EDTA (filter sterilized) • Solution B: 0.5% (w/v) BSA in solution A (prepare fresh and filter sterilize) • OptiPrep (1.078 g/ml): 1 vol. OptiPrep: 3 vol. solution B


• OptiPrep (1.068 g/ml): 1 vol. OptiPrep: 4 vol. solution B * Phosphate-buffered saline (PBS): 0.01 M phosphate, 0.15 M NaCL Dissolve 20.5 g NaH2P04.H20 and 179.9 g NaHPO4.7H2O in

4 litres of water. Adjust the pH to 7.2. Add 701.3 g NaCl and add water to a total volume of 8 litres (10 x PBS). Dilute this stock 1:10 prior to use.

Method 1 Collect the blood into six 10 ml EDTA (K3) Vacutainers. 2 Pool the blood into a 50 ml polypropylene tube and centrifuge at 550 g for 20 min at room temperature. 3 Transfer 10 ml of bufty coat to a new 50 ml tube and mix with 4 ml OptiPrep. Overlay the mixture with 10 ml of 1.078 g/ml OptiPrep. Overlay the 1.078 g/ml layer with 20 ml of 1.068 g/ml OptiPrep and 0,5 ml HBS. 4 Centrifuge at 600 g for 25 min at room temperature in a swinging bucket rotor with no brake. 5 After centrifugation, remove the top 20 ml fraction (the monocytes), without disturbing the 1.068/1.078 interface. 6 Wash the cells three times with PBS and resuspend them in appropriate culture medium.

The method described in Protocol 4 is optimized for use with OptiPrep density gradient media (19), Further purification by adherence is not necessary since monocytes can be isolated with a purity of approximately 90% from the gradient alone. Protocol 4 will yield approximately 3 x 106 monocytes with a purity of greater than 90% (19). Since adherence and/or selection via antibodies are not necessary, the cells are not activated by the isolation and are greater than 95% viable.

3.3 Alveolar macrophages Phagocytosis by alveolar macrophages is the primary defence mechanism against microorganisms and foreign particles in the lung. Further, alveolar macrophages can be potent suppressors of T lymphocyte responses, which is important for the limitation of tissue damage (21), These cells reside in an environment quite different than most macrophages due to the aerobic atmosphere. To collect human cells, the most common method is broncheolar lavagc (22); however, since willing subjects arc not always available, methods have been developed for cell isolation from lung biopsies (23). One such procedure is outlined in Protocol 5. Lung lavages are possible in rodents also (24, 25). However, recovery is limited and in many cases, a more complicated procedure of mechanical and enzymatic dissociation is required (26, 27). 7


Isolation of human alveolar macrophages from tumour lung biopsies Equipment and reagents • Sterile forceps and scalpel blades • Sterile gauze • Sterile heparinized saline: 0.85% NaCl, 8.5 U/ml heparin (Sigma Chemical}

• Sterile 75 x 15 mm Petri dish • Hank's balanced salt solution (HBSS) (Gibco BRL)

Method 1 Immediately place the lung biopsy specimen in sterile heparinized saline at 4 "C. 2 Transfer the tissue to a sterile Petri dish containing 2-4 ml HBSS. 3 Tease and mince the parenchyma using sterile forceps and a scalpel blade. 4 Filter the resulting fragments through several layers of sterile gauze to obtain a single cell suspension. 5 Isolate the mononuclear cells by Ficoll-Hypaque density centrifiigation (see Chapter 2).

Isolation of murine alveolar macrophages by lung lavage Equipment and reagents • Sterile scissors and forceps • Blunt 18-gauge needles attached to 1 cc tuberculin syringe • 50 ml conical centrifuge tube

• • « •

Table-top centrifuge Pathogen-free mice Sterile saline RPMI 1640 containing 10% FCS (Gibco BRL)

Method 1 Sacrifice mice by cervical dislocation and bleed them by aortic section. 2 Excise the lungs from the thoracic cavity using sterile scissors and forceps. Place the lungs on a sterile Petri dish. 3 Cannulate the trachea with a blunt 18-gauge needle and lavage the lungs three times with sterile saline (35 ml/kg body weight) warmed to 37 °C. 4 Collect the cell suspension in a 50 ml conical tube and centrifuge at 450 g for 10 min. 5 Resuspend in RPMI 1640 containing 10% PCS.

8


Isolation of human alveolar macrophages from lung biopsies by Protocol 5 yields 4-8 x 106 macrophages per cubic centimetre of lung tissue, but requires extensive cell manipulation and there is a greater risk of contamination with other cell types than harvesting cells by broncheolar lavage (23), described in Protocol 6. A procedure for isolation of macrophages from whole murine lung is given in Protocol 7.

Protocol 7 Isolation of alveolar macrophages from whole murine lung Equipment and reagents • • • • • • •

Sterile wire mesh screens (PGC Scientific) Tube rotator/rocker Syringe plunger Sterile forceps, scissors, and scalpel 15 ml conical centrifuge tube 75 X 15 mm Petri dishes Pasteur pipettes

RPMI 1640 containing 5% FCS (Gibco BRL) Dissociation medium: RPMI 1540, 5% FCS, collagenase type I (150 U/ml) (Sigma Chemical) HBSS (Gibco BRL) (see Protocol 5) DNase (150 U/ml) (Sigma Chemical) PBS (see Protocol 4)

Method 1 Sacrifice the mouse by cervical dislocation and remove the lungs. 2 Pass the lungs through a Petri dish containing HBSS to eliminate contaminating blood. 3 Mince the lung tissue into 1 mm3 pieces with a scalpel and transfer them to a 15 ml tube containing 15 ml dissociation medium. 4 Seal the tube and place it on the rotator at 37 °C for 90 min. 5 Pour the lung tissue onto a wire mesh screen while discarding the liquid into a beaker underneath. 6 Place the wire mesh screen on top of an open sterile 75 x 15 mm Petri dish and rinse the tissue two times with a Pasteur pipette using 2-3 ml cold HBSS. 7 Gently push the tissue through the screen using the rubber end of the syringe plunger. 8 Rinse the plunger and mesh screen with HBSS. Repeat the procedure three times. 9 Transfer the contents of the Petri dish to a 15 ml conical tube using a Pasteur pipette and let the tube sit on ice for 4 min to let particulate matter settle. Do not leave the suspension on ice longer than 5 min. 10 With a Pasteur pipette, transfer the liquid to a new 15 ml tube leaving aggregates of tissue behind. 11 Centrifuge the cell suspension at 300 g for 10 rain.

9


12 Aspirate the liquid, and resuspend the pellet in 10 ml HBSS. 13 Repeat steps 10 and 11 twice and finally resuspend the pellet in 1-5 ml RPMI 1640 containing 5% FCS. 14 Count the cells and adjust the cell concentration to 2 x 106 cells/ml in RPMI 1640 containing 5% PCS. To enrich for the macrophage population, the cells from Protocol 7 can be passed through a Ficoll-Hypaque density gradient (see Chapter 2). This will increase the macrophage population to 30-70% of the cells (26). The remaining cells are predominantly lymphocytes. To achieve purity of greater than 85%, macrophages can be selected by plastic adherence (see Chapter 2). The viability of the remaining cells is greater than 95%. By this method approximately 1 x 106 macrophages can be obtained per animal (26).

4 Macrophages in haematopoietic tissues The bone marrow is a mix of pluripotent stem cells at different stages of haematopoiesis and mature cells whose function is to drive the former down a wellorchestrated path of development. Resident bone marrow macrophages are found within erythroid clusters and have a morphology and phenotype distinct from monocytes or bone marrowderived macrophages. In mice, each cluster contains an average of 35 cells. Approximately 1% of bone marrow cells are resident macrophages (2.5 x 105 per two femurs) (28). The method described in Protocol 8 is based on that described by Crocker and Gordon (8, 28) and details the isolation of resident macrophages from the bone marrow of mice. In the isolation of resident bone marrow macrophages, it is important that any mechanical manipulations be very gentle. These cells have long plasma membrane processes which branch throughout the marrow stroma. Vigorous pipetting or passage through needles should be avoided as it will result in the loss of the mature macrophage population. Centrifugation should also be avoided to minimize artificial clustering of cells. Large numbers of macrophages can be derived from bone marrow precursors. The isolation of these cells involves harvesting immature cells and culturing them with specific growth factors to continue haemalopoiesis in vitro (29). Utilization of bone marrow-derived macrophages is desirable because a more homogeneous population of cells is obtained. However, bone marrow-derived macrophages require about a week before they are ready to use, and due to the necessity of supplemental growth factors, can be a costly method. Approximately 3-10 x 106 bone marrow cells can be harvested per mouse which are then cultured under conditions which favour growth of the macrophage population (16, 29). Protocol 9 describes the in vitro propagation of bone marrow-derived macrophages from pathogen-free mice. 10


Isolation of resident bone marrow macrophages Equipment and reagents • • • • • • • • • •

30 ml syringes and 25-gauge needles 100 x 15 mm Petri dishes 50 ml polypropylene tubes Sterile circular coverslips (PGC Scientific) 24-well tissue culture plates (Fisher Scientific) 5% CO2 incubator Table-top centrifuge Sterile forceps and scissors Rubber tubing to fit 30 ml syringe Tubing clamp

• Tube rotator/rocker • Pathogen-free mice • Flushing solution: RPMI 1640 (Gibco BRL) containing 0,05% coUagenase type I (Boehringer Mannheim} and 0.001% DNase type I (Sigma Chemical} • RPMI1640 containing 10% endotoxin-free FCS {Gibco BRL) • RPMI 1640 containing 30% endotoxin-free FCS (Gibco BRL} • Ficoll-Hypaque (Pharmacia) • PBS (see Protocol 4)

Method 1 Sacrifice the mice by cervical dislocation or C02 asphyxiation. 2 Make an incision at the top of each hind leg and pull the skin down towards the foot to expose the muscle. 3 Cut off the hind legs and place them on a sterile 100 x 15 mm Petri dish. 4 Remove the muscle from the bones by cutting with scissors then pulling the muscle downward and away with forceps, and remove the foot and the skin. 5 Cut the tibia from the femur at the joint. 6 Fill a 5 ml syringe with flushing solution and attach a 25-gauge needle. 7 Wash the bone marrow cavity free of cells by inserting the needle and injecting 2-5 ml of flushing solution while holding the bone over the Petri dish at a 45° angle with sterile forceps. 8 Collect the bone marrow plugs in a fresh Petri dish and place it on ice while continuing the procedure with the remaining bones. 9 Suspend the bone marrow plugs collected from one mouse in a 50 ml tube in volume of 10 ml flushing solution and incubate the mixture for 1 h at 37 °C, with constant rotation (one revolution per second). 10 Stop the enzymatic digestion of the extruded material by adding FCS to the mixture at a final concentration of 1% (v/v), 11 Pool the digests of material from the bone marrow of two mice. 12 Centrifuge the cells at 100 g for 10 min at room temperature. 13 Gently resuspend the pelleted cells in 3 ml RPMI 1640 without serum.

11


14 Place a 3 ml layer of Ficoll-Hypaque in a 20 ml syringe with rubber tubing attached and clamped closed. 15 Carefully layer 20 ml RPMI containing 30% FCS over the Ficoll-Hypaque cushion using a 10 ml pipette, 16 Layer the 3 ml cell suspension over the RPMI/Ficoll-Hypaque using a Pasteur pipette and let the gradient stand for 1 h at room temperature. 17 Unclamp the tubing and collect 2 ml fractions into a 24-well plate. 18 Inspect the fractions for the presence of cell clusters free of contaminating single cells using a phase-contrast microscope. 19 Pool the fractions containing the clusters. 20 To separate the bone marrow clusters, layer 5 ml of the bone marrow digest over 10 ml RPMI containing 30% FCS in a 50 ml conical tube. 21 Let the gradient stand for 1 h at room temperature. 22 Aspirate the upper 14 ml of medium, leaving 1 ml remaining in the tube—this contains the clusters of cells with macrophages. 23 Wash the cells twice with RPMI 1640 alone by centrifugation for 10 min at 100 g. 24 Resuspend the cells in RPMI 1640 plus 10%FCSusing 0.8 ml per original column number. 25 Add 100 n-1 of cells to sterile circular coverslips in 24-well plates and allow them to adhere at 37 °C for 30 min in a 5% C02 incubator. 26 Add 1 ml RPMI 1640 with 10%FCS(per well). 27 Incubate the cells at 37°C in a 5% C02 incubator for 3 h. 28 Rinse the coverslips five times by washing with 1 ml PBS per well and removing the PBS with a Pasteur pipette. 29 Place 1 ml PBS per well and incubate the cultures for 30 min at room temperature. 30 Rinse the coverslips repeatedly with PBS. The remaining adherent cells are 50% bone marrow macrophages.

Isolation of murlne bone marrow-derived macrophages Equipment and reagents • • • •

12

Sterile forceps and scissors 5 ml syringes (25-gauge needles) 150 x 15 mm Petri dishes Table-top centrifuge

• 25 cm2 and 75 cm2 tissue culture flasks (Falcon) • Sterile rubber policeman • Pasteur pipette


• Pathogen-free mice • PBS (see Protocol 4) . ,. . 14.1% Nycoprep (Accurate Chemical) • Dulbecco's modified Eagle medium (DMEM) (Gibco BRL) • Macrophage colony-stimulating factor (M-CSF) (Genzyme)

• Complete DMEM: medium plus 10% FCS, 2 mM gramme, 15 mM Hepes buffer, 0.02% sodium bicarbonate, 100 U/ml penicillin, 100 ug/ml streptomycin (Gibco

Method 1 Extrude bone marrow plugs as described in Protocol 8, using PBS as the flushing solution. 2 Pool all extruded material into a 50 ml polypropylene tube and resuspend gently using a Pasteur pipette. 3 Centrifuge at 500 g for 10 min at room temperature. 4 Discard supernatant fluids. 5 Resuspend the pelleted cells in 5 ml serum-free DMEM. 6 Place 5 ml of 14.1% Nycoprep in a 15 ml conical tube. 7 Overlay with the 5 ml solution of bone marrow cells and centrifuge at 500 g for 20 min at room temperature with no brake. 8 Remove the cells at the interface with a Pasteur pipette. 9 Transfer to a fresh 15 ml tube and centrifuge at 500 g for 10 min at 4 °C. 10 Resuspend the cell pellet in complete DMEM to a concentration of 5 x 106 cells/ml. 11 Add 1 x 107 cells to individual 25 cm2 tissue culture flasks in 10 ml complete DMEM. 12 Incubate flasks for 24 h at 37°C in a 5% C02 incubator. 13 Harvest the non-adherent cells and transfer to a 75 cm2 tissue culture flask. 14 Add 10 ml complete DMEM containing M-CSF at a final concentration of 500-1000 U/ml. 15 Incubate for four days at 37°C, 5% C02. 16 Add an additional 10 ml complete DMEM with M-CSF (500-1000 U/ml} to each flask, 17 Incubate cells for an additional three days. 18 At the end of seven days, remove or decant culture medium. 19 Wash the adherent cells with 10 ml PBS. 20 Add 5 ml of filter sterilized dispase solution (pre-warmed to 37 °C). 21 Incubate at 37°C for 5 min. 22 Rap the flask sharply against the palm of the hand to dislodge adherent cells. 23 Remove the cells from the bottom of the flask by scraping gently with a rubber policeman.

13


24 25 26 27

Add 10 ml complete DMEM to the flask, Resuspend the cells and place them into a 50 ml tube. Centrifuge the cells 500 g for 10 min at 4°C. Resuspend the cells in 5 ml complete DMEM.

The yield of bone marrow-derived macrophages after seven days of culture should approach 2-3 x 106 macrophages per 10 x 106 cultured bone marrow cells. Other growth factors such as granulocyte-macrophage colony-stimulating factor or interleukin-3 can be used in place of M-CSF. Using these growth factors will result in a yield of 1 x 106 macrophages per 10 x 106 bone marrow cells (16).

5 Fixed tissue macrophages Macrophage subpopulations located in different tissues are interesting to study since they are adapted to specialized activity within their local environment. Because they are closely associated with surrounding tissues, mechanical and/or enzymatic digestion is often necessary. Macrophages can be recovered from liver, gut, brain, bone, and spleen with varying degrees of time, purity, viability, and yield. In other tissues, macrophage experimentation is mostly limited to histological techniques. To make single cell suspension of splenocytes. only gentle disruption is required and fairly large numbers of macrophages can be obtained from a single mouse spleen and isolated by Percoll gradient centrifugation (30, 31), as described in Protocol 10. Isolation of human splenic macrophages requires a more rigorous protocol of tissue dissociation and is discussed in Protocol 11.

Isolation of murlne splenic macrophages Equipment and reagents • • • • • •

75 x 15 mm Petri dish Sterile forceps and scissors Sterile 250 um nylon mesh (PGC Scientific) Dissecting board Table-top centrifuge Syringe plunger

• Pathogen-free mice • 70%ethanol • RPMI1640 (Gibco BRL) • HBSS (Gibco BRL) • RPMI 1640 with 10%FCS(Gibco BRL}

A Isolation of cells 1 Sacrifice the mice by cervical dislocation or CO2 asphyxiation, 2 Pin the mouse to a dissecting board with the left-side up.

14


3 4 5 6 7 8

Wet the mouse with 70% ethanol and make a longitudinal incision exposing the peritoneal cavity. Remove the spleen by lifting it at one end with sterile forceps and cutting away from the body. Place the spleen on sterile nylon mesh. Press the spleen through the mesh with a sterile syringe plunger. Collect the suspension in a 75 x 15 mm Petri dish containing 5 ml cold RPMI 1640. Wash the cell suspension twice with RPMI 1640 by pelleting cells at 300 g for 10 min at4°C Resuspend the cell pellet in 5 ml HBSS for separation by density gradient centrifugation.

Equipment and reagents • 25 ml polycarbonate tube (Beckman) • Beckman type 30 rotor and ultracentrifuge

• Percoll (Pharmacia) • HBSS (see Protocol 5)

B Density gradient separation 1 Dilute stock Percoll (Pharmacia) in HBSS to 280-320 mOsm/kg H20 and 1.070 g/ml density. Various dilutions of stock can be made to lower densities by using the formula: Vy = V, x (p, - p) (p - py), where Vy = volume density VT = volume stock Percoll P] = density stock Percoll py = density diluting medium p = desired final density. 2 To prepare a continuous gradient, pipette 20 ml of 1.070 g/ml Percoll into a 25 ml polycarbonate tube, 3 Spin the Percoll at 30 000 g for 15 min at 4 °C in a Beckman type 30 rotor (decelerate with no brake). 4 Layer up to 1 x 108 mononuclear cells diluted in HBSS on top of the gradient. 5 Spin the gradient at 400 g for 20 min at 4°C (without brake). 6 Aspirate the resulting cellular bands with a Pasteur pipette. 7 Wash the cells three times with 10 ml HBSS per wash. 8 Resuspend the cells in RPMI with 10%FCSfor tissue culture,

5.1 Mechanical and enzymatic digestion of tissue The simplest and least time-consuming method of tissue disaggregation is mincing tissue fragments into small pieces and/or forcing the tissue through metal 15


or cloth screens. Unfortunately, this method can be overly rigorous. Over-mincing cells often leads to poor viability and it may favour survival of only hearty subpopulations of cells (20). However, careful mechanical dissociation or a combination of mechanical and enzymatic manipulation may be the only method to obtain single cell suspensions of tough tissues. When the quandtation of surface markers are desired, special care must be taken to be as gentle as possible with the tissues.

5.1.2 Enzymatic dissociation Enzymatic dissociation is often preferred, but may take experimentation with several enzyme cocktails and incubation times before the right degree of disaggregation is obtained. In most enzymatic protocols a certain level of mechanical dissociation is necessary. The most widely used protease, i.e. collagenase. weakens and/or dissolves the collagen of the stroma, but does not affect the cells. Pronase and DNase also arc used in many protocols. One of the most important steps in any enzymatic protocol is to inactivate these enzymes after tissue dissociation, and to wash the resulting cell suspension thoroughly to remove all proteases (32). The types and amount of enzyme used depends largely on the tissue being dissociated, and the desired cell type being isolated. The following protocols were designed specifically for macrophage isolation from these tissue types, however, it is highly recommended that a few 'practice nans' he performed to optimize viability and purity.

Isolation of human splenic macrophages Equipment and reagents • • • • • •

Tenbroeck tissue homogenizer (Bellco) 100 x 15 mm Petri dish 50 ml polypropylene tubes Sterile gauze 2 in X 2 in (Fisher Scientific) Sterile forceps and scissors MP medium: RPMI1640. 2 ^M Lglutamine, 10 fig/ml garamycin, 1% trypticase soy broth, 10% heat-inactivated newborn calf serum (Gibco BRL)

• Collagenase type VIII (Sigma Chemical Co) • Tris-NH4Cl: add 90 ml of 0.16 M NH4C1 to 10 ml of 0.17 M Tris pH 7.65, and adjust to pH 7.2 with HC1 • Heat-inactivated newborn calf serum (Gibco BRL) • Bovine pancreatic DNase I (Sigma Chemical Co)

Method 1 Cut the splenic tissue (10-20 g) into pieces in a Petri dish containing 5-10 ml MP medium. 2 Load the contents of the Petri dish into a Tenbroeck tissue homogenizer and homogenize the sample with eight to ten strokes.

16


3 Decant the suspension and stroma into a 50 ml polypropylene tube. 4 Wash the suspension twice by centrifugation for 10 min at 400 g in MP medium at 4°C. 5 Resuspend the cells to 3 X 108 cells/ml in MP medium. 6 Add collagenase at 260 U/ml of cell suspension, 7 Incubate at 37°C, 5% CO2 for 30 min with occasional agitation. 8 Pellet the cells by centrifugation for 10 min at 400 g at 4 °C. 9 Resuspend the cells in 1 ml Tris-NHtCl/O.l ml packed cells. 10 Leave the suspension at room temperature for 2 min. 11 Underlay the celts with 5 ml of newborn calf serum, 12 Centrifuge the cells at 400 g for 10 min at 4 "C. 13 Wash the cells twice as described in step 4. 14 Resuspend the cell pellet to 3 x 108 cells/ml in MP medium containing 20 jig/ml bovine pancreatic DNase I. 15 Incubate the suspension for 30 min at 37 °C, 5% C02 with occasional agitation. 16 Dispense the stromal fragments by gently pipetting. 17 Dilute the cell suspension in MP medium (enough for easy filtering) and filter the cells through a sterile gauze pad into a 50 ml polypropylene tube. 18 Centrifuge the cells at 400 g for 10 min at 4°C. 19 Resuspend the cells in 5 ml MP medium containing 20 M-g/ml fresh DNase 1 for further purification by adherence of countercurrent centrifugal elutriation (see Chapter 2),

i. Human splenic macrophages Human monocyte/macrophage populations are most often studied using peripheral blood due to availability and ease of isolation. However, the human spleen is an abundant source of macrophages and these cells can be obtained by a combination of mechanical and enzymatic dissociation (33). This method yields a cell suspension containing approximately 13% macrophages (most of the splenic macrophage population). Macrophages can be further isolated by plastic adherence (see Chapter 2) but for highly enriched cells without selective loss, countercurrent centrifugal elutriation is recommended (see Chapter 2). ii. Kupffer cells The liver is comprised of parenchymal cells (hepatocytes) and non-parenchymal cells. The majority of non-parenchymal cells are sinusoidal cells. Of this subset, 60% are endothelial cells and 40% are Kupffer cells. Hepatocytes can be separated from non-parenchymal cells due to their sensitivity to collagenase (34-36). Kupffer 17


cells can then be separated from endothelial cells by centrifugal elutriation (see Chapter 2). Approximately 1.8 x 106 cells per gram of wet liver tissue can be obtained from mice. These cells are 98% viable and 95% pure Kupffer cells (34), Protocol 12 describes the isolation of Kupffer cells from pathogen-free mice.

Isolation of murlne Kupffer cells Equipment and reagents 150 cm2 tissue culture flask 100 x 15 mm3 Petri dish 15 ml and 50 ml polypropylene tubes Table-top centrifuge 24-gauge cannula (Popper and Sons) Sterile scissors and forceps Sterile steel mesh {PGC Scientific) Sterile rubber stoppers Sterile 75 um sterile nylon mesh (PCG Scientific) • Pathogen-free mice • 70% ethanol

• • • • • • • • •

• Collagenase A(0.176U/mg)(Boehringer Mannheim) • Dissociation buffer: 0,1 mM L-aspartic acid, 0.2 mM L-threonine, 0,3 nxM L-serine, 0.5 mM glycine, 0.6 mM t-alanine, 0.9 mM L-glutamic acid, 3 mM KC1, 0.7 mM NaH2PO4.H2O, 0.5 mM MgCl2, 24 mM NaHCO3,20 mM glucose, 20 mM fructose, 197 mM saccharose, 0.05% collagenase A pH7.4 • Gey's balanced salt solution (GBSS) (with and without NaCl) (Gibco BRL) • Metrizamide {Accurate Chemical Co)

Method 1 2 3 4 5 6 7 8 9 10 11 12 13

18

Sacrifice the mice by cervical dislocation or CO;j inhalation. Pin the mice to a dissecting board, abdomen up. Clean the mouse with 70% ethanol. With sterile scissors, make a ventral midline incision exposing the peritoneal cavity. To perfuse the liver, first cut the vena cava to prevent reflux of blood. Using a 24-gauge cannula, perfuse the liver through the portal vein at a flow rate of 10 ml/min for 5 min with dissociation buffer at 37°C. Remove the liver from the animal with sterile forceps. Mince the liver into small pieces and push it through sterile steel mesh with a sterile rubber stopper. Collect the cell suspension in a 100 x 15 mm Petri dish. Transfer the cells to a 150 cm2 tissue culture flask. Add 100 ml of a 4 to 1 ratio of GBSS dissociation buffer to the cells. Incubate with continuous agitation for 1 h at 37 °C, 5% CO2. Filter the resulting cell suspension through sterile nylon mesh.


14 15 16 17 18 19

Centrifuge at 300 g for 10 min at 4°C in 50 ml polypropylene tubes, Resuspend cell pellets in 10 ml medium. Dispense 5 ml of the cell suspension equally into two 15 ml tubes. Mix this solution with an equal volume of 30% metrizamide in GBSS without Nad Top with 1ml of GBSS (with NaCl). Spin the cells at room temperature at 1400 g for 25 min in a table-top centrifuge without brake, 20 Recover the cells from the interface with a Pasteur pipette. 21 Purify the macrophages by centrifugal elutriation (see Chapter 2). Protocol 13 describes the isolation of human Kupffer cells.

Isolation of human Kupffer cells from liver wedge biopsies Equipment and reagents • Sterile scissors and forceps

• Buffer II: GBSS, 0.8 ng/ml DNase

• 60 n.m sterile gauze (Fisher Scientific) • 15 ml polypropylene tubes

• 1 M NaOH

• Pasteur pipettes • GBSS (Gibco BRL) (see Protocol 12) • Dissociation buffer: GBSS, 0,2% pronase (Gibco BRL), 0.8 M.g/ml DNase (Boehringer Mannheim)

• 16% Nycodenz (Accurate Chemical Co) in isotonic buffer: 0.75% NaCI, 5 mM Tris-HCt pH 7.5. 3 mM KC1. 0.3 mM CaNa2EDTA

Method 1 Mince the biopsy tissue into small pieces (1-2 mm3 in size) in GBSS. 2 Incubate the liver fragments in 75 ml dissociation buffer with continuous stirring at 37°C for 30 min. During incubation period, check and correct the pH to 7.3-7.5 with 1 M NaOH, as needed. 3 Filter the resulting cell suspension through 60 j*m gauze. Complete tissue dissociation may require reincubation of unsuspended tissue for another 15 min in dissociation buffer. 4 Spin the cells at 300 g for 10 min to pellet them. 5 Wash the pellet twice in 50 ml buffer II. 6 Resuspend the pellet in 5 ml buffer II. 7 Layer the resulting cell suspension over 16% Nycodenz.

19


8 9 10 11 12 13

Spin the gradient at 600 g for 20 min at 4 °C in a 15 ml polypropylene tube. Collect the interface cells with a Pasteur pipette. Wash the cells in 10 ml GBSS. Spin the cells at 300 g for 10 min. Resuspend the pellet in buffer II. Enrich the human Kupffer cells by counterflow centrifugal elutriation (see Chapter 2).

///. Osteoclasts Osteoclasts are the macrophage-like cells of the bone. These cells are specialized for bone resorption. Osteoclasts are often multinucleated as a result of frequent cell fusion and the majority of cells stain positively for tartrate-resistant acid phosphatase (TRAP) after three days in culture (37). Although osteodasts exist in low numbers in the bone, the protocol described below outlines steps to enrich for these cells (38). Further purification of these cells is possible by micromanipulation of cells under phase-contrast microscopy (38) and is not presented here. Techniques similar to those described can be used to enrich for Osteoclasts in other mammalian species including humans (37). From 1 cm3 of starting material, approximately 1.2 x 106 cells can be harvested (37). Protocol 14 describes the isolation of marine osteociasts.

Isolation of osteociasts from murlne bone Equipment and reagents • Sterile forceps and scissors • Supplemented medium 199: medium 199 (Gibco BRL), 15% FCS, 100 U/ml penicillin, 100 p-g/ml streptomycin

• 6-well tissue culture plates (Falcon) • Pathogen-free mice • PBS

Method 1 Sacrifice the mice by cervical dislocation or CO2 asphyxiation. 2 Dissect mouse femurs and tibias from two 5-10 day-old mice (see Protocol 8). 3 Remove all soft tissue from the bones. 4 Split the bones longitudinally using scissors. 5 Remove and discard the bone marrow with sterile forceps and cut the bone into small pieces. 6 Suspend the bone fragments in 1 ml supplemented medium 199. 7 Dispense 0.2 ml aliquots into a 6-well tissue culture plate.

20


8 Incubate the fragments at 37°C. 5% CO2 for 45 min. 9 Remove non-adherent cells and bone fragments by rinsing the plates with PBS. 10 Add 5 ml fresh medium. iv. Microglial cells Microglial cells are found throughout the central and peripheral nervous system. These cells express low levels of MHC class I antigen (8) and are thought to be important in response to injury (5), These cells can be adapted for use with mice. However, due to the low yield, several mice must be sacrificed to recover a sufficient number of cells as described in Protocol 15.

Isolation of rat mlcroglial ceils Equipment and reagents • Sterile forceps and scissors • Sterile stainless steel mesh (PGC Scientific) • Table-top centrifuge • Eight-week-old pathogen-free rats • PBS (see Protocol 4)

• HBSS with 3%FCS(see Protocol 5) • Dissociation buffer: 42 mM MgCl2, 23 mM CaCl2, 50 mM KC1,153 mM NaCl, 0.75% collagenase type II (Boehringer Mannheim), 7 x 103 U/ml DNase I (Sigma Chemical)

Method 1 Sacrifice the rats by administering an ether overdose. 2 Perfuse each rat via the carotid artery with 200 ml cold PBS. The central nervous system must be completely perfused for a clear isolation. 3 Remove the brain and spinal cord from one rat and transfer to a Petri dish containing ice-cold HBSS with 3% FCS. 4 Mince the brain and spinal cord with scissors. 5 Pass the tissue through a stainless steel mesh into a Petri dish. 6 Collect the dissociated material, as well as that remaining in the sieve, in a 50 ml polypropylene tube. 7 Centrifuge the material at 170 g for 10 min at 4 °C. 8 Digest each brain/spinal cord for 60 min at 37 °C in 1.4 ml dissociation buffer. 9 Add 5 ml HBSS with 3% FCS. 10 Centrifuge for 10 min at 300 g to pellet the cells. 11 Resuspend the cells in Percoll at 1.098 g/ml diluted in HBSS (final density 1.088 g/ml). 12 Separate the microglia by centrifugation (see Protocol 10).

21


If the CNS material weighs more than 0.5 g, first add the cell suspension to a low density Percoll gradient (1.03 g/ml) and centrifuge to remove the myelin. Recovered microglial cells can be further purified by Percoll gradient centrifugation (see Protocol 10), and collected at the 1.072 g/ml interface. Approximately 4 X 103 cells per CNS are recovered. These are 80% microglial cells (39), Protocol 16 outlines the isolation of human microglia.

Isolation of human microglial cells Equipment and reagents • Stainless steel sieve (PGC Scientific) • Table-top centrifuge • PBS containing 0.2% BSA (see Protocol 4)

• DNase I (Sigma Chemical) • Type II collagenase (Boehringer Mannheim)

Method 1 Process fresh CNS tissue by mechanical disruption and passage through a stainless steel sieve (see Protocol 15). 2 Wash the dissociated material by centrifugation at 400 g for 10 min in 30-50 ml PBS with 0.2% BSA. 3 Digest tissue for 30 min at 37°C with 15 U of DNase per 200 mg of tissue and 3 U of type II collagenase per 200 mg of tissue. 4 Wash cells in 30 ml PBS containing 0.2% BSA at 400 g for 10 min. 5 Resuspend pelleted cells in PBS containing 2% BSA. 6 To isolate microglia, add the cell suspension to a graduated Percoll gradient consisting of Percoll at 1.21,1.088,1.072, and 1.03 g/ml, Microglia are isolated from the 1.072 g/ml layer.

Recovery of microglial cells by Percoll gradient separation yields approximately 5 X 10b cells/g CNS tissue (40). v. Lamina propria macrophages The gastrointestinal tract mucosa is the largest reservoir of macrophages in the body (41). These cells accumulate beneath the epithelium covering the luminal surface of the lamina propria (42). Gut-associated macrophages display the typical characteristics associated with the macrophage phenotype. For example, they stain positively for non-specific esterases, contain abundant cytoplasm, and phagocytese latex beads (43). Protocol 17 describes the purification of lamina propia macrophages from human small intestine. 22


Isolation and purification of lamina propria macrophages from human small Intestine Equipment and reagents • • • •

Sterile forceps and scalpel Platform rotator 2 in X 2 in sterile gauze (VWR Scientific} Storage medium: RPMI 1640 (Gibco BRL) supplemented with 100 U/ml penicillin, 100 ^g/ml streptomycin, 50 (ig/ml gentamycin, 250 ^g/ml amphotericm B, 100 mM pyruvate, 2 mM glutamine, 1 M Hepes • HBSS (Gibco BRL see Protocol 5) containing 200 |Ag/ml DTT (Sigma Chemical)

• HBSS (Gibco BRL, see Protocol 5) containing 0.2 M ethytenediamine tetraacetic acid (Fisher Scientific) and 10 mM 2-mercaptoethanol (2-ME) (Sigma Chemical) • Digestion medium: RPMI 1640, 100 M.g/ml DNase (Sigma Chemical), 75 jxg/ml dispase (Boehringer Mannheim) • PBS (see Protocol 4)

Method 1 Obtain resected intestinal segments and dissect the mucosa (that without detectable Peyer's patches) from the underlying muscularis propria and submucosa. 2 Place the tissue in cold storage medium until tissue digestion. 3 Rinse the mucosa with PBS. 4 Wash the tissue in BBSS plus DTT for 20 min at 37 °C on a platform rotator. 5 Repeat this procedure once with fresh HBSS plus DTT, then twice with HBSS plus ethylenediamine tetraacetic acid and 2-ME to remove mucus epithelium. 6 Rinse the tissue again in PBS. 7 Mince the tissue into 1 mm3 pieces. 8 Digest the sample in RPMI containing DNase and dispase for 45 min at 37°C, 200 r.p.m. 9 Collect the supernatant fluid—this contains the lamina propria macrophages. 10 Strain the cell suspension through sterile gauze to remove the cell clumps. The lamina propria macrophage population can be enriched for by counterflow centrifugal elutriation (see Chapter 2). Approximately 50 x 106 macrophages can be obtained from 25 g of tissue. The viability of these cells is greater than 99% with a purity of 99% (41,42,44). vi. Skin-associated macrophages Langerhans cells are macrophage-like cells that reside in the epidermis. They are poorly phagocytic, and express the F4/80 antigen. Protocol 18 describes the preparation of Langerhans cells from the skin of pathogen-free mice (45,46). 23


Isolation of murine Langerhans cells Equipment and reagents • • • • • • • • • •

Single edge razor blades Sterile gauze 100 x 15 cm Petri dishes Autoclaved paper towels Autoclaved 15 cm filter paper circles Nitex mesh filters (40 jun) (Tetko) See syringes Table-top centrifuge Pathogen-free mice PBS see (Protocol 4)

• Langerhans cells (LC) culture medium: RPMI 1640,10% FCS, 100 ^g/ml streptomycin, 100 U/ml penicillin, 1 x Fungizone, 2 mM L-glutamine, adjust pH to 7.2 • 2.5% trypsin (Boehringer Mannheim) • DNase (0.5 mg/ml) (Boehringer Mannheim) • FCS (Gibco BRL)

• Lymphoprep (Accurate Chemical Co)

Method 1 Sacrifice the mouse by cervical dislocation or CO2 asphyxiation. 2 Clean the mouse thoroughly with sterile PBS, 3 Shave the body on sterile paper towels. 4 Add 18 nil PBS to a 100 x 15 mm Petri dish on ice (two mice/dish).a 5 Place the mouse on a fresh paper towel. 6 Remove and peel apart the ears. 7 Place the skin from the ear area, epidermis-side up, in the Petri dish. 8 Remove the skin from the rest of the mouse. Try to keep the skin in one piece. Do not use the tail or lower legs. 9 Place the skins on filter paper circles moistened with PBS. 10 Clean the skin of any excess fat or major blood vessels. 11 Cut the skin into 4 mm wide slices. 12 Place the skin in Petri dishes with the epidermis-side up, 13 Add 2 ml of 2.5% trypsin to the 18 ml of PBS (final concentration 0.25% trypsin). 14 Incubate the skin for 1 h at 37 °C, 5% CO2. 15 Remove the epidermal layer and place it in a new Petri dish containing 8.5 ml PBS, 0.5 ml DNase, and 1 ml trypsin. 16 IncubateforlOminat37°C,5%C0 2 . 17 Add 2 ml FCS to inactivate the trypsin. 18 Using a 5 ml syringe (no needle), draw and release the epidermal cells until they become a single cell suspension. 19 Filter the cells through 40 turn Nitex mesh into a 50 ml tube.

24


20 21 22 23 24

a b

Centrifuge the cells at 300 g for 10 min at 4 °C. Decant the supernatant fluid. Wash the pelleted cells two times in RPMI1640 with 10% FCS.b Count the cells and wash them once more. To enrich the epidermal cells for Langerhans cells, layer the cell suspension (2-5 x 107 cells/5 ml) on an equal volume of Lymphoprep and centrifuge at 400 g for 20 mm.

Keep skins and cells on ice unless noted otherwise. If the cells clump, add DNase (50 fU/10 ml). The recovered cell population is 5-15% Langerhans cells. Culture the cells in LC medium at 2 x 106 cells/ml (47). Langerhans cells can be further enriched by depletion of the dendritic epidermal T cells by incubation with Thy-1 antibody plus complement (45), or by flow cytometric sorting (48) (see Chapter 3).

6 Macrophages in immune response sites 6.1 Macrophages in infection Specific types of infections give rise to granulomatous inflammation. These include tuberculosis, sarcoidosis, cat-scratch disease, leprosy, brucellosis, and schistosomiasis (49, 50). A granuloma is identified by its distinctive pattern of inflammatory reaction in which aggregates of epithelial-tike macrophages are surrounded by lymphocytes and a few plasma cells. Older granulomas also display an outer layer of fibroblasts and connective tissue. In some cases, fused macrophages, giant cells, are also found (49). Protocol 19 describes the isolation of granuloma macrophages (51) from livers or lungs (52) of infected mice.

Isolation of granuloma macrophages from the livers of Infected mice Equipment and reagents • • • • • •

Tissue homogenizer (Biospec Inc.) Rocking platform 250 tun sterile nylon mesh (PCG Scientific) Dissecting board Pathogen-infected mice MEM (Gibco BRL)

• Collagenase type I (Sigma Chemical Company) • Hypotonic lysis buffer (Tris-NH4Cl) (see Protocol 11) • 70% ethanol

25


Method 1 Sacrifice the mice by cervical dislocation or CO2 asphyxiation. 2 Place each mouse on a dissecting board ventral-side up. 3 Clean the abdomen with 70S6ethanol. 4 Make a longitudinal incision exposing the peritoneal cavity. 5 Remove the liver lobes with sterile forceps. Use only livers that exhibit multiple small whitish granulomas. 6 Collect the livers in a 50 ml tube containing enough MEM to cover all organs. 7 Homogenize the tissue using a low to medium setting on the tissue hotnogenizer for 4-7 min or until tissues appear to be completely in suspension. 8 Place the homogenate in a 50 ml tube and allow the granulomas to sediment for 5 min. 9 Decant the supernatant and resuspend the pellet in the same volume of MEM as initially used. 10 Repeat sedimentation procedure three more times. 11 Resuspend the final pellet in MEM containing 1000 U/ml collagenase at a volume equal to that of the granulomas. 12 Incubate the cells for 30 min at 37°C on a rocking platform. 13 Stand the tube upright and allow the undigested granulomas to sediment 14 Decant the supernatant through nylon mesh into a. new 50 ml tube. 15 Wash the cells three times with MEM by centrifbgation for 10 min at 300 g at room temperature. 16 Lyse the red blood cells by hypotonic shock using Tris-Nr4Cl, 17 Wash the cells again with MEM. 18 Resuspend the cells in MEM plus 10% FCS. The resulting population is 30-45% macrophages, which can be further purified by adherence (see Chapter 2) or by negative selection using antibodies against Thy-1, heat stable antigen, and granulocytes in the presence of 10% rabbit complement for 30 min at 37°C (51, 53). In the negative selection procedure, macrophages can then be obtained by separation or a self-forming Percoll gradient (see Protocol 10), which results in a 95% pure macrophage population.

6.2 Tumour-associated macrophages The same levels of tissue dissociation discussed in Section 5.1 apply to tumourassociated macrophages. Isolation of these cells may require more trial and error since each tumour is different and incubation with enzymes may be longer or shorter. Histology is an important prerequisite to isolation with respect to expected yields and contaminating cell types. The protocols described below are a good starting place and have been used successfully in mammary tumours

26


(54, 55). A more extensive review of enzymatic tissue dissociation is reported by Russell et til. (55). These methods were optimized for murine tumours. However, similar methods can be used for isolation of macrophages from human tumours (55, 56). Protocol 20 describes the isolation of tumour macrophages by mechanical dissociation; Protocol 21 describes their isolation by enzymatic digestion.

Isolation of macrophages from solid tumours (mechanical dissociation) Equipment and reagents • Sterile 250 ^m nylon mesh screens (PGC2 Scientific) • Sterile rubber stoppers • Sterile forceps and scissors • Table-top centrifuge

• • • •

75 mm2 sterile Petri dishes Pasteur pipettes RPMI1640 with 10% FCS 19% Nycodenz (density 1,1 g/ml) (Accurate Chemical Co) (see Protocol 13)

Method 1 Cut the tumour into 2-3 mm3 pieces of tissue. 2 Pass the tissue pieces through a sterile screen mesh into a Petri dish by pressing on tumour sections with a sterile rubber stopper. 3 Collect the cell suspension in a fresh 75 mm2 Petri dish containing 2-3 ml of ice-cold RPMI 1640 with 10% FCS. 4 Layer the resulting cell suspension onto a Nycodenz density gradient 5 Spin the cells at 400 g for 10 min at 4 °C. 6 Aspirate the cells at the interface with a Pasteur pipette. 7 Wash the cells twice with RPMI 1640 and centrifuge at 300 g for 10 min. 8 Resuspend the final pellet in RPMI 1640 with 10% PCS, 9 Purify the macrophage population by adherence (see Chapter 2).

Isolation of tumour3-associated macrophages (enzymatic method) Equipment and reagents • • • • •

Sterile forceps and curved scissors 125 ml Erlenmeyer flask Sterile magnetic stir bar Sterile gauze DMEM (Gibco BRL)

* Digestion medium: DMEM. 50 U/ml collagenase type II (Worthington Biochemial Corp.). 10 U/ml DNase (Calbiochem) • HBSS plus 10%FCS(Gibco BRL) (see Protocol 5)

27

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M, LOPEZ

Method 1 Trim the tumour of any fat, grossly necrotic, or haemorrhagic pieces with sterile scissors. 2 Mince the tumour into approx. 1 ram3 pieces with curved scissors. 3 Digest the tumour mince in a 125 ml Erlenmeyer flask in 50 ml digestion media. Stir with a magnetic stir bar for 20 min at room temperature. 4 Stop stirring and allow the tumour suspension to settle for 2-3 min. 5 Carefully collect the supernatant and wash it immediately in cold HBSS with 10% FCSto neutralize any residual enzyme. 6 Repeat steps 3-5 three more times. 7 Pool the supernatants and filter them through sterile gauze to remove clumps, This cell suspension can then be further purified by centrifugal elutriation (see Chapter 2).

References 1. Rutherford, M. S., Witsell, A., and Schook, L B. (1993). J. Lcuk. Biol, 53, 602, 2. Morahan, P. S., Volkman, A., Melnicoff, M., and Dempsey, W, L. (1988). In Macrophages and cancer (ed, C. H. Heppnerand A. M. Fulton), p. 2, CRC Press, Boca Raton, Florida. 3. Lawson, C. S., Rabinowitz, S., Crocker, P, R,, Morris, L, and Perry, V. H. (1992|. Curr. Top. Micrnbwl Immunol, 181, 1. 4. Gordon, S., Crocker. P. R,, Morris, L, Lee. S. H., Perry, V, H,, and Hume, D. A. (1986). Cibu Found. Symp., 118, 54. 5. Gordon, S, (1995). Biofssuys, 17, 977, 6. Stein, M. and Gordon, S. (1991). Eur.j. Immunol, 21, 431. 7. Daems, W. T., Koerten, H, K,, and Soranzo, M. R. (1976). Adv. Exp. Med. Biol, 73, 27. 8. Gordon, S., Eraser, I., Nath, D., Hughes, D., and Clarke, S. (1992). Curr. Opin. imnwnol, 4, 25. 9. Werb, Z. and Goldstein, I. M. (1987). In 1987 Basic and clinical immunology (cd. D. P. Stites, J. D. Stobo, andj. V. Wells), p. 96. Appleton & Lange, Los Altos, California. 10. Leenen, P.J. M., deBruijn, M. F, T. R., Voerman, j. S. A., Campbell, P, A., and Ewijk, W. V. (1994). J. Immunol. Methods, 174, 5. 11. Zisman, D. A., Ktmkel, S. L, Streiter, R. M.. Gauldie,J.,Tsai, W. C., Bramson.J., et al. (1997). Shock, 8, 349. 12. Kmnaert, P., De Wilde. J. P., Boumonville, B., Husson, C., and Salmon. I. (1996). Ann, Surg., 224, 749. 13. DiNapoli, M. R,, Calderon, C. L, and Lopez, D. M. (1996JJ. Exp. Med., 183, 1323. 14. Newell, S. L and Atkinson,]. P, (1983). J. Immunol.. 130, 834. 15. Mishell, B. B., Shiigi, S. M., Henry, C, Chan. E, L, North,]., Gallily, R., et al. (1980). In Selected methods in cellular immunology (ed. B. B. Mishell and S. M. Shiigi), p. 6. W. H, Freeman and Company, San Frandstro. 16. Kruisbeek, A, M, and Vogel. S. (1995). In Current protocols in immunology (ed.]. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober), p. 41,0.1. Green Publishing Assoc, Inc. and John Wiley & Sons, Inc.. New York, 28

ISOLATION OF MACROPHAGES FROM TISSUES, FLUIDS, AND IMMUNE RESPONSE SITES 17. Fauve, R. M., Jusforgues, H., and Hevin, B. (1983). J. Immunol. Methods, 64, 345. 18. Li, Y. M., Baviello, G., Vlassara, H., and Mitsuhashi, T. (1997)./. Immunol. Methods, 201, 183. 19. Graziani-Bowering, G. M., Graham, J. M., and Filion, L G. (1997) J. Immunol. Methods, 207,157. 20. Center, W. A. J. C. S. General procedures for primary cell culture. Lake Placid, New York, Corning Glass Works. 21. Toossi, Z., Hirsch, C. S., Hamilton, B. D., Knuth, C. K., Friedlander, M. A., and Rich, E. A. (1996). J. Immunol, 156, 3461. 22. Hance, A. J., Douches, S., Winchester, R. J., Fenrans, V. J., and Crystal, R. G. (1984). J. Immunol, 134, 284. 23. Hunninghake, G. W., Gadek, J. E., Szapiel, S. V., Strumpf, I. J., Kawanami, O., Ferrans, V. J., et al (1980). Methods Cell Biol.. 21A, 95. 24. Gilmour, M. I., Park, P., and Selgrade, M. K. (1993). Am. Rev. Respir. Dis., 147, 753. 25. Thoren, S. A. (1992).;. Toxicol. Environ. Health, 36, 307. 26. Hunninghake, G. W. and Fauci, A S. (1976). Cell. Immunol., 26, 89. 27. Stein-Streilein, J., Bennett, M., Mann, D., and Kumar, V. (1983). J. Immunol., 131, 2699. 28. Crocker, P. R. and Gordon, S. (1985).;. Exp. Med., 162, 993. 29. Brunt, L. M., Portnoy, D. A., and Unanue, E. R. (1990). J. Immunol., 145, 3540. 30. Watson, G. A., Fu, Y.-X., and Lopez, D. M. (1991). J. Leuk. Biol., 49,126. 31. Kurnick, J. T., Ostberg, L., Stegagno, M., Khnura, A. K., Orn, A, and Sjoberg, O. (1979). Scand.J. Immunol., 10, 563. 32. Prop, F. J. A. (1982). In Tumor immunity in prognosis (ed. S. Haskill), p. 177. Marcel Dekker, Inc., New York. 33. Buckley, P. J., Beelen, R. H. J., Burns,]., Beard, C. M., Dickson, S. A., and Walker, W. S. (1984).J. Immunol. Methods, 66, 201. 34. Janousek, J., Strmen, E., and Gervais, F. (1993J. Immunol. Methods, 164,109. 35. ten Hagen, T. L. M., Vianen, W. V., and Bakker-Woudenberg, A. J. M. (1996). J. Immunol. Methods, 193, 81. 36. Heuff, G., Van de Loosdrecht, A. A, Betjes, M. G. H., Beelen, R. H. J., and Meijer, S. (1995). Hepatology, 21, 740. 37. Lambrecht, J. T. and Marks Jr., S. C. (1996).Clin.Anat., 9,41. 38. Tong, H. S., Sakai, D. D., Sims, S. M., Dixon, S. J., Yamin, M., Goldring, S. R., et al. (1994). ]. Bone Miner. Res., 9, 577. 39. Sedgwick, J. D., Schwender, S., Imrich, H., Dorries, R., Butcher, G., and Meulen, V. T. (1991). Proc. Natl. Acad. Sri. USA, 88, 7438. 40. Dick, A. D., Pell, M., Brew, B. J., Foulcher, E., and Sedgwick, J. D. (1997). AIDS, 11,1699. 41. Smith, P. D., Meng, G., Shaw, G. M., and Li, L. (1997). J. Leuk. Biol., 62, 72. 42. Nagashima, R., Maeda, K., Imai, Y., and Takahashi, T. (1996). J. Histochem. Cytochem., 44, 721. 43. Beeken, W., Mieremet-Ooms, M., Ginsel, L A, Leijh, P. C. J., and Verspaget, H. (1984). ]. Immunol. Methods, 73,189. 44. Smith, P. D., Janoff, E. N., Mosteller-Barnum, M., Merger, M., Orenstein, J. M., Kearney, J. F., et al. (1997). J. Immunol. Methods, 202,1. 45. Dai, R. and Streilein, J. W. (1997) J. Invest. Dermatol, 108, 721. 46. Dai, R., Grammer, S. F., and Streilein, J. W. (1993). J. Immunol, 150, 59. 47. Xie, Y., Fernandez, M. E., Streilein, J. W., and Lopez, D. M. (1996). Anticancer Res., 16, 9. 48. McKinney, E. C. and Streilein, J. W. (1989). J. Immunol., 143, 1560. 49. Robbins, S. L. and Kumar, V. (1987). In Baste pathology (ed. D. Manke), p. 28. W. B. Saunders Company, Philadelphia, PA. 50. Villanueva, P. O., Reiser, H., and Stadecker, M. J. (1994). J. Immunol., 153, 5190.

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MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ 51. Stadecker, M. J., Wyler, D. J., and Wright, J. A. (1982).J.Immunol., 128, 2739. 52. Chensue, S. W., Ruth, J. H., Warmington, K., Lincoln, P., and Kunkel, S. L. (1995)J. Immunol, 155, 3546. 53. Flores Villanueva, P. 0., Harris, T. S., Ricklan, D. E., and Stadecker, M. J. (1994) J. Immunol, 152,1847. 54. Nelson, J. A. S., Parhar, R. S., Scodras, J. M., and Lala, P. K. (1990).J. Leuk. Bio!., 48, 394. 55. Russell, S. W., Doe, W. F., Hoskins, R. G., and Cochrane, C. G. (1976). Int.]. Cancer, 18, 322. 56. Moore, K. and Mortari, F. (1983). BrJ. Exp. Pathol, 64, 354.

30

Chapter 2 Purification of macrophages Sandra Gessani, Laura Fantuzzi, Patrizia Puddu, and Filippo Belardelli Laboratory of Virology, Istituto Superiore di Sanita, Viale Regina Elena 299-00161 Rome, Italy.

1 Introduction The investigation of cell populations consisting of a single cell type confers a great advantage over the complicated examination of mixed cell populations in many experimental studies. The achievement of enriched preparations of cells of the monocyte/macrophage lineage is particularly troublesome as these cells display a variety of functional and morphological phenotypes and are readily activated in response to environmental signals. Macrophage purification is further complicated when cells of this lineage represent a minority of the whole mononuclear pool (e.g. peripheral blood). Several methods are currently available for macrophage purification and they are based on four general principles: (a) Ability of monocytes/macrophages to adhere to foreign surfaces from which they can be subsequently released by different physical-chemical procedures (1. 2). (b) Density differences between monocytes/macrophages and other cells (3-6). (c) Differences in cell velocity sedimentation (6-8). (d) Differences in sedimentation after elution of cells previously centrifuged (elutriation) (9-11). A comparison among different purification procedures used to obtain a pure population of monocytes/macrophages from different sources is summarized in Table 1. It would be desirable if monocytes/macrophages purification procedures could conform to the same general guidelines that have been applied to the isolation of most other leukocytes in cellular immunology: (a) A high degree of purity is mandatory. (b) The viability and functional integrity of obtained cell suspensions should be assured. (c) The purification procedure should be a negative selection process that in itself does not alter the functional capabilities of the fractionated cell.

31

SANDRA GESSANI ET AL. Table 1 Comparison of monocyte/macrophage purification methods Method

Basis for separation Advantages

Adherence

Functional cell property

Disadvantages

Easy and inexpensive Transient monocyte/macrophage activation Problems in detaching cells for further studies Contamination with other cell types Morphological and functional alterations

Cell size Velocity sedimentationi

Easy and inexpensive Long separation time Streaming phenomenon at the sample/gradient interface

Elutriation

Recovery of high Require high numbers of macrophage numbers mononuclear cells Expensive equipment High purity Monocyte subpopulations may be separated

Cell size and density

Cell density Isopycnic sedimentation

Good macrophage recovery Good purity

Some toxicity of the cell separation medium Require extensive standardization of the experimental conditions

(d) The procedure should have the capacity of generating large numbers of purified cells to assure that certain leukocyte subsets are not inadvertently selected by the fractionation procedure. This is of particular importance since it has become evident that distinct subsets of monocytes/macrophages exist (12) and high yields would facilitate the recovery of all such subsets. It is very well known that macrophages are extremely versatile cells, which adapt and respond easily to environmental signals by changing their functional program (reviewed in refs 13-15). This raises the question of whether the isolation and purification procedure itself can induce drastic changes. In fact, the methods used for monocyte/macrophage separation may have variable effects on cell functions and/or result in the isolation of different cell subpopulations. Thus, it is not unexpected that the chosen method of separation of monocytes/ macrophages may influence subsequent results about their physiology and biochemistry. Likewise, it is very likely that the isolated cell populations can differ depending on the isolation procedure used. Thus, the choice of a particular macrophage purification technique is largely dependent on the requirements of the investigator and the nature of the functions to be studied.

2 Purification of macrophages by adherence-based methods 2.1 Adhesion properties of macrophages Macrophages are large cells which attach tenaciously to solid substrates during short periods of in vitro culture. The adherence of macrophages to solid 32

PURIFICATION OF MACROPHAGES

substrates is an energy- and pH-dependent process that is enhanced by high serum concentration and low pH (16). This property of mononuclear phagocytes has been used extensively to deplete lymphoid cell suspensions of monocytes/ macrophages and to prepare macrophage cultures from different anatomic sites. Adherence-based methods generally use Ficoll-Hypaque isolated peripheral blood mononuclear cells (PBMC), or peritoneal or broncoalveolar lavage cell preparations, followed by positive selection of monocytes/macrophages from these preparations by virtue of their adherence to plastic or glass surfaces, either untreated or coated with different materials (17-21). Although other (non-monocytic) cells can also adhere, their adhesion properties are not as strong as for the macrophages and the majority of these cells can be removed by washings. However, as adherence is not a feature unique to macrophages, procedures that rely solely on adherence will yield enriched monocyte/macrophage populations contaminated with a small, but appreciable, number of other cell types particularly for macrophages isolated from tissues (e.g. fibroblasts and endothelial cells).

2.2 Macrophage adhesion molecules Monocytes/macrophages are capable of binding to other cells, complement components, immune complexes, and extracellular matrices in a very specific manner as they express both the leukocyte class of adhesion receptors [LFA-1, Mac-1 and p!50, 95 (P2 integrin family, involved in cell-cell and complement interactions)] as well as several members of the 'very late activation antigen' (VIA) receptors class ((31 integrin family) which play a role in cell-substrate interactions (reviewed in refs 22, 23). Integrins are able to bind matrix molecules such as fibronectin (FN), laminin, and collagen, recognizing specific amino acid sequences in their ligands. The most well studied is the RGD (arginine, glycine, aspartic acid) sequence found within a number of matrix proteins including FN, fibrinogen, vitronectin, laminin, and type I collagen (22, 23). Several well-known adhesion molecules expressed in monocytes/macrophages (i.e. VLA-1 or CD49a, VLA-4 or CD49d, VLA5 or CD49e) are also found on other haemopoietic cells (primarily lymphocytes), fibroblasts, endothelium, and epithelium (22,23). There are still few examples of macrophage-specific adhesion molecules. In this regard, it has been reported that, unlike other cell types, macrophages attach to tissue culture plastic in the absence of divalent cations (24). This cation-independent macrophage adhesion is mediated by the scavenger receptor (24). Interactions of the above-mentioned receptors with extracellular matrix components and/or cell surface ligands of endothelial and connective tissue cells have important and specific influences on signal transduction processes in monocytes/macrophages.

2.3 Effect of adherence on macrophage gene expression Adherence is a well-known activation signal for monocytes/macrophages and it has been proposed to play a pivotal role in the earliest events in macrophage maturation and activation (13-15). During the process of extravasation, monocytes adhere to capillary endothelium and subsequently to a variety of extracellular 33

SANDRA GESSANI ET AL.

matrix components (25). These early interactions are likely to serve as modifiers of transcriptional activity and to prime monocytes for rapid synthesis of mediators. These observations highlight the important role that adherence, during purification or cultivation of monocytes/macrophages, can play in transcriptional activation. In particular, shortly after adherence, a complex set of regulatory events takes place (26-34). These events are denned by rapid changes of mRNA levels mainly coding for proto-oncogenes and inflammatory mediators. Table 2 summarizes the major transcriptional modifications, described after adherence of monocytes/macrophages to different substrates. The vast majority of adherence effects regards the up-modulation of gene transcription (27, 30-34) even though some negative transcriptional effects have also been described (28, 29). Another important aspect of the macrophage response to adherence is represented by the nature of the surface, which is likely to have selective influences on this process. In this regard, Thorens et al. (26) reported that induction of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA in thioglycollateelicited murine peritoneal macrophages required adherence to FN-coated plastic, whereas c-sis induction could occur by adherence to plastic alone. Similarly, Eierman and colleagues (29) reported that adherence to substrates pre-treated Table 2 Effect of macrophage adherence to different substrates on gene expression Substrates

Time after

Genes"

Effect

References

28,31

adherence Untreated plastic

20 min

c-fosa

t

Untreated plastic

30 min

lkBa

t

34

Untreated plastic

30min

SODa

32

Untreated plastic

0.5-1 n

IL-l<x,b IL-lpa'b

Untreated plastic

2h

IL-6

Untreated plastic

en

c-juna

Untreated plastic

6-24 h

EGR2a

FN-coated surfaces

2-3 h

GM-CSFb

Untreated or FN-coated surfaces

1.5-4.5 h

CSF-1a

T T T T T t T

Untreated or FN-coated surfaces

6h

PDGF (B)a,b

r

"26,31

Untreated, FN-, or collagen-coated surfaces

20-40 min

TNFaa,c

t

28,29,33

Untreated, collagen-, or FN-coated surfaces

SOmin

lL-8a,c

T

32,33

Untreated plastic

40 min

Lysozymea

4h

c-fmsa

1 1

29

Untreated plastic 3

a

27,32,33

30 31 31

26 28, 29

28

Human peripheral blood monocytes. Mouse peritoneal macrophages. c Human alveolar macrophages. d Abbreviations: SOD, superoxide dismutase; IL, interleukin; PDGF, platelet-derived growth factor. b

34


with FN resulted in tumour necrosis factor (TNF) a and CSF-1 mRNA levels approximating those found after adherence to plastic alone, whereas adherence to FN, FN/anti-FN complexes, or collagen resulted in markedly decreased levels of CSF-1 induction and lysozyme down-regulation.

2.4 Effect of adherence on macrophage functional activities It has been reported that adherence and spreading of monocytes to culture dishes induce a number of physical and biochemical changes that have been interpreted as an expression of differentiation and/or activation (35-37). Characteristic morphological changes, including a consistent increase in cell size and the development of a higher cytoplasm to nucleus ratio, are generally observed in human monocytes during the first few days of adherent culture (38, 39). Other studies (40-43) have explored the influence of attachment on a number of parameters generally used as markers of macrophage activation (i.e. secretion of lysosomal enzymes, 5'-nucleotidase activity, superoxide anion generation, and plasminogen activator production). In this regard, it has been reported that adherent macrophages exhibit an increased secretion of lysosomal enzymes (pglucuronidase, p-glucosaminidase, and neutral proteases) and superoxide anion generation as well as a decreased level of membrane 5'-nucleotidase expression (40, 41). Other macrophage functional activities, such as phagocytosis and cytotoxicity, have also been reported to be stimulated following adherence to substrate (44,45). Although macrophages can to some extent be activated during their isolation and purification, the influence of attachment to a foreign surface such as plastic is significant and has important implications in the design and interpretation of experiments examining the biology of the monocytes/ macrophages in vitro.

3 Technical approaches for macrophage purification by adherence-based methods The methods described herein have been used by different laboratories, including ours, to purify monocytes/macrophages from different anatomical sites in human, mice, and other animal species. The procedures outlined below refer mainly to human peripheral blood monocytes present in the Ficoll-Hypaque separated mononuclear cell pool, but the same protocols can be applied to mononuclear phagocytes from other sources. In regard to the adhesion properties of human peripheral blood monocytes, it should be taken into account that adhesiveness of these cells varies from one donor to another and variations in their affinity to plastic surface during their in vitro cultivation have been well documented (42). Monocytes, which initially attach strongly to the plastic surface, retract their plasma membrane and reduce their diameter by 50% during the first day in culture. On the next day (approximately 16-18 hours later), the adherent monocytes round up and cluster in small groups, rendering easy their collection by pipetting the plate without the aid of any additional treatment. 35

SANDRA GESSANI ET AL,

Only a small fraction of the initial population of phagocytic cells does not adhere to the plastic during this period of incubation (46).

3.1 Adherence to uncoated plastic or glass surfaces Hcoll-Hypaque derived PBMC and peritoneal or broncoalveolar lavage cells can be directly seeded in uncoated plastic or glass surfaces. Protocol 1 describes a procedure for the purification of monocytes from Ficoll-Hypaque derived PBMC by this method.

Purification of macrophages by adherence to uncoated plastic or glass surfaces Equipment and reagents • Various tissue culture vessels (Corning Costar or Falcon plastics), including 25 and 75 cm2 flasks, 10 cm diameter Petri dishes, 24-well cluster plates • RPMI medium (BioWhittaker) containing 15% heat-inactivated FBS (BioWhittaker)

Tissue culture sterile Ca2+/Mg2+-free phosphate-buffered saline (PBS): 1.9 mM NaH2PO4 (anhydrous), 8.1 mM Na2HPO4 (anhydrous), 154 mM NaCl, adjust to pH 7.2-7.4 (PBS can be purchased from HyClone)

Method 1 Seed Ficoll-Hypaque derived PBMCa (see Protocol 8) at the concentration of 2 x W6jcm*. 2 Allow cells to adhereb for at least \ h at 37 °C, 3 Remove non-adherent cells by vigorous washings with Ca2+/Mg2+-free PBS (pH 7.4) for at least four times. 4 Add culture medium to the adherent cells and maintain at 37°C until use.c a Seed 5 x 107 in 10 ml, 1.5 x 108 in 30 ml, 1 x 10s in 20 ml, and 3 x 106 in 1 ml of PBMC in 25 cm2 flasks, 75 cm2 flasks, 10 cm diameter Petri dishes, and 24-well cluster plates, respectively. b The most efficient attachment of monocytes to commonly used plastic labware or glass generally occurs after 1-2 h of incubation at 37°C. c Adherent monocytes can then be detached by the methods illustrated below (Section 4).

3.2 Adherence to gelatin-coated surfaces This technique was originally developed by Bevilacqua and co-workers (47) and subsequently modified by Freundlich and Avdalovic (48). These authors reported that peripheral blood monocytes/macrophages express high affinity receptors for FN, which can bind to plasma FN immobilized on gelatin-coated surfaces. The human plasma was used as source of FN, thus providing a simple device to allow reversible adherence of monocytes/macrophages via a magnesium-dependent 36


mechanism. However, the purity of monocyte/macrophage preparations obtained by these techniques was hampered by a consistent contamination with platelets. Based on the finding that macrophages also secrete cell membrane-associated FN (49), a simple method of macrophage purification taking advantage of the capacity of I:N to bind to various types of collagen, as well as to denatured collagen (gelatin), was developed (20). Under these conditions, contamination with platelets is not generally observed. Occasionally, platelets can be seen around monocytes (platelet satellitism), but their number can be easily reduced by rinsing flasks with ethylenediamine tetraacetate (EDTA) at room temperature. A procedure for the purification of macrophages by adherence to gelatin-coated surfaces is given in Protocol 2.

Purification of macrophages by adherence to gelatincoated surfaces Equipment and reagents • Various tissue culture vessels including 25 and 75 cm2 flasks, 10 cm diameter Petri dishes, 24-well cluster plates

• RPMI1640 medium alone and containing 15% heat-inactivated FBS • 2% gelatin in double distilled H20

A Preparation of gelatin-coated plates 1 Add 2 g of type 1 gelatin to 100 ml of double distilled water and autoclave at 110°C for 40 min. 2 Bring the 2% gelatin solution to 50°C. 3 Pipette 0.5 ml into 24-well tissue culture plates, 2 ml into 35 mm diameter plastic dishes, 10 ml to 75 cm2 tissue culture flasks, or 14 ml into 10 cm diameter Petri dishes. 4 Hold the gelatin-coated dishes at 50 "C for 2 h. 5 Remove the excess gelatin by suction and incubate dishes at 37 °C, in a dry incubator, for 48 h before use. These pre-coated flasks can be stored under sterile conditions at room temperature for up to four weeks. B Purification of macrophages 1 Rinse the gelatin-coated culture flasks twice with culture medium, 2 Add an appropriate volume of mononuclear cell suspension (see Protocol 1, footnote a) to each gelatin-coated vessel. 3 Incubate for 1 h at 37 °C, 5% C02, 4 Remove the non-adherent cells by suction and gently wash flasks several times with culture medium pre-warmed at 37°C.

37

SANDRA GESSANI ET AL.

3.3 Adherence to microexudate-coated surfaces Production of microexudate is a property shared by a variety of mammalian cells of different origin. Accordingly, a number of human and murine cell lines can yield conditioned plastic surfaces suitable for purification of monocytes/ macrophagcs (19). The mechanism and specificity of attachment of these cells to microexudates is not completely understood. It is known that the composition of the microexudate is complex and contains FN, vitronectin, and possibly collagen amongst other molecules (50), Monocyte/macrophage adherence to these exudates is likely mediated by receptors for proteins of the integrin family. The presence of FN receptors may be important since neutrophils and lymphocytes bind poorly or not at all to FN-coated surfaces. Although serum proteins adsorb to plastic surfaces and have been considered to be a component of the microexudate, the different behaviour of monocytes/macrophages on coated versus uncoated surfaces in the presence of high serum concentrations indicates that the effect of the microexudate is not due simply to rapid adsorption of a serum component (19). Baby hamster kidney (BHK), mKSA-TU5, a Balb/c mouse kidney cell line, rat fibroblasts (R22CIF), and Chang liver cells arc generally used as conditioning cell lines. Protocol 3 gives a procedure for the separation of macro phages on microexudate-coated surfaces.

Purification of macro phages by adherence to microexudate-coated surfaces Equipment and reagents • Various tissue culture vessels including 25 and 75 cm3 flasks, 10 cm diameter Petri dishes, 24-well cluster plates • Microexudate-coated vessels • RPMI 1640 medium alone and containing 10% or 20% heat-inactivated fetal bovine serum (FBS) • Dissociation medium: 1 mM EDTA (BioWhittaker) in PBS (see Protocol 1)

Hank's balanced salt solution (HBSS): 5.4 mM KC1, 0.3 mM Na2HPO4, 0.4 mM KH2PO4,4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.6 mM MgSO4,137 mM NaCl, 5.6 mM D-ghicose, 0,02% phenol red (optional), adjust to pH 7.4 (HBSS can be purchased from BioWhittaker)

A Preparation of microexudate-coated plates 1

Grow cells (BHK, mKSA-TU5, R22CIF, or Chang liver cells) to confluence in standard 75 cm2 tissue culture flasks or in 10 cm Petri dishes in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, using standard tissue culture practices.

2

Decant the culture medium by overturning or by sucking using a pipette.

•*8


3 4 5

Incubate cell monolayer with 1 mM EDTA in PBS (see Protocol 1) for 10-20 min at 37 °C. Rinse the microexudate-coated culture vessels vigorously by four to six washings with 10 ml of HBSS and once with complete culture medium. Carefully assess, by microscopic inspection under an inverted microscope, that no conditioning cells are left on plastic. Conditioned plastic dishes can be used immediately or stored at 4°C for up to two months without loss of activity.

B Purification of macrophages 1

2 3

Seed 1-1.5 x 108 washed mononuclear cells obtained from Ficoll-Hypaque centrifugation (see Protocol 8) into 20-30 ml of RPMI1640 medium supplemented with 20% FBS. Incubate the flasks at 37 °C for 1 h, to allow the attachment of monocytes. Remove all non-adherent and loosely adherent cells by vigorous shaking the flasks and aspirate released cells.

4

Wash cell monolayer four to five times with medium without serum,

5

Check, under an inverted microscope, that non-adherent cells have been washed off and that most of the adherent cells are spread out Plastic surfaces may also be pre-treated with FBS by overnight incubation at 4°C with 10 ml of undiluted FBS, as originally described by Kumagai and colleagues (51).

3.4 Adherence to collagen matrices Collagen matrices have been used for the cultivation of many cell types and, in general, promote adhesion, cell growth, and/or differentiation (52, 53). Type I collagen has most often been used because it is the most abundant. Protocol 4 outlines a procedure for the separation of macrophages by adherence to collagen matrices.

Purification of macrophages by adherence to collagencoated surfaces Equipment and reagents • 24-well cluster plates • Collagen-coated plates • RPMI1640 medium alone and containing 15X heat-inactivated FBS

• 0,1 M acetic acid l%(w/v) paraformaldehyde in PBS (see Protocol 3)

Method 1 Resuspend macrophages at 106/ml, 2 Aliquot 3 ml into replicate polypropylene culture rubes and keep on ice until use. 3 Add bacteria/protozoa at varying multiplicities of infection (MOI) in 500 ul complete tissue culture medium. 4 Pellet microbes and macrophages together by centrifugation at 2000-3500 r.p.m. at 4°C.a 5 Warm tubes to 37 °C by placing in a water-bath.

106

ANALYSIS OF ANTIGEN PROCESSING AND PRESENTATION

6 Incubate for varying periods of time (typically from 15 min to up to 24 h). 7 Remove a tube at each time point and chill on ice. 8 Remove the majority of non-ingested micro-organisms by resuspending pellet and differential centrifugation at 1000 r.p.m. for 10 min at 4°C.b Discard supernatants, to appropriate disinfectant if pathogens are involved. 9 Fix macrophages with 1% paraformaldehyde as described in Protocol 3, using 500 ul of fixative, 10 Use fixed macrophages as APC source in desired functional assay. a

The required centrifugation will be dependent upon the micro-organism in question rather than the macrophage population. This centrifugation step allows rapid intimate contact between macrophage and microbe, and helps to ensure a uniform rate of uptake. b If organisms cannot be removed efficiently by two to three rounds of centrifugation then proceed directly to step 9. However, excess free organisms may influence later T cell function or be subsequently processed by contaminant APCs in the T cell preparation. 3.2.2 Assessment of infection level/antigen dose Given the above discussion, it Is particularly important to ascertain the actual presentable antigen dose within macrophages exposed to infectious agents. The methodology for accomplishing this will vary depending on the organism, and may involve: (a) Direct staining of macrophage monolayers or cytospins. (b) Lysis of macrophages, followed by re-culture and counting of released organisms (by limiting dilution, [3H]TdR uptake, or other biochemical means). (c) The use of flow cytometry.

In the latter instance, we have found that use of the CFSE and CMFDA Cell Tracker dyes (Molecular Probes Inc.) provide a convenient means of pre-labelling protozoan and bacterial pathogens. However, it should be noted that as these are vital stains and require metabolic activity to yield a fluorescent product, they do not indicate the degree of contamination of the microbe population with dead organisms (see Section 3.23). We have therefore found that flow cytometric methods are limited to assessing the extent that infection alters phenotype, rather than as a definitive means of evaluating antigen uptake. Protocol 8 outlines a rapid procedure for histochemical assessment of microbe uptake by macrophages. When adherent monolayers are used, the importance of substrate in determining adhesive properties can not be underestimated (see also Chapter 2). Although it may be tempting to adhere macrophages to glass coverslips to enumerate pathogen uptake, this may not reflect directly the status of a macrophage monolayer which has adhered to tissue culture plastic and then been washed several times before addition of T cells. 107

P. M. KAYE

Enumeration of microbe uptake by macrophages using direct staining Equipment and reagents • Pre-washed microscope slides (BDH) • Infected macrophages, in suspension or as adherent monolayers in 96-well plates (see Protocols 3 and 7)

Cytospin centrifuge, including holders, filter paper (Shandon) Reagents for appropriate staining of micro-organism

Method 1

Prepare slides of macrophages infected in suspension culture, by making cytospin sample (600 r.p.m. for 10 min).

2

Stain slides with an appropriate method to visualize the organisms in question.

3

For adherent monolayers, prepare and stain replicate wells as will be used to evaluate T cell responses.

4

Determine both the percentage of infected macrophages and the number of organisms per 100 macrophages by microscopic examination. If the organisms are over-dispersed (as is common at low MOI) then a distribution should be established by scoring, for example, macrophages with zero, 1-3, 4-5, 7-10, > 10 organisms. In the case of pathogens which have been ttansfected to express reporter antigens it may be possible to develop direct methods for establishing antigen dosage in infected macrophages. For example, for our studies with OVA-transfected Leishmania, a capture ELISA was used to determine the amounts of OVA incorporated into macrophages following infection versus pulsing with soluble ovalbumin (17). By also determining the quantity of OVA per parasite and the numbers of parasites per macrophage, it is also possible to indirectly estimate the total level of antigen uptake. 3.2.3 The live versus dead enigma An issue which continues to preoccupy and frustrate researchers is whether the presentation of antigens can occur from the various vacuolar compartments inhabited by different pathogens. Viable pathogens are known to make various modifications to the host vacuolar compartment, e.g. mycobacteria may exclude the proton ATPase, and hence modify vacuolar pH, and Leishmania may sequester MHC class II antigens (10). Often, these modifications occur only in the vacuoles surrounding live organisms, not those in which either dead organisms have been engulfed, or in which the organism has subsequently died. The issue then arises as to whether presentation observed from an infected macrophage population truly represents antigens derived from living, as opposed to dead organisms. A compelling discussion on this issue can be found in Wolfram et al. (4). In their

108


studies, optimal presentation of an abundant non-secreted leishmanial protease was achieved by killing the parasite within the vacuolar compartment using exposure to a leishmanicidal drug. These data. as with others previously published (17-19), suggested that to obtain access to antigen sequestered inside parasites, macrophages had to first destroy the parasite's membrane integrity. However, significant presentation was observed even with untreated macrophages infected with 'live' organisms. The leishmanial protease in question is abundant, and Wolfram et al. calculated that death of a single intracellular organism would produce an intravacuolar antigen concentration of approx. 20 ug/ml! 3.2.4 Assessing antigen release and re-presentation A major consideration with intracellular pathogens is the issue of antigen regurgitation, from either living or dead pathogens, and the possibility of representation by other, possibly non-infected cells in the culture. Two approaches are available to assess this functionally. First, stipernatants from infected macrophages can be transferred to other fresh populations, i.e. used as 'soluble' antigen to pulse these secondary cultures (as described in Protocol 3). Secondly, the principles of MHC restriction can be utilized, whereby infected macrophages mismatched at the MHC are added to cultures containing uninfected macrophages syngeneic to the responder T cells. This latter approach has the advantage that because intimate contact is made between macrophages in the co-culture, transfer of small quantities of antigen may be detectable. In addition, it has recently been appreciated that one potent means of antigen uptake is by the phagocytosis of apoptotic macrophages and acquisition of any antigens that they may contain. At least in the case of dendritic cells, this would appear an efficient way for cross-priming T cell responses (20), Protocol 9 describes one means of evaluating the level of antigen transfer between APC populations.

Evaluating antigen transfer between APC populations Equipment and reagents • Flat-bottom 96-weD tissue culture plates (Nunc) • BMM0 (Protocol 2) or macrophages from other tissue sources (see Chapter 1) from two MHC disparate inbred mouse strains

• Complete tissue culture medium (see Protocol 3) • Antigen, e.g. ovalbumin, to which antigenspecific T cell clone or hybridoma is available • Antigen-specific T cell clone or hybridoma

Method 1 2

Purify an optimal number of macrophages, derived from a mouse strain syngeneic to the responder T cell clone/hybridoma. Plate an optimal number of these macrophages in 100 ul of complete tissue culture medium per microtitre well.

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P. M. KAYE

3

Add optimal numbers of T cell (as predetermined) to these cultures in 50 ul of complete tissue culture medium per well. Add an additional 50 ul of tissue culture medium per well containing various numbers of antigen pulsed allogeneic macrophages, with or without fixation (prepared as in Protocol 3 or 7). Allogeneic macrophages with no added antigen should also be included as controls. Incubate for the required period of time to determine T cell activation.

4

5 a

The 'optimal' number of cells should be determined using specific APC assays, as described in Protocol 3. A similar approach can be used to separate events resulting from apparent T cell recognition (as a result of processing) from 'bystander' events, e.g. mediated by cytokines. An example would be specific killing of macrophages exposed to T cells (21).

3.3 Cell biology of antigen processing As discussed above, T cell recognition currently provides the dominant method for determining effective antigen processing. However, valuable information may also be obtained, if not over-interpreted, from the analysis of surrogate markers for processing activity. Specifically, these include assessment of the nature of the vacuolar compartment into which organisms are taken; its maturation or otherwise resulting from fusion with other vacuolar compartments; the relative subcellular distribution of MHC class I and II molecules as well as their chaperones or accessory proteins; the intraphagosomal pH; the enzymatic repertoire of the compartment. Techniques for determining the above are included elsewhere in this volume (see Chapter 3). The most significant advance in the study of the cell biology of antigen processing in recent years is the generation of mAbs which recognize MHCpeptide complexes with a specificity approaching that of the TCR (22, 23). Hence, for the first time it is possible to directly visualize, using immunofluorescence or immunogold, the subcellular site where MHC-peptide complexing takes place. To date, such antibodies are available for cytochrome B and hen egg lysozyme peptides. The relative ease with which these antigens can be transfected into a variety of pathogens promises to yield exciting data in the near future.

4 Analysing antigen presenting function of macrophages 4.1 Correlative studies of cell phenotype Numerous cell surface molecules have been defined in addition to class I and II molecules, as regulators of T cell antigen recognition and activation. Arguably of 1 10


most importance are the co-stimulatory ligands B7-1 and B7-2, whose receptors CD28/CTLA4 may opposingly regulate T tell activation (3). In addition, new molecules of interest continue to be identified, e.g. 4-1BB, TOLL The approach to measure expression of these cell surface antigens is conventional immunofluoresccnce and flow cytometry, confocal microscopy, or immunohistochemistry (see Chapter 3). It is worth noting that, in our hands, directly labelled mAb are preferable to indirect staining methods, particularly where infected pathogens may differentially regulate FcR levels.

4.2 Functional assays of co-stimulation The B7-1/B7-2 mediated co-stimulatory capacity of macrophages can be measured directly. In these assays, T cells are allowed to interact with anti-CD3 mAbs. which alone induce minimal levels of proliferation or cytokme production. The provision of CD28 ligands on the surface of APC or by anti-CD28 mAb induces vigorous responses, A protocol for such an assay is provided in Protocol 10.

Assay of co-stimulatory function of macrophages Equipment and reagents • Flat-bottom 96-well plates (Nunc)

PBS (see Protocol 3)

• BMM0 (see Protocols 1 and 2)

Complete tissue culture medium {see Protocol 3) Naive T cells, harvested from lymph node or spleen of mice syngeneic to the donor of the BMM0

• Specific antibodies to CD3 and CD28: mAbs 145-2C11 and 37.51 (PharMingen) • Carbonate-bicarbonate buffer pH 9.6:

0.015 M NaC03, 0.035 M NaHCO3

Method 1

Coat 96-well flat-bottom plates with 50 ul of 10 ug/ml anti-CD3 mAb in pH 9.6 carbonate-bicarbonate buffer overnight at 4°C. Have appropriate wells with buffer alone or with 20 ug/ml anti-CD28 mAb in pH 9.6 buffer (see Table 1).

2

Wash wells once with PBS.

3

Block for 1 h with 200 ul complete tissue culture medium.

4

Wash twice with complete medium,

5

Add 5 x 104 to 105 naive CD4+ T cells per well in 100 ul complete tissue culture medium.a

6

To assess the involvement of known co-stimulatory receptor-ligand pairs, add specific mAbs or chimeric fusion proteins (e.g. CTLA4-Ig) to specific wells, at doses up to 20-40 ug/ml (maximal inhibition) in 50 ul complete tissue culture medium. Leave these in for the duration of the assay.

Ill

P. M. KAYE

7

Add macrophages in graded numbers to T cell cultures in 50 u1 total volume of complete tissue culture medium per well.

8

Assess proliferation of T cells in all cultures at day two or three by conventional thymidine incorporation (see Protocol 3}.

a

Isolated from spleen or liver by conventional methods (nylon wool columns and anti-CD8 plus complement-mediated depletion, or Magnetic selection). Table 1 Suggested protocol for assessing co-stimulatory properties of macrophages Wells coated with None uCD3a T cells 1

1

2

3

4

T cells + M02

5

6

T cells + M03

7

8

T cells + M04

9

10 1'

T cells +

1

b

M01

1

I I T cells + M0n T cells + anti-CD28J

1 1

1

17

18

19

20

a

Coat with 145-2C11 at 10 ug/ml. Compare either variable numbers of macroptiages of control versus infected macrophages as required. b

e

Assay replicates of three or four wells per group. In triplicate, up to eight macrophage related variables can be assayed on one plate. d Plate bound, added to wells at same time as anti-CDS mAb.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 112

Unanue, E. R. (1992). Curr. Opin, Immunol., 4, G3. Chain, B. M.. Kaye, P. M., and Shaw, M. A. (1988). Immmol. Rev., 106, 33, Jenkins, M. K. and Johnson, J. G. (1993). Curr. Opin. Jmrmmol., 5, 361, Wolfram. M., f u c h s , M., Wiese, M., Stierhof. Y. D,, and Overath, P. (1996). Eur.J. Immunol., 26, 3153. Schneider, S. C. and Sercarz, E. E. (1997). Hum. Immurol, 54, 148. Burgert, H. G., White, J.. Weltzien, H. U., Marrack, H., and Kapplcr.J.W. (1989). J. Exp. Med.. 170, 1887. Shastri, N. (1995). Curr. Opin. Immunol, 7, 258. Steinman, R. M., Pack, M,, and Inaba, K. (1997). Immunol. Rev., 156, 25. Palucrka. K. A., Taequet, N., Saneherc-Chapuis, F,. and Gluckman, J. C. (1998). j.Immunol.,16O, 4587. Lang, T., Hellio, R,, Kaye, P. M., and Antoint.J. C, [ 1994). J. Cell So.. 107, 2137. Liew, F. Y, (1995). Curr. Opin. Immunol., 7, 3%,

ANALYSIS OF ANTIGEN PROCESSING AND PRESENTATION 12. Chen, H. and Rhodes, J. (1996). J. Mol Med., 74, 497. 13. Nakagawa, T., Roth, W., Wong, P., el al. (1998). Science, 280, 450. 14. Carbone, F. R., Hosken, N. A., Moore, M. W., and Bevan, M. J. (1989). Cold Spring Harb. Symp. Quant. Biol., 54, 551. 15. Reise Sousa, C. and Germain, R. N. (1995). J. Exp. Med., 182, 841. 16. Oh, Y. K., Harding, C. V., and Swanson, J. A. (1997). Vaccine, 15, 511. 17. Garcia, M. R., Graham, S., Harris, R. A., Beverley, S. M., and Kaye, P. M. (1997). Eur. J. Immunol, 27, 1005. 18. Lang, T. and Kaye, P. M. (1991). Eur.J. Immunol, 21, 2407. 19. Kaye, P. M., Coburn, C., McCrossan, M., and Beverley, S. M. (1993). Eur.J. Immunol, 23, 2311. 20. Rubartelli, A., Poggi, A., and Zocchi. M. R. (1997). Eur.J. Immuno!., 27, 1893. 21. Smith, L E., Rodrigues, M., and Russell, D. G. (1991). J. Exp. Med., 174, 499. 22. Dadaglio, G., Nelson, C. A., Deck, M. B., Petzold, S. J., and Unanue, E. R. (1997). Immunity, 6, 727. 23. Zhong, G., Reise Sousa, C., and Germain, R. N. (1997). Proc. Not!. Acad. Sci. USA, 94, 13856.

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Chapter 5 Macrophage secretory products Paola Allavena, Giancarlo Bianchi, Walter Luini, Andrea Doni, Pietro Transidico, and Silvano Sozzani Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.

Alberto Mantovani Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy; also at Department of Biotechnology, Section of General Pathology, University of Brescia, Italy.

1 Introduction Mononuclear phagocytes play a fundamental role in tissue remodelling as a first line of resistance against pathogens and in the activation of specific immunity. These functions of cells of the monocyte/macrophage lineages are mediated to a large extent by the production of secretory molecules. The secretory capacity of mononuclear phagocytes is enormous and the repertoire of secreted molecules is vast and diverse, ranging from lipid mediators, to enzymes, cytokines, and their receptors (1). A simplified classification and view of macrophage secreted molecules is provided in Table 1. In a schematic way, two general modes of secretion can be defined. Certain molecules are produced in a tonic, constitutive way, whereas for most secretion is strictly regulated by activation signals. The latter include engagement of adhesion/ recognition molecules, bacterial products (e.g. bacterial lipopolysaccharide), and cytokines (e.g. interferon -y). Within the same class of products, both modes of production can be encountered. For instance, macrophages are a major source of the chemokine MCP-1, when exposed to activation signals (2). At the same time macrophages (but not monocytes) constitutively express the CC chemokine macrophage-derived chemokine (MDC) (3). An extensive coverage of the methodology used for measuring macrophage secretory products is virtually impossible and beyond the scope of this chapter. Here we will focus on selected aspects, familiar to us, and which lend themselves to general considerations. In particular, among cytokines, we will focus on chemokines. As for many macrophage products, antibody-based assays are commercially available. However, the bioassay is of fundamental importance in the evaluation of the functional relevance of immunoreactive material and to assess 115

PAOLA ALLAVENA ET AL. Table 1 Selected secretory products of mononuclear phagocytes Functional group

Selected molecules

Short lived toxic molecules

Reactive oxygen intermediates Reactive nitrogen intermediates

Cyclo-oxygenase/lipo-oxygenase products

Prostaglandins (e.g. PGE2)

Enzymes and coagulation factors

Tissue factor; factor IX, X, V, prothrombin

Complement components

Classical pathway (Cl, C2, C3, C4, C5) Alternative (factor B, D, properdin)

Growth factors

Platelet-derived growth factors (PDGF) Fibroblast growth factors

Leukotrienes (e.g. LTB4) Urokinase-type plasminogen activator; inhibitors

Haemopoietic growth factors

Macrophage colony-stimulating factor

Primary proinflammatory cytokines

IL-1; TNF; IL-6; IL-12; IL-18

Secondary proinflammatory cytokines

Chemokines (e.g. monocyte chemotactic protein 1-4)

Anti-inflammatory cytokines

IL-10; transforming growth factor p

Cytokine receptors

IL-1 type II receptor; TNF receptors

the function of molecules for which an ELISA is not available. Moreover, we will discuss methodology to measure released soluble cytokine receptors.

2 Cytokines and chemokines: chemotaxis Most cytokines have been identified thanks to appropriate bioassays, which have also provided tools to measure these mediators and to standardize them (3). Even when immunometric methods are available, bioassays are invaluable tools to assess the functional relevance of immunoreactive material and to quantitate undefined mediators. Here we will focus on chemotaxis, the eponimous function of the chemokine superfamily (4, 5). N-terminal processing of chemokines results in products with reduced activity or with a different spectrum of action, thus emphasizing the importance of assaying function (4-6). Chemotaxis is defined as the directional locomotion of cells sensing a gradient of the stimulus. Chemotaxis has been extensively studied with leukocytes that are 'professional migrants', but a variety of cell types including fibroblasts, melanoma cells, keratinocytes, and vascular endothelial cells exhibit directional locomotion in vitro. Two main techniques have been used to measure migration in vitro: migration under agarose and chemotaxis across porous membranes. While the former approach may more closely resemble the in vivo conditions, the latter is easier to quantitate and allows analysis of directional versus random locomotion. We will therefore focus on the description of migration through a porous membrane. Both a classic modified Boyden chamber assay (7) and a micromethod (8, 9) will be described. Protocol 1 describes the use of a micromethod for assessing leukocyte chemotaxis. A schematic representation of the micro chemotactic chamber is shown in Figure 1. 116

MACROPHAGE SECRETORY PRODUCTS

A

LOWER COMPARTMENT OF THE CHAMBER

B

POROUS FILTER

C

SILICON TRIMMING

D

UPPER COMPARTMENT OF THE CHAMBER

Figure 1 Schematic representation of the 48-well micro Boyden chemotaxis chamber.

Assessment of macrophage migration using a Boyden chamber Equipment and reagents • 48-well micro Boyden chamber, including filters (polycarbonate 25 x 85 mm, 5 um pores), clamps, and special rubber policeman (Neuroprobe) • Humidified 5% CO2 incubator • Glass slides • Peripheral mononuclear cells (PBMC; see Chapters 1 and 2)

RPMI1640 medium (Biochrom KG) containing 0.2% (v/v) BSA (Sigma) Standard chemoattractants (e.g. fMLP and C5a; Sigma) Diff-Quik stain (Harleco) PBS (Biochrom KG)

117

PAOLA ALLAVENA ET AL.

Method 1 Aliquot 25 ul of an appropriate chemoattractant into the wells of the lower chamber (Figure 1A). The 25 ul volume may have some variations (2-3 ul more or less), depending on the microchamber used. It is advisable to calibrate in advance the lower wells, so that having seeded the chemoattractant, the liquid in the lower wells forms a small convex surface that guarantees a perfect adhesion of the filter avoiding air bubble formation. 2 Put the filter (Figure 1B) onto the lower chamber leaving the opaque surface on top. To avoid confusion concerning the order of the experimental groups in the same filter, it is suggested that a small angle of the filter be cut (e.g. at the upper right edge). 3 Mount the silicon trimming (Figure 1C) and then the upper chamber (Figure 1D). Press and screw tightly the upper chamber to avoid air bubbles. 4 Seed 50 ul cell suspension (1,5 x 106/ml) in the upper wells by leaning the pipette tip on the border of the well and quickly ejecting the cell suspension. 5 Incubate the chamber at 37 °C in 5% CO2 for 1.5 h. 6 Remove chamber from the incubator. 7 Unscrew and turn upside down the chamber. 8 Hold the upper chamber (Figure ID) tightly, and remove the lower chamber (Figure 1A), keeping in place the silicon trimming and the filter. At this point the migrated cells are on the upper surface (bright) of the filter. 9 Remove the silicon trimming. 10 Lift the filter by placing a clamp on each end. 11 Wash the opaque surface of the filter, where the non-migrated cells remain, by gently washing this side with PBS, Do not entirely dip the filter in PBS otherwise the migrated cells will be lost. 12 Remove all non-migrated cells by scraping the opaque surface of the filter against the special rubber policeman. 13 Stain the filter with Diff-Quik according to manufacturer's instruction, 14 Place the filter on glass slides and count the migrated cells present on the bright surface of the filter. Count five to ten microscopic fields at x 1000 final magnification.

3 Leukocyte transmigration The emigration of leukocytes from blood to tissues is essential to mediate immune surveillance and to mount inflammatory responses. The interaction of leukocytes with endothelial cells (EC) can be divided into four sequential steps: tethering, triggering, strong adhesion, and migration. The selectin family of adhesion molecules mediates tethering; strong adhesion is mediated by the 118

MACROPHAGE SECRETORY PRODUCTS

intcgrin family, which need to be activated (triggering), and finally migration is induced by local promigratory factors including some cytokines and chemokines (10, 11). We have studied the adhesive properties and transendothelial migration of leukocytes, but this method may also apply for investigation of other cell types, for instance tumour cells. Protocol 2 describes a radioisotopic assay for monitoring transendothelial migration, based on an assay described in ref. 12,

Assessment of transendothelial migration by radioisotopic detection Equipment and reagents • Single well Boyden chambers (Neuroprobe) • Nitrocellulose filter (12 mm diameter. 5 um pore, Sartorius) • pvP-free polycarbonate filters (12 mm diameter, 5 um pore, Sartorius) • 24-well plates (Falcon, Becton Dickinson) • 3 ml centrifuge vials (Falcon, Becton Dickinson} • Cotton floes (Johnson and Johnson) • Humidified 5%CO2incubator • Gamma counter windowed for 51Cr

• Endothelial cells (EC) were obtained and cultured as described (13} • Tissue culture medium (complete medium}: M199 (BioLife) supplemented with 20% FBS (Hyclone), 50 ug/ml endothelial cell growth supplement (ECGS, Collaborative Research), 100 ug/ml heparin (Sigma) • Peripheral mononuclear cells (PBMC; see Chapters 1 and 2) • 5'Cr (Amersham) 37 MBq, 1 uCi • PBS (Biochrom KG)

Method 1 Coat PVP-free polycarbonate filters with 1 ml of 10 ug/ml fibronectin in PBS (at room temperature for 2 h) in 24-well plates, 2 Aspirate fibronectin and add 105 endothelial cells (EC) in 2 ml of M199 complete medium and grow to confluence (five to six days). 3 Place 0.2 ml of complete medium in the lower compartment of each Boyden chamber. 4 Mount the first uncoated filter and on top the second filter coated with EC. 5 Immediately add 0.15 ml of complete medium. Drying should be avoided. 6 Assemble and screw the upper compartment of the chamber. 7 Label PBMC (100 uCi 51Cr at 370C for 1 h) and seed cells (3-6 x 105 in 0.15 ml of complete medium) into the upper compartment of the chamber. 8 Incubate the chambers at 37°C for 60 min. 9 Remove the chambers from the incubator. 10 Collect the medium containing non-adherent cells in a 3 ml vial (fraction A).

119

PAOLA ALLAVENA ET AL,

11 Gently wash the EC monolayers with 0.5 ml warm medium and collect it (fraction B). 12 Scrape (gently) the EC mono layer and adherent leukocytes with cotton fiocs and transfer to vials (fraction C). 13 Transfer the double filter system to vials together with the medium of the lower compartment (fraction D). 14 Measure radioactivity in each fraction. Fractions A and B represent non-adherent cells. Fraction D represent migrated cells. As migrated cells had first adhered to EC, total number of adherent cells is calculated by summing fractions C and D. The spontaneous adhesion of resting leukocytes to unstimulated RC varies for different cell subsets. For instance, the adhesion of NK cells is usually 5-15%, a value intermediate between that of tiionocytcs (20-40%) and the very low value of T cells and PMN{ 24 hours) cytotoxicity assays, but the mechanism involved has not been completely defined yet. At least one target, the murine mastocytoma cell line P815, has been shown to be susceptible to lysis by NO.

4 Microbicidal activity The microbicidal activity of macrophages is a major line of defence of higher organisms that can not be fully replaced by the sophistication of the specific aim of the immune response. Phagocytic and microbicidal activities have been reproduced in vitro using different micro-organisms as targets, including Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonie, Clamidia psittaci, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium 142

ANALYSIS OF MACROPHAGE LYTIC FUNCTIONS

lepraemurium, Candida albicans, Cryptococcus neoformans, Leishmania donovani, and Toxoplasma gondii (30-34). The methods used to determine monocyte/macrophage intracellular killing rely on: (a) Direct evaluation of the intracellular killing of micro-organisms by specific staining of the infected macrophages to assess the number of intracellular micro-organism (Candida) and/or the number of micro-organisms per vacuole (Leishmania, Toxoplasma). (b) Use of radioactive tracers such as tritiated glucose, leucine, or uridine. These methods involve the measurement of isotope incorporation into the macromolecules of the obligate intracellular parasites. Differential uptake of isotopes in infected and uninfected cultures is a parameter for measuring the ability of macrophages or monocytes to inhibit or kill the parasites. (c) Counting the surviving micro-organisms as colony-forming units on solid media (Candida and bacteria). The phagocytes are challenged with the microorganisms at different effector-to-target cell ratios for various length of time. At the end of the challenge, the killing is stopped by lysing the phagocyte and the surviving micro-organisms are plated and counted. Direct evaluation has the advantage of low cost, simplicity, and rapidity. However, these methods are somewhat subjective and the observer's bias is a potential problem. The radioactive methods are simple, rapid, and objective but they are more expensive and have lower sensitivity. The method of the plate counting is reproducible and inexpensive, but the results are lengthy especially with slow growing micro-organisms like Mycobacteria. Macrophages can kill microbes via both oxygen-dependent and -independent mechanisms. Oxygen-dependent mechanisms include the production of reactive oxygen intermediates (ROI), hydrogen peroxide, and the generation of reactive nitrogen intermediates (RNI), whereas non-oxidative killing involves phagosome acidification plus phagosome-lysosome fusion and the release of antimicrobial peptides and enzymes (e.g. defensins). The expression of most of these effector mechanisms is not constitutive but can be induced by appropriate stimulation with activating signals such as interferon -y. IFNy-induced release of nitric oxide (NO) is a major effector mechanism in the murine system. NO participates in the killing of Cryptococcus neoformans, Leishmania major, Schistosoma mansonii, Toxoplasma gondii, Mycobacterium leprae, M. tuberculosis, and Candida (35). In the human system the situation is more complex. There is some evidence for NO production by human monocytes (36, 37), but the role of NO in microbicidal activity is still controversial. The relative importance of oxygen-dependent or -independent pathways in the microbicidal activity of mononuclear phagocytes varies depending on the assay system and the target micro-organism. The anti-Candida activity of mononuclear phagocytes depends on the production of superoxide anion, one reactive oxygen intermediate that is essential for the oxidative killing of macrophages. 143

MARIA CARLA BOSCO ET AL.

The importance of superoxide anion in killing C. albicans is suggested by the observation of increased susceptibility to fungal infections in chronic granulomatous disease (CGD), a genetically inherited disease characterized by lack of superoxide anion production by phagocytic cells due to mutation of genes encoding for the subunits of NADPH oxidase (38). Formal proof of C. albicans' susceptibility to superoxide anion, reactive nitrogen intermediates, and to the myeloperoxidase-hydrogen peroxide-halide system has been provided. Furthermore, there is evidence that oxygen-independent mechanisms are operative in the killing of C. albicans (39). Similarly, studies on the mononuclear phagocyte's oxygen-dependent and -independent antimicrobial systems revealed that activity against the intracellular protozoa Leishmania donovani and Toxoplasma gondii is principally oxygen-dependent, although oxygen-independent mechanisms can be involved. In contrast, it has been shown that non-stimulated human monocytederived and tissue alveolar macrophages support the growth of Chlamydia psittaci in vitro, but that lymphokine-stimulated macrophages restrict Chlamydia replication utilizing primarily an oxygen-independent antimicrobial mechanism (40, 41). Both oxygen-dependent and -independent mechanisms are involved in the killing of extracellular targets such as the hypha form of Candida and Schistosoma SPP (30, 39). Normal human monocytes rely on oxygen-dependent mechanisms to kill Candida hyphae, as their activity is inhibited by scavengers of hydrogen peroxide and inhibitors of the MPO-hydrogen peroxide-halide system. Studies on monocytes from granulomatous disease patients suggest that oxygen-independent mechanisms should also exist. The mechanisms involved in Schistosomal killing are still unclear. It has been reported that human monocytes can kill the schistosomula of Schistosoma mansoni in standard in vitro cytotoxicity assays in a cell-to-cell contact-dependent manner with the possible contribution of oxygen-dependent mechanisms (42). We present here the classic anti-Ieishmania and the anti-Candida assays. The latter is particularly interesting because it offers the possibility to study the cytotoxic activity against both the intracellular yeast as well as the extracellular fungal hyphae. 4.1 Anti-Le/shman/a activity of monocytes/macrophages Intracellular survival and proliferation are primary mechanisms adopted by many infectious agents for evading the immune response of their vertebrate host. Protozoa of the genus Leishmania reside and multiply in the macrophage phagolysosomes. Leishmaniae exhibit a heteroxenous life cycle that includes two developmental stages: an extracellular, flagellated leptomonad form (promastigote) and a sessile form (amastigote) which is an obligate intracellular parasite. Promastigotes are taken up by macrophages and rapidly convert to the resistant amastigote stage. The parasite changes metabolism from aerobic to anaerobic and loses its extracellular replicative capacity. This form resides and replicates within macrophage phagolysosomes and is susceptible to macrophage-mediated killing (31, 43). 144


Assessment of macrophage anti-Leishmania cytolytic activity Equipment and reagents • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other labware (see Protocol 1) • Centrifuge • Haemocytometer • Standard microscope • Complete media: RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (HyClone), 100 U/ml penicilb'n, 100 ug/ml streptomycin, 2 mM L-ghitamine, 20 mM Hepes (Gibco BRL), for macrophages; 199 medium supplemented with 20% heatinactivated FCS (HyClone). 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM L-ghitamine, 40 mM Hepes, 0.1 mM adenine (in 50 mM Hepes), hemin (5 fig/ml in 50% triethanolamine), and 6-biotin (1 ug/ml in 95% EtOH) (Sigma Chemical), for the maintenance of Leishmaniae • 70% ethanol

Lab Tek tissue culture slides (Flow Laboratories) Tissue homogenizer Steel mesh screens PBS (see Protocol 1) Giemsa Plus stain solution (Trend Scientific Inc.) Balb/c mice (Charles Rivers) Leishmania major parasites Lectin peanut agglutinin (PNA, isolated from Arachis hypogae) (Sigma) Murine peritoneal macrophages, bone marrow-derived macrophages, human macrophages (see Chapters 1 and 2 for monocyte/macrophage recovery and purification strategies), U937 and THP-1 human macrophage precursors cell lines2 Macrophage activators (see Protocol 1)

A Maintenance and preparation of parasites 1 Maintain Leishmaniae in Balb/c mice by injecting 50 of PBS containing 2 x 106 amastigotes into the rear mouse footpads. 2 Sacrifice the mice four to five weeks after infection. 3 Harvest Leismantoe by cutting the infected footpad just around the joint and rinsing for a few seconds with 70% ethanol, followed by PBS. 4 Place the footpad in a Petri dish and remove the necrotic tissue. 5 Disrupt the footpad tissue by scraping on stainless steel screens (50 mesh). 6 Wash the screens several times with 50 ml of 199 complete medium to collect the cell suspension. 7 Transfer the macrophage suspension in a tissue homogenizer to release parasites by disrupting the infected macrophages. 8 Transfer the parasite suspension to a tissue culture flask. 9 Incubate the flask tightly closed at 22 °C, in a dry incubator. These culture conditions allow the transformation of amastigotes into promastigotes, 10 After four to five days, start a new culture with 106 parasites/ml in 199 complete medium.b

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11 Culture for four to six days (depending on the strain) to reach a stationary phase of growth and separate the stationary phase promastigotes from the log phase promastigotes. 12 Resuspend promastigotes to 108 cells/ml in PBS after three washings in the same buffer. 13 Add an equal volume of PNA (100 ug/ml in PBS) to agglutinate log phase promastigotes. 14 Incubate for l h at room temperature. 15 Centrifuge the suspension at 80 g for 5 min and carefully collect the supernatant. 16 Spin the supernatant at 300 g for 10 min and collect the pellet. 17 Wash the pellet by centrifugation at 300 g for 10 min. 18 Resuspend the parasites at a final concentration of 1-2 x 106 parasites/mlc in RPMI complete medium.

B Preparation of effector cells 1

2 3 4 5 6 7

Plate macrophages in Lab Tek tissue culture slides at 1 x 105 cells/well in 0.5 ml complete RPMI. U937 and THP-1 are also differentiated in Lab Tek tissue culture slides at 5 x 104 cells/well in 0.5 ml RPMI complete medium containing PMA (10 ug/ml) or retinoic acid (RA) (10-6 M). Incubate at 37°C to allow the cells to adhere (3-4 h for macrophages, three days for differentiating U937 or THP-1). Remove non-adherent cells by three extensive washings with warm complete RPMI. Add the appropriate macrophage activators in 0.1 ml of medium to a final volume of 0.5 ml. Incubate the cells for 18-24 h at 37°C. Remove supernatant by aspiration. Wash the cells three times with warm complete RPMI.

C Assay 1

2 3 4 5 6

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Add 0.5 ml of parasite suspension (1-2 x 106 parasites/ml to have a 10:1 parasite-tohost ratio) to Lab Tek tissue culture slides containing washed monocyte/macrophage monolayers, Incubate for 4 h at 37°C. Remove non-internalized parasites by three washings with warm RPMI complete medium (eliminate recovered parasites using an appropriate biohazard disposal). Culture the infected cells in medium, replacing every 24 h. Fix and stain the slides with Giemsa Plus reagent at the desired times. Observe the slides at x 1000 with an immersion objective.


7

Count at least 200 macrophages chosen at random in non-contiguous fields. Determine microbicidal activity, defined as decrease in infected macrophages in treated cultures relative to control cultures by the following formula: {{% infected control macrophages - % infected treated macrophages) / (% infected control macrophages)} x 100, Results are expressed as per cent Leishmania-mfectzd macrophages ± SEM standard error of the mean of replicate samples,

a

The cell lines (U937, THP-1) need to be differentiated into non-dividing, plastic adherent monolayers utilizing PMA (10 ug/ml) (Sigma Chemical) or retinoic acid (RA 10-6 M) (BIOMOL Research Lab.), respectively, for three days (44). b A long-term maintenance m vitro causes a decrease in virulence. To maintain the virulence, a monthly passage in vivo is necessary. Promastigotes are very mobile and can be counted more easily in a haemocytometer after fixation with 2% formaldehyde. The infectivity of Leishmania promastigotes varies according to the growth phase: during the logarithmic growth phase the cells are actively dividing and less infectious than in the stationary growth phase. c The concentration should be adjusted to achieve the desired parasite-to-host ratio.

4.2 Anti-fungal activity Candida albicans is an opportunistic pathogen whose relevance has increased in recent years because of the augmented incidence of candidiasis in immunocompromised hosts. C. albicans can be considered a facultative intracellular pathogen because it survives within the macrophage and grows out of this cell by germination into a highly pathogenic form, C. albicans can undergo dimorphictransition in vitro and in vivo from the yeast (Y-Candida] to the hyphal (H-Ctmdida) form. The anti-Candida activity of macrophages is an interesting and important effector mechanism that involves intracellular and/or extracellular killing of parasites. Mononuclear phagocytes are unable to ingest the hyphae because of their large size and rely on extracellular killing mechanisms to eliminate the hyphal form of Candida from infected tissues. Activated macrophages, on the other hand, are endowed with the ability to kill the yeast form of C. albicans intracellularly. Protocols 7-9 detail methods for the detection of phagocytosis, intracellular and extracellular killing of Candida, respectively,

4.2.1 Phagocytosis of C. Albicans Phagocytosis is the first step in the intracellular killing of C. albicans blastoconidia by mononuclear phagocytes. Most of the studies on phagocytosis are performed using conventional dyes, such as trypan blue, methylene blue, and Gicmsa, to estimate the number of phagocvtosed C. albicans. Yeast labelling with flnorescein, also, is a convenient tool to measure phagocytosis (39, 45).

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Detection of phagocytosis of C. albicans by macrophages Equipment and reagents • Haemocytometer • Flow cytometer • Centrifuge • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes. and other labware (see Protocol 1) • PBS (see Protocol I) • Complete medium: RPMI 1640 (see Protocol 6)

• PBS/EDTA: PBS containing 10 mM EDTA • Fluorescein isothiocyanate (FITC) (Sigma Chemical) • Carbonate buffer: 0.25 M Na2C03 pH 10 • Sabouraud dextrose agar (Biolife) • C. albicans organisms • Monocyte/macrophage effector cells, appropriately purified (see Chapters 1 and 2)a

A Maintenance and preparation of C. albicans 1

Maintain C, albicans by weekly transfer on Sabouraud dextrose agar plates at 28°C.

2

Use a sterile loop to transfer a colony from a three-days-old culture to 5 ml PBS in 15 ml polypropylene tube.

3

Wash twice with PBS by centrifugation at 200 g for 10 min.

4

Resuspend yeasts in PBS and count in a haemocytorneter.

5

Label yeasts by resuspending 10 x 106 Candida in carbonate buffer containing 1 mg/mt FITC in 15 ml polypropylene tubes.

6

Incubate the yeasts at room temperature for 2 h.

7

Separate FITC-labelled Candida from free FITC by three washings with PBS (200 g, 10 min). Carefully resuspend labelled Candida in complete medium at 20 x 106/ml.

8

B Phagocytosis assay 1 Spin down 106 monocytes/macrophages effector cells in 4 ml round-bottom polypropylene tube. 2 Resuspend the cell pellet in 0.5 ml complete medium, 3 Add 10 x 106 FITC-labelled Candida suspension in 0.5 ml of medium (effector-totarget ratio 1:10). 4 Incubate for 1 h at 37°C. An additional sample of macrophages/Candido suspension, to be used as control, should be kept for an equivalent time at 4 °C. At this temperature macrophages do not phagocytose the yeasts. 5 Shake the tubes every 10 min or place them on a slowly rotating device in order to keep the cells suspended. 6 Centrifuge the tubes for 10 min at 200 g, 4 "C.

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7 Wash the pellet twice in PBS/EDTA by centrifugation for 10 min at 200 g at 4 °C. 8 Resuspend cells in 0.5 ml PBS/EDTA. 9 Examine monocytes/macrophages by flow cytometiy,b 10 Measure the increase in mean channel fluorescence intensity of monocytes after culture with the labelled organisms, relative to control cells.e 3

Non-adherent monocytes/macrophages (e.g. monocytic cell lines growing in suspension, elutriated or Percoll purified monocytes, monocytes/macrophages harvested from Teflon cultures) are more suitable to be used as host cells in this assay. b Choose logarithmic amplification for F1 (green fluorescence) and linear amplification for side and forward scatter. First run control sample to adjust settings for acquisition. Monocytes can be recognized on the basis of their scattering properties. In the two-dimensional plot of forward versus side scatter, monocytes appear as a homogeneous population distinct from smaller cells, debris, and uningested Candida. Set a gate around the monocyte population. Adjust the green PMT (photomultiplier tube) voltage to yield a green fluorescence intensity of 1-2 {channel 10-100) for control cells (incubated at 4°C). Keep in mind that phagocytosis may increase side scattering of monocytes/macrophages; this requires careful adjustment of gate for every sample. It is advised to run samples shortly after incubation in order to prevent clumping of uningested Candida. c The fluorescence intensity of cells will correlate with the number of Candida ingested. A check should always be made to see that the results obtained by flow cytometry match those obtained by microscopical examination. A critical issue for the correct interpretation of the results of the phagocytosis assays is to differentiate between extracellular, cell surface-adherent, and ingested organisms, all of which will give a signal detectable by flow cytometric analysis. One easy way to discriminate between these forms is to add ethidium bromide (50 [i-g/ml final concentration) ro the cell suspension. Under these conditions, ingested FITC-labellcd C. albicarts will maintain the FITC-based green fluorescence, while the fluorescence of extracellular yeasts attached to the cell surface will be quenched by the ethidium bromide. Alternatively, fluorescence of extracellular Con did a can be quenched by the addition of 0.5 mg/ml crystal violet (39, 46), C. attains can be phagocytosed by human and murine macrophages in the presence or absence of opsonins. However, phagocytosis appears to be optimal when the yeast are opsonized with serum, C, albiccms cells can be opsonized by incubation in 50% AB serum at 37 °C followed by washing with PBS (47, 48). 4.2.2 Intracellular killing of C. Atbicans by macrophages Some of the methods used to determine macrophage candidicidal activity rely on microscopic examination of Giemsa stained slides, because living Candida are stained blue whereas killed cells remain colourless (ghost cells). Other methods measure the incorporation of radioisotopes ([3II]glucose, [3H]leucine, [3H]uridine) as an indication of the candidicidal activity of macrophages. However, most of 149

MARIA CARLA BOSCO ET AL

the studies use the microbiological method of plate counting (colony-forming units, CFU) to measure candidicidal activity by macrophagcs.

Assessment of macrophage-mediated intracellular Candida killing using a colony counting technique Equipment and reagents • MICROTEST tissue culture plates. 96-well, flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Labware) • Device for vacuum aspiration • Repeating dispenser « Combitips 1-5 ml (Eppendorf) • Snaking platform • Complete medium: RPMI 1640 (see Protocol 6)

• • • • • •

Centrifuge Triton X-100 (Sigma Chemical) Distilled water Sabouraud dextrose agar (Biolife) C. albicans organisms Monocyte/macrophage effector cells, appropriately purified (see Chapters 1 and 2) • Macrophage activators (see Protocol J)

A Maintenance and preparation of C. alblcans 1 2 3 4

Maintain C, albicans by weekly transfer on Sabouraud dextrose agar plates at 28 *C. Transfer a colony from a three-days-old culture to 5 ml PBS in 15 ml polypropylene tube using a sterile loop. Wash twice with PBS by centrifugatiort at 200 g for 10 min. Resuspend yeasts at 1 x 105/ml in complete RPMI medium.

B Preparation of effector cells 1 Resuspend monocytes/macrophages at 1 x 106 cells/ml in complete RPMI 1640 medium. 2 Plate 0.1 ml of the macrophage cell suspension (containing 1 x 105 cells) into triplicate wells of a 96-well, flat-bottom microtitre tissue culture plate. 3 Add the appropriate macrophage activators in 0.1 ml of medium to yield the final volume of 0.2 ml/well (see Protocol 1). 4 Incubate the cells for 18-24 h at 37°C with activators. 5 Centrifuge the plate at 250-300 g for 5 min. 6 Remove supernatant by vacuum aspiration. 7 Wash the macrophage effector cells in the plate three times with warm complete medium (see Protocol 1}, C CFU assay 1 Add 0.1 ml of Candida suspension (containing 1 x 104 yeasts) to wells containing washed monocytes/macrophages {effector-to-target ratio 10rl)a and to six wells not containing macrophages to be used as control cultures.

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2

Add 0.1 ml of complete medium to each well to yield a final volume of 0.2 ml/well.

3

Incubate the plates containing macrophages and Candida for 3 h at 37°C.

4

Remove the plates from the incubator and shake vigorously on a shaking platform.

5

Stop the Lntracellular Candida killing by lysing the phagocytic cells with 20 ul/well of a solution of 10% Triton X-100 in water.

6

Carefully mix each well by pipetting and make serial dilutions in distilled water (from 1:20 to 1:100 final dilution).

7

Plate 0.1 ml of serial dilutions on Sabouraud dextrose agar (triplicate samples).

8

Count the surviving colony-forming units (CPU) after 24 h of incubation at 37°C and compare them to control cultures consisting of C. albicans incubated without effector cells.

9

Express the results as the percentage of anti-Candida activity according to the formula: (1 - (CFU of experimental group/CFU of control culture)} x 100.

a

Different effector-to-target ratios should be tested (e.g. from 20:1 to 2.5:1). Usually, the number of target cells is kept constant and an effector-to-target ratio titration is achieved by varying the number of effector cells.

To optimize this test, it is important to use an agerminative strain of C. albicans that grows as a pure yeast form in vitro at 28°C or 37°C in conventional media. This will avoid bias due to hyphae formation during the incubation (49).

4.2.3 Extracellular killing of C. Albicans Protocol 9 describes a method for assessment of extracellular killing of Candida (hyphal form} using the (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium) MTT colorimetric assay.

Assessment of macrophage-mediated extracellular Candida killing using a colorimetric assay Equipment and reagents • Microplate reader • MICROTEST tissue culture plates, 96-well, flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Labware) • Device for vacuum aspiration • Repeating dispenser

• Combitips 1-5 ml (Eppendorf) • Centrifuge . Complete medium: RPMI1640 (see Protocol 6) • PBS (see Protocol 1) • C albicans organisms

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• Monocyte/macrophage effector cells, appropriately purified (see Chapters 1 and 2 ) • Triton X-100 (Sigma Chemical)

• Distilled water . MTT (3-(4,5-dimethylthiazol -2-yl)-2.5-diphenyltetrazolium) (Sigma Chemical) • Isopropyl alcohol (Fluka)

A Maintenance and preparation of C. alblcans hyphae 1 Maintain C. albicans by weekly transfer on Sabouraud dextrose agar plates at 28 °C. 2

Transfer a colony from a three-days-old culture to 5 ml PBS in 15 ml polypropylene tube using a sterile loop.

3 4

Wash twice with PBS by centrifugation at 200 g for 10 min. Resuspend the yeasts at 4 x 105/ml in complete RPMI medium.

5

Plate 0.1 ml (5 x 104 yeasts) per well in 96-welI, flat-bottom microtitre plates with a repeating dispenser.

6

Incubate plates for 3-6 h at 37 °C until 95% of Candida have germinated into hyphae (30-100 n-m in length) that adhere firmly to the bottom of the wells (check hyphae germination by inverted microscope examination)/

7

Remove supernatant from each well by pipette aspiration observing biohazard precautions.

B Assay 1 Add 2 x 105 macrophages in 0.1 ml complete medium to each well containing the Candida hyphae (effector-to-target ratio 5:l).b 2 Centrifuge the plates at 200 g for 10 min. 3 Incubate the plates at 37 °C for 3 h. 4 Aspirate the supernatant fluid from each well. 5 Lyse the macrophages by adding 0,1 ml/well of 1% Triton X-100 in water. 6 Wash remaining Candida hyphae three times with 0.2 ml of distilled water.c 7 Add 0.1 ml of MTT (0.5 mg/ml) in RPMI 1640 medium (without serum) to each well.d 8 Incubate the plates for an additional 4 h at 37 "C. 9 Centrifuge the plates at 300 g for 5 min, 10 Aspirate dry each well. Candida hyphae will appear blue at the bottom of the wells. 11 Solubilize the content of each well with 0.1 ml of isopropyl alcohol. 12 Determine the optical density (OD) of each well at the wavelengths of 540 nm and 690 nm with an automated microplate reader.e 13 Calculate anti-Candida activity using the formula: {1 - (OD of experimental wells/OD of control wells)} x 100, "Hyphal growth can be stopped at this stage, if necessary, by storing the plates at 4 °C. Do not store plates for more than a few hours before use.

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b Different effector-to-target ratios should be tested (e.g. from 10:1 to 1:1) by varying the number of effector cells, c It is necessary, after incubating fungi with leukocytes, to lyse the leukocytes and rid the system of serum before the incubation with MTT to avoid a non-specific reduction of MTT (50, 51). d For the stock solution, MTT is dissolved in RPMI without serum at 0.5 mg /ml, passed through a 0.22 um filter, and kept at 4°C for no more than two weeks. e A well containing only isopropyi alcohol Is used as a blank. Control wells, containing Candida but not macrophages, should be included in each experiment. The use of MTT constitutes a simple, rapid, and inexpensive method of assaying viability of fungal cells. Live, metabolically active fungi cleave the tetrazolium ring of the yellow compound MTT to produce its purple formazan derivative. This assay is particularly useful in measuring the viability of hyphae, because with the dilution and plate counting assays the accuracy is limited by fungal clumping or adherence to the wells and by the possibility of nuclear multiplication and mycelial growth without a corresponding increase in CKI. The assay can be used to measure killing by fungicidal agents and also killing or damage of fungi by leukocytes (50, 51).

References 1. 2. 3. 4.

Krahenbuhl, J. L and Remington.]. S, (1983). J. ImmunoL 113. 507. Bersani, I.., Colotta, F., and Mantovani, A. (1986). Immunology. 59, 323, Pace, J. L, Varesio, L, Russell, S. W., and Blasi, E. (1985), J. Leuk. Biol 37, 475. Varesio. L., Espinoza-Delgado, I., Gusella, L, Cox, G. W., Melillo, G., Musso, T., et al. (1995), (n Human cytokines: their role in disease and therapy Jed. B. R, Aggarwal and R. K. Puri), pp. 55-69. Blackwell Science, New York. 5. Kspinoza-Delgado, I., Bosco, M. C., Musso, T, Gusella, G. L, Longo, D. L, and Varesio, L, 11995).J. Leufc. Biui.,57. 13. 6. Varesio, L, Radzioch, D., Buliam, B., and Gusella, G. L (1992). Curr. Top. Microbiol. Immunol., 181, 209. 7. Wilt rout, R. H..Taramel1i, D., and Holden. H. T. (1981). In Manual of macrophage methudology (ed. H. B. Herscrowitz, H. T. Holder, j. A. Bellanli, and A. Ghaffar), pp. 337-44. Dekker, New York. 8. Jadus, M. R., Irwin, M. C. N., Irwin, M. R., Horansky, R. D., Sekhton, S., Pepper, K. A., et al (1996). mood, 87, 5232. 9. Martin, J. H. J. and Edwards. S. W, (1993).j. Immunol, 150. 3478, 10. Sampson-Johannes, A. and Cavlino.J. A. (1988).}. immunol., 141, 3680. 11. Espinoza-Delgrado, L. Longo, D. L., Gusella, C. L., and Varesio, L. (1990J.J. Immunol., 145, 1137. 12. Galligioni, E., Quaia, M., Spada, A., Favaro, D., Santarosa, M., Talaraini, R., et al. (1993). Int. J. Cancer, 55, 380. 13. Whitman, E. D., Doherty, G. M., Peplinski, G. R., and Norton. J. A. (1995), In Human cytokines: their role in disease and therapy (ed. B. B. Aggarwal and R. K. Pun), pp. 333-51. Blackwell Science, Inc., USA. 14. Meltzer, M, L. (1981). In Manual of matrophage methodology (ed. H. H. HerscowiLz, H. T. Holden, J. A. Bellanti, and A. Ghafiar), pp. 329-36. Marcel Dtkkcr, New York,

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MARIA CARLA BOSCO ET AL. 15. Kaplan, A. M. (1981). In Methods for studying mononudear phagocytes (ed.D. O. Adams, P.J. Edelson, and H. Koren), p. 775. Academic Press, New York. 16. Weinberg, J. B. and Hibbs, J. B., Jr. (1981). In Manual ofmacrophage methodology (ed. H. B. Herscowitz, H. T. Holden, J. A. Bellanti, and A. Ghaffar), pp. 345-55. Marcel Dekker, Inc., New York and Basel. 17. Braun, D. P., Mi-Chung, A., Harris, J. E., Chu, E., Casey, L, Wilbanks, G., et al. (1993). Cancer Res., 53, 3362. 18. Drysdale, B. E., Agarwal, S., and Shin, H. S. (1988). Prog. Allergy, 40, 111. 19. Gusella, G. L., Musso, T., Rottschafr, S. E., Pulkki, K., and Varesio, L. (1995) J. Immunol, 154, 345. 20. Paulnock, D. M. and Lambert, L. E. (1990) J. Immunol, 144, 765. 21. Ho, J. L, Reed, S. G., Sobel, J., Arruda, S., Hui He, S., Wick, E. A., et al. (1992). Infect. Immun., 60, 1984. 22. Ichinose, Y., Bakouche, Q., Tsao, J. Y., and Fidler, I. J. (1988) J. Immunol, 141, 512. 23. McLachlan, J. A., Serkin, C. D., Morrey, K. M., and Bakouche, 0. (1995) J. Immunol, 154, 832. 24. Bosco, M. C., Pulkki, K., Rowe, T. K., Zea, A. H., Musso, T., Longo, D. L, et al. (1995)J. Immunol, 155, 1411. 25. Taniyama, T. and Holden, H. T. (1981). In Manual of macrophage methodology (ed. H. B. Herscowitz, H. T. Holden, J. A. Bellanti, and A. Ghaffar), pp. 323-7. Marcel Dekker, Inc., New York. 26. Hori, K., Mihich, E., and Ehrke, M. J. (1989). Cancer Res., 49, 2606. 27. Nishihara, K., Earth, R. F., Wilkie, N., Lang, J. C., Oda, Y., Kikuchi, H., et al (1995). Cancer Gene Therapy, 2, 113. 28. Klostergaard, J., Leroux, M. E., and Hung, M. (1991). J. Immunol, 147, 2802. 29. Espevik, T. and Nissen-meyer, J. (1986)J. Immunol, 95, 99. 30. Levitz, S. M. and Diamond, R. D. (1985)J. Infect. Dis., 152, 938. 31. Murray, H. W. and Cartelli, D. M.(1983).J. Clin. Invest., 72, 32. 32. Shiratsuchi, H., Johnson, J. L., and Elmer, J.-J. (1991) J. Immunol., 146, 3165. 33. Mauel, J., Biroum-Noerjasin, S. and Behin, R. (1974). In Activation of macrophages (ed. W. H. Wagner and H. Hahn), pp. 261-79. Excerpta Medica. 34. Bermudez, L. E. and Kaplan, G. (1995). Trends Microbiol, 3, 22. 35. Nathan, C. F. and Hibbs, J. B. (1991). Curr. Opin. ImmunoJ., 3, 65. 36. Denis, M. (1994). J. Leuk. Biol., 55, 682. 37. Dugas, B., Mossalay, M. D., Damias, C., and Kolb, J. B. (1995). Immunol Today, 16, 574. 38. Cohen, M. S., Isturiz, R. E., Malech, H. L., Root, R. K., Wilfert, C. M., Gutman, L, et al. (1981).AmJ.Med., 71, 59. 39. Vasquez-Torres, A. and Balish, E. (1997). Microbiol Mol Biol Rev., 61, 170. 40. Rothermel, C. D., Rubin, B. Y., Jaffe, E. A., and Murray, H. W. (1986) J. Immunol., 137, 689. 41. Murray, H. W., Rubin, B. Y., Carriero, S. M., Harris, A. M., and Jaffee, E. A. (1985). J. Immunol, 134, 1982. 42. Lehn, M., Chiang, C. P., Remold, H. G., Swafford, J. R., and Caulfield, J. P. (1991). Am. J. Pathol., 139, 399. 43. Nacy, C. A., Fortier, A. H., Meltzer, M. S., Buchmeier, N. A., and Schreiber, R. D. (1985). J. Immunol, 135, 3505. 44. Ogunkolade, B. W., Colomb-Valet, L, Monjour, L., and Rodhes-Feuillette, A. (1998). Acta Trop., 47, 171. 45. Schuit, K. E. (1979). Infect. Immun., 24, 932. 46. Fartorossi, A., Nisini, R., Pizzolo, J. G., and D'Amelio, R. (1989). Cytometry, 10, 320. 47. Segal, E., Lehrer, N. and Ofek, I. (1982). Exp. Cell Biol 50, 13.

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ANALYSIS OF MACROPHAGE LYTIC FUNCTIONS 48. Thompson, H. L. and Wilton, J. M. A. (1992). Clin. Exp. Immunol., 87, 316. 49. Mattia, E., Carruba, G., Angiolella, L, and Cassone, A. (1982). J. Bacteriol., 152, 555. 50. Puliti, M., Radzioch, D., Mazzolla, R., Barluzzi, R., Bistoni, P., and Blasi, E. (1995). Infect. Immun., 63, 4170. 51. Levitz, S. M., Parrel, T. P., and Maziarz, R. T.(1991).J. Infect. Dis., 163, 1108.

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Chapter 7 Analysis of macrophage activity in vivo Nico van Rooijen and Esther van Kesteren-Hendrikx Department of Cell Biology and Immunology, Faculty of Medicine, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

1 Introduction In mammals, macrophages have developed into multifunctional cells. Apart from their scavenger role in the clearance of non-self materials such as micro-organisms and altered-self materials such as apoptotic cells, senescent erythrocytes, immune complexes, and inflammatory products, they play a crucial role in the regulation of both innate and acquired immunity. Whereas the former activity is based on phagocytosis and intracellular degradation, the latter activity largely depends on the production and secretion of a panel of regulatory molecules such as cytokines, chemokines, and nitrogen oxide (NO). Depletion of macrophages and blocking of phagocytosis form important approaches to study their role in various host defence mechanisms. Available methods for this purpose suffer from: (a) A lack of selectivity with respect to macrophages. (b) General toxicity. (c) Blocking of phagocytosis being attended with activation of cytokine production. (d) Opposite effects on macrophages of high and low doses of the agents, cancelling each other in vivo where these agents will reach some macrophages in a high dose and others in a low dose (1). For that reason we developed a liposome-mediated macrophage 'suicide' technique, based on the intraphagocytic delivery and accumulation of liposomeencapsulated drugs that are not toxic in their non-encapsulated form, but induce apoptotic cell death when intracellular concentrations increase (2).

2 Previous methods for blocking of phagocytosis 2.1 Silica, carrageenan, and dextran sulfate 30 years ago, administration of silica particles of defined dimensions was recommended as a procedure to deplete macrophages in vivo (3). The cytotoxicity of 157

NICO VAN ROOIJEN AND ESTHER VAN KESTEREN-HENDRIKX

silica was found to be correlated with its capacity to disrupt the membranes of secondary lysosomes in macrophages, demonstrated by release of marker enzymes. More recent studies have shown that silica particles interact directly with both the plasma and lysosomal membranes. Results of the latter studies indicated that interaction of silica particles with the plasma membrane leads to Ca2+ influx with resultant cell death and ATP depletion, whereas their interaction with lysosomal membranes leads to release of lysosomal contents but is not followed by irreversible cell injury. That ATP depletion is involved in silicamediated damage to macrophages has since been confirmed. Since the original description of the cytotoxic effects of silica on macrophages, macrophage depletion or blockade of phagocytosis by silica has been used in many studies aimed at the unravelling of macrophage functions in vivo. However, like most other agents that are toxic for macrophages and block their phagocytic capability when administered in a high dose, sublethal doses of silica will stimulate cells of the monocyte/macrophage lineage to produce IL-1, IL-6, TNFa, and NO. Silica-induced production of cytokines by alveolar macrophages, especially TNFa, is a crucial factor in the induction of proliferation of fibroblasts in silicosis. As a consequence, silica-induced blocking of phagocytosis as a method to study macrophage function in vivo, can only be recommended if it can be excluded that one or more of the molecular products of macrophages play a regulatory role. This will be the case only rarely. It has been reported that carrageenan, a sulfated polygalactose is cytotoxic to macrophages (4). Also other polyanionic polysaccharides such as dextran sulfate (DS 500) appeared to inhibit macrophage functions (5). Carrageenan and dextran sulfate have since been used for elimination or suppression of phagocytic activity or to reveal macrophage functions. However it has been confirmed that in addition to their effects on macrophages, carrageenan and dextran sulfate have a strong effect on lymphocytes. Moreover, both carrageenan and dextran sulfate enhanced the macrophage-mediated effects of LPS-induced septic shock and LPSinduced TNFa production. It was found that although treatment of macrophages with carrageenan reduced their phagocytic activity with respect to yeast cells, their ability to kill the yeast cells was increased. This may be explained by the increased production of molecules such as NO, which are thought to play a role in the killing of intraphagocytic micro-organisms. In conclusion, neither silica nor carrageenan or dextran sulfate can be recommended for blocking of macrophage functions. 2.2 Gadolinium chloride Several chemicals are able to suppress the phagocytic capability of macrophages to some extent. A strong blockade of phagocytosis could be achieved by treatment of animals with 'rare earth metals' such as gadolinium (6). In addition to blocking of phagocytosis, intravenous injection of gadolinium chloride in rats also eliminated a part of the Kupffer cell population in the liver, namely the large macrophages situated in the periportal zone of the liver acinus, but not 158

ANALYSIS OF MACROPHAGE ACTIVITY IN VIVO

those in the rat spleen (7). However, intravenous administration of gadolinium chloride in mice did not eliminate Kupffer cells in their liver and addition of gadolinium chloride to cultures of mouse peritoneal macrophages did not affect these cells. Moreover, frequent mitotic figures of hepatocytes in the liver of mice injected with gadolinium chloride suggested a proliferative effect on nonphagocytic cells. In contrast to particulate agents and polymerized complexes, gadolinium chloride, as a small molecule, could have the advantage of a relatively easy transport through capillary walls. For that reason, intravenously injected gadolinium chloride could be able to reach macrophages in many organs apart from liver and spleen. However it has been demonstrated that intravenously injected gadolinium chloride did not affect alveolar macrophages and interstitial macrophages in the rat lung. Although animals treated with gadolinium chloride revealed a significantly lower phagocytic activity of Kupffer cells, a pronounced rise in serum cytokine activity (TNFa and IL-1) was detected (8). Obviously inhibition of phagocytosis was closely related to stimulation of cytokine production, a phenomenon also described for treatment with silica, carrageenan, and dextran sulfate.

2.3 Anti-macrophage antibodies and receptor antagonists Different surface receptors on macrophages, such as scavenger receptors, complement and Fc receptors, sialoadhesin (SER) receptors, and mannose receptors play a role in phagocytosis. As a consequence, both receptor antagonists and antibodies directed against these receptors should be able to suppress receptormediated phagocytosis for at least a limited period of time. However it may be expected that macrophages will rapidly internalize and hydrolyse any blocking molecules so that suppression of phagocytosis will last for a short period of time only and will require high doses of the blocking molecules. Nevertheless, a dosedependent blockade of phagocytosis by mannose and mannose derivatives was shown for macrophages in the rat spleen. In practice, blocking of macrophages by mannose or mannose derivatives has not been applied. Also polyclonal antimacrophage antibodies have the capability to suppress phagocytosis for a certain period of time, and this approach has been used in several studies aimed at the unravelling of macrophage function. Since most studies in which anti-macrophage antibodies were applied for blocking of macrophages were performed well before it was found that macrophages are responsible for the production of a large panel of cytokines, the latter aspect has not been investigated in these studies.

2.4 Competition Administration of a high dose of particles that will be ingested by macrophages may lead to saturation of their phagocytosis capability. In early studies on the role of phagocytosis in the induction of antibody responses, India ink (colloidal carbon particles), or other finely divided compounds such as saccharated iron oxide, thorotrast, or polystyrene latex were used as agents for blocking of 159


phagocytosis. However these particles can not be degraded by macrophages and will be ingested by new macrophages as soon as they are released from dying macrophages. In the contrary, liposomes are artificially prepared spheres, that can be degraded by macrophages. The natural fate of liposomes when administered in vivo, is phagocytosis followed by intracellular degradation of the liposomal phospholipid bilayers as a result of the activity of lysosomal phospholipases. It has been demonstrated that liposome suspensions are also able to saturate the phagocytic activity of macrophages in a way similar to that described for finely suspended carbon particles (9). One should be aware that, dependent on their composition, liposomes may alter the structure and characteristics of cell membranes of macrophages after their internalization. However, liposomes can be prepared from phospholipids that are practically inert; for instance they can be made up merely of lecithin (phosphatidylcholine). Contrary to the blocking of phagocytosis by agents such as silica, carrageenan, dextran sulfate, and gadolinium chloride, liposomes did not stimulate the basic or lipopolysaccharide-induced production of proinflammatory cytokmes and/or nitric oxide (NO) by macrophages. So, the application of liposomes as a phagocytosis blocking agent offers the advantage of minimum side-effects on cytokine production and secretion.

3 The liposome-mediated macrophage suicide technique 3.1 Principles Since liposomes can be used to encapsulate water soluble molecules and phagocytosis is the natural fate of liposomes in the body, we have developed a more sophisticated approach for the in vivo elimination of macrophages by liposome encapsulated drugs (see Figure 1). Mechanism and selectivity of the method is explained as follows: (a) Once ingested by macrophages, the phospholipid bilayers of the liposomes are disrupted under the influence of lysosomal phospholipases. (b) Encapsulated hydrophilic molecules, such as the bisphosphonate clodronate and the diamidine propamidine, will be released within the cell. (c) Since such molecules will not easily escape from the cell by crossing the cell membranes, they accumulate in the cell. (d) At a certain intracellular concentration of the drugs, irreversible damage induces cell death by apoptosis (10, 11). (e) Drug molecules, released in the circulation from dead macrophages or by leakage from liposomes, will not easily enter into cells by crossing cell membranes in the opposite direction. Moreover, they have short half-life times in circulation and body fluids, explaining the fact that non-phagocytic cells are not affected.

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Figure 1 Liposomes are artificially prepared spheres, consisting of concentric phospholipid bilayers, separated by aqueous compartments. They form when phospholipid molecules are dispersed in water. Part of the aqueous solution, together with hydrophilic molecules dissolved in it such as the bisphosphonate clodronate (black squares; see also structural formula) or the diamidine propamidine, will be encapsulated during the formation of the liposomes. Liposomes encapsulating clodronate molecules (squares), are ingested by macrophages via endocytosis (1). After fusion (2) with lysosomes (L) containing phospholipases (arrowheads), the latter are disrupting the bilayers of the liposomes (3). The more of the concentric bilayers are disrupted, the more of the clodronate is released within the cell (4). It has been demonstrated that liposome-mediated intracellular delivery of clodronate or propamidine causes cell death by apoptosis. (N = nucleus of the macrophage.)

Macrophage depletion has been confirmed by immunocytochemical and electron microscopical methods, and by functional assays (12, 13).

3.2 Liposomes liposomes are artificially prepared spheres, consisting of concentric phospholipid bilayers separated by aqueous compartments (2). They form when amphipathic phospholipid molecules are dispersed in an aqueous solution. The phospholipids tend to find a conformation in which their hydrophobic fatty acid chains are prevented from direct contact-with the water molecules. For that reason, phospholipid bilayers are formed of which both outer parts are made up of the hydrophilic head groups, whereas the hydrophobic fatty acid chains are located 161


directly opposite to each other and form the inner part of the bilayer (see Figure 7). Part of the aqueous solution, together with hydrophilic molecules dissolved in it will be encapsulated during the formation of the liposomes, whereas lipophilic molecules may be associated with the phospholipid bilayers. Amphipathic molecules, as the phospholipids themselves, or conjugates made up of a phospholipid molecule and a hydrophilic molecule, attempt to find a conformation with their hydrophobic parts inserted in the bilayers and their hydrophilic parts extended either in the aqueous compartments or on the outer surface of the liposomes. Because liposomes are biodegradable and may be composed of natural, non-toxic, and immunologically inert phospholipid molecules, they have been suggested as promising carriers of drugs (14), Liposomes can be prepared according to different methods. They may vary in their dimensions, composition (different phospholipids and variable cholesterol contents), charge (resulting from the charges of the composing phospholipids), and structure (multilamellar liposomes consisting of several concentric bilayers, separated by aqueous compartments or unilamellar liposomes, consisting of only one phospholipid bilayer surrounding one aqueous compartment),

3.3 Liposome-encapsulated clodronate From several liposome-encapsulated drugs that have been shown to be efficacious in the depletion of macrophages, clodronate remains the best choice due to its low toxicity and short half-life. Protocol 1 details a conventional method for the production of multilamellar clodronate-liposomes {slightly modified from ref, 2),

Preparation of multilamellar clodronate-liposomes Equipment and reagents • Sonicator (Sonicor SC-200-22, 55 kHz; Sonicor Instr. Corp.} • Superspeed centrifuge (Sigma, 3MK) • Apparatus for vacuum evaporation (Buchi) • Pasteur pipettes, sterile • Polycarbonate centrifuge tubes (Nalgene) • Filter (0,45 um pore; FP 030/2, Schleicher & Schuell) • Polycarbonate membrane filter (1.0 um pore; Poretics Products) • Chloroform, analytical grade (Riedel-de Haen) • Nitrogen gas (or other inert gas)

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Sterile phosphate-buffered saline (PBS): dissolve 12.2 mM phosphate and 8.2 g/litre NaCl in Milli Q.(or similar purified water), adjust pH to 7.4; autoclave the solution Stock solution of phosphatidylcholine (egg lectin): 100 mg/ral phosphatidylcholine (lipoid) in chloroforma Stock solution of cholesterol: 20 mg/ml cholesterol (Sigma) in chloroformb 0,7 M clodronate solution: 2.5 g clodronate (Boehrmger Mannheim GmbH) in 10 ml Milli Q,(or similar purified water), adjust pH to 7.1 with 5 M NaOH


Method Add 0.86 ml [8.6 ml]c phosphatidylcholine stock solution to 0.40 ml [4.0 ml] cholesterol stock solution in a 500 ml [2 litre] round-bottom flask. 2 Remove the chloroform by low vacuum (min. 120 mbar) rotation (150 r.p.m.) evaporation. At the end a thin milky white phospholipid film will form against the inside of the flask. 3 Disperse the phospholipid film in 10 ml [40 ml] PBS (for empty liposomes) or 0,7 M clodronate solution (for clodronate-liposomes) by gentle rotation (max. 180 r.p.m.) at room temperature (RT) for 20-30 min (PBS) or 5-10 min (0.7 M clodronate solution). 4 Hold the milky white suspension under nitrogen gas at RT for 1.5-2 h, 5 Shake the solution gently and sonicate it in a water-bath for 3 min.d 6 Hold the suspension under nitrogen gas at RT for 2 h (or overnight at 4°C) to allow swelling of the liposomes. 7 Before using the clodronate-liposomes: (a) Remove the non-encapsulated clodronate by centrifugation of liposomes for 20 min at 25000 g and 10°C, The clodronate-liposomes will form a white band at the top of the suspension, whereas the suspension itself will be nearly clearf (b) Carefully remove the clodronate solution under the white band of liposomes using a Pasteur pipette (about 1% will be encapsulated). (c) Recycle the non-encapsulated clodronate for reuse by filtration using a 0.45 um filter. This recycling procedure should not be repeated for more than five times. 8 Wash the liposomes two to three times using centrifugation at 25 000 g and 10°C for 15 min. Remove each time the upper solution and resuspend the pellet in approx. 80 ml sterile PBS using a Pasteur pipette. 9 Resuspend the final liposome pellet in sterile PBS and adjust to a final volume of 4 ml [40 ml]. The suspension should be shaken (gently) before administration to animals or before dispensing, in order to achieve a homogeneous distribution of the liposomes in suspension. * This stock can be made in advance and stored at -20°C under nitrogen gas. Nitrogen gas is used to prevent oxidation of phosphatidylcholine. b This stock can be made in advance and stored at -20°C. c Instructions are given for preparation of 4 ml and [40 ml] liposome suspension. d In order to limit the maximum diameter of the liposomes, the suspension can be extruded six times using polycarbonate membrane filters with 1.0 um pores. The latter procedure may lead to some loss of encapsulated clodronate and can not generally be recommended, since large liposomes are more efficacious than small liposomes in macrophage depletion. e Clodronate-liposomes can be stored in the original clodronate solution at 4 °C under nitrogen gas. Nitrogen gas is used to prevent denaturation of phospholipid vesicles. This is particularly important in the case of clodronate-liposomes, as they float on the aqueous phase after preparation. PBS liposomes form a pellet on the bottom of the tubes. f There is no problem when the suspension is not completely clear, since the remaining liposomes will be very small ones. The relatively large clodronate-liposomes are efficacious with respect to depletion of macrophages. 1

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Under N2 (in sealed tubes) the clodronate-liposomes can be stored in PBS at 4"C for up to months since the rate of clodronate leakage appeared to be extremely low. If they are kept in the original clodronate solution, they can be stored for longer periods of time. In that case however, before using the liposomes, the procedure should be continued from step 5 (including sonication). There is no indication that the mac roph age-depict ing activity of clodronateliposomes is affected during transport or storage. The amount of clodronate encapsulated in the liposomes has been determined earlier using methods based on murexide clodronate competition for calcium and on measurements of 99mTclabelled clodronate. Protocol 2 describes a rather simple method, modified from a method described by Monkkonen et al (15), for determination of the level of encapsulated clodronate. It is based on the fact that bisphosphonates form a complex with copper (from CuSO4) that can be measured by spectrophotometrical analysis at 240 nm.

Spectrophotometric determination of the amount liposome-encapsulated clodronate Equipment and reagents • 16 ml glass tubes, taps with Teflon inlay (Kimble) • 10 ml polystyrene tubes (Greiner) • Spectrophotometer (UV-160A, Shimadzu) • Pasteur pipettes • Glass pipette 10 ml (piston pipette; Hirschmann) • Pipettes (P20, P200. and P1000, Gilson) • Milli Q or similar purified water • Chloroform, analytical grade (Riedel-de Haen) • 0.7 M clodronate solution (see Protocol 1) • Standard clodronate solution: dissolve 10.0 mg/ml clodronate (Boehringer Mannheim GmbH) in Milli Q and adjust the pH to 7.1 with 5 M NaOH

Phosphate-buffered saline (PBS) (see Protocol 1) Saline-saturated phenol: warm up (65-70°C) 250 g phenol, analytical grade (Janssen Chimica) and add 0.1% (w/v) 8-hydroxyquinoline (Baker) and 200 ml saline {0.8% (w/v) NaCl in Milli Q). Stir this solution for 10 min. Remove the upper (aqueous) phase at RT and add again 200 ml saline. Stir for 20 min. Repeat removing upper phase, adding saline, and stirring three times.a 4 mM CuSO4 solution: dissolve 0.64 g/litre CuSO4. analytical reagent (Merck) in Milli Q. HNO:, solution: dilute 65% HNO3, analytical grade (Merck) 100 times in Milli Q.

A Extraction of clodronate from liposomes 1 Dispense in separate glass tubes: 1 ml of the clodrortate-liposome suspension, 1 ml of standard clodronate solution, and 1 ml of the saline solution.b 2 Add 8 ml of phenol/chloroform (1:2) to each tube. Phenol should be saturated with saline before use.

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3 Vortex and shake the tubes extensively. 4 Hold the tubes at RT for at least 15 min. 5 Centrifuge (1125 g} the tubes at 10°C for 10min. 6 7 8 9 10 11

Hold the tubes at RT until clear separation of both phases (at least 10 min). Transfer the aqueous (upper) phase to clean glass tubes using a Pasteur pipette. Add 6 ml chloroform per tube: re-extract the solution by extensive vortexing. Hold the tubes for at least 5 min at RT. Centrifuge (1125 g} the tubes at 10°C for 10 min. Transfer the aqueous phase (without any chloroform) to 10 ml plastic tubes using a Pasteur pipette. These are the samples for determination of clodronate concentration.

B Determination of clodronate concentration 1

Prepare a standard curve using 0, 10, 20, 40, 50, 70, and 80 u1 of the extracted standard clodronate solution added with saline to a total volume of 1 ml per tube.

2 3

Dilute the samples until they are within the range of the standard curved.c Add 2.25 ml of 4 mM CuSO4 solution, 2.20 ml Milli Q, and 0.05 ml HNO3 solution to each tube, containing 1 ml sample or standard. Vortex all tubes vigorously. Read the samples at 240 nm using a spectrophotometer.

4 5 a

Storage at -20 °C. When pipetting phenol with glass pipette, be sure not to take saline (upper phase). This would dilute the sample. b Attention should be paid to the right controls. If liposornes are suspended in PBS, PBS controls should be included. N,B. Phosphate (depending on concentration) may disturb the assay. C A suspension of clodronate-liposomes prepared according to Protocol 1 contains about 6 rag clodronate per 1 ml suspension. 20 n-1 extracted clodronate-liposorae suspension (thus diluting the sample 50 times) has an average absorption of 0.5 using a 1 cm quartz cuvette.

3.4 Injection of liposornes and access to tissue macrophages A suspension of clodronate-liposomes prepared according to Pruloml 1 contains about 6 mg clodronate per 1 ml suspension. Splenic macrophages can be depleted by intravenous administration of about 0.1 ml of a suspension of clodronateliposomes (prepared according to Protocol 1) per 10 g body weight. Macrophages in the liver (Kupffcr cells) arc more susceptible and can be completely depleted by intravenous administration of 0.02 ml of the suspension per 10 g body weight. See the relevant literature for detailed information on the doses of clodronate-liposomes required to deplete different macrophage (sub)populations in various organs (for references sec next paragraph). Liposomes are not able to cross vascular barriers such as capillary walls. For 165


that reason it is important to choose an administration route allowing an unhindered access of liposomes to macrophages targeted for depletion. Four administration routes for clodronate-liposomes that are frequently used are shown in Figure 2. Given the open blood circulation system in spleen, liver, and bone marrow, intravenous administration of clodronate-liposomes allows the depletion of macrophages in these organs (Figure 2a) (16). Since the peritoneal cavity of mice and rats is drained by parathymic lymph nodes from where the lymph is ultimately carried into the circulation, intraperitoneal administration of clodronate-liposomes may be used for depletion of macrophages in peritoneal cavity, parathymic lymph nodes, liver, and spleen (Figure 2V) (17). Intratracheal instillation of the liposomes can be used for depletion of the alveolar macrophages in the lung (Figure 2c) (18). For obvious reasons, the interstitial macrophages in the lung can neither be depleted by intratracheal instillation nor by intravenous administration. Subcutaneous injection in the draining area of lymph nodes induces macrophage depletion in these lymph nodes. Macrophage depletion may also be induced in the next lymph node stations on the efferent path. However subcutaneously administered liposomes are usually given in a low dose, so that splenic and liver macrophages are not affected by the few liposomes that reach the circulation (Figure 2d) (19). In some organs, such as the testis, where collagen fibres are only loosely packed (20), or in knee joints where phagocytic synovial lining cells can be found (21), depletion can be achieved by direct injection in the organ.

3.5 Selectivity of the approach with respect to macrophages Since the liposome-mediated macrophage suicide technique is based on a rapid internalization and consecutive intracellular degradation of liposomes, it is not surprising that macrophages are the only cells to be affected. Indeed the approach allows the selective removal of mononuclear phagocytes from heterogeneous spleen cell populations in vitro. No effect was found on non-phagocytic spleen cells as measured by growth, protein production, antigen presentation, and antigen-specific T cell proliferation (22). An important question concerns the possible depletion of neutrophil granulocytes as a consequence of treatment with clodronate-liposomes. These play a crucial role in the clearance of many micro-organisms. Neutrophils, however, appeared neither morphologically nor functionally to be affected by clodronate-liposomes administered in vivo (23). Dendritic cells (DC), which are mainly responsible for processing and presentation of antigens to T lymphocytes form another important cell population that might be affected. However, DC isolated from animals treated with clodronateliposomes were not impaired in their ability to induce primary CTL responses (24). Recently it has been shown that two distinct populations of DC are present in the spleen. In addition to the classical population of DC in the T cell areas of the white pulp, a new subpopulation of DC has been described at the border between the marginal zone and the red pulp of the spleen. These so-called marginal DC (25) are able to phagocytose particulates in vivo, express markers characteristic of both DC and macrophages, and have a high turnover rate. In 166


Figure 2 The four most frequently applied administration routes of clodronate-liposomes are shown. (a) Intravenous (IV) administration of clodronate-liposomes may induce depletion of macrophages in spleen (SP), liver (LI), and bone marrow (BM), since liposomes may unhindered leave the blood vessels (BV) here. (b) Intraperitoneal (IP) administration of clodronate-liposomes may at first induce depletion of macrophages in the peritoneal cavity (PE). From there liposomes are drained towards the parathymal lymph nodes (LN2) where macrophages are also depleted. Via the ductus thoracicus, lymph is carried into the circulation. As a consequence macrophages in the organs mentioned in Figure 2a may also be depleted if the injected dose is high enough. (c) Intratracheal (IT) instillation of clodronate-liposomes may lead to a depletion of alveolar macrophages (AL) in the lung (LU), since these have direct access to the alveolar lumen. (d) Subcutaneous administration of clodronate-liposomes in the draining area (DA) of a lymph node (LN1) may induce depletion of macrophages in that lymph node. If there are enough of the liposomes left after lymph node passage, the remaining liposomes, which are carried to a next lymph node station with the efferent lymph, may also lead to macrophage depletion in that lymph node (LN2). N.B. Macrophages in the testis (TE) and phagocytic synovial lining cells in knee joints (SY) may be depleted by direct local injections, but macrophage depletion in many other organs such as in the thymus (TH), eyes (EY), kidneys (Kl), gut (GU), or brain (BR) is difficult and at best it is incomplete.

contrast, DC in the T cell areas are not phagocytic in vivo and have a low turnover rate. Not surprisingly, the marginal DC were completely depleted by a single intravenous injection with clodronate-liposomes, whereas DC in the T cell areas were not affected (25). However given the finding that the antigen presenting capabilities of DC, isolated from spleen of animals that were previously treated with clodronate-liposomes, were not affected, the question remains whether the new DC have to be considered macrophages or DC.

3.6 Duration of macrophage depletion After in vivo depletion of macrophages, repopulation of the depleted tissues with new macrophages depends on the recruitment of their precursors (monocytes) 167


from bone marrow. Monocytes are carried to the tissues by the blood circulation and their final differentiation into tissue macrophages starts as soon as they leave the circulation and enter the organ parenchyme. Repopulation of Kupffer cells in the liver and red pulp macrophages in the spleen starts at about five days after depletion and is completed within 12 days. On the other hand, repopulation of marginal metallophilic macrophages and marginal zone macrophages in the spleen takes much more time, e.g. up to two months for the latter cells (16). Also lymph node macrophages, depleted by subcutaneous injection with clodronate-liposomes in their draining areas require several months for their complete replacement (19). Alveolar macrophages in the lung, depleted by intratracheal instillation of clodronate-liposomes (18), testis macrophages, depleted by direct injection of clodronate-liposomes into the organ (20), and phagocytic synovial lining cells, depleted by intra-articular administration of clodronateliposomes (21) are all replaced within one month.

4 Practical applications of the technique The liposome-mediated macrophage suicide approach was developed initially to serve as an experimental tool for exploration of functional aspects of macrophages in natural and acquired immunity. During these studies, interest in using this approach for transient and organ-specific suppression of macrophage function increased for practical reasons (26). The efficacy of drug and gene targeting to non-phagocytic cells appeared to be enhanced in macrophage-depleted animals, as a consequence of the reduced uptake and degradation of drug or gene carriers by macrophages. Administration of liposome-encapsulated clodronate strongly enhanced the survival of human cells after engraftment in immunodeficient SCID mice. The symptoms of various types of inflammatory reactions could be reduced by treatment with liposome-encapsulated clodronate. In several cases, graft survival and functioning could be improved by the application of liposomeencapsulated clodronate.

4.1 Improved efficacy of carrier-mediated gene transfer Among the available vectors for gene transfer in vivo, replication-deficient, recombinant adenovirus vectors are very efficient ,at transferring genes to target cells. However, both the innate immune system and the acquired immune system may reduce the efficacy of this approach for gene transfer, as macrophages are important participants in both innate and acquired immunity against viruses. Recent studies (27) have shown that depletion of liver macrophages (Kupffer cells) by liposome-encapsulated clodronate, prior to intravenous administration of an adenovirus vector led to a higher input of recombinant adenoviral DNA to the liver, an absolute increase in transgene expression, and a delayed clearance of both the vector DNA and transgene expression. Other recent studies showed that alveolar macrophages in the lung are responsible for a rapid elimination of intratracheally administered adenovirus vectors, that were on their way to the epithelial surface of the respiratory tract (28). Studies focusing on the improve168


merit of the efficacy of carrier-mediated gene transfer, after transient suppression of macrophage activity using clodronate-liposomes, are underway in several laboratories.

4.2 Improved survival of human cells in immunodeficient (SCID) mice Immunodeficient mice are widely used to maintain xenogeneic grafts of human cells. In this way, the role of human cells in host defence mechanisms, pathological disorders, and diseases, as well as in normal human haemopoietic and immunological processes can be studied under in vivo conditions. However, despite of the absence of functional T and B cell-mediated immunological activity against the injected or engrafted human cells in SCID mice, such cells are subjected to some host resistance from elements of the innate immune system. Mice bearing the SCID mutation retain a number of elements of the innate immune system. Macrophages are believed to form the core of the remaining resistance against the grafted human cells. The effects of macrophage depletion in SCID mice on the survival of injected human peripheral blood lymphocytes have been extensively investigated by Fraser et al. (29). Control SCID mice had no detectable human cells within 72 hours. However, animals treated with liposome-encapsulated clodronate maintained a large proportion of human cells in peripheral blood and spleen. After simultaneous implantation of human fetal thymic and liver tissue in control SCID mice (SCID-hu Thy/Liv mice), production of phenotypically normal human T cells into the periphery is induced for prolonged periods, but the cells are rapidly cleared. SCID-hu Thy/Liv mice injected with liposome-encapsulated clodronate showed a transient increase in human cell content in peripheral blood and a large accumulation of human cells in the white pulp compartments of the spleen. These results demonstrate that murine mononuclear phagocytic cells play a crucial role in human cell clearance. Another advantage of macrophage depletion in SCID mice prior to engraftment of human cells has been emphasized by Terpstra et al. (30). The minimal graft size of normal and leukaemic human haemopoietic cells in SCID mice that results in outgrowth of the human cells in the mouse bone marrow appeared to be about tenfold smaller in macrophage-depleted SCID mice. This considerable reduction of the minimal graft size greatly facilitates studies on subsets of human haemopoietic cells, which are not easy to obtain in large numbers. Mice lacking the elements of acquired immunity (e.g. SCID or NIHIII mice) could be made susceptible to the development of the human malaria parasite Plasmodium faltiparum by depletion of macrophages, followed by substitution of mouse erythrocytes by human red blood cells infected with Plasmodium faltiparum (31). In view of the scarcity of animals able to harbour human parasites, this novel model offers new approaches for malaria research. In conclusion, normal and leukaemic human haemopoietic cells as well as human red blood cells all show a greatly improved survival in macrophagedepleted SCID mice.

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4.3 Suppression of inflammatory reactions It is generally believed that inflammatory agents stimulate macrophages to produce and secrete cytokines and/or chemokines. The latter, in turn, induce the recruitment of inflammatory cells, either directly, or indirectly by stimulation of the chemokine production of other (non-phagocytic) cells in the area. Using the liposome-mediated macrophage suicide approach in various studies on inflammatory reactions, it has been confirmed that macrophages play a key role in inflammation and that symptoms of inflammation can be suppressed by depletion of macrophages before administration of the inflammatory agents (see review in ref. 26). For instance: (a) Selective depletion of macrophages produced a significant inhibition of HSV1-induced chorioretinitis. (b) Selective depletion of phagocytic synovial lining cells largely prevented inflammation (synovitis) during immune complex-mediated and collagen type Il-induced arthritis, and significantly reduced the propagation and exacerbation of chronic synovitis in an established arthritic knee joint. (c) Depletion of macrophages suppressed the clinical signs of experimental allergic encephalomyelitis. (d) Depletion of macrophages attenuated symptoms and mortality rate in a model of zymosan-induced systemic inflammation. (e) Depletion of macrophages considerably reduced endotoxin-induced mortality and TNFa production in animals, and reduced endotoxin-mediated fever. (f) Depletion of alveolar macrophages decreased neutrophil chemotaxis to Pseudommas airspace infections. An increasing number of studies aims at the possibilities to reduce the symptoms of inflammatory reactions using clodronate-liposomes. 4.4 Improved graft survival and functioning Graft rejection is frequently preceded by a massive infiltration of both T cells and macrophages. For that reason several studies focused on the effects of macrophage depletion on infiltration of T cells and macrophages in grafted tissues and on graft rejection. Macrophages are found in large numbers in rejected corneal allografts in rats. In animals treated post-operatively with subconjunctival injections of liposomeencapsulated clodronate at the time of transplantation and several times thereafter, grafts were not rejected during the maximum follow-up of 100 days (32). Cellular infiltration in these grafts was clearly reduced and there was a strong reduction in neovasculari/ation of the cornea. Corneal grafts in rats belonging to control groups that had received empty liposomes (containing phosphatebuffered saline only) and those that had been given no additional treatment were rejected within the usual period of 17 days. These results confirm that macrophages play a crucial role in corneal allograft rejection. 170


Other recent studies have shown that macrophages are required for T cell infiltration and rejection of fetal pig pancreas xenografts in NOD mice (33). Also, it has been shown that macrophages have a central role in the development and activation of B cell-cytotoxic T cells that cause B cell destruction resulting in auto-immune diabetes in NOD mice (34). Suppression of macrophage activity by clodronate-liposomes as a tool to improve graft survival and functioning is currently investigated in several laboratories.

References 1. 2. 3. 4. 5.

Van Rooijen, N. and Sanders, A. (1997). J. Leuk, Biol., 62, 702. Van Rooijen, N. and Sanders, A. (1994). J. Immunol. Methods, 174, 83. Allison, A. C, Harington, J. S., and Birbeck, M. (1966). J. Exp. Med., 124, 141. Sawicki, J. E. and Catanzaro, P. J. (1975). Int. Arch. Allerg. Appl. Immunol, 49, 709. Kamochi, M., Ogata, M., Yoshida, S., Matsumoto, T., Kubota, E., Mizuguchi, Y., et al. (1993). FEMS Immunol Med. Microbiol, 7, 153. 6. Husztik, E., Lazar, G., and Parducz, A. (1980). Br. J. Exp. Pathol, 61, 624. 7. Hardonk, M. J., Dijkhuis, F. W. J., Hulstaert, C. E., and Koudstaal, J. (1992). J. Leuk. Biol., 52, 296. 8. Ruttinger, D., Vollmar, B., Wanner, G. A, and Messmer, K. (1996). J. Hepatol, 25, 960. 9. Proffitt, R. T., Williams, L. E., Presant, C. A, Tin, T. W., Uliana, J. A, Gamble, R. C., et al. (1982). Science, 220, 502. 10. Van Rooijen, N., Sanders, A, and Van den Berg, T. (1996). J. Immunol Methods, 193, 93. 11. Naito, M., Nagai, H., Kawano, S., Umezu, H., Zhu, H., Moriyama, H., et al (1996) J. Leuk. Biol., 60, 337. 12. Van Rooijen, N. and Van Nieuwmegen, R. (1984). Cell Tissue Res., 238, 355. 13. Van Rooijen, N., Van Nieuwmegen, R., and Kamperdijk, E. W. A. (1985). Virchows Arch. B (Cell Pathol), 49, 375. 14. Gregoriadis, G. (1995). Trends Biotechnol, 13, 527. 15. Monkkonen, J., Taskinen, M., Auriola, S. O. K., and Urtti, A (1994). J. Drug Target., 2, 299. 16. Van Rooijen, N., Kors, N., Van De Ende, M., and Dijkstra, C. D. (1990). Cell Tissue Res., 260, 215. 17. Biewenga, J., Van der Ende, B., Krist, L. F. G., Borst, A, Ghufron, M., and Van Rooijen, N. (1995). Cell Tissue Res., 280, 189. 18. Thepen, T., Van Rooijen, N., and Kraal, G. (1989) J. Exp. Med., 170, 499. 19. Delemarre, F. G. A, Kors, N., Kraal, G., and Van Rooijen, N. (1990)J. Leuk. Btol., 47, 251. 20. Bergh, A, Damber, J. E., and Van Rooijen, N. (1993) J. Endocrinol, 136, 407. 21. Van Lent, P. L. E. M., Van Den Bersselaar, L., Van Den Hoek, A. E. M., Van De Ende, M., Van Rooijen, N., and Van Den Berg, W. B. (1993). Sheumat. Intern., 13, 21. 22. Claassen, L, Van Rooijen, N., and Claassen, E. (1990). J. Immunol Methods, 134, 153. 23. Qian, Q,, Jutila, M. A., Van Rooijen, N., and Guttler, J. E. (1994). J. Immunol, 152, 5000. 24. Nair, S., Buiting, A. M. J., Rouse, R. J. D., Van Rooijen, N., Huang, L., and Rouse, B. T. (1995). Int. Immunol, 7, 679. 25. Leenen, P. J. M., Radosevic, K., Voerman, J. S. A, Salomon, B., Van Rooijen, N., Klatzmann, D., et al. (1998). J. Immunol, 160, 2166. 26. Van Rooijen, N., Bakker, J., and Sanders, A. (1997). Trends Biotechnol, 15, 178. 27. Wolff, G., Worgall, S., Van Rooijen, N., Song, W. R., Harvey, B. G., and Crystal, R. G. (1997). J. Virol, 71, 624. 171

NICO VAN ROOIJEN AND ESTHER VAN KESTEREN-HENDRIKX 28. Worgall, S., Leopold, P. L., Wolff, G., Ferris, B., Van Rooijen, N., and Crystal, R. G. (1997). Hum. Gene Ther., 8,1675. 29. Fraser, C. C., Chen, B. P., Webb, S., Van Rooijen, N., and Kraal, G. (1995). Blood, 86, 183. 30. Terpstra, W., Leenen, P. J. M., Van Den Bos, C., Prins, A., Loenen, W. A. M., Verstegen, M. M. A., et al. (1997). leukemia, 11, 1049. 31. Badell, E., Pasquetto, V., Van Rooijen, N., and Druilhe, P. (1995). Parasitol. Today, 11, 235. 32. Van Der Veen, G., Broersma, L., Dijkstra, C. D., Van Rooijen, N., Van Rij, G., and Van Der Gaag, R. (1994). Invest. Ophthalmol, 35, 3505. 33. Fox, A., Koulmanda, M., Mandel, T. E., Van Rooijen, N., and Harrison, L. C. (1998). Transplantation, 66, 1407. 34. Jun, H. S., Yoon, C. S., Zbytnuik, L., Van Rooijen, N., and Yoon, J. W. (1999). J. Exp. Med 189, 347.

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Chapter 8 Analysis of gene expression in mononuclear phagocytes Donna M. Paulnock, Joyce E. S. Doan Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, 1300 University Avenue, Madison Wl 53706-1531, USA

Thomas A. Hamilton Department of Immunology, Cleveland Clinical Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-9329, USA

1 Introduction Specific gene expression is a hallmark of differentiated cell populations, as well as of cellular activation within specific cellular subsets. This is particularly true in the context of the mononuclear phagocyte, which has long been described on the basis of changes in cellular phenotype and function associated with activation by proinflammatory or stimulatory agents (1, 2). This chapter will provide a discussion of methods used for analysis of gene expression in the context of mononuclear phagocytes. Even in such a restricted context, however, this represents a broad and diverse subject. Furthermore, gene expression may be modulated both quantitatively and qualitatively at multiple levels, which can be broken down into transcriptional, post-transcriptional, translational, and posttranslational (subcellular localization/secretion) categories. In order to maintain reasonable scope, this chapter will be limited to considerations of experimental methodologies which address the early aspects of gene expression, i.e. transcriptional and post-transcriptional regulation. The major focus of this chapter will be on nucleic acid-based experimental methodologies. The first experimental measurements considered are determination of RNA transcript abundance which can be used for analysis of the frequency with which individual genes are transcribed and for assessment of multiple post-transcriptional mechanisms including mRNA processing, nuclearcytosolic mRNA transport, and cytosolic mRNA decay. Subsequently, analysis of nucleic acid sequences which confer cell and/or stimulus-specific gene transcription patterns will be discussed. The latter will include the identification and characterization of protein factors which recognize regulatory sequence motifs. 173

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The primary methodologies employed for analysis of all these cellular processes can be placed into three broad categories: (a) The measurement of specific RNA levels and metabolism. (b) The use of transfected DNA sequence(s) to identify regulatory motifs in DNA through which gene transcription and RNA metabolism are controlled. (c) The measurement of protein-nucleic acid interactions. Consideration of experimental techniques for addressing the more distal events of macrophage activation, such as specific protein expression or cellular function, that follow the regulation of gene expression are discussed in detail in the other chapters in this volume. In this chapter, we will present the advantages and/or disadvantages of basic strategies employed in each of the three categories of laboratory methodology outlined above, as well as provide detailed methodologies that have been successfully employed in populations of mononuclear phagocytes. Although this unique cellular subset exhibits some features which challenge the application of molecular technologies, essentially all of the pertinent techniques can be successfully adapted to one or more macrophage populations.

2 Detection and quantification of specific RNA levels 2.1 Basic principles Recombinant DNA technology provided the first ability to isolate gene sequences and prepare such molecules in unlimited abundance. The exquisite specificity of pairing between complementary strands of DNA and/or RNA in turn provided the means to measure the presence and quantity of specific genes and/or gene products in living cells and tissues. Perhaps the most common measurement performed in the analysis of gene expression is of the presence or abundance of specific mRNAs. Though this is now accomplished routinely, the major problem in the use of mononuclear phagocytes for determination of specific mRNA levels is presented by their unusually high content of ribonuclease activity, especially in cells which have been exposed to proinflammatory and/or activating stimuli. This problem may be compounded by a relatively low content of total RNA.

2.2 Preparation of total cellular RNA Determination of mRNA levels in whole cell extracts provides a measure of the total amount of specific mRNA. In contrast to analysis of direct transcription using isolated nuclei, preparing RNA from intact macrophages may be appreciably affected by ribonuclease activity. Many investigators routinely use commercially available RNA extraction/isolation methodologies; the parameters of isolation by such a technique are described below. However, it should be noted that these reagents may not provide a fully satisfactory outcome in all macrophage 174

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

populations. Problems encountered may include heterogeneously sized mRNAs (as detected by Northern hybridization) as well as reduced storage life of isolated mRNA. In the event that such changes are observed, an alternative technique may be required to obtain satisfactory mRNA samples. One method to prepare high quality macrophage mRNA, originally developed for preparation of RNA from nuclease-rich tissues, uses guanidine isothiocyanate extraction followed by centrifugation through a caesium chloride cushion (3). This method not only relies upon the denaturing activity of the extraction conditions, but the ability to physically separate RNA from both protein and DNA contaminants. RNA prepared in this way from tissues and even highly activated macrophage cultures can be stored indefinitely without evidence of degradation. Protocol 1 describes a reproducible method for isolation of macrophage RNA, using a commercially available extraction buffer. This technique provides a simple and direct method for the isolation of high quality RNA, free of contaminating DNA, from commonly used macrophage populations. Such cells include macrophages isolated from the peritoneal cavity, the spleen, and the peripheral blood, as detailed in Chapters 1 and 2. For this and all subsequent protocols for analysis of RNA species, it is critical to use only RNase-free reagents and labware, as well as to wear gloves while handling reagents and samples and while performing all manipulations.

Preparation of total cellular RNA from cultured macrophages Equipment and reagents • Sterile and nuclease-free microcentrifuge tubes (1,5-2.0 ml, Life Science Products, Inc.) • Sterile and nuclease-free micropipette tips, with aerosol barrier (Molecular BioProducts, Inc.) • Microcentrifuge (Dupont/NEN) • Macrophage population of interest (see Chapters 1 and 2 for details) • RNA STAT-60 extraction buffer (Tel-Test 'B', Inc.) • Chloroform • Isopropanol • 75% ethanol • 500 mM MgCl2

DEPC-treated H2O: add 1 ml of diethyl pyrocarbonate (DEPC, Sigma Chemical Co.) per litre of water, stir for 1 h at room temperature, and autoclave for 1 h (to hydrolyse any remaining DEPC)a 10 mM dithiothreitol (DTT, Sigma Chemical Co.) RNase-free DNase 1.2.5U/ml (Sigma Chemical Co.) Prime RNase inhibitor (5' -» 3', Inc.} 50 mM Tris-HCl pH 7.4 DNase mix (per reaction): 2 ul of 500 mM MgCl2,1 ul of 100 mM DTT, 4 ul DNase I, 2.5 ul RNase inhibitor, and 74.7 ul of 50 mM Tris-HCl

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Method 1 Remove growth medium from adherent macrophage cultures by aspiration, or pellet non-adherent cells and remove supernatant fluid." 2 Add RNA STAT-60 (2 ml per 5-10 x 106 cells) directly to the macrophage monolayer or pellet. 3 Lyse cells by passing extract repeatedly through a micropipette tip until solution is no longer viscous. 4 Transfer lysate in 1 ml aliquots to microcentrifiige tubes. 5 Vortex lysates vigorously. 6 Incubate at room temperature for 5 min. 7 Add 200 ul of chloroform to each tube and vortex briefly to mix. 8 Incubate 2 min at room temperature. 9 Centrifuge samples for 15 min at 12 000 g (13000-14000 r.p.m.} and 4°C in a microcentrifuge. 10 Transfer the aqueous (clear) phase from each gradient to a fresh microcentrifuge tube, taking care to not disturb the interphase. 11 Store the organic phase at 4°C until retrieval of RNA is confirmed, then discard." 12 Add 500 ul isopropanol to each tube and invert several times to mix, 13 Incubate tubes for 5-10 min at room temperature (for a large original cell number), or 30 min to 24 h at 40C (for a small original cell number) to precipitate the RNA. 14 Centrifuge samples as in step 9. 15 Carefully remove the supernatant, using a micropipette tip to avoid dislodging the RNA pellet. 16 Wash the RNA pellet once with 1 ml of 75% ethanol for each 1 ml of RNA STAT-60 originally used, by centrifugation as in step 9. 17 Invert tubes to detach pellets from the wall of the tubes. 18 Incubate at room temperature for 5-10 min. 19 Centrifuge the samples for 5 min. under the same conditions as in step 9. 20 Remove the supernatant fluid from each tube. 21 Open the tubes and allow the pellets to dry at room temperature until they become translucent (5-10 rntn).r 22 Resuspend each sample in DEPC-H2O, pooling like samples in a total volume of 16 ul. 23 Add 84.2 ul of DNase mix to each pooled RNA sample.e 24 Incubate samples for 30 min at 37°C. 25 Repeat the RNA extraction as described in steps 2-21 using the following volumes for each sample: 300 ul RNA STAT-60, 60 ul chloroform, 150 ul isopropanol, and 300 ul ethanol.

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26 Resuspend each pellet in 20 ul DEPC-H2O. 27 Determine RNA concentrations and purity by spectrophotometric analysis, reading A230, A260. and A2K() of a 1:500 dilution of each sample; the final RNA concentration should be as close to 2 mg/ml as possible.e 28 Store the purified samples at -20 °C until use. a

DEPC is a suspected carcinogen; appropriate safety precautions should be observed during its use. b RNA can also be purified from individual tissues by this method. c After completion of the procedure, the remaining gradient components can be discarded, using appropriate chemical safety procedures. d Do not over-dry the pellets, as this will hinder their re suspension. e The DNase mix can be prepared in bulk, but should be made fresh immediately prior to use, f For spectrophotornetric readings: A260 x 40 x 0.5 = RNA concentration in mg/ml (assuming a path length of 1 cm for the spectrophotometer). A260:A280 = measure of protein contamination; the ideal range is 1.7-2.0, A260:A230 = measure of contamination with guanidine thiocyanate; the ideal range is 1.8-2.0. If the concentration of RNA in any sample is greater than 2 mg/m1, dilute those samples with DEPC-H2O, take a new A260 reading on the spectrophotometer.

2.3 Nuclear run-on analysis The direct determination of transcriprional activity for a specific gene is most often accomplished using nuclear run-on (or run-off) assay. This involves the analysis of specific RNA transcripts which arc generated in vitro in isolated nuclei incubated in the presence of radiolabelled ritaonucleotides. In order for transcription to occur, RNA polymerases must bind to the promoter region of DNA. The more actively a gene is being transcribed, the more polymerases found on that given promoter. The goal of nuclear run-on analysis is to determine the extent to which a given gene is being transcribed under different experimental conditions. To achieve this, macrophages exposed to experimental stimulation are lysed in non-ionic detergent and intact nuclei rapidly prepared. The nuclei are isolated such that the RNA polymerases in the process of transcribing genes are halted at whatever point in the process they are located. The isolated nuclei are subsequently put into an in vilru environment, in the presence of radioactive nticleotides, that favours the continuation of transcription. The RNAs subsequently transcribed arc radioactive. In this system, no new transcripts are initiated; only those RNA molecules that were already initiated at the time of nuclei harvest are continued (4). following a short incubation (30-60 minutes), total RNA is isolated and used to hybridize with filters bearing defined cDNA fragments. Because no new transcription complexes are initiated in isolated nuclei, the intensity of the hybridization signal is proportional to the number of transcription initiation complexes formed prior to cell lysis and generally gives a reasonable estimate of changes in specific gene transcription frequency. 15y comparing the transcriptional 177


activity of given genes from cells that were differentially stimulated, one can gain insight into the effect that a given signal has on RNA synthesis. Protocol 2 provides one method for purification and analysis of nuclear RNA transcripts. In this procedure, the majority of nuclease activity is discarded with the cytosolic fraction and the RNA products of transcription do not need to retain original length for proper analysis. Thus, the degradation of RNA is an infrequent problem in this assay. The assay is only modestly quantitative and the outcome can be influenced by multiple variables including rates of elongation in vitro as well as premature transcription termination.

Detection of nuclear RNA by nuclear run-on assay Equipment and reagents • Nitrocellulose membrane (Schleicher and Schuell) • Commercial slot blot apparatus (Bio-Rad) • Hybridization oven (Unitherm Co., Inc.) • Apparatus for UV cross-linking of membranes (Stratagene) • Water-baths at 30°C,65°C, and 370C • [32P]UTP (3000 Ci/mM specific activity, Dupont/NEN): 1 mCl is sufficient for up to ten samples • Liquid scintillation counting cocktail and glass scintillation vials of choice • Liquid scintillation counter (Packard Instruments) • Pasteur pipettes, sterilized by autoclaving • Sterile microcentrifuge tubes (0.5 ml and 1.5 ml) • Radiographic imaging film (Kodak) • Cassettes for auto radiography (Fisher Scientific) • Isolated nuclei from macrophage population of choice (see Protocol 8) • Bacterial plasmid containing the gene insert of interest, and the appropriate control DNA (see Protocol 4) • 1 M Tris pH 8.0 (autoclave to sterilize) • 1 M MgCl2 • RNase-free DNase at 10 U/ul (Boehringer Mannheim)

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• 3 M KC1 • Nucleotide bases: 100 mM in lithium salt solution (Boehringer Mannheim) • 10 x SET buffer: combine appropriate volumes of 20% SDS, 0.5 M EDTA, 1 M Tris pH 7.8 to give a final concentration of 10% SDS, 50 mM EDTA, and 100 mM Tris pH 7.4 (use autoclaved solutions and autoclaved bottle for preparation—do not autoclave final solution) • Proteinase K solution: 20 mg/ml in 1 x SET buffer (store at -20°C) • PCI (phenol, chloroform, isoamyl alcohol) solution: first combine 24 parts chloroform and one part isoamyl alcohol. Prepare final PCI solution by combining 1 part Cl with 1 part phenol.a PCI can be stored at room temperature until used. • 10 M ammonium acetate (sterilize by passage through 0.2 um filter) • Isopropy1 alcohol • 2 M and 3 M NaOH • 0.48 M Hepes buffer (free acid form, Sigma Chemical Co.) • 100% ethanol • Salmon sperm DNA: boil a freshly prepared 5 mg/ml solution for 10 min, cool on ice for 1 min » 20% (w/v) SDS (Sigma Chemical Co.) • 0,25 M EDTA


• 5 M NaCl • 1 M phosphate buffer pH 6.5: combine 342.5 ml of 2 M NaH2PO4 and 157.5 ml of 2 M Na2HPO4, add H2O to a final volume of 1 litre • 20 X SSC: 3 M NaCl, 0.3 M sodium citrate pH 7.0 • 50 x Denhardt's buffer: combine 5 g Ficoll, 5 g polyvinyl pyrrolidone, and 5 g BSA Fraction V (all from Sigma Chemical Co.): add H2O to a final volume of 500 ml • TE buffer: 10 mM Tris pH 8.0, 1 mM EDTA • 5 x run-off buffer (made fresh): for 0,5 ml (sufficient buffer for six samples), combine 1 M Tris pH 8 (11.25 ul). 1 M MgCl2 (5.625 (ul), 3 M KC1 (112.5 ul), 100 mM each of ATP, CTP, and GTP (5.625 u1 of each cold base}, 315 ju.1 H20 (filter sterilized through 0,2 um filter; do not use DEPC water); vortex to mix

1 M TES pH 7.4 (Sigma Chemical Co,): filter sterilize and store at room temperature; if it becomes cloudy, make a fresh solution Freezing buffer: combine 0.5 M Tris pH 8.3, 5.0 M NaCl, 1 M MgCl2, 0.25 M EDTA, and glycerol to give a final concentration of 50 mM Tris, 40% glycerol, 5 mM MgCl2, and 0.1 M EDTA {all initial stocks should be sterilized by autoclaving) Pre-hybridization buffer (100 ml): combine 5 ml of salmon sperm DNA, 2 ml of 50 x Denhardt's buffer, 5 ml of 1 M phosphate buffer pH 6.5, 25 ml of 20 x SSC, 5 ml of 20% SDS, and 58 ml H2O Hybridization buffer (100 ml): combine 1 ml of 1 M TES pH 7.4.1 ml of 20% SDS, 4 ml of 0.25 M EDTA, 6 ml of 5 M NaCl, 2 ml of 50 x Denhardt's buffer, and 88 ml H2O

A Preparation of membranes hybridized with plasmid DNA (done prior to the day of the run-on experiment) 1 Make a solution of 1 ug plasmid in 20 ul of TE buffer, 2 Add 0.1 vol. of 3 M NaOH (final concentration, 0.3 M NaOH) to each plasmid preparation. 3 Incubate at 65°C for 1 h (this creates nicks and denatures plasmids to yield linear structures for hybridization). 4 Place plasmid preparations on ice and add an equal volume of 2 M ammonium acetate pH 7.0, prepared from 10 M stock. 5 Calculate volume of each plasmid preparation that will yield 7.5 ug of DNA. 6 Soak nitrocellulose membrane in water followed by 10 x SSC for approx, 1 min each. 7 Place membrane in slot blot apparatus and apply vacuum. 8 Apply 7.5 ug of each plasmid DNA to membrane by spotting with a micropipette tip.b 9 After spotting, wash DNA through in each spot with a total of 500 ul of 10 x SSC (apply 200 ul, 200 ul. and 100 ul volumes in sequence). 10 Remove filters from apparatus and permit to dry for 20-30 min at room temperature. 11 Cross-link the plasmid DNA to the membrane.

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12 Store membranes at room temperature until use.c 13 24 h prior to beginning the run-on assay, pre-hybridize the filters by incubating in pre-hybridization buffer overnight at 65°C.d B Nuclear run-on assay 1 Defrost isolated macrophage nuclei, 2 Defrost [32P]UTP using appropriate radiosafety protocols. 3 Make up sufficient 5 x run-on buffer in sterile microcentrifuge tube to give 60 ul per sample. 4 Bring 150 |j,l of isolated nuclei preparation to 200 ul with freezing buffer in a 0.5 ml microcentrifuge tube, preparing a separate sample for each DNA to be assessed. 5 Add 60 ul of run-on buffer to each sample. 6 Add 100 mCi of labelled UTP to each sample, in a final volume of 30 ul. 7 Cap tube and vortex briefly to mix. 8 Incubate at 30°C for 30 min. 9 At the end of this incubation, add 15 ul of RNase-free DNase I. 10 Incubate again at 30°C for 5 min. 11 Add 36 p.1 of 10 x SET buffer and 10 u1 of proteinase K (10 mg/ml stock). 12 Heat tubes transiently to 65 °C, if needed. to redissolve SDS. 13 Incubate at 37 °C for 45 min. 14 Add 360 u1 (approximately equal volume) of PCI at completion of incubation and vortex to mix. 15 Spin 5 min in microcentrifuge at 12 000 gat room temperature to form a gradient. 16 Remove the upper (aqueous) phase containing the RNA using a sterile Pasteur pipette (avoid touching interface or lower phase). 17 Transfer the RNA to a fresh microcentrifuge tube and hold on ice. 18 Add 100 ul of 1 x SET buffer to the original tubes. 19 Re-extract the interface and lower gradient phases as in steps 14 and 15. 20 Remove the aqueous phase from the second gradient using a sterile Pasteur pipette as in step 16. 21 Pool the aqueous phases from the two extractions in a single tube. 22 Add 135 ul of 10 M ammonium acetate and 595 ul of isopropyl alcohol to each tube. 23 Placetubesat -70°Ctbr20min. 24 Remove tubes from the freezer and thaw on ice. 25 Spin tubes for 10 min at 4°C, 12000ginaraicrocentrifuge to pellet the RNA. 26 Remove 'hot' supernatant at the completion of the centrifugation and collect for proper radioactive disposal, 27 Resuspend the RNA pellet in 180 ul of 1 x STE buffer.

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28 Add 20 ul of 2 M NaOH to each tube. 29 Incubate on ice for 10 min. 30 Add 200 ul of 0.48 M Hepes to each tube. 31 Precipitate with 880 (ul of ethanol overnight at -20°C (or on dry ice for 10 min), 32 Pellet the precipitated RNA by centrifugation for 20 min at 4°C, 12 000g, 33 Resuspend pellet in 1 ml of run-on hybridization buffer. 34 Count 10 ul of the resuspended RNA in scintillation fluid using a glass scintillation vial. 35 Add 5-10 x 106 c.p.m. of radioactivity per membrane in 2-5 ml.d 36 Incubate membranes at 65#°C for 36-48 h with frequent mixing. 37 At the completion of the incubation, rinse the membranes briefly in a 1:1 mixture of 0.1 x SSC and 0.1% SDS at room temperature and remove the wash fluid (fluid from steps 37 and 38 should be disposed of as radioactive waste). 38 Wash membranes twice, for 30 min each, at 65 °C in a 1:1 mixture of 0.1 x SSC plus 0.1% SDS. 39 Expose membranes to radiographic film for appropriate times and develop for autoradiography. a

The phenol used in this procedure should be redistilled from commercially available products as follows. Melt phenol at 65 °C and equilibrate. Place in a separatory funnel with equal volume of 0.1 M Tris pH 8.0 and mix by shaking. Let stand for 10 min until the mixture separates—the phenol is in the lower (aqueous) phase. Retrieve phenol and re-extract a total of four times, or until the pH of the aqueous phase is approx. pH 7.6. Extracted phenol should be used immediately or frozen at -20°C until use. Appropriate safety precautions should be observed during phenol extraction, including protective eye wear. b Pre-hybridized membranes can only be used once, and the radio labelled RNA is used in great excess; thus, it is most efficient to spot as many genes as possible on each slot blot. c Generally, membranes are prepared over two to three days, using DNA that has been purified over the course of a few weeks (e.g. for five to ten genes). If needed, membranes can be cut with a razor blade prior to hybridization. d It is generally advisable to keep the volume of hybridization buffer as small as possible, to ensure the greatest interaction of the fluid with the membrane during incubation.

2.4 Quantification of specific mRNAs Levels of specific mRNAs may be assessed quantitatively using numerous methodologies, including Northern analysis, KNase protection assay, and reverse transcription-polymerase chain reaction (RT-PCR). Northern hybridization is the least sensitive method but provides both the size of the RNA species (based upon electrophoretic mobility) and sequence specificity. This method is also the easiest to use to obtain quantitative determination of specific RNA species. General 181

JOYCE E. S. DOAN ET AL

protocols available for Northern hybridization of RNA from other cell populations can be readily applied to analysis of RNA species purified from macrophages (5, 6). Greater sensitivity can be achieved using RNase protection analysis, in which cellular RNA is hybridized with radiolabelled single-stranded hybridization probes specific for selected genes. Protected fragments are then separated by electrophoresis and hybridized radioactivity is quantirated by autoradiography. This approach requires less total RNA and exhibits high sensitivity but does not generally provide a direct measure of RNA size. In addition, variations in probe labelling can make it difficult to compare quantification measurements. A variety of commercially prepared kits currently are available to allow the analysis of a spectrum of specific RNA species by RNase protection assay. Finally, analysis of specific RNA species using reverse transcription-polymerase chain reaction (RT-PCR) methodologies is the most sensitive approach and can detect even a single mKNA molecule. This sensitivity is, however, offset by potential complicating features, include possible contamination with genomic sequences, the impact of primer selection, cycle time on the efficiency of PCR amplification, and the difficulties inherent in quantitative analysis of products amplified by this method. Nevertheless, RT-PCR analysis can be an effective and rapid method for screening RNA isolated from a small number of macrophages for the expression of a large array of genes. In addition, specific methods are available to allow the quantilation of the amplified mRNA (7). Protocol 3 describes one method for the assessment of cytoplasmic mRNA species using the RT-PCR method.

Analysis of specific gene expression by RT-PCR Equipment and reagents • Horizontal gel apparatus, including comb with 4 mm teeth (International Biotechnologies, Inc.) • Electrophoresis power supply (Bio-Rad) • UVti-ansilluminator(FotoDyneCo.) • Polaroid camera with shield fitted to the transilluminator (FotoDyne Co.) • Polaroid 3000 black-and-white film (Fisher Scientific) • MMLV reverse transcriptase (200 U/ul, Life Technologies) • MMLV reverse transcriptase 5 x buffer (provided with MMLV-RT) • 0.1 M DTT • Prime RNase inhibitor

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• dNTP mixture: 25 mM each dATP, dCTP, dGTP, dTTP (each dNTP obtained separately from Life Technologies) • Oligo dT primers (Roche Biomedical) • Taq DNA polymerase: 5 U/ul (Molecular Biosciences, Inc.) • Sterile double distilled H2O (ddH2O) • RT mix, prepared per reaction as follows (final concentrations are given in parentheses): 6 ul MMLV-RT 5 x buffer (1 x), 1 ul Prime RNase inhibitor (lU/ml), 1.2 ul dNTP mixture (1 mM each), 2 ul oligo dT primers (0.1 ug/ug total RNA), 3 ul of 0.1 M DTT (10 uM). 1 ul MMLV-RT (200 U/10 ug total RNA)


Mg-free Taq polymerase 10 x buffer (provided with Taq polymerase} 25 mM MgClz DEPC-H20 (see Protocol 1) PCR mix, prepared per reaction as follows (final concentrations are given in parentheses): 33.08 ul ddH2O, 0.625 ul dNTP mixture (312 uM), 5 u1 Taq 10 x buffer (Mg-free) (1 x), 6 ul of 25 mM MgCl2 (3 mM),a 1 u1 pooled (5' + 3') primers (500 nM), 0.3 u1 Taq DNA polymerase (1.5 U) Oligonucleotide sense and antisense primers specific for the mRNA of interest (Life Technologies)" Chill-Out reagent (MJ Research) Nuclease-free, PCR-compatible tubes or plates (MJ Research) Sterile, nuclease-free micropipette tips with aerosol barriers

Programmable thermocycler (MJ Research) Electrophoresis grade agarose (Fisher Scientific) Hthidium bromide staining solution: add 5 u1 of a 0.5 mg/ml ethidium bromide stock solution to 50 ml H20 (store at room temperature; protect stock and working solutions from light)1 50 x TAE running buffer: combine 242 g Tris base, 57,1 ml glacial acetic acid, and 37.2 g sodium EDTA (disodium salt, dihydrate, Sigma Chemical Co.), add ddH2O to 1 litre 10 x DNA loading dye: 0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene cyanol, 30% (v/v) glycerol (all from Sigma Chemical Co.) in 10 x TAE buffer 1 kb DNA ladder molecular weight standard (Life Technologies)

A Preparation of cONA 1 Prepare RT mix in bulk, making 10% more than the volume needed. 2 Dilute 10 ug of total cellular RNA, prepared as in Protocol 1, to 15,8 ul with DEPC-H20 3 Incubate RNA at 65°C for 5 min. 4 Transfer samples to 37°C and incubate for approx. 2 min. 5 Add 14.2 ul of the RT mixture to each tube. 6 Pipette the mixture several times to combine. 7 Incubate at 37°C for 70 min. 8 Transfer samples to 95 °C for 5 min to deactivate the MMLV-RT. 9 Transfer samples to ice and incubate for 5 min. 10 Spin samples briefly (5-10 sec) in a microcentrifuge to collect all liquid. 11 Add 170 ul sterile ddH2O to each tube and store the cDNA at -20°C until use.d B Amplification of cDNA 1

Program thermocycler for appropriate amplification conditions.e

2

Keeping all reagents on ice, prepare PCR mixture in bulk, making 10% more than the volume needed.

3

Aliquot 46 ul of the PCR mixture per cDNA sample into PCR-compatible tubes or microtitre plate wells.

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4 5 6 7

Add 4 u1 of target cDNA to each aliquot of the PCR mixture, Overlay samples with approx. 15 ul of Chill-Out.' Once samples are prepared, start the thermocycler, When the thermocycler reaches temperature. place samples on the temperature block and allow amplification to proceed,

C Electrophoretic analysis of amplified products 1 Prepare a 1.8% solution of agarose using 1 x TAE buffer. 2 Pour a gel of appropriate size for the number of samples and allow it to polymerize. 3 Combine 1 ul of PCR-amplified product with 10 ul of 10 x DNA loading dye and mix well. 4 Add 10 ul of combined product (or 5 ul of 1 kb ladder) to individual wells of the gel. 5 Electrophorese the samples for 20-30 min at 150 V, or until the two visible bands are well-separated and the leading band has migrated approx. half of the gel length.55 6 After turning power supply off, remove gel carefully from the electrophoresis apparatus. 7 Stain the gel by submerging it for 1-5 min in the ethidium bromide solution.' 8 Rinse the stained gel for 1-5 min in water. 9 Place the gel on the transilluminator and observe amplified products using UV light. 10 Record the labelled products by photography, using appropriate exposure conditions. J

The concentration of MgCl2 needed varies with both primer pair and amplification conditions and should be optimized for each primer pair used. l> Primer pairs specific for a variety of genes expressed in macrophages are commercially available from several manufacturers (including ClonTech and R&D Systems). Primers appropriate for RT-PCR use also can be designed by individual laboratories, using a computer program such a Oligo 4.0 (National Biosciences, Inc.) or the equivalent. r To dispose of the ethidium bromide, add 50 ml of household bleach (5% sodium hypochlorite) to 100 ml of the staining solution, let the mixture sit at room temperature for 24 h, then dispose of it down the drain, along with a 10 x volume of water,

d DEPC-H2O inhibits the polymerase chain reaction and should not be used to dilute the cDNA samples. e

Appropriate amplification conditions are provided by the manufacturer when commercially available primers are purchased. Amplification conditions for lab-designed primer pairs, including the optimal annealing temperature and the number of amplification cycles needed,

should be determined empirically. r

The addition of Chill-Out prevents sample evaporation during PCR amplification and due to its chemical properties (i.e. becoming solid when incubated at 4 °C) allows for efficient sample retrieval. If this is not available, a similar volume of mineral oil can be used for sample overlay. % Observe appropriate safety conditions and follow manufacturer's instructions for electrophoresis. Wear gloves throughout the procedure.

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3 Gene transfer 3.1 Basic principles Perhaps the most powerful application of recombinant DNA technologies is the ability to precisely manipulate the genotype and phenotype of a cell. This is accomplished by the intracellular delivery of DNA molecules of defined sequence which have the capacity to function in the recipient cell. This technology provides the opportunity to evaluate the biological information and function specified by the nucleotide sequence of a gene and can frequently distinguish the function of normal and mutant gene products. In addition, the regulatory properties of specific nucleotide sequences can also be assessed. While these methods have led to the development of highly sophisticated strategies to change the genotype of whole organisms (either by addition or deletion of genetic material), the following discussion will target specific issues relating to gene transfer methodologies in cultured mononuclear phagocytes. Gene transfer strategies generally fall into two categories: those which involve short-term or transient expression, and those in which the transferred gene is permanently integrated in the genome of the recipient cell. Each of these approaches has distinct advantages and disadvantages both in general terms and with respect to mononuclear phagocytes. The primary advantage of transient transfection is its potential application in the largest selection of cell types. It has been difficult to obtain stable integration of the transgene material in many differentiated cell types including macrophages. In many cases, however, these cells can be transiently transfected. Furthermore, the transient gene transfer assays can be conducted with much greater speed because selection and expansion of stably transfected clones is not required. Although the focus in the following section is on methods for the transient transfection of macrophage populations, each of these methods can be used to generate stable transfectants, assuming the plasmid DNA carries an appropriate selectable marker.

3.2 Experimental strategies Gene transfer has been applied in a wide variety of settings for analysis of gene expression. Perhaps the most common use is to identify the functional role of a transfected normal or mutant gene. For this purpose, the coding sequence of the target gene can be cloned into one of numerous commercially developed expression plasmids which provide constitutive or inducible transcription promoters, as well as non-coding sequences necessary for transcription termination and transcript processing events such as capping, polyadenylation, and nuclearcytoplasmic transport. These properties all contribute to achievement of high level transgene expression in the transfected cell population. Transfected cells are then assessed for changes in specific cellular phenotypes as a result of the introduction and expression of the normal or mutant gene. Gene transfer has also been used successfully to study the functional attributes of non-coding DNA sequences which are frequently critical in controlling ex185


pression of specific genes. Evaluation of transcriptional control frequently requires the analysis of regulatory nucleotide sequences in gene promoters/enhancers. Likewise, primary transcript processing, nuclear-cytosolic transport, subcellular localization, translational activity, and mRNA stability may be controlled by sequences in intron regions, 5' and/or 3' untranslated regions, and coding regions of a specific transcript. The experimental strategies employed commonly use plasmids in which the sequence of interest is linked with a reporter gene which is placed under regulatory control of the test sequence. Thus reporter gene transcription is determined by enhancer/promoter sequences, which generally are not transcribed themselves, while post-transcriptional controls involve sequences located within primary transcripts or mature mRNAs. Manipulation of each category of sequence allows precise identification and characterization of regions which control one or more aspects of the expression pattern of a specific gene. The choice of reporter gene maybe influenced by the nature of the aspect of gene expression which is under study as well as the availability of specific assessment methods. Generally speaking, the choice of reporter gene is determined experimentally in each situation and can involve read-out of gene expression via spectrophotometric (e.g. p-galactosidase) (8), fluorescent (e.g. firefly luciferase) (9), or radioactive label (e.g. chloramphenicol acetyltransferase) (10) measurements.

3.3 Transient transfection Many cell types, including macrophages, are difficult to transfect, even using transient strategies. One important consequence is that the proportion of cells in a culture which receives the transgene is small (often less than 20%). Under these circumstances, the outcome measures do not fully represent the behaviour of the whole population. Indeed, measurement of the impact of a transfected gene on the behaviour of an endogenous gene is likely to be uninterpretable. This problem can be overcome by co-transfecting cells with multiple DNAs, at least one of which functions as a reporter gene by virtue of the sensitivity to the action of the transgene. Successfully transfected cells will receive copies of all DNAs and the outcome measurement (reporter gene product function) will thereby be restricted to transfected cells. One drawback of this latter property is that transfected cells take up large amounts of DNA and consequently express large amounts of the transgene product. While this has the advantage of increasing sensitivity, it may produce a non-physiological circumstance where the level of gene product markedly exceeds the amount present in normal cells. Transient transfections are also limited with respect to the number of different genes which can be transferred at one time. When concentrations of input DNA are too high, generalized cell toxicity may result, undermining the value of the experimental data. Primary cells, including normal macrophages obtained from animals or humans, are most often limited to transient transfection only and that is often achieved with only low frequency. Thus long-term cultured cell lines of macrophage 186


lineage are often requisite to successful application of gene transfer technologies in the analysis of macrophage gene expression. There are a wide variety of transfection strategies and many have been applied in mononuclear phagocytes, though with variable success in each case. The most commonly reported methods include the use of DEAE dextran, electroporation, or liposomal preparations to achieve effective gene transfer. The liposomal reagents are the most recently developed tool for transfection procedures. Many of these are now available commercially and are highly efficient; their use should substantially expand the application of this technology to mononuclear phagocyte populations and other cell types. Protocol 4 describes one method for the preparation of plasmid DNA for use in transfection experiments; Protocols 5-7 then provide three different methods for transient transfection of macrophages using this supercoiled plasmid DNA, all of which have been used successfully to transfect macrophages.

Purification of supercoiled plasmid DNA Equipment and reagents • 20-gauge and 22-gauge needles • 3 ml syringes • 15 ml conical polypropylene centrifuge tubes • Microcentrifuge tubes (1.5-2.0 ml) • Heat-sealable tubes for ultracentrifiigation and sealing device (Amicon) • Test-tube support stand and clamp to secure centrifuge tubes for sample retrieval • Ehrlenmeyer flasks of appropriate size for bacteria] culture • 250 mi polypropylene centrifuge bottles with screw caps {Fisher Biotechnologies} • UV lamp • Bacteria containing plasmid of choice for purification • Glucose buffer: 50 mM glucose, 25 mM Tris-HCl pH 8.0. 10 mM EDTA

• Lysozyme (Sigma Chemical Co.) • 0.2MNaOH • 2% (w/v) SDS prepared in H20 • 3 M sodium acetate pH 4.8 • 7.5 M ammonium acetate • Isopropanol • 70% ethanol • TE buffer: 10 mMTris-HCl pH 7.5. 1 mM EDTA • OptiPrep (60% iodixanol: Life Technologies) • 0.5% DAPI (Sigma Chemical Co.) • Terrific broth (TB) (1 litre): prepare one stock solution of 12 g tryptone, 24 g yeast extract, and 0.5 ml of 80% glycerol plus H2O to 900 ml, and one stock solution of 2.31 g KH2PO4 (anhydrous) and 12.54 g K2HPO4 plus H2O to 100 ml. Autoclave both stocks, cool to 60°C, and combine.

Method 1 Inoculate 250 ml TB with 1 ml of the bacterial culture in exponential phase of growth, 2 Incubate the culture for 16 h at 37 °C with aeration.

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3 Centrifuge the 16 h culture fluid at 2000 g for 20 min at room temperature, using two 250 ml centrifuge bottles. 4 Remove and discard the supernatant fluid from each bottle. 5 Resuspend each of the cell pellets in 10 ml glucose buffer. 6 Add lysozyme to a final concentration of 5 mg/ml in each bottle. 7 Incubate at room temperature for 10 min. 8 Add 20 ml of a 1:1 mix of 0.2 M NaOH and 2% SDS to each bottle. 9 Swirl gently and incubate again at room temperature for 10 min. 10 Transfer the culture bottles to ice. 11 Add 15 ml of 3 M sodium acetate to each bottle and incubate for 10 min. 12 Centrifuge the samples at 25 000 g for 30 min at 4 °C, 13 Transfer each supernatant preparation to a fresh 250 ml bottle; if the supernatant is not clear, centrifuge again as in step 12 to ensure the purity of the final preparation, 14 Add 0.6 vol. of isopropanol to each sample. 15 Incubate at -20°C for at least 30 min. 16 Centrifuge samples at 20 000 g for 30 min at 4°C to pellet the DNA. 17 Wash each DNA pellet briefly by adding 20 ml of 70% ethanol and centrifuging as in step 16. 18 Carefully remove the ethanol wash solution by aspiration, 19 Allow the DNA pellets to dry briefly. 20 Resuspend the washed and dried DNA pellets in 10 ml TE buffer. 21 Add OptiPrep to a final concentration of 27% (v/v), and DAPI to 0.005% (v/v), to each sample.a 22 Aliquot samples into ultracentrifuge tubes, using a 5 ml syringe and 18-gauge needle, and heat-seal according to manufacturer's instructions (fill each tube completely; if additional volume is needed, use a solution of TE/27% OptiPrep/0.005% DAPI). 23 Centrifuge samples for 16-24 h at 300 000-350 000 g and 4°C. 24 Secure tubes for sample retrieval using a clamp mounted on a test-tube support stand. 25 Illuminate the tubes with long-wave ultraviolet light in order to visualize the band

27 28 29 30

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of supercoiled plasmid DNA.b 26 Carefully puncture the top of each tube with in place. Retrieve the plasmid DNA by aspiration, using a 3 ml syringe and 20-gauge needled Pool identical samples in 15 ml conical centrifuge tubes, Add 0.5 vol. of 7.5 M ammonium acetate and 1 vol. of 100% ethanol, based on the volume of plasmid DNA retrieved, to each tube and mix by inversion, Incubatefor20~30minat-70°C.


31 Aliquot samples into 1.5 ml microcentrifuge tubes. 32 Centrifuge at 12000-15000 r.p.m. and 4°C in a microcentrifuge for 30 min (if no precipitate is visible) or 5 min (if precipitate is readily visible). 33 Resuspend pellets in an appropriate volume of TE buffer." 34 Measure A260 and A280 to determine the concentration and purity of plasmid samples.e a

This is easiest to execute in 5 ml increments; use fresh TE to adjust volumes as necessary. Protective eyewear should be worn while using a UV light source, L To aspirate plasmid DNA efficiently, insert the needle, bevel down, just below the band of plasmid DNA. Rotate the needle so the bevel faces upward, then extract the illuminated band by gently pulling on the syringe plunger. d The volume of TE required to resuspend the pellets will vary with the individual plasmid, but generally will be at least 1 ml per 250 ml of original bacterial culture volume, b

eAssuming a spectrophotometer path length of 1 cm, the concentration of the plasmid in solution is equivalent to A260 x 50 x dilution factor. The A2fi0:A280 ratio provides a measure of plasmid purity, and should be between 1.7-2.0.

DEAE dextran-medlated transfection of macrophages Equipment and reagents • Humidified CO3 incubator suitable for cell culture (NuAire Corp.) • Sterile 60 mm tissue culture dishes (Becton Dickinson Labware) • Macrophage population of choice (see Chapters 1 and 2) • Supercoiled plasmid DNA of choice (see Protocol 4) • 10 x Tris-buffered saline (TBS) pH 7.6: 24.2 g Tris base, 80 g NaCl, and H2O to 1 litre • TBS-D: TBS plus 0.1% glucose • TBS-DD: TBS containing 0.1% glucose and 20 n.g/ml DEAE dextran (prepare fresh)

20% (w/v) glucose solution 300 ug/ml DEAE dextran (Sigma Chemical Co.) 10 x phosphate-buffered saline (PBS) pH 7.4: 14.8 g Na2HPO-4, 4.3 g KH2PO4, 72.0 g NaCl, and H2O to 1 litre Dimethylsulfoxide (DMSO, Sigma Chemical Company) Complete tissue culture medium: RPMI 1640 (Life Technologies) containing 10% endotoxin-free fetal bovine serum (FBS, Life Technologies)

Method 1 Plate macrophages into 60 mm tissue culture dishes at a concentration of 5 x cells in a total volume of 6 ml complete medium. 2 Incubate at 37 °C, 5-7% C02 overnight.

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3 At the time of transfection, prepare sufficient TBS-DD to provide 1.75 ml for each plate to be transfected. 4 Add the appropriate amount of plasmid DNA to the TBS-DD solution, mix by inversion, and warm to 37°C,a 5 Remove the growth medium from the macrophage monolayers by aspiration." 6 Wash each monolayer gently with 2 ml pre-warmed TBS-D by gently rocking the dishes manually from side to side. 7 Aspirate the wash buffer from the dishes and replace with 1.5 ml of the plasmidcontaining TBS-DD mixture per plate, 8 Incubate plates at 37 °C and 5-7% C02 in a humidified incubator for 2-4 h.c 9 Remove the transfection mixture by aspiration and add 1 ml of a PBS/10% DMSO mixture.b 10 Incubate for 1 min at room temperature. 11 Remove the PBS/DMSO by aspiration, and gently wash the monolayers three times with TBS-D as in step 6. 12 Remove the wash buffer by aspiration and add 6 ml of complete medium to each plate. 13 Incubate dishes at 37 °C for 18-24 h prior to further stimulation and/or assessment of reporter gene expression. a

The optimal plasmid concentration will vary based both on the target macrophage population as well as the plasmid, and should be determined experimentally in each case. b Work with only two dishes at a time, to prevent drying and detaching of the macrophage monolayer. c The duration of the transfection should be experimentally determined in order to maximize transfection efficiency and reporter gene expression and minimize toxicity to the target cells.

Transfection of macrophages using lipophilic reagents Equipment and reagents • Humidified C02 incubator suitable for cell culture • Sterile 6-well tissue culture plates (Becton Dickinson Lab ware) • Table-top centrifuge for spinning cells (Dupom/NEN)

• Macrophage population of choice (see Chapters 1 and 2) • Supercoiled plasmid DNA (see Protocol 4) • Lipophilic transfection reagent of choicea • RPMI 1640 tissue culture medium: serumfree and supplemented with 15% FBS

Method 1 Add 0.625 ml of serum-free RPMI 1640 medium to each well of a 6-well tissue culture plate.

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2 Add the appropriate volume of the lipophilic transfection reagent to each well and incubate the plate at room temperature for 30 min.b 3 During this incubation, prepare serum-free medium containing plasmid DNA at a final concentration of 15 n-g/ml. 4 Add 0.625 ml of this mixture to each well of the 6-well plate at the completion of the incubation. 5 Incubate the plate for an additional 15 min at room temperature. 6 During the 15 min incubation, harvest the recipient macrophages as appropriate (see Chapters 1 and 2). 7 Wash the macrophages once with serum-free RPMI 1640 by centrifugation at 300 g, 10 min, 4 °C. 8 Resuspend the cells at 6 x 106 to 2 x 107 cells/ml in serum-free medium. 9 Add 250 u1 of this cell suspension to the appropriate wells of the 6-well plate.e 10 Incubate the plate at 37°C, 5-7% CO2 for 4 h. 11 Add 3 ml of pre-warmed RPMI 1640 supplemented with 15% FBS to each well in the plate and return cells to the incubator, 12 Incubate the transfected macrophages for 24 h prior to further stimulation and/or assessment of reporter gene expression. J

A number of effective lipophilic transfection reagents are now commercially available. Several of these reagents should be tested to determine the best reagent for the macrophage population under study. b The optimal amount of the lipophilic transfection reagent to be used will vary based on the macrophage population and must be determined empirically. c The number of cells required for transfection will vary from population to population, but will likely be within the given range.

Transfection of macrophages by electroporation Equipment and reagents • Sterile disposable electroporation cuvettes, 4 mm gap size (BTX, Inc.) • Sterile 60 mm tissue culture dishes • Table-top centrifuge • Electroporation apparatus (BTX, Inc.) • Macrophage population of choice (see Chapters 1 and 2)

Supercoiled plasmid DNA of choice (see Protocol 4) RPMI 1640 tissue culture medium supplemented with 20% FBS Phosphate-buffered saline (PBS, see Protocol 5)

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Method 1 Wash recipient macrophages twice at room temperature in a large volume of serum-free RPM11640 medium by centrifugation for 5-10 min each at 250-500 g. 2 Remove the supernatant fluid from the final cell pellet by aspiration. 3 Resuspend the macrophages in cold PBS to a final concentration of 3 x ifl6 cells/ml or higher/ 4 Transfer 300 ^,1 of this cell suspension into an electroporation cuvette, keeping everything on ice. 5 Add plasmid DNA to the macrophage suspension to a final DMA concentration of 50 ug/ml. 6 Tap the cuvette gently several times with an index finger to disperse the plasmid among the cells.b 7 Incubate the macrophage/DNA suspension on ice for 5 min. 8 Remove the cuvettes from the ice and dry the outside. 9 Place the cuvettes in the electroporation apparatus and pulse the cells twice, using appropriate conditions.1 10 Immediately following the second pulse, dilute the cell suspension with 5 ml of RPMI1640 supplemented with 20% FBS. 11 Plate the resulting cell suspension in a 60 mm tissue culture dish. 12 Incubate the transfected cells for 24 h at 37°C, 5-7% C02, prior to further stimulation and/or assessment of reporter gene expression. a

The number of cells needed for each electroporation reaction will differ for individual cell populations and should be determined experimentally. b The concentration of plasmid DNA needed to achieve efficient transfection will vary, and should be optimized in each case. c The voltage and capacitance settings to use for successful electroporation will vary with each macrophage population and should be optimized in each case. The following is a successful electroporation protocol used for transient transfection of the murine macrophage cell line RAW 264.7 using the BTX 600 electroporation apparatus: 2.5 kV resistance; 400 fiF capacitance; 24 Ohm resistance timing (R4); charging voltage set to 140 V (peak pulse voltage will be approximately 130 V). Some or all of these parameters may need to be adjusted for particular macrophage populations.

3.4 Stable transfection While transient transfection methods are most commonly applied to analysis of macrophage gene expression, especially in primary cells, the preparation of cell lines in which genetic material has been stably integrated Into the genome can provide perhaps the most reliable result with respect to gene expression. A thorough discussion of the systems available for transaction and drug selection 192


of stably transfected macrophages is beyond the scope of this chapter; suffice it to say that these experiments involve careful consideration of the choice of vector, of the nature of the selectable marker, of the process to be used to deliver the DNA, and to select and maintain the transfected cells. However, several advantages and disadvantages to application of this procedure to mononuclear cells can be noted here. The major advantages of preparing stable transfectants include the enhanced reproducibility of experimental outcome and the more physiological manner in which transferred genetic material is expressed. Perhaps most importantly, the impact of transgene expression can be evaluated on the entire cell population allowing measurements of endogenous gene behaviour. Balanced against these obvious advantages is the considerable time needed for the generation of stably transfected cell lines. In addition, there are some strategic disadvantages. Because transgenes integrate at different sites in the genome in each cell population, there is inherent variability in transgene behaviour due to integration site idiosyncrasies. This will be evident not only between cell lines but may also impact transgene behaviour within a single cell since there may be multiple integration events within each cell. Such heterogeneity may be averaged by examining bulk cell populations derived from the original transfection process. These bulk cultures will exhibit an 'average' effect which may nullify individual variability. However, uncloned cell lines are inherently unstable and over time may exhibit changes in behaviour which reflect changes in relative abundance of individual cell genotypes. A second strategic problem may be the potential toxicity or growth inhibitory effects of particular transgenes which preclude the generation of cells exhibiting stable expression. Anecdotal information from a number of investigators suggests this problem has been observed in several macrophage systems where the generation of stable transfectants has been attempted, although few quantitative analyses are available concerning the extent of this problem. Toxicity may be overcome in some circumstances by the use of vector systems in which the transgene expression is under control of exogenous stimuli and such systems are worth consideration in designing an experimental strategy for the production of stably transfected cells.

4 Measurement of protein-nucleic acid interactions 4.1 Basic principles Protein-nucleic acid interactions are the means by which regulatory nucleotide sequences either in DNA or in RNA molecules effect the functions which they specify. Nucleic acid binding proteins interact with either DNA or RNA but generally not both. Individual sequence motifs serve as recognition/binding sites for proteins which, either directly or through interaction with additional proteins, produce changes in the function of the original sequence. Thus transcription factors modulate the transcription rate of genes which contain cognate specific 193


binding sites while the metabolism of RNA molecules (processing, transport, translation, and decay) is mediated by factors which recognize and bind regulatory sequences in the gene transcripts. Binding/recognition specificity for interactions between proteins and nucleic acids is determined precisely by relatively short nucleotide sequence motifs, on the order of 10-25 residues. The specific nucleotide requirements in the motif define the consensus sequence for particular families of protein factors; some nucleotide positions are invariant while others may vary. This produces classes of sites recognized by families of protein factors. In many cases, allowed sequence heterogeneity may translate into functional heterogeneity through allosteric modulation of binding protein function. Such functional heterogeneity among sites introduces substantial complexity and specificity into the consequences of interaction. For example, multiple sequences can be recognized by protein members of the NFk

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