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

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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

Fixed tissue macrophage

Bone marrow

Monoblast, promonocyte, monocyte, macrophage

Lamina propria

Fixed tissue macrophage

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

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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

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

• 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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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 MACROPHAGES FROM TISSUES, FLUIDS, AND IMMUNE RESPONSE SITES

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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 MACROPHAGES FROM TISSUES, FLUIDS, AND IMMUNE RESPONSE SITES

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.

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MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

• 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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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.

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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.

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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 OF MACROPHAGES FROM TISSUES, FLUIDS, AND IMMUNE RESPONSE SITES

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

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

MARY ELLEN HANDEL-FERNANDEZ AND DIANA M. LOPEZ

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

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

(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

PURIFICATION OF MACROPHAGES

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

PURIFICATION OF MACROPHAGES

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

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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

PURIFICATION OF MACROPHAGES

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
39

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A Preparation of collagen-coated matrices 1 2 3 4

Dissolve lyophilized type I collagen at 1.5 mg/ml in 0.1 M HOAc at 4°C by stirring overnight Dispense 250 nl of type I collagen solution to each well of 24-well cluster plates. Simultaneously, add 25 \d of 10 x culture medium and 15 ^,1 of 0.142 M NaOH to each well to bring the pH of the mixture to 7.6. Incubate plates for 1-2 h at 37DC (gelation usually occurs in 30-60 min), then wash well with culture medium. Collagen-coated dishes can be kept hydrated at 37°C until use.

B Purification of macrophages 1 2 3 4 5

Seed 3 x 106 Ficoll-Hypaque derived PBMC (see Protocol 8) per well in 1 ml of RPMI containing 15% FBS. Incubate the plate 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. Wash cell monolayer four or five times with medium without serum, Check, under an inverted microscope, that non-adherent cells have been washed off and that most of the adherent cells are spread out.

4 Methods for detachment of adherent macrophages Different procedures, including mechanical, enzymatic, and pharmacological treatments are currently used to detach adherent monocytes/macrophages (19, 20, 48, 51, 55-57}, However, it remains difficult to detach the adherent cells from the surface and to transfer them without great losses in number or viability and without inducing some alterations in their functions.

4.1 Mechanical detachment This method generally is used to remove monocytes/macrophages adhered to uncoated plastic surfaces. Cells can be removed soon after washing of the nonadherent cells or after different period of culture.

Mechanical detachment of macrophages Equipment and reagents • Sterile rubber policemen (Corning Costar) • Ca^/Mg^-free PBS (see Protocol 1)

40

• RPMI 1640 medium containing 15% FBS

PURIFICATION OF MACROPHAGES

Method 1 Prepare a cell monolayer as described in Protocol 1. 2 Collect the culture medium (containing the non-adherent macrophage population) in a 50 ml conical tube and leave it on ice.a 3 Rinse the plate with cold Ca2+/Mg2+-free PBS by gentle shaking, then collect the wash to the same conical tube. 4 Add Ca2+/Mg2 '"-free PBS to completely cover the cell surface and incubate plates on ice for 10 min. 5 Gently scrape the adherent cell monolayer with a rubber policeman. 6 Collect the cell suspension and add it to the culture medium containing tube. 7 Rinse again the plate with Ca2+/Mg2+-free PBS to collect residual cells, especially those remaining at the edge of plate, and add the fluid to the same tube containing the culture medium and the cell suspension, 8 Centrifugefor8rninat250gat4°C. 9 Remove the supernatant fluid and resuspend the cell pellet in culture medium. 10 Evaluate cell viability by trypan blue exclusion or alternative method. a

A general feature of macrophage cultures is the presence of adherent, loosely adherent, as well as non-adherent populations. This step will avoid the loss of macrophage populations that grew in suspension or that loosely adhered to plastic.

4.2 Recovery of adherent cells by EDTA treatment This method is generally employed to detach monocytes/macrophages adhered to microexudate-, gelatin-, or FCS-coated surfaces. In fact, cells adhered to these surfaces, in contrast with those adhered to uncoated substrates, can be rather easily removed by KDTA at low concentration (19, 20, 51, 55). The requirement for divalent cations for intcgnn family proteins to bind to their ligands may partly explain the reversibility of attachment of monocytes/macrophages to microexudates by EDTA treatment (50). However, this method can also be applied to cells adhered to untreated plastic tissue culture dishes. Protocol 6 describes one procedure for the detachment of adherent macrophages by EDTA treatment.

Detachment of macrophages by EDTA treatment Reagents • 10 mM EDTA in PBS (see Protocol 1) • RPMI 1640 medium alone or containing 20% FBS

• l:l mixture of 10 mM EDTA in PBS pH 7.4 and RPMI 1640 medium containing 20% FBS (mixture A)

41

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Method 1 Prepare a monolayer of adherent monocytes as described in Protocol 1. 2 Collect the non-adherent cell fraction as described in Protocol 5, step 2. For 75 cm2 flasks, add 10 ml of mixture A. Incubate cells for 15 min at 37°C, 3 Collect the detached cells (generally more than 90%) by gentle shaking of the flask and pipetting. 4 Transfer the cell suspension to 50 ml conical centrifuge tubes. 5 Centrifuge at 400 g for 5 min at 20 °C. 6 Wash the cell pellet with SO ml of RPMI 1640 medium prior to subsequent analysis. The efficiency of dislodgment of adhered cells by EDTA method tan be improved at low temperature (4-20 °C). No mechanical scraping is generally required for further detachment under these conditions.

4.3 Recovery of adherent macrophages by lignocaine treatment The local anaesthetic lignocaine, has been reported to provide reversible inhibition of cell monolayers with negligible toxicity, whereas other anaesthetics were found to be too toxic (56). A modification of the original protocol (57), which allowed greater recovery and viability, is described in Protocol 7.

Detachment of macrophages by treatment with lignocaine Reagents • 30 mM lignocaine in isotonic PBS pH 6.7: add 1 vol. of lignocaine (2% in NaCl, for clinical use) to 1.2 vol. of 120 mM

phosphate buffer pH 6.7 (the final solution will contain 30 mM lignocaine, 60 mM NaCl, and 60 mM phosphate buffer)

Method 1 Add 3-4 ml of 30 mM lignocaine solution in isotonic PBS to 25 cm2 flasks or 10 ml to 75 cm2 flasks. 2 Incubate cells at 22 °C for 15 min. 3 Shake flasks gently to dislodge adherent cells. 4 Remove residual attached cells by gently streaming medium onto the raonolayer using a Pasteur pipette. 5 Pool the suspended cells in 15 ml conical tubes. 6 Wash three times with medium by centrifugation (200 g for 10 min at 22 °C). 7 Resuspend cells in 5-10 ml of medium and assess viability.

42

PURIFICATION OF MACROPHAGES

5 General considerations on the adherence-based methods Although all of the protocols outlined above can be used successfully, it must be acknowledged that techniques based upon the ability of monocytes/macrophages to adhere to different surfaces may exhibit several disadvantages. They include poor cell viability, low yield, and contamination of cell preparations with other cell types. Based on the assumption that macrophages are not the only cell types capable of adhering to plastic or glass surfaces, adherence techniques alone cannot yield a 100% pure macrophage population. The recovered adherent cells generally contain cell types other than macrophages in small, but appreciable, numbers. In addition, macrophages are a heterogeneous group of cells that may differ in their adherence capacity. Thus, adherence-based techniques may not result in a recovery of all subpopulations in proportion comparable to that present in their starting cell sample. In the light of the well-known effect of adherence on macrophage activation, this method cannot be utilized for purification of macrophages to be used in a short-term assay, if the cells are to be considered 'non-activated'. Macrophages isolated by adherence-based methods should be allowed to revert to a non-activated status for at least 24 hours. Many of the activation parameters have returned to their basal levels at that time (26-28, 30, 32, 34). Alternatively, if activation functions of monocytes/macrophages are to be studied, it may be more advantageous to perform these studies on cells purified by physical methods. In fact, macrophages obtained by these procedures generally are considered to be in a 'non-activated' state. The use of physical methods of separation are discussed further in Section 6.

5.1 Effects of different surfaces on macrophage morphology and functions The surface to which the monocytes/macrophages adhere can influence their morphology, survival, differentiation, and functional activities. In this regard, differences in cell phenotypes have been shown to be induced by cultivation on different substrates. In particular, it has been reported that in vitro differentiation of human peripheral blood monocytes to macrophages is dependent on the conditions of monocyte culture (39). Cultivation of monocytes on plastic, glass, or microexudate-coated glass gives rise to cells exhibiting epithelioid-like morphology. In comparison, cultivation of monocytes on collagen matrices results in cells resembling human resident tissue macrophages. The two cell populations differ in morphology, phagocytic activity (39, 58), receptors (39), and surface antigen expression (39). In addition, culture of monocytes on plastic or glass surfaces induces the expression of non-specific, extracellular cytotoxic activity against tumour target cells (45, 58). In contrast, monocytes cultured on collagen-coated surfaces do not develop a significant cytotoxic activity (45). It has also been reported that culture of monocytes on plastic surfaces, but not on FNcoated surfaces, results in some changes in the biosynthesis of polysaccharides 43

SANDRA GESSANI ET AL.

(59). The nature of the substrate has also been shown to modulate the expression of some calcium-binding proteins (MRP8 and MRP14), involved in the process of substrate adherence (60). In particular, adherence of monocytes to laminin, FN, or collagen results in an enhanced surface expression of MRP8 and MRP14, whereas adherence to untreated plastic surfaces is completely ineffective and resembles culture in suspension (60). Additional effects of adherence to different substrates have been described by Gudewicz and co-workers (61). These authors reported that adherence to collagen matrices primes monocytes to an increased response to phorbol ester stimulation of respiratory burst and arachidonic acid metabolism. In contrast, no such priming effect is observed in monocytes adhered to FN or laminin.

5,2 Effects of different detachment procedures on macrophage physiology In early studies, results obtained after adherence of monocytes/macrophages to solid surfaces with subsequent removal of cells by a rubber policeman (62), were not completely satisfactory because of the variability in the number of viable cells recovered and of their poor responsiveness to activating stimuli (54, 63). However, membrane damage can be significantly reduced by incubating the adherent monocytes with ice-cold PBS prior to their removal by scraping (64). Subsequently, it was found that it is possible to coat Petri dishes with a microexudate from different cells before incubation of mononuclear cells and to remove adherent monocytes/macrophages by EDTA (51). This method, however, has the drawback of needing plastics conditioned by a previously resident adherent cell line. A modification of this method has been subsequently described based on pre-treatment of plastic surfaces with serum (52). Effective release of monocytes/macrophages has also been achieved by means of lignocaine solutions. Initial studies in which the lignocaine method was employed reported a variable release of monocytes/macrophages and, in particular, very low viability of the recovered cells (56). The finding that the pH of the lignocaine solution was a critical determinant of the recovery, viability, and reproducibility of the separation procedure permitted development of a modified lignocaine method that has been more successful (57 and Protocol 7). No consistent difference among the various detachment techniques is generally observed with regard to cell recovery, viability, adherence, morphology, neutral red uptake, phagocytosis of yeast cells, non-specific esterase staining, chemotaxis, and contamination by T and B lymphocytes. Methods such as EDTA treatment may be less damaging to the cell membrane than scraping. However, this compound has been reported to remove or alter the structure of adhesion molecules on the cell surface, rendering them unrecognizable to specific monoclonal antibodies (65). Moreover, the metabolism of macrophages released from glass or plastic by EDTA treatment may be depressed and cells should be allowed to recover from the separation procedure for one to several hours before physiological measurements are attempted (18). 44

PURIFICATION OF MACROPHAGES

6 Physical methods of macrophage purification Physical methods for the purification of individual type of cells, including monocytes/macrophages, have increased markedly in variety and sophistication during the past three decades (3-11). The successful development of isopycnic (buoyant density) and velocity sedimentation methods, alone or in sequential combination has resulted in numerous reports of useful macrophage separations. In addition, separation of cells by velocity sedimentation can be accomplished in the absence of gradients by a procedure termed elutriation. The physical properties of cells that govern their sedimentation in gradient centrifugation, velocity sedimentation, and elutriation are density and diameter (Figure 1). However, these physical properties have different importance in the various methods. Cell size has a greater impact than density in velocity sedimentation whereas cell density becomes the most important parameter in isopycnic centrifugation. Isopycnic or buoyant density sedimentation of cells in continuous gradients requires sufficient force and/or time for cells to relocate to respective density bands within the gradient. Since separation is effected on the basis of respective cell densities, and the densities of many cell types overlap broadly, the application of this method has some limitations, A further disadvantage is the high centrifugal force often used and the possible toxicity of the gradient medium employed. Nevertheless, in combination with sedimentation velocity, this method has provided highly enriched populations of viable cells. Sedimentation at unit gravity is reliable and technically easy to achieve and has the advantage of using

Figure 1 Physical methods for cell separation. The physical properties of cells that govern the cell separation in isopycnic centrifugation and velocity sedimentation are density and size. The different impact of these two physical features in various methods is schematically illustrated. 45

SANDRA GESSANI ET AL.

inexpensive equipment. The apparatus can accommodate a limited number of cells (maximum 108 cells) layered on to a gradient of serum, BSA, or Ficoll. Problems arise at the sample/gradient interface at high cell numbers where cells stream down into the gradient (streaming phenomenon). A major disadvantage of sedimentation at unit gravity is the long separation time required. A variety of methods based on physical properties of macrophages can be used for the purification of these cells. In this section we will cover the most commonly used procedures, including isopycnic centrifugation (for small number of cells) and elutriation (for large number of cells).

6.1 Purification of macrophages by isopycnic gradient centrifugation The density of a cell is a major physical characteristic, which can be used for purification and separation of cell populations by means of isopycnic centrifugation. It reflects the average chemical composition of a cell rather than surface characteristics or size. The various major cellular components all differ substantially in density. However, since most cells have roughly similar proportions of these components, the density range of cells is relatively narrow. In fact, most mammalian cells are characterized by densities between 1.055-1.110 g/ml. Separation is accomplished simply by centrifuging a cell with sufficient centrifugal force and for a sufficient period of time for it to arrive at its isopycnic density in the gradient (i.e. the location in the gradient where the density of the cell is the same as the density of the gradient). Many kinds of gradient media that are suitable for use with the isopycnic centrifugation of monocytes have been generated using different substances (4, 5, 7). They include Ficoll-Hypaque, Percoll, albumin, dextran, metrizamide, and Nycodenz. Major requirements for a density medium are the absence of toxicity, low viscosity at high density (1.10 g/ml), and little osmotic pressure in solution. In constructing gradients for isopycnic centrifugation, it is important that the most dense portion of the gradient be more dense than any cells to be separated and that the least dense portion of the gradient should be of as low density as possible.

6.2 Isolation of whole mononuclear cells from peripheral blood by the Ficoll-Hypaque gradient This simple and rapid method takes advantage of the density differences between mononuclear cells and other elements found in blood samples. Mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer as they have a lower density. In contrast, red blood cells and granulocytes have a higher density than Ficoll-Hypaque and collect at the bottom of Ficoll-Hypaque layer. Platelets are separated from the mononuclear cells by subsequent washing or centrifugation through a FCS cushion gradient, which allows penetration of mononuclear cells but not platelets. 46

PURIFICATION OF MACROPHAGES

Isolation of mononuclear cells by Flcoll-Hypaque gradient separation Equipment and reagents" • 10 on long metal needles • Buffy coats or heparinized bloodb • Ca2+/Mg2+-free PBS (see Protocol 1)

• Ficoll-Hypaque solution (Seromed. density 1.077 g/ml) • RPMI 1640 medium containing 15% FBS

Method 1 Dilute blood from a buffy coat (30-40 ml of blood) 1:2 with Ca2+/Mg2+-free PBS and mix well. 2 Place 30 ml of the blood/PBS mixture in a 50 ml conical centrifuge tube. 3 Slowly layer 15 ml of the Ficoll-Hypaque solution (density 1.077 g/ml) underneath by using a needle. 4 Centrifuge gradient for 30 min at room temperature at 280 g with no brake. 5 Using a sterile pipette, remove the upper layer that contains plasma and most of the platelets. 6 Using new pipette, recover the mononuclear cell layer and transfer this to another 50 ml conical tube. 7 Bring the volume to 50 ml by adding an appropriate amount of Ca2+/Mg2+-free PBS, 8 Centrifuge at room temperature for 5 min at 280 g with low brake. 9 Remove the supernatant by using a pipette and discard. 10 Vigorously resuspend the cell pellet in 50 ml of Ca2"/Mg2+-free PBS. 11 Centrifuge at room temperature for 10 min at 90 g. 12 Wash the cell pellet again (as described in step 9-11). 13 Resuspend the final cell pellet in 10 ml of RPMI 1540 medium containing 15% FBS. 14 Count the viable cells by trypan blue exclusion. a All reagents must be at room temperature before use since gradient density varies according to temperature. b Peripheral blood human monocytes can be separated from heparinized blood samples or from buffy coats of normal bank donors with sodium citrate as an anticoagulant. Blood bank buffy coats are obtained after centrifugation of blood bank bag (original volume 400 ml) and removal of the upper layer of the content. The separated fraction (25-40 ml) contains more than 90% of the leukocytes of the blood donation, 10% of the erythrocytes. and 5% of the plasma. Macrophages tan be further purified from mononuclear cells by using adherence to different surfaces (see Protocols 1-4), Percoll gradient (see Protocol 9), or counterflow centrifugal elutriation (see Protocol 10). 47

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6.3 Purification of monocytes by Percoll gradient Percoll is a polyvinylpyrrolidone-coated silica which is supplied as a sterile colloidal suspension with characteristics which vary slightly from batch to batch. In many cases, monocytes/macrophages are separated on discontinuous (preformed) Percoll gradients. In this case, a relatively large number of density layers is arbitrarily fixed. Consequently, the number of cell subpopulations harvested depends on the number of Percoll solutions which are overlayed in the tubes. An improved cell separation can be obtained by using continuous (or self-generated) density gradient of Percoll. A Percoll solution of a given density is centrifuged and high and low densities are distributed around this given density. The specific cell density of macrophages is influenced by several factors (species, age, sex, strain). For this reason, it is recommended to first determine this specific cell density in an analytical step. This can be done by centrifuging simultaneously a continuous gradient containing coloured beads of well-known cell density together with another gradient of cell preparation to be studied. Subsequently, a preparative step using a discontinuous density gradient allows separation of all the cell subpopulations according to their actual specific density. Protocol 9 describes one procedure for Percoll gradient separation of monocytes.

One-step continuous Percoll gradient separation of monocytes Reagents • PBMC purified by Ficoll-Hypaque gradient (Protocol 8) • Ca2+/Mg2+-free PBS (see Protocol 1) • RPMIl 640 medium supplemented with 15% FBS • Iso-osmotic Percoll. To prepare an osmotically balanced stock solution of

Percoll (Pharmacia) mix 9,25 parts of concentrated Percoll with 0.75 parts (v/v) of 10 x Ca2+/Mg2+-free PBS. Adjust the osmolarity of the Percoll solution, as well as of the RPMI 1640 medium supplemented with 15% FBS, to 285 mOs with 10 x Ca2+/Mg2+-free PBS or distilled water, respectively.

Method 1 Prepare a 46% Percoll solution by mixing 4.6 parts of the iso-osmotic Percoll solution with 5.4 parts of iso-osmotic complete medium. 2 Resuspend 5-10 x 107 PBMC in 5 ml of iso-osmotic complete medium. 3 Place in 10 ml centrifuge tube, 4 Slowly layer 5 ml of 46% Percoll solution underneath PBMC layer. 5 Centrifuge for 30 min at room temperature at 500 g with gradual acceleration and no brake. The monocyte-enriched fraction is collected at the interface, whereas lymphocytes are at the bottom of the tube.

48

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6 Carefully remove the supernatant fluid using a pipette and discard this layer. 7 Recover the monocyte layer using a fresh pipette (avoid the Percoll layer) and transfer cells to a 50 ml conical tube. 8 Bring the total volume to 50 ml by adding Ca2+/Mg2+-ftee PBS. 9 Centrifuge for 5 min at room temperature at 280 g with low brake, 10 Resuspend the cell pellet in 50 ml Ca2+/Mg2+-free PBS and repeat step 9. 11 Resuspend the cell pellet in RPMI1640 medium containing 15% FBS. 12 Count the viable cells by trypan blue exclusion.

6,4 Importance of the control of experimental conditions in the preparation of a gradient Density separation of cells depends on relatively small density differences and the actual buoyant density of cells can vary readily with various external factors. Thus, close attention must be paid to the technical aspects to obtain good resolution and reproducible density distribution. Imprecise control of the experimental conditions, as briefly summarized below, is a very common source of error in cell separation by means of centrifugation in density gradient (3. 4). The major sources of error include the variables: (a) Acceleration and deceleration. Too rapid acceleration and/or deceleration can result in the production of artefacts for a variety of reasons. Acceleration of density gradients must be gradual because of Coriolis forces and because shallow gradients are not stable during too rapid acceleration. The full speed should be reached over the course of 45 sec. accelerating to a force of 5 g over the first 15 sec, 25 g over the next 15 sec, and reaching the desired speed over the remaining 15 sec. More rapid acceleration, particularly during the early part of centrifugation, results in artefacts. During deceleration, gradients undergo some degree of rotation (swirling) within the centrifuge tube. If the deceleration is too rapid or if the gradient is too shallow and consequently unstable, swirling may be vigorous and may cause mixing of different zones of the gradient. (b) Temperature. Small changes in the temperature can cause large changes in viscosity. Since the velocity of a cell in a density gradient is a function of the viscosity of the gradient at the location of the cell, precise control of the temperature is important. (c) Gradient or band capacity. It is important for the investigator to be aware that each gradient is characterized by an upper limit with respect to the number of a particular kind of cell that will sediment ideally. Practically, gradient or band capacity is one of the most important and commonly ignored considerations in planning the separation of cells by gradient separation. When 49

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the band capacity is exceeded, gradients can become locally unstable and well-defined peaks become broader. If too many cells are layered over a gradient, ideal separation no longer occurs and predictable cell separation is not possible. The band capacity is not linearly related to the cross-sectional area of the tube and appears to be more closely related to the circumference of the centrifuge tube. The circumference/volume ratio is larger for small tubes; consequently, larger amounts of cells are often better prepared simply by using several smaller gradients simultaneously. (d) Wall effect. This effect results when cells layered on a gradient in a cylindrical tube and centrifuged in the conventional manner hit the tube walls and sticks rather than moving to their buoyant density. This frequently results in the loss of cells in their usual location. In order to minimize the loss of cells resulting from the wall effect artefact it is best to use a centrifuge that allows one to place the gradient as far as possible from the centre of revolution (5). (e) Clumping. The formation of aggregates or interaction of cells in small units is enhanced by high concentrations of most macromolecules; this results in cell banding at the average density of the aggregate, rather than their own buoyant density, with an overall loss of resolution. Aggregation can be minimized by appropriate choice of medium (3% BSA or 10% FBS), by working at sufficiently diluted concentrations of cells, by lowering the pH, and temperature (0-4 °C). (f) Continuous versus discontinuous gradients. In spite of their widespread use, discontinuous gradients have inherent in their design several disadvantages as compared to continuous gradients. The presence of interfaces in discontinuous gradients results in the accumulation of cells at high concentrations at these interfaces. This determines the incorporation of different kinds of cells in the same aggregates. In other words, discontinuous gradients do not permit cells to be distributed according to their natural densities or rate of sedimentation. (g) Osmolarity. Since the density of a cell depends on its water content, and since cells act as osmometers, the actual buoyant density will vary with the osmolarity of the medium. Buoyant density values are not meaningful unless this latter value is precisely controlled. The 'true' density distribution of a cell population will only be obtained in a density gradient with constant physiological osmolarity throughout the gradient. 6.5 Counterflow centrifugal elutriation Counterflow centrifugal elutriation (CCE) is a negative-selection isolation technique that can be used to isolate large numbers of purified monocytes. This technique has been described for almost 50 years (66). However, it is only over the last 25 years that it has been increasingly employed, due to the venue of the Beckman centrifugal rotor, which has improved elution parameters (67). The principle behind the use of CCE for cell purification is based on differences between cell populations in sedimentation velocity in relation to cell volume. 50

PURIFICATION OF MACROPHAGES

Cell shape as well as cell density can play a role in the effectiveness of cell purification methods. However, the latter two aspects have a less significant impact on the effectiveness of tell separation by CCE than on techniques based on isopycnic separation. Cells loaded in the elutriation chamber are subjected to two opposite forces: the centrifugal force (rotor speed) and the centripetal force (the flow rate) (Figure 2), Providing that the centrifugal force is maintained (i.e. a constant rotor speed), the increase in the centripetal force obtained by the steady increments of the elutriation medium flow rate forces the cells to exit the chamber according to their volume. One major disadvantage of the commonly used elutriation system is that the percentage of cell loss becomes very high when the machine is loaded with less than 108 mononuclear cells. This means that more than 100 ml of human blood is required to perform a good monocyte separation (68). This fact poses a limitation in the use of CCE for separation of

Figure 2 Separation chamber of the elutriation apparatus. A schematic representation of cell separation in the 5 ml elutriation chamber, indicating the opposite forces to which cells are subjected, is illustrated.

51

SANDRA GESSANI ET AL.

small numbers of cells. However, to date, CCE is the most suitable separation technique to isolate monocytes/macrophages in cell suspension with high yield and purity providing a good preservation of their functional properties. Recently, a semi-closed CCE system has been developed (using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber), to purify macrophages from mononuclear cell concentrates of healthy donors and cancer patients (69). The high capacity of the rotor allows the harvest of very high numbers of monocytes in a single run and does not require a Ficoll density centrifugation step. Protocol 10 describes the purification of macrophages by CCE.

Purification of macrophages by counterflow centrifugal elutrlation (CCE) Equipment and reagents • CCE apparatus, including a variable-speed peristaltic pump regulated by a micrometer speed control and a pressure gauge • Elutriation medium: Ca2+/Mg2+-free PBS containing 5% FBS

• Ficoll-Hypaque derived PBMC (Protocol 8) • 70% ethanol, or 6% hydrogen peroxide, or 0.2% diethyl pyrocarbonate • Sterile distilled water

Method 1 Assemble the apparatus as shown in Figure 3. 2 With the rotor stationary, start the pump and set the speed such that about 40 ml/ min of the sterilizing agent (i.e. 70% ethanol, 6% hydrogen peroxide, or 0.2% diethyl pyrocarbonate) is drawn into the system (300-500 ml in total). 3 Rinse the apparatus successively with 500 ml of sterile distilled water and 500 ml of elutriation buffer. 4 Clear the elutriation chamber system of all air by adjusting the elutriation rotor speed to 750 r.p.m. and the peristaltic pump to a flow rate of 14 ml/min. 5 Increase the rotor speed to 2000 r.p.m. and adjust the flow rate to 6 ml/min. 6 When the CCE system has been calibrated to the above settings, inject the mononuclear cell suspension (4-5 ml in elutriation buffer) into the system with a sterile syringe connected to the three-way valve. 7 Turn off the pump to stop the flow of elutriation medium and allow the cells to enter the system. 8 Turn on the three-way valve at the completion of cell entry. Extreme caution must be used to prevent any air bubbles from entering the elutriation chamber, since this will create a back pressure that will cause fluid flow through the elutriation chamber to cease.

52

PURIFICATION OF MACROPHAGES

9 Start to collect cell fractions in 50 ml aliquots in centrifuge conical tubes immediately after cell loading. 10 Gradually increase the flow rate to allow different cell populations to leave the elutriation chamber. 11 Collect 50 ml fraction in 50 ml centrifuge tubes. 12 Analyse each fraction to determine the cell type collected in each fraction, 13 At completion of the elution, wash the apparatus with sterile Ca2+/Mg2+-free PBS to eliminate residual cells and serum proteins, followed by 70% ethanoi

Figure 3 Schematic representation of the complete elutriation system. A diagrammatic representation of a complete elutriation system and of the different elutriation steps is illustrated.

The first fractions collected will contain platelets and cell debris followed by erythrocytes eventually contaminating the mononuclear cells. Next, fractions of lymphocytes (pure lymphocyte fraction), followed by small monocytes and lymphocytes (intermediate monocyte fraction), and finally purified monocytes (purified monocyte fraction) will be obtained. This latter fraction will also contain small proportion of natural killer (NK) cells and large lymphocytes (about 2%). 53

SANDRA GESSANI ET AL.

7 Additional issues to be considered in monocyte/macrophage purification In this section, we will discuss some additional aspects that should be taken into consideration when using different methods for the purification of monocytes/ macrophages from different sources.

7.1 Macrophage heterogeneity It has long been recognized that macrophages isolated from different anatomical sites are heterogeneous with respect to many different properties including size, morphology, density, expression of cell surface proteins, and functional capabilities (reviewed in refs 12, 70). Because macrophage function is dependent, at least in part, on signals received from the immediate microenvironment, it has been suggested that macrophage heterogeneity may arise from the unique conditions within specific tissues. However, macrophages isolated from a given tissue also display heterogeneous functions, thus indicating that multiple mechanisms are probably involved in the generation of macrophage heterogeneity (12, 70). The variety of functions displayed by monocytes makes dissection of heterogeneity difficult, although subsets of monocytes that differ in size (71) or density (72 have been isolated. In particular, these cells show differential hydrogen peroxide production (73), phagocytosis (74), cytotoxicity (75), cytokine release (76), and markers for activation or differentiation (77). A potential problem in understanding the significance of this heterogeneity is that the isolation procedure may select for certain subpopulations of cells, making extrapolation to the in vivo situation uncertain (73, 75, 78, 79). Isolation of monocytes/ macrophages has been shown to have effects upon the levels of expression of certain cell surface molecules (64, 80). Thus, some perturbation of monocyte/ macrophage function might be the result of the purification technique used. Size fractionated monocytes isolated by elutriation show heterogeneous phagocytic capacity (81), cytokine production (77), prostaglandin secretion (77), and activation by GM-CSF for tumour killing and IL-la production (81). The large monocyte subpopulation has been shown to be more phagocytic than the small one (72, 74), more responsive to chemotactic stimuli (73), and exhibited enhanced cytotoxicity against certain target cells (73). In contrast, studies performed by velocity sedimentation separation show that large monocytes release more IL1(3, prostaglandin E2, and TNFa in response to bacterial lipopolysaccharide (LPS) than do small monocytes (79). Large monocytes also produce more H202 than small monocytes (73). Monocyte subpopulations separated on the basis of their density through discontinuous gradients also differ from one another with respect to various biochemical characteristics. In fact, high density cells express high 5'-nucleotidase activity but low acid phosphatase and peroxidase activity, while the low density cells express low 5'-nucleotidase activity and high acid phosphatase and peroxidase activity (72, 77). Density defined monocyte subpopulations also differ in their expression of various functional activities. In fact, 54

PURIFICATION OF MACROPHAGES

high density monocytes are less active than low density cells in antibodydependent cell-mediated cytotoxicity reactions (75, 77) and less effective accessory cells in the generation of mitogen-proliferation responses (77). Monocytes are also heterogeneous with respect to their expression of a large number of cell surface proteins including Fc (FcR) and complement (CR) receptors, the structures recognized by some lectins, and various molecules defined by monoclonal antibodies (12, 70). In this regard, it is of interest that phenotypic and functional characterization of Fey receptor I (CD64)-negative monocytes defines a minor human monocyte subpopulation with high accessory and antiviral activities (82). Furthermore, it has been reported that CD14low/CD16+ small monocytes, representing around 10% of the monocytes in the human peripheral blood mononuclear cells, produce less TNFex, IL-1, and IL-6 than CD14high monocytes (83, 84). However, the CD14low/CD16+ monocyte subset has a higher expression of major histocompatibility complex (MHC) class II proteins (83).

7.2 Comparison of the efficacy of different purification techniques with respect to the source of macrophages Mouse peritoneal macrophages represent an extensively used model for studies on macrophage functions. In fact, the peritoneal cavity provides an accessible site for harvesting fairly high numbers of resident macrophages, as they represent the major cell population present in the peritoneum (approximately 70%). However, the number of macrophages obtained from the peritoneum in noninflammatory conditions is generally insufficient for large scale studies and differs among various species (85). To increase the yield of macrophages, a sterile peritoneal exudate can be induced by injection of an eliciting agent. Notably, macrophages obtained from a stimulated animal differ from those found in a resident population in that many of the cells are recently immigrated from the circulation and are likely to be morphologically, biochemically, and functionally different. In this regard, differences have been reported in the adherence properties of peritoneal macrophages isolated from elicited mice. In fact, these cells attach less to microexudate-coated plates as compared to resident macrophages (19). Furthermore, differences in function can also be associated with differences in the eliciting agent used (86). Satisfactory purification of rodent peripheral blood monocytes in suspension has not been achieved so far, since in rat and mice, these cells occur as a minor population of the peripheral blood leukocytes overlapping with lymphocytes in size and density. This problem appears to be restricted to rodent blood. Recently, a two-step procedure for the isolation of monocytes from rat blood, characterized by a high yield and purity, has been described (87). In the human system, the peripheral blood represents the major source of macrophages. Although these cells are present in the pool of mononuclear cells at low abundance (10-15%), their purification can be easily achieved by using physical methods (isopycnic centrifugation or elutriation) which allow a consistent enrichment of the monocyte fraction. 55

SANDRA GESSANI ET AL

7.3 Non-adherent versus adherent culture of macrophages In the light of the well-known effect of adherence in macrophage activation, attention should be paid to denning the culture conditions of cells isolated by physical methods. As described in Section 2, if activation functions of macrophages are to be studied, it is advantageous to perform these studies on nonadherent macrophages. A variety of non-adherent systems have been established using monocytes isolated by centrifugal elutriation or density gradient centrifugation. The use of culture dishes made of hydrophobic Teflon (fluorinated ethylene propolyne) film is one of the most common ways to maintain monocytes in a partially adherent state (88, 89). In both adherent and non-adherent systems, monocytes can be maintained in long-term culture, differentiate into culture-derived macrophages, and undergo similar changes in the expression of various cell surface antigens (90). However, some differences have been described between these systems. Non-adherent monocytes respond differently to stimuli than adherent cells. For instance, neither CSF-1 mRNA nor protein is induced by IPS in non-adherent monocytes (91), while LPS treatment of adherent monocytes induces CSF-1 secretion (28). Morphologically, non-adherent monocytes are smaller than adherent cells (90). In addition, a lower activity of some enzymes of intermediary metabolism has been detected in suspension cultures as compared to adherent monocyte cultures (92).

7.4 Problems caused by LPS contamination during the course of macrophage purification and culture LPS is a major factor in the modulation of macrophage functions and is capable of affecting the macrophage physiology even at very low concentrations (reviewed in ref. 93). LPS contamination during purification steps may result in marked effects on the adhesion properties of these cells. For example, it has been shown that the presence of low levels of LPS results in the recovery of weakly adherent subpopulations exhibiting a high cytolytic capacity (94). Low concentrations of LPS also may result in the production of some macrophage-derived cytokines in unstimulated or interferon (IFN) 7 primed macrophages (95, 96) that in turn can influence macrophage activities. The response of monocytes/macrophages to LPS is also influenced by the state of differentiation (95, 96) and appears to be different depending on specific monocyte subsets (80). Finally, LPS-stimulated monocytes may exhibit a down-modulation of chemokine receptors (97), which might result in an impaired chemotactic response of these cells. In consideration of these well-known effects of LPS on macrophage physiology, it is extremely important to avoid LPS contamination during all stages of purification and culture by paying special attention to the use of endotoxin-free reagents and materials.

8 Conclusions In this chapter, we have described the main procedures for macrophage purification currently used in various laboratories and we have reviewed some critical 56

PURIFICATION OF MACROPHAGES

points to be considered in approaching the separation of these cells from different tissues. Monocytes/macrophages are extremely responsive to a variety of stimuli, which can markedly affect their phenotype and functional activities. Thus, slight variations in the protocols or reagents used for purification may result in major differences in the experimental results, especially when the cell cultures are to be tested shortly after their isolation. Each of the different methods used for macrophage purification has its advantages and disadvantages (Table 1) and the specific choice may depend on the macrophage functions to be studied as well as on practical considerations. In general, methods based on velocity sedimentation, elutriation, or isopycnic sedimentation are more suitable than those based on cell adherence, if the cells have to be maintained in a non-activated state at early times after separation. Especially when the same research objective is faced by different groups, it is highly recommendable that reference protocols for macrophage purification are followed by the different groups involved in the study. Notably, most of the currently used standard methodologies for macrophage purification are somehow deficient in one or more of the general criteria desirable for an ideal separation (high purity and yield, functional integrity, maintaining of the correct proportions of monocyte subsets). In order to obtain a 100% pure population of monocytes/macrophages, the use of combined methods of purification should be considered. In this regard, immunomagnetic procedures based on the use of antibody-coated magnetic beads would be useful for removing specific subsets of contaminating cells. Research interest in macrophages is likely to increase because of the accumulating evidence on their crucial role in the regulation of the host response in physiological and pathological conditions. The need for purified macrophage cultures for experimental studies increases the need for a comparison of the results obtained in different laboratories. It is to be 'hoped' that these factors will stimulate experimental efforts aimed at improving macrophage purification procedures as well as at defining reference protocols for specific research purposes.

Acknowledgements We are indebted to Alberto Mantovani, Carlo Federico Perno, Stefano Fais, and Colomba Giorgi for providing scientific material and for critical reading of the manuscript. We thank Sabrina Tocchio, Anna Maria Fattapposta, and Cinzia Gasparrini for their excellent secretarial assistance. Work in the authors laboratory was supported in part by grants from the Italian Ministry of Health (40B/H and 40B/D).

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Chapter 3 Characterization of macrophage antigens and receptors by immunochemistry and fluorescent analysis: expression, endocytosis, and phagocytosis Leanne peiser, Peter J. Gough, Elizabeth Darley, and Siamon Gordon Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford 0X1 3RE UK

1 Introduction This chapter describes techniques to identify macrophages and measure their endocytic and phagocytic capabilities. These methods can assist in the analysis of the physiological and pathological situations in which macrophages play a central role including homeostasis, wound repair, and immunity. The expression of cell surface proteins and other molecules varies greatly among macrophage populations as a result of heterogeneity in their differentiation, activation state, or response to external influences in their microenvironment or cell culture conditions (1-3). Immunochemkal and fluorescent techniques can be used to investigate the basal expression of antigens, the changes induced in response to a particular stimulus, and to detect antigens in tissues. The function of cellular proteins can also be investigated using immunochemical and fluorescent techniques. In the case of endocytic receptors, appropriately labelled ligands can be added to the cell, their intracellular trafficking traced, and compared with that of other macrophage and non-macrophage receptors. Changes in activity of known receptors and potential functions of novel molecules can be tested in a range of assays with soluble and particulate ligands. In vivo and in vitro investigation of macrophages or specific molecules is essential for an overall understanding of their biological function, as is highlighted in the analysis of knockout and transgenic mice that lack, under-, or overexpress selected molecules. This chapter details basic techniques used to detect macrophage-specific anti61

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gen markers in tissue and in vitro, and to assay endocytic and phagocytic abilities of isolated cells, with emphasis on selected plasma membrane receptors involved in innate immune recognition. The techniques described can be applied to a variety of macrophage-like cell lines such as RAW 264.7 murine macrophages, THP-1 human monocytic-like cells, and isolated murine or human primary macrophage populations. These include: murine resident peritoneal macrophages or cells elicited by Bio-Gel polyacrylamide beads, thioglycollate broth, or Mycobacterium bovis BCG; murine bone marrow-derived macrophages and dendritic cells; human blood monocytes and monocyte-derived macrophages and dendritic cells (see Chapters 1 and 2). Methods used to examine other characteristic macrophage functions, including release of secretory products, are discussed elsewhere in this volume (see Chapters 5-7) and in useful general compendia (4).

2 Immunochemical labelling of monocytes and macrophages 2.1 Introduction Immunochemical labelling of monocytes and macrophages can be used to identify macrophage populations in a complex mixture, like blood. The cells can be identified using macrophage-specific antibodies and then separated from the others by flow cytometry or magnetic beads. Table 1 is a list of macrophageTable 1 Selected macrophage markersa Marker"

Species0

CD68 (macrosialin)

Human

EBM-11

Dako

Mouse

FA-11

Ref.33

Human

ICRF44

Serotec

Mouse

5C6

Serotec

Human

YFC118.33

Serotec

Mouse

C71/16

Serotec

Human

UCHM1

Serotec

Mouse

rmC5-3

PharMingen

Human

CR3/43

Dako

Mouse

TIB120

ATCC

Human

AT10

Serotec

Mouse

2.4g2

PharMingen

F4/80

Mouse

F4/80

Serotec

SR-A (scavenger receptor)

Mouse

2F8

Serotec

Sialoadhesin

Mouse

3D6.112

Serotec

CDllb(CR3)

CD18 CD14 MHCIId

CD32

Clone

Supplier/reference

a See refs 28-30 for other markers and reagents. b Several of these markers may also be expressed on dendritic cells, neutrophils, selected endothelial, or other cells. c All Ab reagents listed in this table for human antigens are mouse, for mouse antigens, rat. d Note if antibody is polymorphic or monomorphic. Mouse antibodies can be difficult to detect by indirect methods in mouse tissue.

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Figure 1 Imniunoflu ore scent staining and Dil-AcLDL labelling of murine bone marrow-derived macrophages, Murine bone marrow-derived macrophages were plated in a 24-well plate containing 13 mm glass coverslips at a density of 2 x 105 cells per well. (A-D) Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-macrosialin (FA-11) (A and B), or anti-F4/SO (F4/SO) (C and D), followed by FITC -conjugated goat anti-mouse IgG. Cells were viewed by fluorescence microscopy and photographs of the same field taken under phase-contrast (A and C) or fluorescence (B and D) illumination. The staining highlights the predominantly intracellular localization of macrosiaim compared to the cell surface expression of the F4/80 antigen. (E-F) Cells were labelled by incubation with Dil-AcLDL at a concentration of 5 ug/ml for 3 h at 37°C, washed four times with PBS, and subsequently fixed in 4% paraformaldehyde. Coverslips were viewed by fluorescence microscopy and photographs taken under phase-contrast (E) and fluorescent (F) illumination.

specific markers used to sort and identify macrophages. The level of expression of a particular antigen which may vary markedly during maturation from monocyte to macrophage, can be quantified by flow cy tome try, or visualized by microscopy. The distribution of an antigen in macrophages can be examined, moreover dual and triple detection of multiple antigens by microscopic methods can compare their distribution and allow for the identification of the endocytic compartments in which they are found. Hgure 1 A-D shows bone marrow culture-derived macrophages stained with mac roph age-specific markers, F4/80 or FA.ll ; note the 63

LEANNE PEISER ET AL.

differences in antigen distribution with regard to surface and intracellular expression.

2.2 Practical considerations for immunofluorescent staining of macrophage populations in vitro 2.2.1 Direct versus indirect immunochemical labelling There are two methods to detect antigens expressed by macrophages (5). Coupling of a detectable label (fluorochrome or enzyme) to an antigen-specific antibody (Ab) can be used for direct identification of the corresponding antigen. Indirect immunochemical staining utilizes an unlabelled primary Ab which is detected by a labelled secondary Ab raised against IgG of the species that produced the primary reagent. Background labelling, caused by non-specific binding of antibodies to the cell, should be low with direct detection, but staining can be less sensitive than by the indirect method. Cell labelling with conjugated antibodies is quicker; however the coupling of markers to antibodies is time-consuming, expensive, and may result in a loss of activity. The indirect staining technique offers greater versatility of reagents with no loss of activity of the unlabelled primary Ab and is more sensitive than direct labelling as the signal is amplified due to the primary Ab having multiple binding sites for the secondary Ab. However, there may be higher background staining (see Section 2.2.4), which can be overcome by blocking non-specific binding sites with an appropriate serum. A variation of both protocols is the use of a biotinylated primary or secondary Ab, which is detected by labelled biotin-binding proteins or anti-biotin antibodies. Titrate primary Abs to obtain an optimal specific signal-to-background noise.

2.2.2 Dual and triple immunochemical staining The simultaneous study of multiple antigens can be achieved by dual and triple labelling. The use of direct immunochemical detection is preferable, however, if not available, indirect methods can be adapted for use. The primary antibodies limit the number of detectable parameters as these should either be differently labelled or raised in a different species. For indirect detection the secondary Abs must distinguish between the various primary Abs so they should vary in species or IgG subclass. A biotinylated form of one of the primary Ab may be used (see Section 2.1.1) to facilitate detection if the primary Abs are from the same species. Controls are essential. Single-stained cells must always be included to control for the 'spill over' (bleed-through) for example, of fluorochromes into the range of different fluorescent detectors (see Section 3.1.2).

2.2.3 Permeabilization of cells If the antigen is predominately expressed on the cell surface, the use of nonpermeabilized cells is recommended as there is then less background binding of Ab to intracellular proteins. Permeabilization allows the visualization of intra64

CHARACTERIZATION OF MACROPHAGE ANTIGENS

cellular and cell surface antigens. Cells can be permeabilized after fixation (see Section 2.3.3) by 0.1% (v/v) Triton X-100 or 0.25% (w/v) saponin (see Protocol 3). Comparisons between permeabilized and non-permeabilized cells can give an estimate of the distribution of the antigen within a cell.

2.2.4 Secondary antibodies Macrophages express several receptors that bind the Fc region of both the primary and secondary Abs. We recommend using Fab2' fragments as secondary Abs to reduce this and to block the cells with normal serum or IgG from the animal used to raise the secondary reagent. Labelled secondary Abs are commercially available from Sigma, Serotec, or other commercial suppliers. Table 2 contains a list of fluorochromes commonly attached to proteins or Abs. Secondary Abs coupled to alkaline phosphatase, peroxidase, and biotin allow for enzymatic detection of the antigen. These are useful for light microscopic analysis of macrophages in tissues. Endogenous enzyme or biotin can be a problem in different tissues. Table 2 Fluorochromes and their spectra Fluorochrome

Excitation (nm)

Emission (nm)

Supplier

Fluorescein isothiocyanate (FITC)

492

Sigma

Phycoerythrin (PE)

480-565

Texas Red (TRSC)

596

520 578 620

Rhodamine (TRITC)

570 680

Sigma

Dil

550 650 550

'570

Lucifer yellow

425

530

Indodicarbocyanine (Cy5)

Sigma Molecular Probes Molecular Probes Molecular Probes Sigma

2.2.5 Antibody specificity controls Indirect immunochemical staining must include a control for the specificity of the primary Ab. Isotype-matched control Abs should be of the same subclass as the primary antibody, but not directed against any cell antigens. Controls for directlyconjugated Abs are more difficult, but an identically labelled non-reactive isotypematched control should be used. These controls are especially important because of antigen non-specific Fc receptor (CD16, CD32, CD64) binding (see Section 2.2.4). Blocking some Fc receptors with the 2.4G2 antibody (PharMingen) can reduce this background. Where available, excess free antigen, for example a peptide, could be used to show specificity of labelling by a putative antibody.

2.3 Preparation of in vitro macrophage cultures for immunochemical staining 2.3.1 Cell culture Chapters 1 and 2 of this book cover the isolation and in vitro cultivation of macrophages. Section 1 contains a list of macrophage populations on which these 65

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techniques are commonly used. All human cells and serum must be treated as potentially contagious, with HIV or hepatitis especially, and must be handled and disposed of accordingly. When preparing isolated macrophages for immunochemical staining, we recommend plating them on glass coverslips. Protocol 7 describes one method for coverslip staining.

Preparation of isolated macrophages for immunochemical staining Equipment • 13 mm, no.1 thickness glass coverslips (BDH)

• 24-well dishes (Falcon, Becton Dickinson)

A Preparation of coverslips 1 The coverslips should be sterilized and cleaned before use by washing in ethanol and flaming. 2 To prevent macrophages attaching to the undersurface of the coverslip, place coverslips in the wells of a 24-well plate, cover with culture medium, and press down onto the well bottom before adding the cells.a B Staining of coverslips 1 Staining of a coverslip can be done either in a well of a 24-well dish or by floating the coverslip on a drop of antibody pipetted onto Parafilm, It is quicker and easier to stain the coverslip in the well and it does require less handling of the fragile coverslip, though larger volumes of antibody for staining are needed. Staining on a drop of Ab cannot be used when Triton X-100 has permeabilized the cells as the presence of detergent reduces the surface tension, making it difficult to float the coverslips. 2 The coverslips can be mounted on glass slides for easy handling; plastic coverslips with suitable optical properties can also be mounted on glass. If analysing the cells by flow cytometry do not plate the macrophages on coverslips, but on a substratum from which the cells can be detached (see Section 2.3.2). a

The suggested plating density for each well is 2-5 x 105 macrophages.

2.3.2 Detachment of macrophages By their nature, macrophages are uniquely adherent cells and are difficult to detach from plastic dishes. Cultivation in scrum makes it easier to detach macrophages compared with serum-free protein-containing medium such as OptimemI (Gibco). Dishes may be coated with proteins such as fibroncctin, microexudate, or gelatin (plus serum as a source of adhesion molecules; see Chapter 2). It is often beneficial to culture the macrophages on bacteriological, i.e. unmodified 66

CHARACTERIZATION OF MACROPHAGE ANTIGENS

plastic surfaces. The complement receptor type 3 (CR3) is responsible for most of the adhesion to serum-coated bacteriological plastic, while the macrophage scavenger receptor (SR-A) mediates most macrophage EDTA-rcsistant adhesion to serum-coated tissue culture plastic dishes. Our laboratory uses a combination of EDTA and Lidocaine-HCl, to avoid the use of degradative enzymes, like trypsin, that may destroy antigen epitopes and are ineffective (6). Lidocaine-HCl can also be added directly to serum-containing culture medium. Scraping cells from the bottom of the dish may damage fragile and more spread cells in the population; rather pipette the Lidocaine/EDTA vigorously over the cells. Procedures can also be performed at 4°C, with pre-cooled cells. The viability of the cells should be tested in all cases. Pro toco! 2 describes the use of Lidocaine for detaching macrophages.

Detaching cultured macrophages from plastic and glass surfaces using EDTA/Lidocaine Equipment and reagents • Phosphate-buffered saline (PBS) (Sigma): combine 8.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4.12H2O. 0.2 g KC1 (all Sigma) in 1 litre dH20, and pH to 7.3

Polypropylene tubes (various sizes) Cultured macrophages (prepared as described in Chapter 2) in flasks or tissue culture dishes

• Detachment buffera combine 10-15 mM Lidocaine-HCl (Sigma) and 10 mM EDTA in PBS

Method 1 2 3 4 5 6 7 8 a

Aspirate the culture medium from the cells. Wash the monolayers twice with PBS. Add the detachment buffer. Use approx. 10 ml buffer per 100 cm2 of tissue culture dish or flask. Incubate the cells at 37 °C for 5-30 min. Remove the cells by vigorous pipetting and place in an appropriate tube. Harvest the cells by centrifugation at 1500 g for 5 min. Aspirate the supernatant fluid. Resuspend the cells in PBS or fixative as appropriate (see Protocol 3).

The Lidocaine may be added directly into the culture medium,

2.3.3 Fixation Fixatives routinely used for immunochemical labelling can be divided into two categories, organic solvents and cross-linking reagents. Organic solvents, such as acetone and alcohols, remove the lipids, dehydrate the cells, and precipitate intracellular 67

LEANNE PEISER ET AL.

proteins. In comparison, cross-linking reagents form intermolecular bridges, normally through free amino groups, creating a network of linked antigens. Both methods result in some antigen modification, thereby affecting the ability of the antibody to recognize the antigen; it is this that largely dictates which fixative is used. Cells fixed by cross-linking fixatives, such as formaldehyde, paraformaldehyde (see Protocol 2), and glutaraldehyde, retain their architecture well and are best for subcellular localization studies by light or confocal microscopy and flow cytometry. Glutaraldehyde may be used at relatively low concentrations such as 0.25-0.5% to preserve antigenicity, although antigens like F4/80 are stable to 1.25% glutaraldehyde. Organic solvents permeabilize the cells, which may be an advantage, but care must be taken when using plastic containers and they should not be used on non-adherent cells for flow cytometry as the fixative can cause cell aggregation and blockage of the flow cytometer. Protocols 3 and 4 describe a reliable procedure for fixation and indirect staining of cultured macrophages.

Preparation of 4% paraformaldehyde Equipment and reagents • 0.22 p.m filter (BDH)

5 M NaOH

• Paraformaldehyde (BDH)

PBS {see Protocol 2)

• 1 M Hepes (Gibco)

Method 1 2 3 4 5

Dissolve 4 g of paraformaldehyde in 90 ml PBS containing one to two drops of 5 M NaOH by mixing at 56°C. Make the solution up to 100 ml by adding 10 ml of 1 M Hepes. Check the pH to ensure that it is approximately 7. Filter the paraformaldehyde using a 0.22 |j.m filter. Chill to room temperature or 4°C, as appropriate, before use and use immediately.a

a

The paraformaldehyde should be made fresh. It also can be prepared as 8% stock, stored in aliquots at -20°C, and diluted with PBS before use.

Indirect immunofluorescent staining of cultured macrophages Equipment and reagents • Flow cytometer

PBS (see Protocol 2)

* 4% paraformaldehyde (see Protocol 3)a

Saponin (Sigma) or Triton X-100 (Sigma)

CHARACTERIZATION OF MACROPHAGE ANTIGENS

• Blockingbuffer:b 10% normal serum of the species of secondary antibodyc diluted in PBS

* Primary antibody • Labelled secondary antibody (see Section 2.2.4)

Methodd 1 Detache adherent cells from the culture dishes (see Protocol 2)f and place in appropriate size tube. 2 Harvest the cells by centrifugation at 1500 g for 5 min. 3 Resuspend the cell pellet in fresh 4% paraformaldehyde and leave on ice for 10 min. 4 Harvest the cells by centrifuging them at 1500 g for 5 min. 5 Remove the paraformaldehyde by aspriration. 6 Resuspend the cells in 1-2 ml blocking solution to quench any remaining fixative. If staining an intracellular antigen, add permeabilization agentsg at this point to the blocking solution and keep present in all subsequent incubation steps. 7 Harvest the cells as in step 4 and resuspend them in the blocking solution. 8 Incubate at room temperature for 3 min. 9 Harvest the cells as in step 4. 10 Resuspend the cells in blocking solution containing the correct dilution of primary Ab. Use a concentration of approx. 5 x 106 cells/ml blocking solution. Use antibodies at the manufacturer's recommended concentration or titrate (10 ^g/ml is a good starting concentration). 11 Incubate cells for 1 h at room temperature. 12 Harvest the cells as in step 4. 13 Aspirate the antibody and wash the cells in blocking solution by centrifugation for 5 min at 1500 g. 14 Repeat the washing step above three times. 15 Resuspend the cells in blocking solution containing the secondary antibody (titrate each antibody to obtain the correct dilution). 16 Incubate for 1 h at room temperature. 17 Harvest the cells as in step 4. 18 Aspirate the secondary antibody and wash cells in blocking solution as in steps 13 and 14. 19 Resuspend cells in 500 ^.1 of PBS and analyse on a flow cytometer. If analysing the cells by microscopy place the coverslip, cell-side down, on a drop of mounting medium (see Section 3.2) on a glass slide. a Other fixatives, like acetone, may be used for microscopic analysis, but we recommend paraformaldehyde. Paraformaldehyde is best to use for flow cytomerry. b This is to block non-specific IgG binding sites.

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c

Use the normal serum of the primary antibody if doing direct immunofluorescent labelling. If staining unfixed cells then perform the staining at 4°C and begin at step 9. f If staining cells attached to a coverslip, begin at step 3, leaving out step 4. Wash the coverslips in a well of a 24-well dish (see Protocol 1). f Approx. 1-5 x 106 cells should be stained in analysing results by flow cytometry. See Protocol t for the recommended number of cells to be plated on a toverslip, g We recommend 0.25% (w/v) saponin or 0.1% (v/v) Triton X-100 be added to the blocking buffer. d

3 Detection techniques for fluorescent analysis of macrophages 3.1 Flow cytometry 3.1.1 Applications of flow cytometry How cytometry can measure the size and granularity of cells from their light scattering properties, in addition to determining the expression of antigcnic markers by fluorescence detection. Flow cytometry may also be used to measure the relative uptake of fluorescent endocytic and phagocytic tracers (see Sections 5.2 and 5.3). Macrophages can be distinguished and sorted from other cell types in a complex cell mixture on the basis of size and granularity, using the forward and side scatter parameters, and by the expression of macrophage-restricted antigens using fluorescent antibody labelling.

3.1.2 Practical considerations for detecting macrophages by flow cytometry A few points on the analysis of macrophages by flow cytometry will be mentioned here. However, for detailed information on the techniques required see refs 7 and 8. Due to their large size, macrophagcs have a large forward scatter making it easy to sort them from lymphocytes in mixed cell populations. Macrophages arc heterogeneous with respect to their size within a given isolated population, which results in a broad range of forward scatter values when compared with other cell types. Double and triple labelling of macrophages for the flow cytomcter requires adjustment of the flow cytometer to compensate for the broad emission spectra of the commonly used fluorochromes (see Table 2). The broad emission spectrum of the fLuorochromes is responsible for the overlap into the spectrum of other fluorochromes. To help in the accurate setting of the colour compensation, it is important that there is little cross-reactivity between reagents and the macrophages cultured in phenol red-free medium to minimize acquired autofluorescence. The colour compensation should be set up with control macrophages labelled with only one of the fluorochromes used. It is recommended that a large 70

CHARACTERIZATION OF MACROPHAGE ANTIGENS

number of control cells are prepared since balancing compensation can require many cells. A good description on how to set up a flow cytometer for double and triple labelling can be accessed at: http://www.molbiol.ox.ac.uk/ pathology/tig/facs.html Macrophage adherence to plastic test-tubes during incubation is a problem so use less sticky polypropylene vessels and incubate and wash the cells at 4°C. Ensure that the macrophages are in a single cell suspension as clumps can alter the population distribution on the flow cytometer.

3.2 Fluorescent microscopy Immunofluorescence microscopy is useful for studying the subcellular localization of a particular antigen and examining the endocytic and phagocytic pathways of macrophages. The recent increase in the availability and use of confocal microscopes has allowed greater insight into the behaviour of a number of intracellular proteins. However, microscopy cannot quantify the levels of a particular antigen expressed by a cell. In addition, measurements of the proportion of cells expressing a particular antigen requires time-consuming counting of large number of cells for statistic significance. Similar to flow cytometric analysis, fluorescence microscopy can be hindered by 'bleed-through' of signal from antibodies labelled with different fluorochromes. This may partially be corrected by titration of antibodies during labelling to reduce the signal to levels where spill over is less. However, care should always be taken when interpreting results of double or triple labelling experiments, examining the co-localization of antigens within a given cell. Following staining, the coverslips should be mounted onto glass slides using mounting medium. We recommend the use of mountant containing agents that slow the bleaching of the fluorochrome during viewing on the microscope such as Vectashield (Vector Laboratories). A small quantity (5 ul) of mountant should be spotted onto the slide and the coverslip lowered slowly after one edge has been placed on the slide to exclude air bubbles. After the slide is flat it should be gently pressed to minimize the movement of the coverslip whilst viewing under oil immersion.

4 Immunohistochemical staining of macrophages in mouse tissues The protocols described below are for mouse tissue, though they can be adapted for use with human and rat. Detection of antigen in tissue embedded in wax can be enhanced by empirical methods including microwaving, pressure cooking, and protease treatment, but will not be described here (9, 10). Staining procedures must be optimized for each antigen and Ab. All diseased specimens, especially human tissue, are potentially hazardous. 71

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4,1 Preparation of tissue 4.1.1 Frozen versus perfused tissue Tissue may either be taken fresh from the animal and frozen immediately, or the animal may be perfused with fixative and then dissected. Perfused tissue yields better morphology than fresh tissue, however, the procedure requires practice to ensure the best result. Also, one is limited to a single fixative, whereas with fresh tissue it is possible to use a number of different fixatives to ensure optimal conditions for antibody binding. Tissue processed by wax embedding methods may also be used, but generally the stages of alcohol dehydration and the temperature required for infiltration and embedding render the tissue unsuitable for all but the most robust antibody staining.

4.1.2 Practical considerations for the preparation of fresh tissue It is important to harvest and freeze tissue as quickly as possible to preserve antigen. If processing more than one animal, sacrifice individually and dissect immediately. It is also preferable to use some form of ayo-protectant around material to be frozen as this allows for a longer storage time without marked deterioration of the tissue. Cryo-protectants may vary in quality and storage ability, but the example given (see Protocol 5) has proved satisfactory over a long period of time. Freezing the tissue in an iso-pentane bath is preferable, though liquid nitrogen may be used. However, with liquid nitrogen freezing generally occurs too quickly, which may result in the distortion and cracking of the material. In an iso-pentane bath, the block freezes from the bottom leaving a small area at the top to freeze last, which allows for the expansion or contraction of the material. Once frozen, the tissue may be left for some months, but will eventually lose moisture and change texture, which makes cutting more difficult. A procedure for preparation of fresh tissue for immunohistochemistry is given in Protocol 5.

Preparation of fresh tissue for immunohistochemistry Equipment and reagents • Disposable moulds (Raymond A, Lamb) • Iso-pentane (BDH) • Tissue-Tek OCT compound (Bayer Diagnostics)

Dry ice (C02) Freshly obtained mouse or human tissue

Method 1

Before dissection, place a small amount of iso-pentane in a small Pyrex dish.a

2

Place the dish in a polystyrene box containing a layer of 'dry ice' (CO2).

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3

Cover box to lower temperature quickly.

4

Fill a mouldb with Tissue-Tek, ensuring as few air bubbles as possible.

5

Place the tissue carefully in mould,c excluding air bubbles, and orientating it so that the required plane faces the bottom of the mould as this is the face that is presented to the cryostat blade.

6

Add more Tissue-Tek, if required, to ensure the tissue is completely surrounded.d

7

Place the mould in the iso-pentane and leave to freeze for at least 5 min.

8

Store frozen blocks at -20 "C.

a

Do NOT use alcohol as it is miscible with Tissue-Tek and will not allow the block to freeze properly. It will then be impossible to cut as the texture will never harden to the same consistency as the tissue. b Moulds can be plastic, metal, or foil. The foil mould is made by wrapping foil around an object, such as the end of a pen and sealing one end. Tap on the bench to flatten the end. then remove the pen. c Upon dissection the tissue may be washed in PBS, but any fluid should be absorbed on a piece of tissue. Tissue-Tek will not adhere to wet material, which will detach from the block whilst cutting, therefore drying of the tissue is essential. d This eliminates the possibility of'freezer-burn' if the blocks are to be stored for some time.

4.1.3 Perfusion of tissue Protocols 6 and 7 outline one method for the perfusion of tissue in intact animals, prior to tissue dissection. The buffered lysine and para formaldehyde used for perfusion can be prepared and stored at 4°C overnight, but they should only be mixed together when needed as the fixative does not keep (see Protocol 6). If the tubing becomes detached from the heart during the perfusion procedure it is not easy to reattach without losing fluid from the old incisions. Thus, fixation will be incomplete and the procedure should be abandoned. Care should also be taken so the line is not pushed through the heart and out the other side!

Preparation of perlodate-lyslne paraformaldehyde for perfusion of mouse tissuea Reagents • Lysine monohydrochloride (Sigma): dissolve 6.85 g in 187.5 ml dH20 • Sorensen's salt (Na2HPO 4 2H0) (Sigma): dissolve 0.9 g in 50 ml dH2O

Paraformaldehyde (BDH): dissolve 10 g of paraformaldehyde in 100 ml dH2O and ten drops of 1 M NaOH at 56°C, then cool to room temperatureb

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• Sodium periodate (Sigma) • 1 M NaOH • 0.1M phosphate buffer: 7.8 g NaH2PO4,2H20 (BDH) dissolved in 250 ml

dH20 and 34 g Na2HPO42H2O (BDH) dissolved in 1 litre dH2O. Mix the dissolved NaH2PO4.2H2O and Na2HPO4.2H2O and dilute 1:1 to make the 0.1 M working solution.

Method 1

Buffer the 187.5 ml lysine monohydrochloride with the 50 ml Sorensen's salt to give a pH of 7.4.

2

Mix the buffered lysine monohydrochloride and the paraformaldehyde.

3

Make the volume to 500 ml with 0.1 M phosphate buffer.

4

Add 1.07 g of sodium periodate to the lysine/paraformaldehyde.c

a

Adapted from ref. 11. This paraformaldehyde solution must not be confused with the solution described in Protocol 3. c The pH will fall to about 6.4-6.6 on mixing. This appears not to have an adverse effect on antigen preservation, but tissue preservation may not be ideal. Some variation in pH can be found at this point and improved preservation is obtained by bringing the pH to about 6.8 using a few drops of 20% NaOH. b

Perfusion of mouse tissue Equipment and reagents • • • • • •

Dissection instruments and board Perfusion fluid (see Protocol 6] Heparinized PBS (20 U/ml) 20 ml syringe 50 ml syringe Length of fine Portex plastic tubing (size 23G) (Fischer Scientific)

Butterfly needle Container for organs 70% alcohol 0.1 M phosphate buffer (see Protocol 6} Sucrose (Sigma)

Method 1 Fill the 20 ml syringe with heparinized PBS and the 50 ml syringe with periodatelysine paraformaldehyde solution (see Protocol 6). 2 Stretch the plastic tubing between fingers until quite thin. 3 Cut obliquely across the tubing at the thin section and attach the butterfly needle to the other end. 4 Connect the 20 ml syringe to the butterfly needle and test to ensure good flow.

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CHARACTERIZATION OF MACROPHAGE ANTIGENS

5 After sacrificing the animal, secure the animal on the dissection board and spray with 70% alcohol. 6 Thread the plastic tubing under the animal's left hind leg, for stability, and in a curve round to the heart area. 7 Make an incision to expose the abdominal organs and one down each side of the ribs to expose the heart.a 8 Introduce the obliquely cut end of the plastic tubing into the left ventricle of the heart.b 9 Make an incision in the inferior vena cava, just above the right kidney, to allow the fluid to drain. 10 Run the heparinized saline through the body at an approx. rate of 2 ml/min.c 11 When the body fluid appears to be clear upon exit from the kidney area, change the syringe to run the fixative through the animal at approx. 2 ml/min.d 12 Continue to perfuse the whole 50 ml of fixative (perfusion fluid} to ensure good fixation. 13 At the completion of perfusion, dissect the animal. 14 Post-fix the organs in the same fixative for about 4-6 h. 15 Place the organs in 20% sucrose in 0.1 M phosphate buffer overnight. 16 Block the tissue (see Protocol 5, steps 4-8), taking care to ensure all moisture is absorbed on a tissue before embedding. a Leaving the uppermost portion of the ribs gives support to the heart against the pressure of the fluid. b Do not use gloves as this will cause the tubing to slip out of the heart. c lt is critical not to hurry with this procedure as forcing the fluid through too quickly will irretrievably damage the organs and cells. If the tubing becomes detached from the heart during the perfusion procedure it is not easy to reattach without losing fluid from the old incisions. Thus, fixation will be incomplete and the procedure should be abandoned. Care should also be taken so the line is not pushed through the heart and out the other side! d Shortly after starting to run the fixative through the animal may be seen to 'twitch'; this an indication of the fixative reaching al! areas.

4.1.4 Cutting the tissue blocks Tissue blocks are cut on a cryostat.; usually at a thickness of 5-10 ^.m. Depending on the density and hardness of the material, some tissues may require a slight change of temperature in the cryostat chamber to cut satisfactorily. For example, bone may require cutting at a lower temperature than liver. The temperature required is resolved by trial anci error, however, as a guide, a recommended Temperature for the cryostat chamber is -20 °C and that of the chuck, -130C to -18°C, The sections are collected onto slides and may be stored at -20^C. When 75

LEANNE PEISER ET AL.

block cutting is finished, a small amount of Tissue-Tek should be used to cover the exposed surface to prevent 'freezer-burn' before returning to the freezer. Some thawing is inevitable when the Tissue-Tek is placed on the exposed surface after cutting, this results in some loss of antigen so if re-cutting, cut well into the block before collecting sections. Once frozen, slides should not be allowed to thaw until required for use. It is safest to assume that freeze/thawing slides will significantly reduce antigen detection, su when collecting some slides for use return the rest to the freezer as soon as possible.

4.2 Immunochemical staining of tissue See Figure 2 for examples of immunohistochemical staining of human and mouse tissues with macrophage-restricted markers. All DAB procedures should be carried out on a tray and on a ventilated table or in a fume hood. When finished, the tray and all contents should be placed in the sink and filled with a strong solution of bleach making sure everything is submerged or filled with the solution. It should then be left overnight, after which the bleach can be flushed away with copious amounts of water and the contents disposed normal disposal means.

Figure 2 Immunohistochemistry of macrophages in human and murine tissues. (A) Antiscavenger receptor (SR-A) (31) staining of Kupffer cells in human liver. (B) CD68 (EBM-11) staining of tingible body macrophages in a germinal centre from human tonsil. (C) F4/80 (F4/80) staining of macrophages in the red pulp of mouse spleen. (D) Sialoadhesin (3D6) (32) staining of the subcapsuiar sinus area of a mouse lymph node. (C and D) Pictures courtesy of L. Martinez-Pomares,

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Immunochemical staining of mouse tissue Equipment and reagents • Slides (BDH) • • • • • • • • • • • •

Coverslips (BDH) 20 ml syringe 0.45 (im filter Two 21-gauge needles 0.5-10 (ul pipette and tip Primary Ab 2% paraformaldehyde (see Protocol 3, but use 2 g in 100ml)a PBS tablets (Sigma) Triton X-100 (BDH) Normal serum (Sigma) Secondary Ab (Sigma) Imidazole (Sigma): 0.34 g imidazole dissolved in 500 ml PBS

Quench solution for endogenous peroxidases: 50 ml of 0.1 M phosphate buffer (see Protocol 6), 50 ul of 1 M sodium azide (BDH), and 0.09 g glucose (BDH); warn to 37°C and add 20 ul glucose oxidase (Sigma) just before use DABb (Park Scientific) Avidin/biotin block (Vector Laboratories) 0.1% cresyl violet acetatec (Raymond A. Lamb): 1 g cresyl violet acetate in 1000 ml dH2O, heat to boiling point and cool while stirring continuously; filter the cooled liquid in a 0.22 um filter ABC reagent (Vector Laboratories) DPX (BDH) H202

A Immunofluorescent labelling 1 Thaw the slide mounted with frozen tissue for 30-60 min.d 2 Fix the fresh sections by incubating with 2% paraformaldehyde on ice for 10 min. 3 Wash and permeabilize the slide by treating with PBS containing 0.1% Triton for 5 min. 4 Repeat step 3 three times. 5 Quenche the slide with the quench solution for 15 min at 37 °C, 6 Wash the slide three times with PBS for 5 min each timef 7 Block the tissue by incubating with 2-5% normal serum for 30 min at room temperature. 8 Dilute the primary antibody to the appropriate concentration in normal serum. 9 Add the antibody to the slide and incubate for 60 min at room temperature, 10 Remove the primary antibody as wash the slide with PBS (see step 6). 11 Add the secondary antibody, diluted to manufacturer's specifications, and incubate for 30 min. 12 Remove the secondary antibody and wash the slide with PBS (see step 6). 13 Incubate the slide with the ABC reagent for 30 min. 14 Remove the secondary antibody as wash the slide with PBS (see step 6). 15 Stain with DAB (see part B).g

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B Dlamlnobenzidine detection 1 Place the imidazole into the beaker and draw 20 ml of the solution into a syringe. 2 Push the two needles into the rubber stopper of the DAB vial. 3 Inject the imidazole into the vial through one needle and mix the two compounds thoroughly. 4 Pipette 6 ul H202 into beaker. 5 Withdraw the DAB solution from the vial using the syringe, 6 Inject the DAB solution through the 0.45 |xm filter into beaker containing H2O2. 7 Remove the coverplates from the slides and lay slides on racks in trays, which are all stacked up on one side of the vented table and kept solely for this purpose. 8 Cover the tissue with DAB and monitor by eye for a reaction using the negative control to assess colour. 9 After the desired reaction has taken place, remove slides from rack and place in a staining rack in a dish of PBS to stop further colour development. 10 When all slides have been reacted, wash each slide twice with PBS, then once with distilled water. 11 Dip the slides a couple of times in cresyl violet acetate to counterstain. 12 Wash the slides with distilled water (see part B, step 10). 13 Dehydrate the tissue and place a coverslip over the tissue. a Other fixatives may be used such as acetone and methanol. b DAB is a carcinogen and should be treated with respect. It is most dangerous in powder form and should not be removed from the container. c Methyl green may be used as a counterstain in place of cresyl violet, d With perfused tissue sections proceed directly to step 4. e Quenching is often carried out using 1% H2O2 in methanol, but we have found best results using glucose/glucose oxidase, f The slide may be blocked in avidin and biotin following this step by incubating it with avidin for 15 min, followed by three washes with PBS, then incubate with biotin for 15 min, followed by another three washes with PBS. g The slide may be stained with alkaline phosphatase-conjugated secondary antibodies in place of DAB detection. For information, see ref. 10.

5 Fluorescent analysis of macrophage endocytic function 5.1 Introduction to the endocytic pathway Macrophages are able to ingest particulate and soluble matter efficiently by phagocytosis and endocytosis. These processes are essential for macrophage 78

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antigen presentation and removal of pathogens. Below is a brief introduction to some of the components of the endocytic pathway, for a comprehensive review see refs 12 and 13. Endocytosis includes pinocytosis, receptor-mediated endocytosis, and phagocytosis. Pinocytosis is the non-specific sampling of the extracellular milieu and occurs by the random invagination and pinching off of plasma membrane resulting in vesicle formation. It is a constitutive process with no sorting of contents of the vesicle or its membrane (12). Receptor-mediated endocytosis utilizes specific receptors that bind ligands and concentrate them inside the cell. The receptors collect in micro-domains of the plasma membrane called coated pits. These pits are underlined with clathrin and adaptor proteins that bind to the receptor's cytoplasmic tail by the recognition of specific motifs on the tails, for example NPVY found on the LDL receptor and a di-leucine motif found on Fc receptor. The receptor-ligand complexes are internalized into a clathrin-coated vesicle. The clathrin is rapidly shed, the vesicle fuses with the early endosome resulting in the sorting of the receptor, ligand, and membrane through the endocytic pathway. The later endocytic compartments are characterized by decreasing pH and increasing delivery of lysosomal enzymes, like cathepsins, to the compartments (13,14). There are two models for the movement of membrane and vesicles through the endocytic pathway. First, it is proposed that early endosomes mature into late endosomes and subsequently lysosomes (15). The second and favoured model is based on vesicular transport. Here endocytic compartments such as the early and late endosomes are stable, preformed, and vesicles shuttle back and forth to deliver components between them (16, 17). Mechanisms that tightly control docking and vesicle fusion are required to maintain the correct flow and function of the pathway. Proteins implicated in this control are the Rabs, small GTPbinding proteins; and the SNARES. Each compartment is thought to have a specific SNARE to direct the docking machinery and confer specificity. v-SNARES are found on vesicles and t-SNARES on the compartment membrane. The SNAREs bind the NSF/SNAP complex, which is essential for vesicle fusion (18). Macropinocytosis is a form of specialized pinocytosis, which occurs when a membrane ruffle folds over itself, engulfing material. This form of internalization can mediate the uptake of soluble and particulate material. For example, Salmonella in epithelial cells and macrophages is internalized by this mechanism (19, 20). Potocytosis is another form of specialized endocytosis. Receptors, such as the folate receptor, that utilize this method of uptake often have glycosylphosphatidylinositol glycan (GPI)-linked anchors and cluster in imaginations of the plasma membrane called caveolae (21). Phagocytosis is the ingestion of particulate matter by a cell. Macrophages, monocytes, and neutrophils are the most efficient phagocytes. Macrophages express many phagocytic receptors which fall into two broad categories. The first are the opsonin-dependent receptors, which require the coating of the particle by opsonin before the receptor can recognize it. Such receptors are the complement receptor type 3, Fc receptors, Clq receptor, and CD14, which bind 79

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particles coated by iC3b, antibodies, Clq, and lipopolysaccharide-binding protein, respectively. The other group of receptors, the pattern-recognition receptors, are able to bind structures on the particle surface directly and include the mannose receptor (MR), CD36, and SR-A (22). Following ingestion, the particle is contained within a phagosome, which matures by fusing with components of the endocytic pathway. The maturation is characterized by decreasing pH in the phagosome due to the action of the Na+/H+ ATPase and the delivery of proteolytic enzymes from the fusion with lysosomes. For a detailed review on the mechanism of phagocytosis see ref. 23. The mechanisms of endocytosis and phagocytosis are markedly different. Phagocytosis depends on the recruitment of bulk plasma membrane to facilitate particle ingestion whereas endocytosis represents small invaginations of the membrane. The proteins involved are different: actin; actin-binding molecules such as a-actinin, vinculin, paxillin; and signalling molecules like Syk tyrosine kinases are required for phagocytosis. Each endosomal compartment is characterized by marker proteins which may be used to identify the individual compartments (see Table 3). Also, tracers can be loaded into specific compartments and physical changes like the fall in pH measured. As the various receptors have different pathways through the endocytic network, uncharacterized receptors must be traced through the pathway by examining their co-localization with known receptors or tracers inside specific compartments. The alterations in the rate of endocytic and phagocytic uptake in response to changes in the environment can be tested. Table 3 Endocytic and phagocytic compartments have characteristic markers Compartment

Marker

Supplier

Early endosomes

Transferrin receptor (CD71) LDL receptor Na/H ATPase Rab5

Serotec American Diagnostics, Inc. Affinity Bioreagent, Inc. Santa Cruz Biotechnology

Late endosomes

LAMP 1/2

Developmental Studies Hybridoma Bank Dako Serotec Santa Cruz Biotechnology Dako

CD68 (macrosialin) CD63 Rab 7 MHCII Lysosomes

Phagosomes

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LAMP 1/2 CD63 Cathepsin D Cathepsin B

Developmental Studies Hybridoma Bank Serotec Sigma Serotec

MARCKS Actin (phalloidin) Talin Vinculin Protein kinase C (PKC)

Santa Cruz Biotechnology Sigma Sigma Sigma Sigma

CHARACTERIZATION OF MACROPHAGE ANTIGENS

5.2 Practical considerations for testing macrophage endocytic function 5.2.1 Cell culture Plate the cells as detailed in Section 2.3.1. Functional assays do not require the cells to be plated on coverslips, except for microscopic analysis. Assays can be modified for cells in suspension, but adherence may influence functions profoundly. Both the phagocytic and endocytic assays should be performed in triplicate. If analysing cells by flow cytometry, culture the macrophages in medium free of phenol red; this minimizes the autofluorescence of macrophages, caused by unknown endogenous products or substances acquired from the culture medium and can obscure tracer or marker fluorescence. 5.2.2 Receptors Endocytosis in macrophages can be mediated by ubiquitously expressed receptors, such as the transferrin receptor, or macrophage-restricted receptors including the MR and SR-A. See Figure 1 for microscopic analysis of bone marrow-derived macrophages loaded with Dil-acetylated LDL and Figure 3 for flow cytometric analysis of macrophages loaded with the same tracer. The expression of many macrophage-specific receptors is regulated by the stage of differentiation of the macrophage and its activation state. Regulation may affect the levels of receptor expression, in addition to the rate of receptor trafficking and its processing in the endocytic pathway. Ubiquitously expressed receptors, like the transferrin receptor, may give information on the basal rate of endocytosis (24). When testing the endocytic function of macrophages choose a well-documented receptor, like the transferrin and LDL receptor or MR. If testing the capabilities of a novel receptor, comparison with known receptors can give a wealth of information. Assays can be readily adapted to measure binding, internalization, and degradation of ligand. Receptor-specific binding will be saturable, i.e. reaches a plateau, when background is subtracted. 5.2.3 Ligands When investigating a particular receptor the appropriate cognate ligand must be chosen. There are a large number of commercially available labelled ligands, but coupling of fluorochromes to proteins is quick and easy (see Protocol 9). Specificity can be shown by competition with saturating amounts of unlabelled ligand, which controls for alterations caused by the labelling procedure. A suitable ligand should not be degraded too quickly once internalized, especially when loading the late endosomes and lysosomes. Dextran is a good marker as its poly-(a-D-l-6-glucose) linkages make it resistant to degradation. See Table 4 and Figure 1 and 2 for examples of commonly used ligands for analysis of the endocytic pathway. Suitable ligands for measuring pinocytosis, such as lucifer yellow, must not be recognized by any macrophage receptors. Horseradish peroxidase is commonly used as a pinocytic marker, however, it is not ideal for macrophages as it has mannose residues which are recognized by the MR. 81

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Figure 3 Row cytometric analysis of FITC-E. coll uptake by murine bone marrow-derived macrophages. FITC-E. coli (A-C) and Dil-AcLDL uptake by human monocyte-derived macrophages (D-E). (A-C) Bone marrow-derived macrophages were incubated with FITC-E. coli for 60 min at ratios of bacteria:macrophages of 25:1 (B) and 50:1 (C). Macrophages were gated during data analysis on the basis of their forward scatter and side scatter properties as established from previous experiments and all subsequent analysis was performed on this gated population (A). The histograms in (B) and (C) show fluorescence per cell (FL-1 channel) on the x-axis in arbitrary fluorescent units, and numbers of cells in a given fluorescence channel on the y-axis. Dotted lines indicate autofluorescence of cells not incubated with FITC-E. coli. This shows that the uptake of bacteria by macrophages increases with bacterial load. (D-F) Human monocyte-derived macrophages were incubated with Dil-AcLDL at a concentration of 5 ug/ml for 3 h in the presence (E) or absence (F) of poly inosinic acid (poly I) at a concentration of 25 ug/ml prior to detachment and flow cytometric analysis. (D) Forward scatter and side scatter properties of the cells after gating to remove cellular debris. Histograms (E) and (F) show fluorescence per cell (FL-2 channel) on the x-axis in arbitrary fluorescent units and numbers of cells in a given fluorescence channel on the y-axis. Dotted lines indicate autofluorescence of cells not incubated with Dil-AcLDL. This shows that virtually all macrophage uptake of Dil-AcLDL is mediated by a receptor which is inhibitable by poly I, another ligand for SR-A.

Selected fluorochromes undergo pH-dependent shifts in their excitation and/or emission spectra so the acidification of endocytic compartments can be monitored. Protocol 9 outlines a procedure for fluorescent labelling of proteins. 5.2.4 Double loading of the endocytic pathway Loading cells with two tracers can be used to differentiate the early and late endocytic compartments and allow for a more accurate investigation of the pathway. Dual loaded cells can be used in conjugation with immunochemical labelling to determine the intracellular location of an antigen. 82

CHARACTERIZATION OF MACROPHAGE ANTIGENS Table 4 Commonly used endocytic tracers and phagocytic particles Probe

Supplier

Texas Red dextran (70000 MW)

Molecular Probes

Unknown

FITC holo-transferrin

Molecular Probes

Transferrin receptor

Di-l LDL

Perlmmune

LDL receptor

Di-l acetylated LDL

Per Immune

SR-A, CD36, MARCO

HRP

Sigma

Mannose receptor on macrophages or fluid phase in other cell types

Lucifer yellow

Sigma

Fluid phase

Mannosylated BSA

E-Y Lab

Mannose receptor

Latex beads (polystyrene; ± carboxylation)

Sigma

Unknown

Sheep erythrocytes

Diamedix, Miami, FL CR3 if coated with complement FcR if coated with IgG

Receptor

FITC-E. coli bioparticles

Molecular Probes

Multiple

FITC-S. aureus bioparticles

Molecular Probes

Multiple

Zymosan

Sigma

MR and p-glucan receptor

Fluorescent labelling of proteins or particles Equipment and reagents • G25 or G50 Sepharose column (Pharmacia or Sigma) • 5.3% Na2C03 in H20 (Sigma) • 4.2% NaHC03 in H20 (Sigma) • 0.15 M NaCl in H2O (Sigma)

FTTC isomer 1 (Sigma) Dimethylformamide (Sigma) Texas Red sulfbnyl chloride (Molecular Probes)

A Coupling of FITC to proteins \ Mix 5.8 ml of 5.3% Na2CO3 with 10 ml of 4,2% NaHCO3. 2 Make the bicarbonate buffer by adding 1 vol. of the mixture in step l to 9 vol. of 0.15 M NaCl, and adjust the pH to 9.5. 3 In separate tubes, dissolve the FITC and the proteina to be labelled in the bicarbonate buffer, at final concentrations of 1 mg/ml and 5 mg/ml respectively.b 4 Add the FITC to the protein solution, use 0.3 ml FITC for each ml of protein. 5 Incubate in the dark for 2 h at room temperature. 6 Equilibrate a G50 or G25 Sephadex column with PBS according to manufacturer's protocol. 7 Pass the FITC/protein mixture over the column. 8 Elute the FITC-conjugated protein with PBS. Collect 0.5 ml fractions.

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9 Determine the OD of the conjugated protein fractions at 280 nm and 495 nm and pool all positive fractions. The ratio of OD495/OD280 should be approximately 1. 10 Determine the conjugated protein concentration using the following formula: OD2go - (OD495 x 0,35)

B Coupling of Texas Red to proteins 1 Prepare the bicarbonate buffer, but adjust the pH to 9 (see part A). Dissolve the protein in the bicarbonate as in part A, step 2. Dissolve 1 mg Texas Red for every 10 mg protein in one drop of dimethylformamide. Add this to the protein. Incubate for 1 h in the dark at room temperature without stirring. Separate the conjugated protein on a Sepharose column (see part A, steps 6-8). Determine the optical density of the pooled fraction at 596 nm and 280 nm. The ratio of OD59S/OD2SO should be approximately 0.8. Determine the conjugated protein concentration using the following formula: OD2ga - (OD596 x 0.18) a b

Dialyse or exchange buffer by running a desalting column. For labelling particles, use zymosan at 109 particles/ml and live bacteria at 107 particles/ml.

To load late endocytic compartments, label the cell with dextran for an hour, followed by a chase of 8-16 hours to allow the dextran to accumulate in the later compartments. A brief incubation with transferrin can then be used to load the early compartments (see Protocol 10B).

Quantitatlon of macrophage endocytic function Equipment and reagents • Flow cytometera • Titertek® 96-well plate fluoroplate (Flow Laboratories) • Huorimetric plate reader (Fluoroscan II, Labsy stems)

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Endocytic tracers (see Table 3) labelled with fluorochromes (see Protocol 9) PBS (see Protocol 2) Optimem-I (Gibco) RPMI(Gibco Laboratories)

CHARACTERIZATION OF MACROPHAGE ANTIGENS

• Detachment buffer (see Protocol 2) • 1% Triton X-100 (Sigma) in 10 mM Tris buffer diluted from a 1 M stock: dissolve 60,57 g of Tris base (Sigma) in 400 ml H2O; adjust the pH to 7.5 using cone. HC1

• 4% paraformaldehyde (see Protocol 3) • BCA™ Protein Assay Reagent Kit (Pierce Chemical Co.) • Adherent macrophages as a monolayer in 24-well tissue culture platesb

A Loading macrophages with a single tracer 1 Remove the culture medium from the cells. 2 Wash the macmphage monolayer twice in PBS. 3 Add 250 ul volume of Optimem-I containing 100 ug/ml tracer. 4 Incubate the macrophages for 1 h at 37 °C. Keep a control sample on ice.c 5 Stop the endocytic uptake by placing the macrophage monolayers on ice. 6 Remove the tracer by aspiration and wash the cell monolayers at least four times in ice-cold PBS. 7 If measuring the amount for uptake by flow cytometer, detach and fix the cells (see Protocol 2 and 3) or if measuring the fluorescence on a fluorimetric plate reader, follow steps 9-11. 8 Lyse the cells with 250 JJL! Trition X-100 in 10 mM Tris buffer. 9 Incubate the cells on ice for 30 min. 10 Scrape the macrophages from the bottom of the dish. 11 Transfer the lysed cells to a Titertek 96-well plate fluoroplate and read on a plate reader. 12 Remove 30 IJL! and determine the protein concentration of the lysate using a BCA Protein Assay Reagent Kit, 13 Express the result as a function of the protein concentration or number of cells depending on analysis. B Dual endocytic tracer loading of macrophages 1 See part A, steps 1-5. 2 Remove the first tracer by aspiration. 3 Wash the cells well in warmed RPMI (or the usual culture medium for the macrophages). 4 Chase the tracer into lysosomes, by incubating the macrophages overnight at 37 °C. 5 Remove the culture medium by aspiration. 6 Wash the macrophages twice in PBS. 7 Add 250 ul Optimem-I containing 100 ug/ml of the second tracer.15 Place a control sample on ice. 8 Load the early endocytic compartments by incubating the macrophages at 37 °C for 10-15 min.e

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9 10 11 12

Remove the second tracer by aspiration, Cool the macrophages quickly by placing them on ice. Wash the macrophage monolayers well with ice-cold PBS, Follow part A, steps 8-14, remembering to take fluorometric readings for both the fluorochromes.

a

This assay is easily adapted for analyses by flow cytometry or a plate reader. The reagents required for both are listed. b See Protocol 1A for the preparation of the monolayers, however, do not add the glass coverslips. c This controls for the non-specific sticking of the tracer to the extracellular surface of the cells and the tissue culture dishes. Subtract this value, after analysis, for a correct measurement of total endocytic uptake. d This tracer should be labelled with a different fluorochrome than that used for the first tracer. e Macrophages are very endocytic cells so incubation for longer times will start to load later endocytic compartments as well as the earlier ones.

Some commercially available reagents have intense labelling which 'bleedthrough' into the spectra of the other fluorochromes. This may hamper colocalization studies so appropriate single-labelled controls are essential. This problem can be alleviated using confocal microscopes with two lasers which individually excite the fluorochromes. Protocol 10 describes one method for quantitation of macrophage endocytic function,

5.3 Practical considerations for testing macrophage phagocytic function 5.3.1 Assays Commonly, phagocytic assays involve the addition of particles to macrophages followed by microscopic analysis of the number of particles bound and internalized by a cell. This type of analysis is time-consuming as large numbers of cells have to be counted manually to obtain statistically significant results. Therefore, we suggest adapting the assays so that the results may be analysed on a plate reader or flow cytometer which can collect information on large numbers of cells. There are two types of bask assay. The first determines the number of particles associated with the macrophages, while the other monitors decreasing numbers of particles in the extracellular medium (25). Either assay is acceptable, but we will only discuss the former. Uptake assays can be adapted to measure cellular responses, such as the respiratory burst, by appropriate bulk or single cell methods (26) and to determine the survival or killing of ingested live organisms. Appropriate safety precautions must be taken in handling living micro-organisms, in all procedures. 86

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5.3.2 Receptors and ligands The ligands expressed on a chosen particle will determine the receptors used for ingestion of that particle and so must be appropriately chosen if investigating a particular receptor. Complex ligands, like bacteria, may be recognized by more than one receptor. Bacteria are easily fluoresceinated using Protocol 9 or some are available commercially (see Table 4). If not investigating phagocytosis mediated by a particular receptor then complex ligands can be used (see Figure 3B). Latex beads, the receptors for which are unknown, are readily taken up and are suitable particles for phagocytosis. They are available in a wide range of sizes and can be coated by absorption or, in the case of carboxylated polystyrene latex beads can be coupled directly to protein ligands to target them to specific receptors; some examples used previously are mannose BSA and lipoarabinomannan. However, even apparently single ligands may also be recognized by multiple receptors. Besides latex beads, zymosan, derived from the cell wall of Saccharomyces cerevisiae, is a commonly used particle. It is highly mannosylated and is recognized by a number of receptors including MR,B-glucanreceptors, and CR3, with or without opsonization (it readily activates the alternative pathway of complement). It is commercially available, though easy to prepare and label with fluorochromes (see Protocol 9). Zymosan should be boiled before use to destroy contaminating phospholipases. Erythrocytes coated with opsonins are widely used to analyse the function of opsonic receptors. Smaller particles may be taken up by macropinocytosis so when using latex beads ensure that the size used is larger than 1 um in diameter and test the ability of phagocytic inhibitors on particle uptake. Inhibitors of ingestion, like cytochalasin B and D, and inhibitors that block ligand binding should always be used as controls for phagocytosis.

5.3.3 Opsonization and the presence of serum Some ligands require opsonization by complement and antibodies. Macrophages themselves may also produce opsonins like complement and fibronectin that could potentially influence uptake. The presence of serum can opsonize particles, so unless analysing general phagocytosis, use a serum-free protein-containing medium. If analysing specific opsonic receptors, coat the particles with the opsonin before the assay. Bacteria are easily opsonized by incubating them in an appropriate serum for 30 min at 37°C. Complement is only present in fresh serum and is destroyed by heat inactivation. Specific IgM and complement target CR3, but beware of IgG contamination of the IgM. IgG-coating targets the Fc receptors. Polyclonal Abs can be raised or where available, monoclonal Abs against erythrocyte antigens or hapten, for example, used with an appropriate isotype matched Ab control.

5.3.4 Ingestion versus attachment The differentiation of intracellular particles and those bound to the extracellular surface is crucial in any phagocytic assay and there are numerous modifications 87

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to existing methods available for this purpose. First, fluorescence on any extracellular particles can be quenched with appropriate agents such as ethidium bromide, crystal violet, and trypan blue. It is not easy to control for total quenching of the extracellular fluorescence. In addition, the quenching agent must not be cell permeable. When measuring the internalization of live bacteria, antibiotics such as Gentamycin are often added to the culture medium to kill any non-phagocytosed bacteria. Like the quenching agents, the antibiotic should not gain access to the cell where it can kill intracellular bacteria. There is some question, however, as to whether the antibiotic may kill bacteria tightly bound to the plasma membrane or whether endocytically active cells, such as macrophages, also take up the drug. An alternative approach is to cleave or lyse the bacteria from the extracellular surface, for example, lysostaphin can lyse S. aureus and lysozyme can lyse Micrococcus lysodeikticus. Erythrocytes are easily lysed by brief osmotic shock (water or hypotonic solutions). Lastly, immunofluorescent techniques can be used to distinguish intra- and extracellular bacteria, with only external bacteria detected by antibodies (see Protocol 11). The distribution of the bacteria, with respect to the numbers found inside and bound to the cell, can be obtained by comparison between antibody staining of permeabilized and unpermeabilized cells. 5.3.5 Time, temperature, and dose Macrophages are highly professional phagocytic cells and particle ingestion occurs rapidly. Generally, incubation times range between ten minutes and one hour. However, the kinetics of uptake should be determined by performing a time course experiment before embarking on these assays. At time zero there should be no uptake. This would also be true at 4°C as the membrane is not fluid enough to mediate uptake. Following any uptake assay, the macrophages should be quickly cooled to 4°C to stop any further internalization by the macrophage and ideally, the rest of the protocol should be performed in the cold. The optimal dose of particles should always be determined, especially when using live and virulent bacteria as too many bacteria may lyse or kill the macrophages. An initial particle to macrophage ratio of between 1:1 and 20:1 is recommended. The rate of ingestion should reach zero-order kinetics with increasing dose and is an important test of any assay method. Particle contact with the macrophages may be enhanced by centrifuging them directly onto the cells, in special holders available commercially. If performing the assay on non-adherent cells, tumble the bacteria and macrophages together for optimal contact. Protocol 11 describes a method of analysis of pahgocytic uptake using adherent populations, though is easily adapted for nonadherent cells.

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Phagocytic uptake of bacteria by macrophagesa Equipment and reagents • Flow cytometer • Culture plate adaptors for table-top centrifuges • Labelled phagocytic particle (Table 4) • Anti-particle antibody (see Section 5.3.4) • Labelled secondary antibody (see Section 2.2,5}

Detachment buffer (see Protocol 2) 4% paraformaldehyde (see Protocol 3) PBS (see Protocol 2) Blocking buffer (see Protocol 4} Adherent macrophage monolayers in 24well tissue culture platesb

Method 1 Prepare a pre-mixc of 750 ul tissue culture medium containing the appropriate number of ligandd and mix well, 2 Remove culture medium from the cells by aspiration. 3 Wash the monolayers twice in 500 ul PBS to remove any non-adherent cells. 4 Add 250 ul of the pre-mix to each well. 5 Spin the macrophages for 5 min at 1500 g. 6 Incubate the macrophages at 370C for 1 h,d 7 Remove the particles from the cell by aspiration. 8 Wash the cells well in ice-cold PBS to remove as many extracellular particles as possible, 9 Detach and fix the macrophages (see Protocols 2 and 3}. 10 Stain the extracellular particles with the anti-particle antibodye (see Protocol 4). 11 Analyse the cells by microscopy or flow cytornetry (see Sections 3.1 and 3.2). a

Adapted from ref. 27. See Protocol 1A for the preparation of the monolayers, however, do not add the glass coverslips. c It is suggested to make a pre-mix so that the particles are evenly distributed between the triplicate wells to lessen systematic error. d See Section 3.3,5 for information on determination of dose and time of uptake. e Remember not to permeabilize the macrophages. b

6 Conclusion This chapter deals with a few basic methods to characterize macrophages. We describe protocols to detect macrophage antigens in mouse tissue and in vitro and to analyse endocytosis and phagocytosis, two hallmark macrophage functions. Though these assays are simple, they are easily modified to ask further

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questions regarding phenotypic expression of markers, functions, and cell activation. There is considerable progress in this field with the development of new fluorescent molecules that do not photobleach as quickly as their older counterparts. Green fluorescent protein (GFP) and improved probes can be detected directly in living systems without the use of secondary reagents and new methods are becoming available to study intracellular Ca2+ compartmentation and DNA transcription in situ. These should be readily applicable to the analysis of macrophages and related cells.

Acknowledgements The authors would like to thank all the members of the Gordon Laboratory for their contributions, especially L. Martinez-Pomares and A. McKnight for advice. L. P. holds a Goodger, Harry Crossley, and Patrick and Margaret Flanagan Scholarship. P. J. G. holds a Goodger Scholarship, and S. G. is supported by the Wellcome Trust for Imaging.

References 1. Martinez-Pomares, L., Platt, N., McKnight, A. J., da Silva, R. P., and Gordon, S. (1996). Immunobiology, 295, 407. 2. Gordon, S., Lawson, L., Rabinowitz, S., Crocker, P. R., Morris, L., and Perry, V. H. (1992). In Macrophage biology and activation (ed. S. Russell and S. Gordon), Vol. 181, p. 1. Springer-Verlag, Berlin. 3. McKnight, A. J. and Gordon, S. (1998). Adv. Immunol, 68, 271. 4. Herzenberg, L. A., Herzenberg, L. A, Weir, D. M., and Blackwell, C. (ed.) (1997). Weir's Handbook of experimental immunology (5th edn), Section 26. Blackwell Scientific Publications, UK. 5. Harlow, E. and Lane, D. (1988). Antibodies: a laboratory manual. Cold Spring Harbour Laboratory, New York. 6. Rabinovitch, M. and de Stefano, M. J. (1976). Proc. Natl. Acad. Sci. USA, 85, 2805. 7. Ormerod, M. G. (1994). How cytometry: a practical approach (2nd edn). IRL Press, Oxford. 8. Shapiro, H. M. (1995). Practical flow cytometry (3rd edn). Wiley-Liss, New York. 9. Polak, J. M. and van Noorden, S. (1988). An introduction to immunocytochemistry: current techniques and problems (revised edition). Oxford University Press, Oxford. 10. Bancroft, J. D. and Stevens, A. (1990). Theory and practice of histological techniques (3rd edn). Churchhill Livingstone, London. 11. McLean, I. W. and Nakane, P. K.(1974).J. Histochem., 22, 1077. 12. Steinman, R. M., Mellman, I. S., Muller, W. A, and Cohn, Z. A.(1983).J. CellBiol, 96, 1. 13. Mellman, I. (1996). Annu. Rev. Cell Dev. Biol., 12, 575. 14. Pearse, B. M. and Robinson, M. S. (1990). Annu. Rev. Cell Biol., 6, 151. 15. Stoorvogel, W., Strous, G. J., Geuze, H. J., Oorchot, V., and Schwartz, A. L (1991). Cell, 65, 417. 16. Griffiths, G. and Gruenburg, J. (1991). Trends CellUBiol, 1, 5. 17. Rabinowitz, S., Horstmann, H., Gordon, S., and Griffiths, G. (1992). J. Cell Biol., 116, 95. 18. Bennett, M. K. (1995). Curr. Opin. Cell Biol., 7, 581. 19. Alpuche-Aranda, C. M., Racoosin, E. L., Swanson, J. A., and Miller, S. I. (1994) J. Exp, Med., 179, 601.

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CHARACTERIZATION OF MACROPHAGE ANTIGENS 20. Garcia-del Portillo, F. and Brett-Finlay, B. (1994). Infect. Immun., 62, 4641. 21. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y., Glenney, J. R., and Anderson, R. G. W. (1992). Cell, 68, 673. 22. Peiser, L. and Gordon, S. (in press). Encyclopedia of life sciences. Macmillan Reference Limited, London. 23. Greenberg, S. and Silverstein, S. C. (1993). In Fundamental immunology (ed. W. E. Paul), p. 941. Raven Press, New York. 24. da Silva, R. P. (1997). In Weir's Handbook of experimental immunology (5th edn) (ed. L. A. Herzenberg, L A. Herzenberg, D. M. Weir, and C. Blackwell), Chapter 168. Blackwell Scientific Publications, UK. 25. van Furth, R. and van den Berg, B. M. (1997). In Weir's Handbook of experimental immunology (5th edn) (ed. L. A. Herzenberg, L A. Herzenberg, D. M. Weir, and C. Blackwell), Chapter 167. Blackwell Scientific Publications, UK. 26. Baorto, D. M., Gao, Z., Malaviya, R., Dustin, M. L., van der Merwe, A., Lublin, D. M., et al. (1997). Nature, 389, 636. 27. de Boer, E. C., Severs, R. F. M., Kurth, K.-H., and Schamhart, D. H. J. (1996). Cytometry, 25, 381. 28. Schlossman, S. F. (1995). Leucocyte typing V: white cell differentiation antigens. Proceedings of the Fifth International Workshop and Conference, Boston, USA, 3-7 November, 1993. Oxford University Press, Oxford. 29. Leenen, P. J. M., Kraal, G., and Dijkstra, C. D. (1997). In Weir's Handbook of experimental immunology (5th edn) (ed. L A, Herzenberg, L. A. Herzenberg, D. M. Weir, and C. Blackwell), Chapter 174. Blackwell Scientific Publications, UK. 30. Garni-Wagner, B. A. and Todd, R. F. (1997). In Weir's Handbook of experimental immunology (5th edn) (ed. L. A. Herzenberg, L. A. Herzenberg, D. M. Weir, and C. Blackwell), Chapter 175. Blackwell Scientific Publications, UK. 31. Gough, P. J., Greaves, D. R., Suzuki, H., Hakkinen, T., Hilrunen, M. O., Turunen, M., et al. (1999). Arterio. Thromb. Vasc. Biol., 19, 461. 32. Martinez-Pomares, L., Kosco-Vilbois, M., Darley, E., Tree, P., Herren, S., Bonnefoy, J.-Y., etal. (1996). J. Exp. Med., 184, 1927. 33. Smith, M. J. and Koch, G. L (1987). J. Cell. Sci., 87, 113.

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Chapter 4 Analysis of antigen processing and presentation P. M. Kaye Department of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine (University of London), Keppel Street, London WC1E 7HT UK

1 Introduction This chapter seeks to identify the key issues relating to the study of macrophages as antigen-presenting cells (APC) and presents protocols for the major assays used to measure antigen processing and presentation. In addition, it discusses some of the theoretical and practical issues related to the processing and presentation of intracellular pathogens for which macrophages are the major host cell. The methods presented are, for the most part, those used in the author's laboratory or by colleagues. Whilst these provide working examples, it is stressed that the nature of individual host-pathogen interactions may necessitate alterations on a case-by-case basis. Potential areas for such modifications are highlighted, as appropriate.

2 Preliminary considerations in study design 2.1 A definition of antigen processing and presentation The recognition of specific antigenic peptides by the T cell receptor (TCR) occurs in the context of host proteins encoded by genes of the major histocompatibility complex (MHC), the H-2 antigens of mice and the HLA antigens of man. CD4+ T cells recognize antigens which generally enter the endosomal compartments, in the context of MHC class II antigens, whereas CD8+ T cells utilize MHC class I molecules for the recognition of 'endogenous' antigens synthesized by the cell itself (e.g. as a result of viral infection). Conceptually, it is usually helpful to separate the linked events of antigen processing and presentation such that: (a) 'Processing' reflects the events by which antigenic proteins are degraded to their constituent peptide epitopes, and these are transported from the sites of production to the plasma membrane for recognition by the TCR. 93

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(b) 'Presentation' which involves the recognition of MHC-peptide complexes by the TCR, and the simultaneous (usually) recognition of additional 'accessory', 'adhesive', or 'co-stimulatory' signals which enable productive TCR-mediated signalling and cell activation (1). By these broad definitions, antigen processing becomes principally a subcellular event and the role of MHC molecules one of peptide transport. The endpoint of antigen processing is cell surface peptide display. Conversely, antigen presentation becomes the study of cellular (APC and T cell) interactions involving plasma membrane receptor-ligand interactions and has a strictly functional read-out as some parameter of T cell activation (usually proliferation, cytokine release, or cytolysis). Experimentally, it would be ideal if these two linked functions could also be readily separated. Tools are becoming available with which to do this (in very defined experimental situations; see Section 3.3) but in many experimental designs, dissecting these two aspects of macrophage function is confounded by the inability to detect MHC-peptide complexes at the cell surface by means other than T cell recognition. Before discussing details of these events, it is therefore worthwhile considering briefly the impact that the available source of T cells may have on study design and interpretations.

2.2 T cell choice restricts functional interpretation In an ideal setting, the assay of antigen processing would be performed independently of T cell recognition, i.e. solely as a cell biological/biochemical series of events culminating in MHC-peptide expression at the plasma membrane. However, such an approach relies upon the availability of methods for both the biochemical and immunochemical detection of specific MHC-peptide complexes. Only recently have such methods become available, and they have to date found limited application in the study of intracellular pathogens. Since the general catabolic activity of macrophages, particularly the action of lysosomal hydrolases, may mask or even be independent of those events which generate antigenic peptides (2), the only reliable and readily available assay of processing has been T cell recognition. Hence, the confounding aspects of 'presentation' need to be limited. Over the years, the general rules (N.B. always there to be broken!) which govern T cell activation have been dissected. In addition to the engagement of TCR by MHC-peptide complexes, naive T cells also require a complex series of secondary events, involving LFA-1-ICAM-1, CD28/CTLA4-B71/2, MHC-CD4/8 ligation and signalling (3). In contrast, activated T cells often are less stringent in their absolute requirements for such additional signals, though they modulate subsequent responses here too. For the study of antigen presentation, low precursor frequencies will normally rule out using naive T cells, even when complex pathogens are used. The advent of TCR transgenic mice, however may circumvent this to a degree, particularly if the antigens for which TCR transgenic mice already exist can be transfected into the pathogen of interest (4). Polyclonal T cells, derived from mice immunized with crude antigens in adjuvant or from mice 94

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infected with pathogens may have some limited uses, but the variable processing characteristics of individual antigens is likely to result in the determination of an 'average' response. Even in the case of polyclonal responses to defined recombinant antigens, there may be epitope-specific variation in processing (5). The preceding discussion clearly leans towards the use of defined epitope-specific responses, especially for the study of processing. Nevertheless, one epitope does not the whole pathogen make! T cell clones may seem an ideal choice, notably because peptide specificity is also likely to be known. However, cloning/restimulation protocols vary enormously and the state of activation, requirements for co-stimulation, and the presence of contaminating residual APCs needs to be carefully evaluated. Perhaps the most appropriate T cell population for studying antigen processing per se is the T cell hybridoma. Numerous, well-established protocols exist for the fusion of primary T cell populations/clones with the widely available BW5147 thymoma (ATCC No TIB48). Numerous derivatives of this line, including those which lack endogenous TCRa and B chains, or coexpress CD8 are also available (6, 7). In most investigators' hands, cytokine (generally IL-2) production by such hybridomas is the assay least confounded by other variables of presentation, though it goes without saying that this should be determined on a case-by-case basis.

2.3 'In vivo veritas' The ultimate goal of performing direct in vivo analysis of immune function, with due reference to microenvironmental constraints, is perhaps no more compelling than for the study of macrophage function. An exciting era awaits as tools to enable the study of antigen processing in vivo become available (see Section 3.3). Yet, at least for the next few years most of us will be forced towards some degree of in vitro analysis of APC function. Interpretation of the data obtained from such in vitro studies should, however, be tempered where possible by some degree of reference to macrophages in their natural environment. In vitro conditions may provide for the accumulation of metabolites, the artificial creation of cytokine environments, exaggerated or under-represented levels of parasitism, and so on. Detailed discussion of the various methods for in situ analysis are, however, beyond the scope of this chapter.

2.4 The dendritic cell issue In the early 1970s Steinman and Cohn described a population of dendritic leukocytes which anatomically resided within the T cell areas of lymphoid tissue and in vitro possessed potent APC function (reviewed in ref. 8). In the years following their discovery, debate raged about whether indeed any other cell types were capable of stimulating T cell responses, or whether trace contamination with DC was in fact responsible. Whilst this polarized view has become somewhat moderated in the 1990s with the acceptance that other cells, including macrophages, can process and present antigens, the lessons from the early DC era particularly regarding the importance of cell purity should not be forgotten. 95

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Indeed, rather perversely, many of the DC purification procedures currently in use allow an unacceptable degree of contamination by other, supposedly 'irrelevant' cell types. A major experimental challenge lies not in devising a protocol which can exclude unwanted cell types, but in adhering to it, and confirming its efficiency within each experiment. More often than not, this will involve timeconsuming phenotypic analysis at each stage of the procedure. These issues are discussed in more detail in Chapter 3. 2.5 Pathogens are not simple antigens Most of the seminal observations on antigen processing and presentation have been made by the study of simple protein antigens or by attaching such antigens to inert particulate carriers, e.g. latex beads. The interest in the study of antigen processing and presentation of intracellular pathogens has been fuelled both by the potential for APC function to be a target of immune evasion strategies and, from a more theoretical standpoint, by the degree of stress which such pathogens can place on cell biological processes (in much the same way as in vivo infection models have been used to stretch T cell function to extremes to refine the Thl/Th2 paradigm). Pathogens bring with them, however, a number of complexities which need to be accounted for in the process of experimental design. For example, though we may strive to use homogeneous pathogen populations, e.g. in infectivity or even viability, this may be an elusive goal. Furthermore, the biological processes of pathogens may be disrupted by the addition of inhibitors of mammalian cell function. Pathogens are also enzymatically rich, leading to potential modifications of host enzyme portfolio, or may contain diverse defence mechanisms which may directly or indirectly influence APC function. Whilst these issues represent the excitement of studying antigen presentation with pathogens, they also represent the most common source of erroneous conclusions. Some of these issues will be addressed further in later sections.

3 Analysis of class I and II antigen processing 3.1 Pathways of processing For both MHC class I- and class II-restricted antigen processing, three simple experimental parameters need to be described, namely dose dependency, kinetics, and sensitivity to various inhibitors which define the biochemical/cell biological route of processing. Complete protocols for the determination of these parameters for class I- and class II-restricted presentation of a simple protein antigen, e.g. ovalbumin, are given below. 3.1.1 Generation of murine bone marrow-derived macrophages We have had success in assays of class I and class II processing using bone marrow-derived macrophages. However, with minor modification, the subsequent protocols for addressing antigen processing should be applicable to any 96

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other monocyte/macrophage population cultured in vitro or harvested ex vivo. It should not be assumed that all macrophages will process similarly. Not only may kinetics of antigen uptake vary, but the vacuolar compartments and enzymatic machinery may be subtly different. Furthermore, it has now been realized that cytokine regulation of macrophage function extends to that of regulation of processing enzymes. introducing another major confounding variable should ex vivo macrophage populations be compared from inflammatory versus quiescent tissue sites. Protocol 1 describes one method for the derivation of bone marrow-derived macrophages for studies of antigen processing and presentation.

Preparation of bone marrow macrophages Equipment and reagents • Surgical instruments: scissors, scalpel, forceps • SO ml polypropylene centrifuge tubes (Falcon) • Centrifuge equipped with Sorval H1000B rotor • Bacteriological grade 90 mm Petri dishes (Bibby Sterilin Ltd.) • C02 incubator • BALB/cmice • 70% alcohol • DMEM medium with 4.5 mg/litre glucose (Gibco BRL), supplemented with 1 mM L-glutamine, 2 mM sodium pyruvate, 100 ug/ml streptomycin, and 100 U/ml penicillin

• L cell conditioned medium: to prepare this L929 flbroblasts (European Collection of Animal Cell Culture) are grown to confluence in 25 cm3 tissue culture flasks, using supplemented DMEM medium plus 10% HI-FCS. Supernatants were harvested, sterile filtered (0.2 um), and stored frozen at -20°C. • Complete bone marrow macrophage growth medium: supplemented DMEM with 20% (v/v) heat-inactivated fetal calf serum (Gibco BRL) and 10-20% L cell conditioned medium. as a source of CSF-1. Generally confluent L929 fibroblast conditional medium contains sufficient CSF-1 to support macrophage growth/differentiation at concentrations of 10-20% (v/v). However, new batches should be titrated for activity.

Method 1 2 3 4 5 6

Dissect femurs from naive BALB/c mice and trim to remove extraneous muscle tissue. Briefly rinse femurs in 70% alcohol to ensure sterility. Remove epiphyses using sharp scissors. Flush femurs with 2-3 ml of ice-cold DMEM medium using a 25-gauge needle. Collect femur flushes into 50 ml polycarbonate centrifuge tube. Wash cells three times by centrifugation (1200 r.p.m,, 4°C, 10 min in Sorval H1000B rotor). 7 Thoroughly resuspend cell pellet after each wash by vigorous 'flicking' of the tube, rather than vortexing. 97

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8 Count cells and plate out in 90 mm bacteriological Petri dishes at 5 x 106 cells/dish in 10 ml complete bone marrow macrophage growth medium. 9 After three days incubation at 37°C, 5% CO2, aspirate 8 ml medium from each dish and replace with complete bone marrow growth medium. 10 Culture for a further three days at 37 °C, 5% CO2. 11 After three days, gently swirl dishes to resuspend non-adherent cells and then remove all medium. 12 Replace with 10 ml of supplemented DMEM with 20%FCS.but no CSF.1, and return to 37°C, 5% C02 for two days. This 'rest' culture allows macrophages to leave the cell cycle over the subsequent two days and mature uniformly, 13 To harvest macrophages, place Petri dishes at 4°C for 1 h. 14 Gently pipette the medium to dislodge > 95% of cells from these plates.a 15 Wash harvested macrophages (approx. yield 2-4 x 106/dish) once prior to use with supplemented DMEM. 16 Confirm phenotype of harvested cells as detailed in Chapter 3. Typically, such '6 + 2' macrophages would be > 95% phagocytic towards zymosan or latex beads, show high levels of MHC class I expression, heterogeneous class II expression (20-40% of cells positive at low to intermediate density), low expression of LFA-1 (CDlla/CD18) and ICAM-1 (CD54), and negligible levels of B7-1 (CD80) and B7-2 (CD86). The presence of CD80/86hi, class IIhi cells may indicate dendritic cell contamination. a

This simple harvesting procedure will not be appropriate if tissue culture grade Petri dishes are inadvertently used. In such circumstances, trypsin-EDTA (Sigma) or enzyme-free dissociation media (Sigma) will be required.

Batches of L cell conditioned medium may vary. In particular, many if not all L929 cells also produce trace amounts of GM-CSF, a cytokine which may modify macrophage functional differentiation or (at high concentrations) enhance dendritic cell maturation (9). It is therefore advisable to check for GM-CSF levels. The period of culture in L cell conditioned medium, or the period of rest, may have subtle influences on phenotype and function. Thus, although it is tempting to allow variations of a day or two in the initial culture period (e.g. 6-8 days + 1-2 of rest), this may produce some variability in results when such cells are used in functional assays. 3.1.2 Induction of class !! expression on bone marrow-derived macrophages For most experimental purposes, it is desirable to have more uniform expression of MHC class II antigens. Not only does this usually enhance the capacity of macrophages to present to class II-restrictcd T cells, but it negates to a degree concerns about heterogeneity of presenting cell function within the macrophage 98

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population. The latter may be a particular problem in the case of particulate antigens where antigen uptake may not be normally distributed in the population (see Section 3.2.1). Achieving more uniform class II expression is usually simply a matter of incubating cells in the presence of IFNy. For bone marrow macrophages, induction of class II requires 24-48 hours, though the kinetics of the response to IFNy may vary with other macrophage populations (e.g. it is notably slower with resident peritoneal macrophages, reaching maximal levels only after five to seven days in the presence of IFNy). Protocol 2 outlines the appropriate conditions for induction of MHC class 11 antigen expression on bone marrow macrophages (BMM0).

Induction of MHC class II antigens on bone marrow macrophages Equipment and reagentsa • 4 ml polypropylene culture tubes (Falcon 2063) • CO2 incubator • '6 + 2' bone marrow-derived macrophage (BMM0, see Protocol 1}

• Complete tissue culture medium: supplemented DMEM (see Protocol 1) with 10% FCS • Recombinant murine IFNy (Genzyme)

Method 1 Adjust BMM0 to 106/ml in complete tissue culture medium. 2

Aliquot 3 x 106 per tube to the required number of polypropylene culture tubes.

3

Add recombinant murine IFNy to a final concentration of 100 U/ml.b

4

Incubate cells for 48 h at 37 °C, 5% CO2.

5

Resuspend BMM0 by gently flicking tube.

6

Remove an aliquot of cells and determine levels of class II expression by flow cytometry (using methods described in Chapter 3).

7

Wash remaining BMM0 twice in complete tissue culture medium, prior to use in functional assays.

a

AlI possible caution should be taken to avoid LPS contamination in culture medium, reagents, glassware, etc, b If recombinant IFNy is not available, 10% (v/v) supernatant from ConA-stimulated splenocytes {5 ug/ml ConA for 72 h, with a cell concentration of 5 x 106/ml will usually suffice. However, other cytokines present may have adverse effects on macrophage function.

The time at which class II should be induced may vary depending on the experimental design. For example, in a direct assay of processing function, optimal levels of class II are usually desired prior to antigen uptake, such that class II

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transcription and subcellular redistribution are not limiting factors. In contrast, in studies aimed at addressing more specific questions, class II induction may be delayed until after antigen uptake. Such was the method adopted to determine whether newly synthesized class II molecules were able to be transported to the parasitophorous vacuole surrounding Leishmania parasites (10). IPS contamination should be avoided in all work. High concentrations of LPS may inhibit class II expression to a degree, and also regulate co-stimulatory ligand expression (3). LPS will also, even in nanogram levels, synergize with IFNy to activate microbicidal activity and the production of NO. NO may be an inhibitor of T cell proliferation (11).

3.1.3 Pulsing macrophages with protein antigens to measure class ll-restricted processing Performing checkerboard titrations is the best initial approach to establishing the optimal conditions for later, more refined experimental designs. Checkerboard titrations may also prove valuable for evaluating the homogeneity of an APC population. A sigmoid titration curve, with a linear relationship between macrophage number versus T cell activity (e.g. proliferation, cytokine production) extending for 1-2 logs in macrophage number, is generally indicative of the population displaying uniform function. In contrast, a rapid loss of functional activity within a small titration range may indicate that APC function actually resides in a minor subset of cells which is rapidly titrated out. Antigen dose responses are also informative, and will generally produce sigmoid or bellshaped curves. Protocol 3 describes a method for simultaneously analysing antigen dose and optimal macrophage number. Subsequent experiments are best performed with both parameters at cell/antigen concentrations giving responses just below maximum. In contrast, when macrophages are to be modified, e.g. by infection, numbers should be ideally used that give 50% maximal T cell activity (allowing both enhancement and depression of function to be observed). The above protocol allows simultaneous determination of the optimal macrophage number and antigen dose required for T cell recognition. The fixation steps (steps 10-13) are not an absolute requirement and unfixed antigen pulsed macrophages can be taken directly from step 8 to step 14. However, fixation provides the most reliable way of terminating processing, and hence for the analysis of processing kinetics (by varying the duration of incubation prior to fixation; step 7 above). Nevertheless, fixation may have some deleterious effects on APC efficiency, probably due to the fixation sensitivity of certain co-stimulatory molecules. It should also be noted that extremely low levels of aldehyde fixation may enhance APC-T cell interactions by the formation of Schiff bases (12). Fixation also obviates any interference in T cell response by secreted products of macrophages, e.g. NO or PGE2. If fixation is not used, inhibitors of these metabolites (5 ug/ml indomethacin and 0.4 mM NMMA, respectively) should be added to media from step 7. If the efficiency of macrophages compared to other APC is to be compared then this protocol will only be suitable if all populations are adherent (e.g. a 100

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comparison with transfected fibroblasts). If this criterion is not satisfied, other antigen pulsing procedures will be required (e.g. see Protocol 7).

Pulsing macrophages with soluble antigens for analysing class ll-restricted processing Equipment and reagents • Flat-bottom 96-well tissue culture plates (Nunc) • Recombinant murine IFNy (Genzyme) • Phosphate-buffered saline (PBS), comprising 10 mM sodium phosphate pH 7.5. 0.9% (w/v) NaCl • Complete tissue culture media: Dutch modified RPMI (Gibco) or DMEM, 4.5 ing/litre glucose (Gibco}. Either medium should be supplemented with 1 mM L-glutamine, 2 mM sodium pyruvate. antibiotics, and 10% HI-FCS.

Specific antigens for stimulation (e.g. soluble protein antigens such as ovalbumin, KLH), to be used over a 1-100 (j.g/ml final concentration range 1% paraformaldehyde: make a 3% (w/v) stock of paraformaldehyde (Kodak) in PBS, as described in Chapter 2, and store at -20°C; prepare 1% stock in PBS using 3% stock as base 0,1 M L-lysine (Sigma) in PBS A source of antigen-specific polyclonal T cells or a T cell hybridoma

Method 1

Harvest IFNy activated BMM0 (see Protocols 1 and 2) and wash once prior to assay.

2

Resuspend the cells at 106/ml in complete tissue culture medium for the final T cell assay.a

3

Make half-log10 dilutions (of the cell suspension), to 104/ml, using tissue culture medium.

4

Plate 100 ul of each dilution into flat-bottom 96-well tissue culture plates (Nunc, Falcon). Plate sufficient cells at each concentration to allow for assay in triplicate, and with varying antigen concentrations (see step 6).

5

Incubate plates at 37°C, 5% C02 for 2 h to allow macrophages to adhere.

6

Add 100 ul of the antigen, of choice, serially diluted in complete tissue culture medium,

7

Incubate for 2-4 h with antigen at 37°C, 5% C02.

8

Wash wells gently three times by removal/replacement of 200 u1 warm complete medium. A multichannel pipette is most convenient for this procedure.

9

Wash once with serum-free tissue culture medium.

10 Add 50 ul of 1% (w/v) paraformaldehyde in PBS and incubate for 15 min at RT to fix the cells. 11 Remove paraformaldehyde by aspiration. 12 Add 50 ul of 0.1 M L-lysine in PBS to quench the fixation reaction (15 min at RT),

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13 Wash fixed, antigen pulsed macrophages three times with complete tissue culture medium. 14 Add 100 u1 of this medium after washing is complete. 15 Add 2-4 x 105 polyclonal antigen-specific T cells or 105 antigen-specific T cell hybridoma cells in 100 u1 complete medium.b 16 Incubate at 37°C, 5% CO2 for 24 h (T cell hybridomas) or 90 h (polyclonal T cells). 17 Assess T cell responses.e a

We routinely prefer Dutch modified RPMI for use with primary T cell cultures, and DMEM for use with T cell hybridomas. b It is important to also include cultures containing only T cells and antigen, at all doses used. For primary T cells, this control functionally assesses the degree of T cell purity, whereas for all types of T cells it excludes mitogenic activity in the antigen preparation. c To assess T cell responses, either harvest culture supernatant fluid for IL-2 determination (T cell hybridomas) or pulse cells with 0.5 uCi [3H]TdR (5 Ci/mol of specific activity, Amersham) for 6 h followed by scintillation counting (for polyclonal T cells).

Confirmation that observed T cell activity is due to class II-restricted presentation should be made. In the case of cloned antigen-specific T cells/hybridomas, MHC class II mismatched BMM0 can be used. More commonly, mAbs (~ 20 ug/ ml) specific for relevant class 11 gene products can be added to the cultures, 15 minutes prior to the addition of T cells (Protocol 3, step 15}, and left in for the duration of the assay. 3.1.4 Biochemical/cell biological aspects of class II processing Having established the optimal cell and antigen concentrations, it is customary to determine the nature of the class II processing requirements for the antigen in question. This has historically involved the use of specific inhibitors of various lysosomal hydrolases, and modulators of intracellular pH. Common examples include chloroquine, pepstatin, and leupeptin (2). Novel inhibitors for various aspects of this pathway arc continually being developed, but if histoiy does indeed repeat itself, it is worth bearing in mind that the specific inhibitor of today may well have other important effects tomorrow! Protocol 4 outlines the basic scheme for assessment of the characteristics of antigen processing for MHC class II-restricted antigens. Gene targeted mice are likely to have an increasing role in the study of macrophage APC function. Gene knockouts for various cathepsins already exist, and macrophages from such mice can be readily compared to wild-type control cells. The use of bone marrow-derived macrophages may be an important consideration when poor survival rates or neonatal mortality occur. Preliminary studies on cathepsin L knockout mice have again highlighted the differential processing which can occur in APCs from different tissue sites (13). 102

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Assessment of the biochemical characteristics of class IIrestrlcted antigen processing Equipment and reagents • CO2 incubator • BMM0(see Protocol 1) • Complete tissue culture medium (see Protocol 2)

• Inhibitors of antigen processing, e.g. chloroquine, pepstatin. leupeptin (Sigma) • Soluble antigen (e.g. ovalbumin) for which antigen-specific T cells are available

Method 1 2 3 4 5

Prepare BMM0 and put them into culture as described in Protocol 3, steps 1-5. Add enzyme inhibitors or modifiers of cellular function diluted in complete tissue culture medium alone or in combination to the adherent monolayers/ Incubate the macrophage monolayers (5% C02 for t h at 37°C). Add antigen diluted in complete tissue culture medium containing the inhibitor to treated cells (see Protocol 3).b Assess efficiency of treatment by determining the influence on antigen-specific T cell stimulation (see Protocol 3).

a

Optimal concentration should be empirically determined by titration with appropriate diluent controls. b It is important to ensure that all antigens and wash buffer contain the appropriate inhibitor until the fixation step (Protocol 3, step 10) is reached.

3.1.5 Processing of soluble antigens for class l-restricted presentation Various methods have been devised for the introduction of soluble antigens into the cytosol and hence the conventional 'endogenous' class I processing pathway. Perhaps that most frequently used is osmotic lysis of pinosomes (14). Protocol 5 describes a method for the loading of soluble antigens into macrophages, using this technique. Controversy still exists as TO whether 'exogenous' protein antigens can be processed for class I-restrictcd presentation by BMM0, in the absence of obvious means for cytosolic entry. In most cases where this has been achieved directly, the very high concentrations of soluble antigen required may allow peptide contaminants to directly load class I molecules (15). Functional testing for peptide contaminants can be made by adding high concentrations of soluble antigen to fixed macrophages, and/or by dialysing protein antigens before use. Coupling of soluble antigens to latex beads, thereby introducing antigen by phagocytosis may improve processing efficiency, though whether this proceeds via conventional TAP-dependent pathways after 'bursting' of occasional phagosomes (15) 103

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or via a novel route (16) requires confirmation by the use of inhibitors or gene targeted macrophages. In contrast to BMM0, DC appear efficient at processing exogenous antigen for class I-resiricted recognition, possibly as a consequence of their ability to perform constitutive macropinocytosis. In our hands, class I processing of soluble antigens by macrophages is always inefficient, and DC are usually suspected when individual preparations of cells show otherwise unexplained high efficiency of class I processing (Kaye, unpublished). The outcome of assays in which stimulation of class I-restricted responses arc measured generally is assessed by monitoring EL-2 production (using CD8" T cell hybridomas)(19)or CTL activity of primed T cells against appropriate target cells (14).

Loading soluble antigens into macrophages by osmotic lysis of pinosomes Equipment and reagents • Centrifuge equipped with Sorval H1000B rotor (Falcon) • 50 ml polypropylene tubes • CO2 incubator • '6 + 2' BBM0 in DMEM (see Protocol 1} • Antigen (e,g, ovalbumin, Sigma} for which class I-restricted T cells are available

• RPMI medium (Gibco) • Hypertonic medium: 0,5 M sucrose, 10% polyethylene glycol 1450 (Sigma), 10 mM Hepes in RPMI medium • Hypotonic medium: 60% (v/v) RPMI medium in

Method 1 Dissolve soluble antigen (e.g. ovalbumin), in hypertonic medium. 2 Make dilutions of antigen in this medium over a range from 0,1-10 mg/ml. 3 Obtain macrophages from desired source (see Chapter 1). 4 Seed 5-10 x 106 macrophages at 106/ml in RPMI to several conical polypropylene tubes. 5 Centrifuge to pellet cells (1200 r.p.m., 10 mm, RT). 6 Remove medium by vacuum aspiration. 7 Resuspend each cell pellet gently in 1 ml of pre-warmed (37°C) hypertonic medium containing antigen at a specific concentration. 8 Incubate macrophages with antigen at 37°C, 5% CO2 for 10 min. 9 Dilute the cell suspension with 15 ml of hypotonic medium. 10 Incubate an additional 3-5 min at 37°C. 11 Wash cells three times by centrifugation in excess normal strength RPMI, to remove exogenous antigen. 12 Use cells in functional assays as desired.

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ANALYSIS OF ANTIGEN PROCESSING AND PRESENTATION

Protocol 6 outlines the most successful protocol we have developed for assessing the stimulation of class I-restrkiced responses by BMM0, The comparison between "osmotically loaded" macrophages and control "untreated" maerophages pulsed with soluble antigen and with specific peptidc epitopes allows the relative efficiency of these routes of antigen uptake to be evaluated.

Stimulation of class l-restricted responses by BMM0 Equipment and reagents • 96-wellflat-bottomtissue culture plates (Nunc) • '6 + 2'BMM0(see Protocol 1) loaded with specific or irrelevant antigen by osmotic lysis (see Protocol 5) • Complete tissue culture medium: supplemented DMEM plus 10% HI-FCS (see Protocol 2)

• CD8f class I-restricted T cell hybridoma • Specific and irrelevant soluble antigens, e.g. ovalbumin, hen egg lysozyme (Sigma) • Specific peptides corresponding to know class l-restricted epitopes of the chosen antigens

Method 1 Plate antigen loaded BMM0 (as in Protocol 5) or untreated BMM0 into 96-well plates as in Protocol 3, steps 2-5. 2 Pulse untreated macrophages with soluble antigen or peptides as described in Protocol 3, steps 6-9.a 3 Assay IL-2 production from CD8+ T cell hybridomas, or CTL activity of primed T cells against peptide pulsed or transfected target cells, as described above. a The dose range for soluble antigens should extend up to 1 mg/ml. Peptides (recognized by the responding T cells) should be tested over 3-4 log range of concentration.

3.2 Modified protocols for use with pathogens As previously stated, each pathogen may bring with it its own set of specific requirements. These often become most apparent when a pathogen is transfected to express a normally soluble 'reporter' antigen (e.g. ovalbumin) and then processing of the same epitopes are compared after these two modes of delivery (17, 19}.

3.2.1 Antigen dose Soluble protein antigens can be readily titrated into APC assays within a concentration range from nanogram to milligram, normally without discernible adverse effects. Such titrations usually produce bell-shaped clones, with high dose inhibition usually attributed to complex immunoregulatory events. In contrast, adherent macrophage monolayers have a relatively narrow capacity to ingest particulate matter. At high multiplicity of infection, it may be possible to obtain 30-50 protozoa or bacteria per macrophage with greater than 95% infection levels. However, unlike soluble antigens, dose response characteristics may be 105

P, M. KAYE

influenced by numerous complex or trivial factors. As M01 is reduced, both the number of organisms per macrophage (the true parameter to be evaluated) and the percentage of macrophages infected (a confounding variable) decrease. At MOls of 10:1, it is not uncommon to observe < 50% infection levels within a macrophage monolayer. Hence, under these conditions, apparent processing efficiency (as measured in a functional T cell assay) becomes a function of both actual processing efficiency and also presenting cell number (and, as a result, MHC density). Varying the MOI may also pose inherent problems at the top end of the dose response, Protocols 1 and 2 are based on antigen pulsing into macrophage monolayers, following by exhaustive removal of excess antigen. At high MOI, the adherence properties of macrophages may vary considerably, and after washing of such monolayers, a rather patchy distribution of macrophages with lower than expected MOI may remain. Furthermore, the ability to effectively remove particulate organisms from 96-well plates is rather poor at high MOI, increasing the chance of antigen uptake and hence new processing occurring over an extended time period. In practice, therefore, in routine pulsing experiments, the effective range of infection levels which can be tested is rather small, A variation of the antigen pulsing protocols we have found useful in extending the effective assay range, involves infection of non-adherent BMM0 in 'non-stick' polypropylene tubes and is given in Protocol 7. Various safety considerations may need to be introduced, e.g. to reduce aerosols or the volume of biohazardous waste material.

Pulsing macrophages with participate antigens In suspension Equipment and reagents • 4 ml polypropylene culture tubes (Falcon 2063) • Centrifuge equipped with Sorval H1000B rotor • BMM0 macrophages obtained as described in Protocols 1 and 2, or other macrophages as described in Chapter 1

• Water-bath • Complete tissue culture medium (see Protocol 3) • Ice > 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.

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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

ANALYSIS OF ANTIGEN PROCESSING AND PRESENTATION

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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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

ANALYSIS OF ANTIGEN PROCESSING AND PRESENTATION

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

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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{<5%). With NK cells and monocytes, only a proportion (usually 30%) of adherent cells effectively has the ability to transmigrate during the assay. When EC are activated with IL-1 a greater number of cells adhere, but usually the same proportion transmigrate. It should be noted that IL-1 does not change the state of confluence of the monolayer, as determined by staining. The identification of adhesion molecules involved in the interaction of leukocytes and EC is performed by the addition of blocking mAbs—most of which are commercially available—specific for the adhesion structures expressed by leukocytes or EC. Studies with specific mAb have demonstrated that the adhesion and transmigration through resting EC is mediated by the LFA-l/lCAM-1,2

Figure 2 Reverse transendothelial migration assay. Cross-section of the compartments of a transmigration apparatus. A modified Boyden chamber is used. An upper filter is coated with extracellular matrix (ECM) and the lower filter is coated with a monolayer of endothelial cells (EC) placed upside down. 51Cr-labelled leukocytes are seeded in the upper compartment and incubated for 3 h at 37°C.

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pathway; while IL-1 activated EC involved both LFA-l/ICAM-1 and VLA-4/VCAM-1 for monocytes and lymphoid cells, neutrophils use only the first pathway, being VLA-4 negative. In addition, PECAM (CD31) is a molecule expressed both by leukocytes and EC, and plays a major role during transmigration (10, 14, 15). Chemoattractants can be seeded in the lower compartment to increase leukocyte transmigration.

4 Reverse transmigration Some leukocytes (e.g. dendritic cells, lymphocytes) have a peculiar trafficking pattern from tissues into the lumen of blood or lymphatic vessels. To mimic in vitro this basal-to-apical process of migration we established a transmigration assay, schematically represented in figure 2 and described in ref. 16, that we named reverse transmigration assay. Protocol 3 describes the essential aspects of this assay.

Assessment of leukocyte reverse transmigration in vitro Equipment and reagents • See Protocol 2

- Stripping buffer: 20 mM NH4OH, 0.5% (v/v) Triton X-100

Method 1 Culture EC to confluent monolayers on fibronectin pre-coated PVP-free polycarbonate filters in 24-well plates as described in Protocol 2. 2 Treat one-half of the filters with 1 ml of stripping buffer for 30 sec. 3 Quickly remove the stripping buffer and the digested EC monolayer and wash twice with complete medium. Cover the exposed ECM with 1 ml of complete medium. Drying should be avoided. 4 Place 0.2 ml of complete medium in the lower compartment of each Boyden chamber. Mount the first filter, coated with EC, upside down, and on top the second filter coated with ECM (see Figure 2). 5 Assemble and screw the upper compartment of the chamber. 6 Immediately add 0.15 ml of complete medium. Drying should be avoided. 7

51

Cr-labelled leukocytes (see Protocol 2) are seeded (3-6 x 105 in 0.15 ml of complete medium) into the upper compartment of the chamber.

& Incubate the chamber at 37 °C for 60 min. 9 Remove the chambers from the incubator. 10 Collect the medium present in the upper chamber (fraction A) and wash the ECM layer with 0.5 ml warm medium (fraction B}.

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11 Collect the ECM adherent cells with cotton floes (fraction C), and the transmigrated cells in the lower compartment (fraction D), 12 Measure radioactivity in each fraction. Fraction A and fraction B represent the nonadherent cells. Fraction C and D represent the adherent cells. Fraction D represents the migrated cells. Please note that in the reverse assay the transmigrated cells comprise only the radioactivity present in the lower compartment.

5 Soluble cytokine receptors Soluble receptors leave cells producing them and can be active in the cellular microenvironment as well as in body fluids (17. 18). The discovery that membrane bound receptors are also released in body fluids has dramatically changed our understanding of the ligand-receptor interactions as well as, more in general, of the mode of action of hormones and cytokines, Ligand concentrations can be modified by soluble receptors, by down-regulation of the number of membrane bound receptors which are released, and may compete with membrane bound receptors for the ligands, thus effectively reducing the level of free active ligand. In addition, soluble receptors can render cells in tissues able to respond to ligands for which they express an incomplete set of receptor molecules. Soluble receptors are generated by expression of an alternative mRNA splice which generates an appropriate transcript encoding a soluble isoform, by proteolytic cleavage, or by the action of phospholipase C (Table 2). The latter enzyme is active on PI linked receptors and is only involved, to date, in the release of the soluble ciliary neurotrophic factor (CNTF) receptor (19). Table 2 Mechanisms of generation of soluble cytokine receptors Proteolyttc cleavage

TNF, Rl and II IL-1RII IL-2Ra IL-6Ra M-CSFR (fms) c-kit

Alternative spllcinga

(IL-1RII) IL-4Ra IL-5Ra GM-CSFRa (IL-6Ra) IL-7R IL-9 LIF-R c-kit

a

The parenthesis for IL-1RII and IL-6Rn indicates that, while an alternative spliced RNA has bean demonstrated, available in vitro information suggests a major role for proteolytic shedding. The soluble form of c-kit originates from proteolytic cleavage of an alternatively spliced form.

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Alternative splicing regulates the generation of mRNA encoding soluble forms of various cytokine receptors, including receptors for IL-4, IL-5, IL-6, IL-7, the type IIIL-1 receptor, the interferon a/B receptor, and the GM-CSF receptor. Alternative splicing yields different soluble isoforms of cytokine receptors via different mechanisms. The simplest mechanism involves exclusion of the exon encoding the transmembrane portion of the receptor. This is the most common mechanism and is exemplified by the GM-CSF receptor a chain. A second mechanism consists of the inclusion in the mature mRNA transcript of an exon called the 'soluble exon', which causes protein chain termination before the transmembrane exon. This is the mechanism of generation of soluble receptors for IL4, IL-5, and LIF. The first cytokine receptor for which differential splicing was shown to generate membrane bound and soluble forms of the receptor was the mouse IL-4 receptor. The mRNA encoding soluble version of the IL-4R contains a 114 bp insertion upstream of the transmembrane domain, which resulted in extra six amino acids and premature termination. The resulting soluble receptor lacks the transmembrane and cytoplasmic domains. For the IL-7 receptor, a deletion of the sequences encoding the transmembrane domain alters the translational reading frame, resulting in 27 novel amino acids and premature termination. Proteolytic cleavage is involved in the generation of soluble forms of the TNF receptor p55 (type I) and p75 (type II), of the IL-1 type II decoy receptor, and of the IL-2 receptor a chain. IL-6 receptor a chain can be released both by proteolysis of the membrane bound isoform or by alternative splicing. The relative contribution of these two mechanisms to the soluble IL-6 receptor found in biological fluids under normal and pathological conditions is unknown. The soluble form of c-kit is generated by both proteolytic cleavage and alternative splicing. There is evidence that expression of the membrane bound versus soluble isoforms of cytokine receptors can be differentially regulated (20). Release of the type II IL-1 decoy receptor has been extensively investigated. Anti-inflammatory agents (glucocorticoid hormones, IL-4, IL-13) augment expression of the type II decoy receptor and consequently its release (20, 21). It has been calculated that in monocytes exposed to dexamethasone, the number of surface receptors increased from 3 x 103/cell to ~ 12 x 103 over a period of 24 hours and that over the same time ~ 20 x 103 receptors are released (21). Thus, under these conditions, augmented release is associated with, and dependent on, augmented expression of the membrane bound isoforms and it is gene expression- and protein synthesis-dependent. A second rapid pathway of regulation of the type II receptor release is shared with the TNF receptors. TNF causes rapid shedding of the p55 and p75 receptor as well as of the type II IL-1 decoy receptor. It is of interest that work with specific blocking antibody suggest that the proteolytic cleavage of the p75 receptor is induced by binding of TNF to the p55 receptor. Chemoattractants and agents which mimic elements in the signal transduction pathway of G-protein coupled receptors (phorbol esters, calcium ionophores) as 123

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well as TNF but not other pro- or anti-inflammatory cytokines, cause rapid release of the type 11 decoy receptors as well as of the TNF receptors (22). The enzyme systems involved in the regulation of shedding of cytokine receptors have not been molecularly identified. There is evidence for the TNF and IL-1 type II decoy receptors that spontaneous release is to some extent dependent upon enzymes belonging to the serine protease group (23, 24), However, recent evidence using inhibitors strongly suggests that matrix metalloproteinases play a major role in the activation of release of the type II IL-1 receptor as well as of the IL-6 and TNF receptors (23).

6 Cross-linking of soluble receptors Cross-linking of soluble receptors with radiolabelled ligands provides a direct experimental approach to identification of these released monocyte products (23, 25). Protocol 4 describes a method for detection of soluble cytokine receptors, based on information from the IL-1 receptor system (IL-1R) (26). Both receptor size and reactivity with appropriate specific antibodies are used to identify soluble receptors following chemical cross-linking.

Identification of soluble cytokine receptors using radiolabelled ligands Equipment and reagents • 15 ml polypropylene tubes (Falcon, Becton Dickinson) • 1.5 ml Eppendorf tube(Eppendorf) • Amicon filtration unit; cut-off 10000 (Beverly) • Bio-Rad Gel dryer (Bio-Rad) • Kodak X-Omat films (Kodak) • RPMI 1640 medium (Biochrom KG)

Peripheral monocytes, purified as described in Chapters 1 and 2 Disuccinimydilsuberate (DSS) (Pierce) Stimuli: PMA (50 ng/ml) or fMLP {(10-7 M) (Sigma) Ligand: 125I-Iabelled IL-1B (specific activity, 180 Ci/ug; NEN Life Science Products), or other specific receptor ligand

Method 1

Place 20 x 106 purified human monocytes in 15 ml polypropylene tubes in I ml of RPMI 1640 medium,

2

Add appropriate stimuli (e.g. 50 ng/ml PMA or 10-7 M fMLP dissolved in RPMI 1640 medium) and incubate at 37°C for 20 min.

3

Recover medium by pipette.

4

Concentrate medium ten times by membrane filtration using appropriate molecular weight.

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5 6 7 8 9

Add 100 ul of concentrate medium to 1 nM 125I-labeIled ligand (e.g. 11-10) in 1.5 ml Eppendorf tube and incubate for 4 h at 4°C. Add 1 nM disuccinimydilsuberate (DSS) and incubate at 4°C for 30 min. Separate samples on 8% SDS-PAGE under reducing conditions. Expose dried gels to photographic film for one to three days, as needed. Process films for autoradiography using standard methods.

Soluble cytokine receptors released by mononuclear phagocytes either constitutively or after exposure to a variety of signals can be assessed conveniently by cross-linking using this method. Alternative detection methods for cell-free receptors include Western blotting or ELISA. The latter is the approach of choice to measure soluble cytokine receptors in body fluids. Cross-linking is a simple and straightforward approach with high sensitivity which allows recognition of soluble receptors for which a sensitive ELISA is not available. Moreover, it provides direct evidence that the soluble receptor can recognize the appropriate ligand. Using cold ligand, the specificity and relative affinity of the soluble receptor can also be evaluated (26).

References 1. Nathan, C. F. (1987). J. Chin. Invest, 79, 319. 2. Colotta. F., Borte. A., Wang, J. M, Tattanelli, M., Maddalena, F., Polentarutti, N., et al. (1992). J. Immunol, 148, 760, 3. Godiska. R., Chantry, D., Raport, C. J., Sozzani, S., Allavena, P.. Leviten, D.. et al (1997). J. Exp.Med., 185, 1595. 4. Baggiolini, M and Moser, B. (1997). J. Exp. MM, 186, 1189. 5. Mantovani, A., Allavena, P., Vecchi, A., and Sozzani, S. (1998). Int. J. (,1in. Lab. (to., 28, 77. 6. Proost, P., De Meester, L, Schols, D., Striryf, S., Lambeir, A. M., Wuyts, A., et al. (1998). J. Biol. Chem., 273, 7222,. 7. Dejana, E., Langumo, L. R., Polentarutti, N., Balconi, G., Ryckuwaerl, J. J., Larrieu, M. J., et al. (1985), J. Chin. Invest., 75, 11. 8. Falk, W., Goodwin Jr., R. H., and Leonard, E. J, [1980) J. Immunol. Methods, 33, 239. 9. Bussolino, F., Wang, J. M., Defilippi, P., Turrini, F, Sanavio, F., Edgell, C. J., et al. (1989). Nature. 337, 471. 10. S p r i n g e r .T.A. (1994), Cell, 76, 301. 11. Adams, D. H. and Shaw, S. (1994). lancet, 343. 831. 12. Bianchi. G., Sironi, M,, Chibaudi, E., Selvaggini, C., Elices, M., Allavena, P., et al (1993), J. Immunol., 151, 5135. 13. Sironi, M., Breviario, F., Proseipio, P., Biondi, A., Vecchi, A., Van Damme,J., etal. (1989J.J. Immunol., 142, 549. 14. Butcher, E. C. (1991). Cell, 67, 1033. 15. Mantovani, A., Russolino, !•'., and Dejana, E. (1992). FASEB ]., 6. 2591, 16. D'Amico, G., Bianchi, G., Bernasconi, S.. Bersani, L, Piemonti, L, Sozzani, S., etui. {1998).Blood, 92. 207. 125

PAOLA ALLAVENA ET AL. 17. Fernandez-Botran, R., Chilton, P. M., and Ma, Y. (1996). In Advances in immunology (ed. F. J. Dixon), p. 269. Academic Press, San Diego. 18. Heaney, M. L. and Golde, D. V. (1996). Blood, 87, 847. 19. Kishimoto, T., Taga, T., and Akira, S. (1994). Cell, 76, 253. 20. Colotta, F., Dower, S. K., Sims, J. E., and Mantovani, A. (1994). Immunol. Today, 15, 562. 21. Colotta, F. and Mantovani, A. (1994). Trends Pharmacol. So., 15, 138. 22. Colotta, F., Orlando, S., Fadlon, E. J., Sozzani, S., Matteucci, C, and Mantovani, A. (1995) J. Exp. Med., 181, 2181. 23. Orlando, S., Sironi, M., Bianchi, G., Drummond, A. H., Boraschi, D., Yabes, D., et al. (1997). J. Biol. Chem., 272, 31764. 24. Porteu, F. and Nathan, C. (1990). J. Exp. Med., 172, 599. 25. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., et al. (1993). Science, 261, 472. 26. Re, F., Muzio, M., De Rossi, M., Polentarutti, N., Giri, J. G., Mantovani, A., et al. (1994). ]. Exp. Med., 179, 739.

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Chapter 6 Analysis of macrophage lytic functions Luigi Varesio, Maria Carla Bosco, Luca Carta Laboratory of Molecular Biology, G. Gaslini Institute, Largo G. Gaslini 5, Geneva, Italy

16148

Tiziana Musso Microbiology Institute, University of Torino, Via Santena 9, 1012 Torino, Italy

1 Introduction 1.1 Biological perspective Mononuclear phagocytes, comprising circulating monocytes and tissue macrophages, are phylogenetically ancient cells deriving from the primordial amoeba that was enclosed into a complex pluricellular organism. During the course of evolution, macrophages retained some of the basic features of the amoeba, such as mobility and phagocytosis, and acquired new immunoregulatory properties as a result of the interaction with the emerging immune system. Engulfing bacteria and fighting with competitors are among the primordial functional activities of macrophages that pre-existed the development of the immune system and that are triggered by classical environmental stimuli such as bacteria, toxins, organic molecules, pH, osmotic changes, etc. The need to communicate with the immune system and with the neighbouring cells caused profound modifications in the number and nature of the signals recognized by macrophages, that learned to recognize distress signals derived from lymphocytes, such as lymphokines and antibodies, or from tissues, such as hormones, adhesion molecules, and matrix proteins. Mononuclear phagocytes established themselves as ubiquitous cells to cany on an effective defence of the host. As monocytes, they circulate in the blood and, upon extravasation, they colonize virtually every tissue where they differentiate into macrophages. Because of the close association with tissue cells, it was important that macrophages remained in a resting state in which the offensive functions could be kept silent to avoid damage to the neighbouring cells. In fact, mononuclear phagocytes, as well as monocytes, require activation by tissue-, immune system-, or environment-derived signals, either alone or in combination, to express lytic properties. Activated macrophages can exert anti-cellular 127

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activities and be toxic for other eukaryotic cells, as shown by the ability to kill tumour target cells as well as fungi (1, 2). Since the discovery of the important role played by macrophages in the defence against parasites and tumours, attempts were made to dissect the mechanisms responsible for the acquisition and the expression of effector functions and to develop experimental systems to study and measure the lytic activity against prokaryotic and eukaryotic targets. In vitro cytotoxicity assays have provided scientists with the tools to probe these functions. The association between response to stimulation and expression of toxicity against cellular targets led scientists to utilize these functions as a parameter for macrophage activation. Models were proposed in which the expression of cytolytic activity against tumour cells was the end-point of macrophage activation. The difficulty of utilizing a biological assay as reference was the fact that the assays existed in almost as many different versions as the number of laboratories utilizing them, and the general validity of the results was undermined. These difficulties were overcome in two ways. On one hand, major efforts were made to standardize the assays (3) as well as to understand how different experimental methods should measure distinct expression of macrophage activity. On the other hand, the mechanisms responsible for the lytic activity were elucidated and the phenomenological observations of lytic activity was associated to activation of biochemically defined pathways and/or secretion of toxic mediators. It is now recognized that: (a) The lytic activity of macrophages is due to the concomitant expression of several toxic pathways/molecules. (b) Each assay will be more sensitive to the expression of certain lytic mechanisms. Therefore, it is no longer surprising that each assay may measure different levels of macrophage activation and various degrees of response to activating stimuli. Monocytes/macrophages can be readily obtained from various anatomical locations in a large number of animal species, humans included; can be separated from other cell types with some degree of efficiency; and are available in sufficient numbers for several types of studies. While short-term maintenance of macrophages in culture is common, long-term cultivation of primary isolates has been rather unsuccessful both because of the relatively short life span and the inability of replication in vitro of mature, differentiated monocytes/ macrophages. Moreover, the results obtained from in vitro studies may not always completely reflect the in vivo activities of this cell type. In fact, the physical state of the macrophage donor, along with the nature of the material used to obtain the cells and the culture conditions employed, have a profound influence on macrophage functions in vitro. Therefore, it becomes sometimes convenient to utilize established macrophage cell lines as a source of functional monocytes/ macrophages. This chapter introduces a series of procedures to assess monocyte/macrophage lytic functions in vitro, including various techniques for the analysis of 128

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cytolysis and cytostasis of tumour cells and methods. to evaluate killing of intracellular and extracellular micro-organisms by macrophages.

1.2 Technical notes

1.2.1 Cells Mononuclear phagocytes from diverse species and anatomical regions can be utilized to measure lyric activity. Circulatory monocytes can be obtained from various vertebrates, although the limiting factor is the size of the donor and, hence, the volume of blood. Blood is a very convenient and major source of mononuclear phagocytes in humans primates and large mammals. Primary macrophages can be obtained from the peritoneal cavity, spleen, liver, lung, skin, brain, etc. In rodents, the most convenient source is the peritoneal cavity. The yield of peritoneal macrophages ranges from 106 resident to 0.5-1 x 107 thioglycollateinduced. Mononuclear phagocytes can be derived from bone marrow cultured in appropriated growth factors. A good health condition of the mice and, hence, the condition of the animal facility is a determining factor for reproducibility and consistency of the results. Infection of the mice can cause high baseline activation of the recovered macrophage populations as well as energy and lack of response to the activating signals in vitro. Mononuclear phagocytes recovery is discussed more completely in Chapters 1 and 2. Murine cell lines that can exert tumouricidal and microbicidal activities were developed These cell lines were fundamental to elucidate the molecular basis of the mechanisms responsible for the expression of toxicity. Mycoplasma contamination is a source of major problems and artefacts in cell culture-dependent assays including cytotoxicify assays. Frozen stocks must be tested for mycoplasma contamination and care should be taken to avoid contamination of passaged cell lines. Mycoplasma contamination can affect macrophage reactivity as well as tumour target sensitivity to killing and the outcome of the assay. Sudden changes in the expected pattern of reactivity are often an indication of ongoing mycoplasma infection.

1.2.2 Purity The purity of the effector cells is essential for the interpretation of the results. The methods to purify mononuclear phagocytes are discussed elsewhere in this volume (see Chapters 1 and 2). Primary cultures can easily be contaminated by few NK cells but sufficient to produce enough interferon to activate macrophages. This is a particular concern for human monocytes, where the starting leukocyte population has between 5-10% monocytes, and less of a problem for peritoneal macrophages that represent from 50-60% of the resident population to over 90% in the thioglycollate-induced exudate.

1.2.3 Reagents A major source of variability is represented by the endotoxin contamination of the reagents used for the assays. Test should be performed to monitor the levels of endotoxins in the system, that should be kept below 0.1 pg/ml. 129

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Common tissue culture media and reagents will be indicated in the protocols. Suppliers will be indicated only when a particular source is recommended.

1.2.4 Activation Expression of lytic activity by macrophages requires that the cells are activated either in vivo or in vitro. Depending on the source of macrophages, the activation protocols change and the extent of the lytic activity of macrophages may vary as well (3). For example, human monocytes can be activated by treatment with interleukin-2 or IFNy alone, whereas murine macrophages require the combination of the two cytokines to express tumouricidal activity. In general, rodent macrophages are optimally activated to express high levels of cytotoxic activity by co-stimulation with multiple signals. A broad discussion of macrophage activation is beyond the scope of this chapter, but useful information can be found in refs 4-6.

2 Macrophage-mediated anti-tumour activity The anti-tumour activity of macrophages was studied by several laboratories and a variety of techniques for the detection of macrophage-mediated lysis or growth inhibition (stasis) of tumour cell were developed. We will describe in detail established techniques routinely used to assess macrophage-mediated cytotoxicity of tumour cells in vitro. Every procedure outlined can utilize, as effector cells, rodent macrophages or human peripheral blood monocytes, purified by adherence or by countercurrent centrifugal elutriation, as detailed in other chapters of this manual (see Chapters 1 and 2), as well as monocyte/macrophage cell lines.

2.1 Morphological tumour cell counting assay Protocol 1 provides a description of the microscopic tumour cell counting assay, which consists in monitoring gross morphological changes in target cell cultures. This assay measures both tumour cell lysis and stasis thus providing a good assessment of net macrophage-mediated anti-tumour activity (7). In addition, this technique allows a direct measurement of the anti-tumour activity avoiding the drawback of assays relying on incorporation or release of radioactive labels. This method is largely used in studies with adherent target cells, although it can be modified for quantifying the cytotoxic effect exerted by activated macrophages on non-adherent tumour cells (7). Although relatively laborious, this assay is quite accurate.

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Measurement of macrophage anti-tumour activity by the tumour cell counting assay Equipment and reagents • Microtest tissue culture plates, 96-well, flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Labware) • 25-75 cm2 tissue culture flasks or 60-100 mmz tissue culture plates, polystyrene (Corning-Costar) • Sterile plastic pipettes, 1-10 mt (CorningCostar) • Centrifuge conical tubes, 15-50 ml polystyrene (Falcon Plastics) • Cell centrifuge • Standard microscope • Haemocytometer • Device for vacuum aspiration • Repeating dispenser • Combitips 1-5 ml (Eppendorf) • Complete medium: RPMI1640 (Hyclone Laboratories) or DMEM (1CN Biomedicals, Inc.) supplemented with 10% heatinactivated, endotoxin-free, fetal calf serum (FCS) (Hyclone Laboratories), 2 mM L-glutamine, 100 U/ml penicillin, and 100 ug/ml streptomycin (Celbio S.r.l.) • Hanks' balanced salt solution (HBSS) (Gibco BRL)

Ca2+-, Mg2-free Dulbecco's modified phosphate-buffered saline (PBS) pH 7.2-7'A, containing 8.0 g NaCl/litre, 0,20 g KCl/litre, 0.12 g KH2P02/litre, and 0.91 g Na2HPO4/litre 0.05% (w/v) trypsin, 0.02% EDTA solutions (Hyclone Laboratories) 10% (v/v) Giemsa stain solution (BDH Laboratory Supplies) Methanol Monocyte/maerophage effector cells, appropriately purified (see Chapters 1 and 2) Appropriate tumour target cells maintained in complete medium by serial passages on 100 mm tissue culture dishes or in 75 cm2 tissue culture flasks: e.g. the adherent, spontaneously transformed, murine cell line 3T12 of Balb/c origin for murine macrophage activity or the HT29 human colon carcinoma cell line for human monocyte-mediated anti-tumour activity Appropriate macrophage activators: e.g. IFNy (Genentech, Inc.), IL-2 (Roche), or LPS from Escherichia coli (Sigma)

A Preparation of effector cells 1 Resuspend monocytes/macrophages at 4 x 106 cells/ml in complete medium. 2 Plate 0.1 ml of the macrophage cell suspension (containing 4 x 105 cells) into triplicate wells of a 96-well, flat-bottom microtitre tissue culture plate, using a repeating dispenser. Add the appropriate macrophage activators in 0.1 ml of medium to yield a final volume of 0.2 ml/well. 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 medium or HBSS as follows: add 0.2 ml/well of warm HBSS using a repeating dispenser, centrifuge the plate at 250-300 g for 5 min, and aspirate medium.

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B Preparation of tumour target cells 1 Wash the tumour target cells, cultured in 25-75 cm2 flasks or in 60-100 mm plates until confluence, once with Ca2+-, Mg2+-free PBS and aspirate the medium, 2 Trypsinize the cells by adding 1 ml of 0.05% (w/v) trypsin in 0.02% EDTA solution for about 2-5 min at 37°C. 3 Observe the cells under inverted microscope and, when monolayers show the first signs of disruption, add 10 ml of complete medium to the flask or plate to inhibit the trypsin. 4 Pipette the cell suspensions several times to separate clumps. 5 Wash the cell suspensions twice in 50 ml of medium each time by centrifiigation for 5 min at 300 g in 50 ml conical tubes. 6 Resuspend cells at a final concentration of 1 x 10s cells/ml.a C Cytotoxicity assay 1 Add 0.1 ml of target cell suspension (containing 1 x 104 cells) to wells containing washed monocyte/macrophage monolayers. Tumour cells should also be added to the wells not containing macrophages. 2 Add 0.1 ml of complete medium to each well to yield a final volume of 0,2 ml/well. 3 Incubate plates containing macrophages and effector cells at 37°C in a humidified incubator for 56-72 h. 4 Remove supernatant from all wells by vacuum aspiration. 5 Wash the plates gently once with PBS. 6 Fix the cells with 100% methanol for 5 min. 7 Stain fixed monolayers by incubating for 7 min with 10% Giemsa stain. 8 Quantify the number of tumour cells remaining in the wells by visual counting clear 'plaques'b in three randomly selected fields of the stained monolayei* at x 430 magnification (x 43 objective with x 10 eyepieces).d 9 Net cytotoxicity is calculated from the average of triplicate samples and determined by the following formula: {1 - (experimental 3T12 cell number per well/total 3T12 cell number per well} x 100 where the experimental 3T12 cells are tumour cells cultured with macrophages, and total 3T12 cells are tumour cells cultured alone without macrophages. a The concentration of the target cells should be adjusted to achieve the desired effector-totarget cells ratio. b

Tumour cells alone continue to proliferate and form a completely confluent monolayer that stains uniformly dark. Activated macrophages destroy tumour cells, inducing a clear 'plaque' in the darkly stained tumour cell monolayer. c The 3T12 cells are easily distinguished from macrophages because they differ in shape, morphology, and by the presence of large nuclei with nucteolt. d lt is calculated that each well of 96-well plates contains 185 microscopic fields under these observation conditions. The average number of rumour cells in randomly selected fields is multiplied by 185 to calculate the total number of tumour cells per well

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2,2 Macrophage-mediated cytolysis: release of radioisotopes The morphological assay is quite laborious and does not distinguish between cytostasis and cytolysis. As a result, other methods based on the use of radioisotopes were developed to provide a more convenient tool to assess macrophage-mcdiated tumour cell killing. These techniques employ the quantitative release of intracellular radioisotopes as a measure of tumour cell destruction. Three different and commonly used isoropic release assays that measure cytolysis arc described in this section. 2,2.1 51Cr release assay Protocol 2 describes one method for the 51 Cr release assay, a convenient and reliable method that employs a cytoplasmic label, the gamma emitter 51Cr. This assay can be used to quantify lysis of susceptible leukaemia, lymphoma, or mastocytoma cells by mouse peritoneal macrophages and murine macrophage cell lines (8-12). However, the relatively high 'spontaneous release' by labelled target cells cultured alone or with resting macrophages (1 -2% per hour) restricts the usefulness of 51Cr to relatively short-term assays (18-24 hours). In addition, its use is limited by the relatively low number of susceptible targets that incorporate the label.

Measurement of macrophage-me dialed cytolysis by the 51 Cr release assay Equipment and reagents • Tissue culture plates. 96-well, U-bottom, polystyrene (Corning-Costar) • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other Jabware (see Protocol ]) • Gamma counter • Centrifuge • Standard microscope • Haemocytometer • Device for vacuum aspiration • Repeating dispenser • Sodium chromate 51Cr-labelled, specific activity 200-500 mCi/mg (New England Nuclear Corp.)

Combitips (Eppendorf) Complete medium (see Protocol 1) Ca 2 ' -, Mg 2 ' -free Dulbecco's modified PBS (see Protocol I) 0.05% (w/v) trypsirt, 0.02% EDTA solution (Hyclone Laboratories) Macrophage effector cells, appropriately purified (see Chapters 1 and 2) Tumour target cells: e.g. the P815 murine mastocytoma cell line, maintained in complete medium by serial passages on 100 nun tissue culture dish or in 75 cm2 tissue culture flasks Macrophage activators (see Protocol ])

A Preparation of effector cells 1

Resuspend monocytes/macrophages at 2 x 106 cells/ml in complete medium.

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2 3 4

Pipette 0.1 ml aliquots of the cell suspension (containing 2 x 105 cells) into triplicate wells of 96-well U-bottom tissue culture plates using a repeating dispenser. Add macrophage activators (as described in Protocol 1). After 18-24 h incubation, wash cells extensively (as described in Protocol 1).

B 51Cr labelling of target cells 1 Wash the P815 murine mastocytoma rumour target cells once with complete medium and resuspend them at a concentration of 5 x 106/ml in complete medium, 2 Label with 51Cr by incubating 5 x 106 cells in 1 ml of medium with 500 uCl of 51Cr in a 50 ml conical tube for 1 h at 37°C with occasional shaking.a 3 Wash radiolabelled tumour cells twice with 50 ml complete medium to remove non-incorporated label. 4 Return labelled cells to the incubator for 1 h at 37°C to allow the release of nonincorporated label into the medium. This 'cold chase' decreases spontaneous release of label during the assay. 5 Wash the cell suspension once more just before addition to macrophage cultures. 6 Adjust concentration to 2.5 x 104 cells/ml in complete medium. C Cytotoxlcity assay 1 Add 0.2 ml of target cell suspension (containing 5 x 103 51Cr-labelled cells} to wells containing washed monocyte/macrophage monolayers to give a 40:1 E:T cell ratio. Labelled tumour cells should also be added to wells without macrophages to determine the spontaneous releaseb and the total incorporated counts.c 2 Incubate the test plates in a humidified atmosphere containing 5% CO2 at 37°C for 18 h. 3 At the end of the incubation time, centrifuge the plates at 300 g for 5 min. 4 Collect 0.1 ml of supernatant/well with an automatic pipette and assess the radioactivity of each sample in a gamma counter. 5 Express the results of the cytotoxicity assay as the percentage of specific 51Cr release, calculated from the average of triplicate samples, according to the following formula: { (experimental c.p.m. - spontaneous c.p.m.) - (total c.p.m. incorporated in target cells - spontaneous c.p.m.) }x 100, Experimental c.p.m. is the radioactivity released in wells containing macrophages and target cells; spontaneous c.p.m. is the radioactivity released by target cells cultured alone; total release is the radioactivity from lysed target cells. a

Any kind of leukaemia, lymphoma, or mastocytoma cell, either in suspension or adherent, can be used as tumour target cells in this assay. b The spontaneous release in the 18 h 51 Cr release assay is typically 30-35% of total radioactivity. c Total incorporated counts are estimated by adding 20 ul/well of 10% SDS or 50 ul/well of 0.1 M NaOH or 1% (v/v) Triton X-100 to lyse cells. The average counts of three wells containing the target cells is used.

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There are a few variations of the 51Cr release assay for macrophage-mediated cytotoxicity, A faster assay with a lower spontaneous release employs TNFasensitive target cells, such as the WEHI 164 murine fibrosarcoma or the MOPC315 murine plasmasytorna cell lines (see Section 3) (9, 12-14). In this procedure tumour cells at the concentration of 1 x 10 6 /ml are pre-treated with 1 ug/ml of actinomycin D for 3 h at 37 °C in a CO2 incubator, washed twice with complete medium, and then radiolabelled as described above, 51Cr-labelled cell concentration is adjusted so that 5 x 103 cells in 0.1 ml are added to each well of the 96-well plate containing 2 x 105 effector cells in 0.1 ml. The release of radiolabel from tumoui' target cells is determined after a six hour incubation period. 2.2.2 [3H]TdR and [125l]dUrd release assays Nuclear labels, such as [3H]thymidine (TdR) and p^ljiododeoxyuridine (dUrd), have a low spontaneous release (less than 10-15% of the total incorporated counts over 72 hours) and have been widely used in assays of 48 hours or longer (15-18). Macraphage cytotoxicity assays with pH]TdR and |125I]dUrd pre-labelled tumour cells require actively dividing target cells for incorporation of the label into DNA, and the level of radioactivity may vary in different cells. In addition, the release of nuclear labels requires both cell death and autolysis which results in delayed detection of cell killing and requires assays of longer duration to quantitatively reflect cytolysis. Because [3H]thymidine (TdR) is reutilized to some extent after release by target cells and/or it is taken up by macrophages, the percentage of lysis determined does not yield values that reflect the real extent of tumour cell killing and the artificially low release may give misleading results (17-19). [125I]dUrd, on the other hand, is not reutilized but can inhibit target cell proliferation and is toxic for certain target cells (4, 15, 16). Protocol 3 describes a cytotoxicity assay that can be used with either [3H|TdR- or 125 [ I]dUrd-labelled target cells

Assessment of macrophage-mediated cytotoxicity using [3H]TdR and [125l]dUrd release assays Equipment and reagents • MICROTEST tissue culture plates, 96-well, flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Labware) • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other labware (see Protocol 1) • Beta counter • Gamma counter • Equipment as in Protocol 2

• [Methyl-3H]thymidine ([3H]TdR), specific activity 2 Ci/mmol (Amersham Life Science); or [125I]iododeoxyuridine ([125I]dUrd). specific activity 2000 Ci/mmol (New England Nuclear Corp.) • OptiPhase 'HiSafe' three liquid scintillation cocktail (Wallac, EG&G Comp,) • Ca2+-, Mg2+-free Dulbecco's modified PBS (see Protocol 1)

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• Complete medium (see Protocol 1) • 0.05% (w/v) trypsin, 0.02% EDTA solution (Hyclone Laboratories) • Macrophage effector cells, appropriately purified (see Chapters 1 and 2)

• Appropriate target cells: e.g. the human melanoma cell line A375 or the murine fibrosarcoma and adenocarcinoma cell lines L929, mKSA TU5 (TU5), AMC 60, and MCA 26 • Macrophage activators (see Protocol 1)

A Preparation of effector cells 1

Resuspend monocytes/macrophages at 2 X 106 cells/ml in complete medium,

2

Plate effector cells in triplicate at a concentration of 2 x 105/well onto flat-bottomed, 96-well plates, using a repeating dispenser.

3

Add macrophage activators (as described in Protocol 1}.

4

After 18-24 h incubation, wash the macrophages extensively (as described in Protocol 1).

B Labelling of target cells 1 Culture the human melanoma cells A375 in 25-75 cm2 flask or in 60-100 mm plates.a 2 Label cell cultures in logarithmic growth phase (about 40-60% confluent) by adding 0.5 (uCi/ml [3H]thymidine or 0.3-0.4 uCi/ml [125l]iododeoxyuridine for 16-24 h in medium.b 3 Wash radiolabelled tumour cell cultures tree of non-incorporated label about 4-6 h before use. 4 Incubate labelled cells in complete medium for 1 h at 37 °C to allow non-incorporated label to be released into the supernatant.1 5 For harvest of target cells, wash monolayers once with Ca2+~, Mg2+-free Dulbecco's modified PBS. 6 Add 1 ml of 0.05% trypsin in EDTA to the cultures for about 1-2 min at 37°C, 7 Observe cells under inverted microscope and, when monolayers show the first signs of disruption, add 10 ml of complete medium to the flask. 8 Pipette the cell suspension several times to separate clumps. 9 Wash the cell suspension twice in 50 ml of medium each time. 10 Resuspend cells at a final concentration of 5 x 104 cells/ml. C Cytotoxiclty assay 1

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Add 1 x 104 pre-labelled target cells in 0.2 ml of complete medium into each well of a 96-well plate containing 2 x 105 effector cells (E:T cell ratio of 20:1). Labelled tumour cells should also be added to wells without macrophages to determine the spontaneous release (release by tumour celts alone) and the total incorporated counts.d

ANALYSIS OF MACROPHAGE LYTIC FUNCTIONS

2

After 48-72 h in culture, harvest 0,1 ml of supernatant from each well with an automatic pipette.

3

When [3H]TdR is used, place supernatants in vials containing 5 ml of scintillation fluid and count released radioactivity in a beta counter; when [125I]dUrd is employed, assess directly the amount of radioactivity of each sample in a gamma counter.

4

Calculate lytic activity as described in Protocol 2,

a

Almost every adherent mouse flbrosarcoma or adenocarcinoma can also be used as tumour target cells in this assay. b Target cells should have no more than 10000 c.p.m. per 4 x 104 cells. [JH]TdR or [125I]dUrd incorporation above this level may be toxic. c This 'cold chase' decreases the label in cytoplasmic pools and, in tum, decreases spontaneous release of label during assay. d Spontaneous release is usually 10-20% of total incorporated radioactivity. Total incorporated counts are estimated by adding 20 ul/well of 10%SDS or 50 ul/well of 0.1 M NaOH to lyse cells. The average counts of three wells containing the target cells are used.

2.2.3

111

Indium release assay

111

Indium ( 1 1 1 ln) is an isotope widely used in nuclear medicine, recirculation, and lymphocyte-mediated cytolysis studies. It is a gamma emitter that labels most cell types efficiently with no decrease in cell viability, and localizes in the cytoplasm with up to 80% of the incorporated label quickly released upon target cell destruction. Spontaneous release is very slow (0.25-0.5% per hour) and this isotope can be used for both short- and long-term cytotoxicity assays (20-24). '"In release assay can be used to quantify macrophage-mediated cyLolysis of a variety of target cells, both adherent and non-adherent, that generally exhibit similar labelling and spontaneous release profiles and whose destruction well correlates with levels of "'In release. Because of its high labelling efficiency and low toxicity, as low as 2-10 c.p.m. can be used for cell labelling, allowing assays employing as few as 103 target cells. The 24 hour 111 In release assay can substitute 51 Cr in every application and has the advantage of a much lower spontaneous release that allows the extension of the cytolytic assay to 72 hours. The possibility to perform short- and long-term cytotoxicity assays using the same isotope offers a good system to examine the kinetics and mechanism of macrophage tumouricidal activity at different levels of activation. For these reasons, we routinely use the 111Inrelease assay for testing both human monocyte and murine macrophage tumouricidal activity (20, 21, 23) For use in cell labelling, 111In must be complexed to 8-hydroxyquinoline (oxine) to form the 111lIn-oxinechelate ( 111 InOx). This chelate is highly lipophilic and labels cells by rapid, passive diffusion. Even though the half-lifeof111InOx is only 2.8 days, each commercial preparation can be used over a two week period because of the high activity of the isotope. In order to standardize the labelling 137

MARIA CARLA BOSCO ET AL.

technique, a known amount of 111ln0x is employed to label target cells. This is done by calculating the concentration of the isotope at the time of labelling from a calibration table and then by adding the appropriate volume containing the desired number of microcoiries of the isotope. The cell lines of choice for this procedure are the P815 murine mastocytoma cell line for murine macrophageme dialed cytolysis, and the HT29 human colon carcinoma cell line for human monocyte-mediated killing. The recommended labelling time should be strictly observed because longer incubation can lower the viability of some cell lines. Moreover, only cell suspension of good viability should be labelled with "InOx because considerable amounts of the label, which is passively lipophilic. is incorporated by dead cells, therefore elevating baseline controls during the assay. Protocol 4 describes an efficient method for monitoring macrophage-mediated tumour cell lysis using 111indiuin release.

Measurement of macrophage cytotoxlclty using the Indium release assay

111

Equipment and reagents • Tissue culture plates, 96-well, U-bottom, polystyrene (Corning-Costar) • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other labware (see Protocol 1) • Centrifuge • Standard microscope • Haemocytometer • Gamma counter • Device for vacuum aspiration • Repeating dispenser • niln-oxyquinoline ( 111 InOx), specific activity 50 mCi/ug In (Amersham Life Science)

• Combitips (Eppendorf) • Complete medium (see Protocol 1) • Ca2+-, Mg2+-free Dulbecco's modified PBS (see Protocol 1) • 0.05% (w/v) trypsin, 0.02% EETTA solution (Hyctone Laboratories) • Macrophage effector cells, appropriately purified (see Chapters 1 and 2) • Tumour target cells: e.g. the P815 murine mastocytoma cell line and the HT29 human colon carcinoma cell line, maintained in complete medium by serial passages on 100 mm tissue culture dish or in 75 cm2 tissue culture flasks • Macrophage activators (see Protocol 1}

A Preparation of effector and target cells 1 Prepare monocytes/macrophages as described for the 51Cr release assay (see Protocol 2). 2 3

To prepare the target cells, trypsinize the adherent tumour cell monolayers as detailed in Protocol 3, partB, steps 5-8). Wash the cell suspension once with complete medium.

4 5

Resuspend the cells at a concentration of 5 x 106 cells/ml. Radiolabel the target cells by incubating 5 x 106 cells with 40 n-Ci of 1HInOx in a

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6 7

50 ml conical tube for 15 min at room temperature in 1 ml of complete medium with occasional shaking.3 Wash the cells three times with 50 ml of complete medium. Adjust the labelled target cells to 2.5 x 104 cells/ml.b

B Cytotoxlclty assay 1 2

3 4 5 6

Add 5 x 103 labelled target cells in 0.2 ml of complete medium to triplicate wells containing 2 x 105 macrophage effector cell to give a 40:1 E:T cell ratio. Incubate the cells in a humidified atmosphere containing 10% CO2 at 37 °C. The cytolytic activity of human monocytes is measured in a 48 h 111In release assay, whereas killing by murine macrophages in a 24 h assay. At the end of the incubation time, centrifuge the plates at 250-300 g for 5 min. Remove 0.1 ml of supernatant fluid from each well. Count the released radioactivity present in each sample in a gamma counter. Calculate levels of cytotoxic activity as described for the 51Cr release assay (Protocol 2).c

3

Because the half-life of mln0x is only 2.8 days, the appropriate volume containing the desired number of uCi of the isotope is evaluated by calculating the concentration of 111InOx at the time of labelling from a calibration table. b Most cells labelled with this technique incorporate 2-10 c.p.m. per cell, with no reduction in viability. c The 111In release test is reliable when the spontaneous release is less than 10% of the total incorporated radioactivity. 2.2.4 Special considerations In performing the isotope release assays described, several effector-to-target cell ratios should be employed to give a more accurate assessment of the lytic activity. However, it is important to consider that the killing by macrophage is density-dependent, so one should never go below a certain cell density (about 3000 cells/mm2). Performing the assay in round-bottom wells permits a closer interaction between the cells than would be possible with the same number of tells in flat-bottom wells. However, increasing the number of cells per well can overcome this problem. When studying the cytolysis of adherent target cells, it is imperative to use a solubilizing agent, such as 1% SDS, for calculation of the total incorporated radioactivity. It is of extreme importance to avoid any kind of endotoxin contamination when performing the tests, and to use reagents endotoxin-free and monocyte/ macrophage preparations 95-96% pure. We wash the effector cells extensively after the activation period to eliminate macrophage activating agents during the cytotoxicity phase of the test. Some laboratories shorten the time of the assay by mixing effector and target cells in the presence of the activating stimuli (25). 139

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2,3 Macrophage-mediated cytostasis of tumour cells: incorporation of radioactive labels The cytostatic effects of activated macrophages on tumour cells can be evaluated by a post-labelling technique using [3H]thymidine as the radiolabel (17, 26-28). 1'or this assay, in vivo- or in vitro-activated macrophages are mixed with suspensions of tumour target cells, usually leukaemia or tymphoma cell lines (for example, the K562 human erythroleukaemia and the WEHI-3B murine myelomonocytic leukaemia), at a synchronized phase of the growth cycle (usually 18 hours after subculture of the target cells). Tritiated thymidine is then added after an additional incubation period, and thymidine incorporation by target cells is taken as a measure of cellular proliferation, Protocol 5 describes a method for the assessment of macro ph age-media ted tumour cytostasis.

Assessment of macrophage-medlated cytostasis of tumour cells based on target cell incorporation of radioactive label Equipment and reagents • MICROTEST tissue culture plates, 96-well. flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Labware) • Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other Labware (see Protocol 1) • Equipment as in Protocol 2 • Automated harvesting device • Glassfibrefilters(Whatman) • Beta counter • [Methyl-3H]thymidme ((3H]TdR), specific activity 2 Ci/mmol (Amersham Life Science)

• OptiPhase 'HiSafe' three liquid scintillation cocktail (Wallac, EG&G Comp.) • Complete medium (see Protocol]) • Ca 2 - -, Mg2+-free Dulbecco's modified PBS (see Protocol!) • Macrophage effector cells, appropriately purified (see Chapters 1 and 2) • Appropriate tumour target cells: e.g. the K562 human erythroleukemia cells, the L929 murine transformed cell line, or the VEHI-3B murine myelomonocytic leukaemia • Macrophage activators (see Protocol 1)

Method 1

Prepare monocyte/macrophage effector cells at 2 x 106/ml, as described in Protocol 2.

2

Prepare appropriate unlabelled tumour target cells at various concentrations in a total volume of 0,2 ml of complete medium.

3

Combine effector (E) and target (T) cells by adding various concentrations of target cells in 0,2 ml of medium to triplicate wells of a flat-bottomed, 96-well microtitre plate, containing washed monocyte/macrophage monolayers (E:T cell ratio between 50:1 and 2:1). Tumour cells should also be added to wells not containing macrophages.

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4 5

6 7 8 9

Incubate the plate at 37°C in a humidified CO2 incubator for 24-48 h. Add 0.5 uCi of [3H]TdR to each well for the final 2-4 h of culture. Wells containing E + T cells and control wells containing target cells alone should be pulsed with [3H]TdR. Harvest the cells onto glass fibre filters, using a semi-automated harvesting device. Dry the filters and place them in vials containing 5 ml of scintillation fluid. Determine radioactivity in harvested cells using a liquid scintillation beta counter.a Use the amount of isotope incorporated by tumour cells alone as a baseline for calculating the degree of growth inhibition by macrophages.

a

Alternatively, the contents of the wells can be combined with 2 ml of 10% TCA, precipitated proteins filtered onto Whatman GF/C filters and washed with 10% TCA, followed by ethanol. Filters are then dried, mixed with 3.5 ml of scintillation fluid, and counted.

3 Target cell sensitivity The basic techniques presented for the in vitro analysis of mononocyte/macrophage tumouricidal functions, although described for a particular experimental situation, can be easily modified to several experimental protocols. However, it is extremely important to keep in mind that the different tumour cell lines that could be used as targets in these procedures have differing susceptibilities to killing by macrophages. There are reports showing that tumouricidal macrophages can distinguish between normal and neoplastic cells and lyse neoplastic cells even under co-cultivation conditions (1). To date, it has been shown that several cytotoxicity mechanisms contribute to macrophage killing of tumour target cells, which are essentially the same as the mechanisms of macrophage killing of infectious organisms. In general, macrophage-mediated tumour cell lysis can occur by both a direct and an indirect process. The direct process involves the direct interaction of effector and target cells, whereas the indirect mechanism can involve the release of soluble cytotoxic molecules, such as lysosomal enzymes, reactive oxygen species (ROS), nitric oxide, and cytotoxic cytokines (TNFa and 1L-1) (1). In addition, macrophages can also recognize targets that are coated with antibodies and attack them through antibody-dependent cellular cytotoxicity (ADCC). The role of the different cytotoxic mechanisms in macrophage anti-tumour activity depends on different factors, such as the sensitivity of target cells to the different kinds of damage, the activation state of monocytes/macrophages, and the presence of antibodies that elicit an ADCC reaction in the system. Therefore, depending on the assay system used, one might detect preferentially the action of one or another of these mechanisms. This explains why macrophage-mediated cytotoxicity in one system can be quite different from that observed in another system. One major mediator of macrophage killing of tumours is TNFa, a cytokine 141

MARIA CARLA BOSCO ET AL.

that has been shown to exert direct toxic effects against neoplastic but not normal cells and to cause either lysis or growth inhibition of both autologous and heterologous tumour cells. The mechanism of TNFa cytotoxicity is at least partially mediated through generation of ROS. The susceptibility of a cell to killing by TNFa may be influenced by its content of antioxidant enzymes that specifically detoxify ROS to prevent cellular damage, such as catalase, superoxide dismutases (SOD), and glutathione peroxidase. SOD (CuZnSOD and MnSOD) are metalloproteins that participate in the inactivation of free oxygen radicals reducing superoxide to H2O2 and protecting cells from oxidative damage, whereas catalase and glutathione peroxidase scavenge H2O2. Several studies have shown that exposure to TNFa induces cells to synthesize MnSOD, which is essential for cellular resistance to TNFa toxicity. However, a wide spectrum of human and murine tumour cells fail to make the enzyme in response to TNFa, therefore becoming susceptible to cytolysis by TNFa, although not all transformed cells are affected by TNFa, and those that are susceptible are not sensitive to the same degree (29). Cellular susceptibility is further enhanced by pre-treatment with inhibitors of RNA and protein synthesis, such as actinomycin D and cycloheximide, and the killing is achieved in a shorter period of time. The cytotoxicity systems based on a short-term assay, such as 51Cr release assay, are usually TNF-dependent and exploit target cell susceptibility to killing by TNFa secreted by activated monocytes/macrophages. The tumour cell lines most commonly utilized in this assay are the WEHI 164 murine fibrosarcoma and the MOPC-315 murine plasmacytoma lines. Because of the high sensitivity of these cell lines to TNFa and the rapidity of this test, this procedure is also currently utilized as a biological assay for measuring TNFa production. The human A375 melanoma cell line has been used in [125I]iododeoxyuridine and [3H]thymidine release assay. The WEHI-3B murine myelomonocytic leukaemia and the L929 murine transformed fibroblast lines are sensitive to the cytostatic effects of TNFa, and are more commonly used in post-labelling technique with [3H]thymidine. Many TNFa-resistant tumour cell lines, on the other hand, such as the human A549 lung, 293 kidney, ME-180 cervical, and HT29 colon carcinoma cell lines, the P815 murine mastocytoma, and the murine mKSA TU5 tumour cells are lysed by monocytes/macrophages in long-term (> 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

ANALYSIS OF MACROPHAGE LYTIC FUNCTIONS

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

146

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.

ANALYSIS OF MACROPHAGE LYTIC FUNCTIONS

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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

• 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

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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)

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

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

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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

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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.

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

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

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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

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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
4.2 Sources and characteristics of binding factors The processes controlling gene transcription have been defined in substantial detail and generally involve the activity of a broad collection of proteins, many of which function through direct interaction with DNA (11). There are many DNA binding factors which are constitutively active and are involved either in the regulation of constitutively transcribed genes or are part of general transcription machinery of the cell. With regard to inducible gene expression, however, many transcription factors are inactive in unstimulated cells and are the substrates of signalling pathways initiated by exposure of cells to exogenous stimuli. Active DNA binding factors are almost always localized in the nucleus. Inactive forms are often maintained in the cytosol and undergo nuclear translocation as part of the activation process. There are many distinct mechanisms involved in regulation of DNA binding activities. Though not a property of all transcription factors, many function in the form of dimers, being composed of either two homologous proteins or two distinct proteins. The dimerization principle provides opportunity for combinatorial complexity both within one transcription factor family as well as between structurally distinct gene families. As understanding of transcriptional mechanisms increases in complexity, many additional layers of protein-protein interaction are likely to emerge which will provide additional specificity to the overall process. Thus protein-nucleic acid interactions provide nucleotide sequence recognition specificity as well as specificity for interaction with other protein components of larger functional complexes. 194

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As is the case with transcriptional control, post-transcriptional control mechanisms involving protein-RNA interactions are impressively broad in functional scope. Perhaps the best studied examples involve the complex processes of translational initiation, elongation, and termination. Less detailed information is available regarding the nature of primary transcript processing, nuclearcytosolic transport, and mRNA decay. Unlike most DNA binding factors, RNA binding proteins can be found in both nuclear and cytosolic locations and often exhibit shuttling characteristics based upon the physiologic status of the cell.

4.3 Measurements of DNA and RNA binding proteins As with measurements of nucleic acids, assessment of DNA and RNA binding protein activities in macrophages also can be influenced by several potential problems. For example, the analysis of proteins in mononuclear phagocytes can be more difficult due to their frequent content of protease activities. Because proteins exhibit highly variable sensitivity to different proteases, the degree to . which this constitutes a problem will depend upon the specific factors being measured. Nevertheless, it is wise to assume that DNA binding activities which are measured in macrophages might be only a fraction of the product found in the intact cell. Ribonuclease activities present in the cytosolic fractions also may hinder the ability to measure RNA binding activities present in such preparations though in practice this appears to be less of a problem than might be anticipated. Finally, DNA binding proteins generally are extracted with high salt from isolated nuclei. The process of cell harvest and lysis provides the opportunity for relatively rapid separation of cytosol and nuclear fractions. However, the use of high salt extraction may create additional problems in assays of proteinnucleic acid binding activity, as the relative sensitivity of protein-nucleic acid interactions to salt concentration can lead to profound changes in complex formation or binding characteristics in response to even small changes in salt. Thus it is useful to vary the experimental conditions of extraction to ensure that the optimal interactions are being assessed. In spite of these potential problems, however, protein-nucleic acid interactions can be successfully monitored in macrophages. Protein binding to both DNA and RNA sites is generally measured using one of several electrophoretic mobility assays. The most common measurement is the electrophoretic mobility shift assay, or EMSA, in which the mobility of a radiolabelled oligonucleotide is shifted based upon interaction with one or more protein factors. The nature of the oligonucleotides which are used as probes in EMSA are particularly critical to the experimental outcome and its ultimate interpretation. While EMSA can be performed using large pieces of DNA or RNA (on the order of hundreds or even thousands of nucleotide residues), the complexity of the emerging result is often very high and frequently uninterpretable. This may be due principally to the potential for formation of many different complexes, each potentially behaving independently of the others and creating remarkable heterogeneity in the number and size of individual complexes. Thus it is often desirable to identify potential 195

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motifs through functional or sequence analysis first and then to prepare smaller oligonucleotides to search for specific binding activities. Optimal sizes for experimental analysis will vary but a general rule of thumb would be to limit oligonudeotide length to 50 residues or less when possible. Complexes detected by EMSA may contain not only the proteins which directly contact and bind nucleic acid but also other factors which participate in the complex. Such proteins may be functionally important. Additional methodologies provide the ability to make more precise identification of proteins involved in direct contact with nucleic acid. A frequently employed strategy involves covalent cross-linking of protein with its bound radio lab el led oligonudeotide by UV irradiation. The products then can be resolved by denaturing polyacrylamide gel electrophoresis, either with or without nuclease digestion, providing high resolution of the proteins and estimates of the protein's molecular size. A separate approach to obtain similar information involves analysis of clectrophoretically separated protein samples transferred to nylon membranes following reactions with radiolabelled oligonudeotide. This experimental strategy, termed either Northwestern (RNA) or Southwestern (DNA) T allows direct determination of" nucleic acid binding activity on separated proteins providing measure of both size and sequence specificity. Though the proteins are generally separated under denaturing conditions, a significant number of activities are recovered following transfer to the membrane. Protocols 8-10 outline a basic scheme for the identi-

Preparation of nuclear extracts from macrophages Equipment and reagents • • • • • •

Sterile 60 mm tissue culture dishes Table-top centrifuge Microcentriruge 1 ml syringes 25-gauge needles Macrophage population of choice (see Chapters 1 and 2} • NE buffer: 10 mM Tris pH 7.4,10 mM NaCl, 6 mM MgCl2 • DTT • Phenylmethyl sulfonyl fluoride (PMSF, Sigma Chemical Co.) • Aprotinin (Sigma Chemical Co.) • Leupeptin (Sigma Chemical Co.)

Pepstatin (Sigma Chemical Co.) PBS (see Protocol 5} Sodium orthovanadate (Na3VO4): prepare a small amount of a 0.1-0.5 M stock solution in water immediately prior to use Buffer C; 20% (v/v) glycerol, 20 mM Hepes pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA Freezing medium: 50 mM Tris pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA Trypan blue (0.4% solution) for vital dye staining (Life Technologies) Serum-free RPMI1640 tissue culture medium

Method 1 Plate an appropriate number of the macrophages in 60 mm tissue culture dishes to create a confluent monolayer of adherent cells at the initiation of the experiment.a

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2 Serum starve the cell cultures for 24-72 h at 37 °C once they reach confluency.b 3 Stimulate the serum-starved macrophages as required for the experiment underway. 4 Remove medium from the monolayers and spin in a centrifuge for 5-10 min, 4°C, 250-500 g to collect any cells growing in suspension. 5 Add 4 ml ice-cold 1 x PBS to the remaining monolayers and harvest the adherent macrophages by an appropriate method (see Chapters 1 and 2). 6 Resuspend the pelleted suspension cells using the same 4 ml wash fluid and collect all the cells by centrifugation as in step 4. 7 Resuspend the cell pellets in 2-5 ml of NE buffer. 8 Incubate the cells on ice for 5 min. 9 Collect the cells by centrifugation as in step 4. 10 Resuspend the cell pellets in 800 ul of NE buffer supplemented to 1 mM with DTT, 0.4 mM with PMSF, and 100 uM with Na3VO4. 11 Lyse the resuspended macrophages by passing the cells repeatedly through a 25gauge needle attached to a 1 ml syringe. Continue to homogenize the cells until the nuclei are released from the cytoplasm of lysed cells, as monitored by staining with trypan blue.c 12 Collect the released nuclei by spinning at 12 000-15 000 r.p.m. at 4°C for 10 sec in a microcentrifuge. 13 Remove and discard the supernatant fluid. 14 Centrifuge the nuclei once more for 2 min under the same conditions as in step 12, using 1 ml of NE buffer, 15 Remove the supernatant fluid and resuspend the nuclear pellet rapidly in three pellet volumes of buffer C supplemented to 3,6 ug/ml with aprotinin, 1,2 ug/ml with pepstatin, and 1.2 ug/ml with leupeptin, and with DTT. PMSF, and Na3V04 as in step 10d 16 Incubate the suspension on ice for 20-30 min. 17 Centrifuge the mixture for 3 min as in step 12 and recover the supernatant fluid. 18 Determine the protein concentration of the extracts. 19 Store the supernatant fluid in 25-50 ul aliquots at -70°C.e a Expand cell cultures (for macrophage cell lines) or purify sufficient primary macrophages to ensure that the cell number at the start of the extraction will be at least 5 x 107 total cells. b The period of serum starvation should be sufficient to render the macrophage population quiescent, but should not have a deleterious effect on cellular morphology or viability. c Nuclei should be as close to 100% free of any cellular debris as possible, as visualized by trypan blue staining. d Alternatively, the nuclear pellet can be resuspended in 100 ul of freezing buffer at this point and stored in liquid nitrogen for use in nuclear run-on experiments (see Protocol 2). e Nuclear extracts are stable for four to six weeks under these conditions.

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fication of nuclear DNA binding factors. Protocols 8 and 9 first detail methods for preparation of the two critical components of EMSA analysis, namely the nuclear extracts and the radiolabelled oligonucleotide probes. Protocol 70 then describes the use of these reagents for HMSA studies. Information concerning additional methodologies for identification of DNA binding proteins is not provided here, but can be obtained from more extensive methodology texts (12).

Preparation of oligonucleotide probes for EMSA Equipment and reagents • Heat-resistant float for holding microcentriruge tubes • 1.5 ml microcentrifuge tubes • Commercial Sephadex G50 Nick column for oligonucleotide purification (Pharmacia) • Horizontal electrophoresis apparatus appropriate for agarose gels • Microcentrifuge • Liquid scintillation counter • Commercially prepared synthetic oligonucleotide primers specific for the upper and lower strands of the DNA region of interest (Life Technologies or other suitable manufacturer)

Liquid scintillation cocktail and glass scintillation vials of choice Sterile water 10 x DNA loading dye (see Protocol 3} Eiectrophoresis grade agarose TAE buffer [see Protocol 3) T4 polynucleotide kinase (PNK), 10 U/ml (New England Biolabs) 10 x PNK labelling buffer: 700 mM Tris pH 7.5-7.8. 100 mM MgCl2, 50 mM DTT (store at-20°C) TE buffer (see Protocol 3} 0.5 M EDTA •y-32P-labelled ATP (3000 Ci/mM, DupontNEN)

A Probe annealing 1 Combine the upper and lower primers of a specific DNA sequence in a microcentrifuge tube at a concentration of 2-20 ug/ul, using sterile water to dilute the DNA as needed.a 2 Bring 1.5-2 litres of water to a rolling boil in a large beaker over a Bunsen burner. 3 Place the tube containing the DNA in the float and place into the boiling water. 4 Incubate the DNA in the boiling water for 30 sec to 1 min. 5 Turn the flame on the burner off and leave the tube containing the DNA in the water overnight (or until the water reaches room temperature) to anneal the primers. 6 Spin tubes briefly to recover all of the volume. B Probe labelling N.B, Observe appropriate radiation safety protocols during this procedure. 1 In a microcentrifuge tube, combine 100-500 ng of the double-stranded oligonucleotide (prepared as in part A) with 2 ul of 10 x PNK buffer, 5 ul of 10 Ci/ml [32P]ATP, 1 ul PNK. and sufficient H2O to yield 20 ul final volume.

198

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

2

Incubate this mixture for l h at 37°C.

3 4

Add 3 ul EDTA and 77 ul TE buffer. Purify the labelled probe by passage through the oligonucleotide DNA purification column, according to the manufacturer's instructions, using 400 jo-1 of TE buffer to elute the DNA. Count the radioactivity in 1 ul of the labelled probe, using a glass scintillation vial.b Use probe immediately for EMSA analysis (see Protocol 10) or store at -20°C for up to two weeks,

5 6 a

The probe volume to be used will be determined by the nM concentration of each of the upper and lower strand oligonucleotides. The final DNA concentration of the complementary primers should be equal at 2-5 ug/ul, b Ideal probe activity for EMSA is generally between 2 x 107 and 1 x 109c.p.m./(u.g.

Electrophoretlc mobility shift assay (EMSA) Equipment and reagents • Glass plates appropriate for pouring 12-16 cm. length polyacrylamide gels using a 1.5 mm spacer • 1.5 mm thickness gel combs containing 12-15 wells • Micropipette tips for loading gel samples • Apparatus for electrophoresis of polyacrylamide gels, including gel boxes with cooling system and appropriate electrophoresis power supply (Bio-Rad) • 3MM filter paper (Whatman) • Get dryer (Bio-Rad) • Film cassettes with intensifying screens (Fisher Biotechnology) • Radiolabelled oligonucleotide probe (see Protocol 9) • Nuclear extracts prepared from appropriately stimulated macrophages (see Protocol 8) • EMSA binding buffer: 40 mM KC1,1 mM MgCl2, 0,1 mM EGTA, 20 mM Hepes pH 7.9. and 4% (w/v) Ficoll (prepare a 5 x stock, filter to sterilize, and store at room temperature for up to one year)

Autoradiographic film (Kodak) 1 nig/ml poly dI:dC stock solution (Pharmacia) prepared in water and stored at -20°C

EMSA mix: for each probe used, combine 4 u1 of 5 x EMSA binding buffer, 3 ul poly dI:dC stock solution, 4 ul of 2 mg/ml BSA, 0.2 ul of 100 mM DTT, and approx. 1-2 ng (usually 1-2 p.1} of radiolabelled probe, with an appropriate amount of water to yield 20 ul of mix per reaction (prepare enough for two extra reactions) 30% acrylamide. bisacrylamide mixture (29:1, Bio-Rad) 1.5 M Tris pH 8.8 10% (w/v) electrophoresis grade SDS 10% ammonium persulfate (APS, Bio-Rad): make this fresh weekly, store at -20°C TEMED (Bio-Rad) 10 x TBE electrophoresis buffer: 108 g Tris base (89 nM). 55 g boric acid (89 nM), 40 ml of 0.5 M EDTApH 8.0 (2 mM); adjust volume to 1 litre with H2O 10 X DNA loading dye (see Protocol 3)

199

JOYCE E. S. DOAN ET AL.

Method 1 Pour a 4-6% non-denaturing acrylamide gel, using appropriate volumes of the 30% acrylamide mix, 10% SDS, 1.5 M Tris pH 8.8, APS, and TEMED and allow it to polymerize.a 2 Label one 0.5 ml microcentrifuge tube for each EMSA reaction to be performed and put the empty tubes on ice. 3 Assemble sufficient EMSA mix, including the radiolabelled probe, for each reaction and hold on ice. 4 Add the nuclear extracts (5-6 ug/tube) and either water or cold competitor probe (at 50-500 x the concentration of the labelled probe) in a total volume of 4 ul to the appropriate empty microcentrifuge tubes. 5 Add 15 ul of the EMSA mix to each tube. 6 Incubate on ice for 60 min.b 7 During the incubation, prepare the gel for electrophoresis by removing the comb and rinsing the wells with deionized water to remove any unpolymerized acrylamide. 8 Place the gel in the electrophoresis apparatus and pre-run it for 15 min at approx. 15 mA (100-200 V) in 0.25 X TBE. 9 Dilute the 10 X DNA loading dye to 1 x concentration with TEE buffer. 10 Add 1 ul of 1 x loading dye per sample, 11 Halt the electrophoresis and load all samples. 12 Restart electrophoresis and run the gel at 100 V for approx. 20 min, with cooling. 13 Increase the voltage to 175-200 V, and continue to run the gel until the fastest migrating band (xylene cyanol) is approx. 5 cm from the bottom of the gel (2-4 h). 14 Disassemble the electrophoresis apparatus and transfer the gel to a correctly sized piece of Whatman filter paper.c 15 Cover the gel with saran wrap and dry on the gel dryer, according to manufacturer's instructions. 16 Expose the dried gel to radiographic film using an intensifying screen and process for autoradiography.d a Recipes for the preparation of polyacrylamide gels can be found in ref. 13. The gel can be poured up to 24 h before electrophoresis. For overnight storage, it should be wrapped tightly in plastic wrap, with comb in place, and stored at 4°C. N.B. Acrylamide is a potent neurotoxin that can be absorbed through the skin. Observe correct safety precautions and wear gloves when preparing gels, h At this point, antibody specific for a DNA binding protein of interest can be added to samples to confirm the identity of the factor under investigation. If the antibody Jigand is present, the antibody will bind to the protein and alter its electrophoretic mobility, causing a so-called 'supershift' of the detected protein. For 'supershift' analysis, add 1-2 y.g of specific antibody to EMSA samples after 10 min of incubation, add an equivalent amount of water to ail other tubes as a control, and continue the incubation for the total of 60 min.

200

ANALYSIS OF GENE EXPRESSION IN MONONUCLEAR PHAGOCYTES

c

The buffer remaining in the electrophoresis apparatus will contain some free probe and should be disposed of as radioactive waste.

d

The length of exposure required will vary for each nuclear extract-probe interaction and according to the specific activity of the probe in any given experiment. Typical exposure times range from 12 h to three days at -70°C,

References 1. Warren, M K, and Vogel, S. N. (1985).J. Immunol., 134, 982. 2. Rutherford, M. S., Witsell, A., and Schook, L. B. (1993). J. Leufc. Biol. 53, 602. 3. Glisin, V., Crkvenjakov, R., and Byus, C. (1973). Biochemistry, 13, 2633. 4. Greenberg, M. E. and Ziff, E. B. (1984). Nature, 11, 433. 5. Wynn. T. A., Nicolct, C. N., and Pnulnock, D, M. (1991). J. Jmrnunol.. 147. 4384, 6. Ohmori, Y. and Hamilton, T. A. (1993), J, Binol. Chem., 268, 6677. 7. Michael. J. P., Luk, K.-C., Williams, B., and Lifson.J. D, (1993). Bio Techniques, 14, 70. 8. Lokuta. M. A., Maher. J., Noe, K, H,, Pitha. P. M., Shin, M. L, and Shin, H. S. (1996). J. Biol. Chem.,271, 13731. 9. Nicolet, C. M. and Paulnock, D. M. (1994). J. Immunol., 152, 153. 10 Neumann,]. R., Morency, C. A., and Russian, K. O. (1987). Bio Techniques, 5, 444. 11. Faisst, S. and Meyer, S. (1992). Nucleic Acids Res., 20, 3, 12. Ausebel, F. M., Brent, R,, Kingston, R, B., Moore, D, D., Seidman, J. G., Smith, J. A., and Struhl, K. (ed.) (1990), Current protocnk in mdecular biology. John Wiley and Sons, NY. 13. Sambrook, J., Fritch, E. F., and Maniatis, T. (ed.) (1989). In Muleculur duning: a laboratory manual (2nd edn), Chapter 18, p. 18.47. Cold Spring Harbor Laboratory Press. NY.

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List of suppliers

Accurate Chemical, 300 Shames Drive, Westbury, NY 11590, USA. Affinity Bioreagents Inc., Cambridge Research Biochemicals, Gadbrook Park, Northwich, Cheshire CW9 7RA, UK. Affinity Bioreagents Inc., 14818 West 6th Avenue, Suite 13A, Golden, CO 80401, USA. American Diagnostics Inc., PO Box 1165, Greenwich, CT 06836-1165, USA.

Anderman and Co. Ltd., 145 London Road, Kingston-upon-Thames, Surrey KT2 6NH, UK. Tel: 0181 541 0035 Fax: 0181 541 0623 Bayer Diagnostics, Strawberry Hill, Newbury, Berkshire, UK.

Beckman Coulter (UK) Ltd., Oakley Court, Kingsmead Business Park, London Road, High Wycombe, Buckinghamshire HP11 1JU, UK. Tel: 01494 441181 Fax: 01494 447558 URL: http://www.beckman.com Beckman Coulter Inc., 4300 N Harbor Boulevard, PO Box 3100, Fullerton, CA 92834-3100, USA. Tel: 001 714 871 4848 Fax: 001 714 773 8283 URL: http://www.beckman.com

Becton Dickinson and Co., 21 Between Towns Road, Cowley, Oxford 0X4 3LY, UK. Tel: 01865 748844 Fax: 01865 781627 URL: http://www.bd.com Becton Dickinson and Co., 1 Becton Drive, Franklin Lakes, NJ 07417-1883, USA. Tel: 001 201 847 6800 URL: http://www.bd.com Bio 101 Inc., c/o Anachem Ltd., Anachem House, 20 Charles Street, Luton, Bedfordshire LU2 OEB, UK. Tel: 01582 456666 Fax: 01582 391768 URL: http://www.anachem.co.uk Bio 101 Inc., PO Box 2284, La Jolla, CA 92038-2284, USA. Tel: 001 760 598 7299 Fax: 001 760 598 0116 URL: http://www.biol01.com

Bio-Rad Laboratories Ltd., Bio-Rad House, Maylands Avenue, Hemel Hempstead, Hertfordshire HP2 7TD, UK. Tel: 0181 328 2000 Fax: 0181 328 2550 URL: http://www.bio-rad.com Bio-Rad Laboratories Ltd., Division Headquarters, 1000 Alfred Noble Drive, Hercules, CA 94547, USA. Tel: 001 510 724 7000 Fax: 001 510 741 5817 URL: http://www.bio-rad.com Blospec Products, Inc., PO Box 788, Barthesville, OK 74005-0788, USA.

203

LIST OF SUPPLIERS

Calbiochem-Novabiochem Corp., 10394 Pacific Center Court, San Diego, CA 92121, USA. CP Instrument Co. Ltd., PO Box 22, Bishop Stortford, Hertfordshire CM23 3DX, UK. Tel: 01279 757711 Fax: 01279 755785 URL: http://www.cpinstrument.co.uk Dako Ltd., Denmark House, Angel Drove, Ely, Cambridge CB7 4ET, UK. Dako Ltd., 6392 Via Real, Carpintera, CA 93013, USA. Developmental Studies Hybridoma Bank,

Department of Biological Sciences, The University of Iowa, 436 Biology Building, Iowa City, IA 52242, USA. Dlamedix Corp., 2140 North Miami Avenue, Miami, FL 33127, USA.

Fisher Scientific UK Ltd., Bishop Meadow Road, Loughborough, Leicestershire LEU 5RG, UK. Tel: 01509 231166 Fax: 01509 231893 URL: http://www.fisher.co.uk Fisher Scientific, Fisher Research, 2761 Walnut Avenue, Tustin, CA 92780, USA. Tel: 001 714 669 4600 Fax: 001 714 669 1613 URL: http://www.fishersci.com Fisher Scientific , 3970 Johns Creek Court, Suite 500, Suwanee, GA 30024, USA. Fluka, PO Box 2060, Milwaukee, WI 53201, USA. Tel: 001 414 273 5013 Fax: 001 414 2734979 URL: http://www.sigma-aldrich.com Fluka Chemical Co. Ltd., PO Box 260, CH9471, Buchs, Switzerland. Tel: 0041 81 745 2828 Fax: 0041 81 756 5449 URL: http://www.sigma-aldrich.com

Dupont (UK) Ltd., Industrial Products Division, Wedgwood Way, Stevenage, Hertfordshire SGI 4QN, UK. Tel: 01438 734000 Fax: 01438 734382 URL: http://www.dupont.com Dupont Co. (Biotechnology Systems Division), PO Box 80024, Wilmington, DE 19880-002, USA. Tel: 001 302 774 1000 Fax: 001 302 774 7321 URL: http://www.dupont.com

Hybaid Ltd., Action Court, Ashford Road, Ashford, Middlesex TW15 1XB, UK. Tel: 01784 425000 Fax: 01784 248085 URL: http://www.hybaid.com Hybaid US, 8 East Forge Parkway, Franklin, MA 02038, USA. Tel: 001 508 541 6918 Fax: 001 508 541 3041 URL: http://www.hybaid.com

Eastman Chemical Co., 100 North Eastman Road, PO Box 511, Kingsport, TN 376625075, USA. Tel: 001 423 229 2000 URL: http://www.eastman.com

HyClone Laboratories, 1725 South HyClone Road, Logan, UT 84321, USA. Tel: 001 435 753 4584 Fax: 001 435 753 4589 URL: http://www.hyclone.com

E-Y Laboratories, Bradsure Biologicals, 15 Church Street, Market Harborough, Leicestershire LEI 6 7AA, UK. E-Y Laboratories, 107 North Amphlett Boulevard, San Mateo, CA 94401, USA.

Invitrogen Corp., 1600 Faraday Avenue, Carlsbad, CA 92008, USA. Tel: 001 760 603 7200 Fax: 001 760 603 7201 URL: http://www.invitrogen.com

204

Genzyme Corp., One Kendall Square, Cambridge, MA 02139, USA.

LIST OF SUPPLIERS

Invitrogen BV, PO Box 2312, 9704 CH Groningen, The Netherlands. Tel: 00800 5345 5345 Fax: 00800 7890 7890 URL: http://www.invitrogen.com Labsystems, Life Sciences International Ltd., Unit 5, The Ringway Centre, Edison Road, Basingstoke, Hampshire RG21 6YH, UK. Life Technologies Ltd., PO Box 35, Free Fountain Drive, Incsinnan Business Park, Paisley PA4 9RF, UK. Tel: 0800 269210 Fax: 0800 838380 URL: http://www.lifetech.com Life Technologies Inc., 9800 Medical Center Drive, Rockville, MD 20850, USA. Tel: 001 301 610 8000 URL: http://www.lifetech.com Merck Sharp & Dohme, Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, UK. URL: http://www.msd-nrc.co.uk MSD Sharp and Dohme GmbH, Lindenplatz 1, D-85540, Haar, Germany. URL: http://www.msd-deutschland.com Millipore (UK) Ltd., The Boulevard, Blackmoor Lane, Watford, Hertfordshire WD1 8YW, UK. Tel: 01923 816375 Fax: 01923 818297 URL: http://www.millipore.com/local/UK.htm Millipore Corp., 80 Ashby Road, Bedford, MA 01730, USA. Tel: 001 800 645 5476 Fax: 001 800 645 5439 URL: http://www.millipore.com Molecular Probes, Cambridge BioScience, 25 Signet Court, Stourbridge Common Business Centre, Swarm's Road, Cambridge CBS 8LA, UK. Molecular Probes, 4849 Pitchford Avenue, Eugene, OR 97402-9165, USA.

New England Blolabs, 32 Tozer Road, Beverley, MA 01915-5510, USA. Tel: 001 978 927 5054 Nikon Inc., 1300 Walt Whitman Road, Melville, NY 11747-3064, USA. Tel: 001 516 547 4200 Fax: 001 516 547 0299 URL: http://www.nikonusa.com Nikon Corp., Fuji Building, 2-3, 3-chome, Marunouchi, Chiyoda-ku, Tokyo 100, Japan. Tel: 00813 3214 5311 Fax: 00813 3201 5856 URL: http://www.nikon.co.jp/main/ index_e.htm Nycomed Amersham pic, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK. Tel: 01494 544000 Fax: 01494 542266 URL: http://www.amersham.co.uk Nycomed Amersham, 101 Carnegie Center, Princeton, NJ 08540, USA. Tel: 001 609 514 6000 URL: http://www.amersham.co.uk Park Scientific, 24 Low Farm Place, Moulton Park, Northampton NM3 1HY, UK. Per Immune Inc., 1330 Piccard Drive, Rockville, Maryland 20850-4396, USA. Perkln Elmer Ltd., Post Office Lane, Beaconsfield, Buckinghamshire HP9 1QA, UK. Tel: 01494 676161 URL: http://www.perkin-elmer.com PGC Scientific, PO Box 7277, Gaithersburg, MD 20898-7277, USA. Pharmacia Biotech (Blochrom) Ltd., Unit 22,

Cambridge Science Park, Milton Road, Cambridge CB4 OFJ, UK. Tel: 01223 423723 Fax: 01223 420164 URL: http://www.biochrom.co.uk

205

LIST OF SUPPLIERS

Pharmacia and Upjohn Ltd., Davy Avenue, Knowlhill, Milton Keynes, Buckinghamshire MK5 8PH, UK. Tel: 01908 661101 Fax: 01908 690091 URL: http://www.eu.pnu.com PharMingen, Becton Dickinson, Between Towns Road, Cowley, Oxford OX4 3LY, UK. PharMingen, 10975 Torreyana Road, San Diego, CA 92121, USA. Pierce, 44 Upper Northgate Street, Chester CHI 4EF, UK. Pierce, 3747 North Meridian Road, PO Box 117, Rockford, IL 61105, USA. Popper and Sons, Inc., 300 Denton Avenue, New Hyde Park, NY 11040, USA. Promega UK Ltd., Delta House, Chilworth Research Centre, Southampton SO16 7NS, UK. Tel: 0800 378994 Fax: 0800 181037 URL: http://www.promega.com Promega Corp., 2800 Woods Hollow Road, Madison, WI 53711-5399, USA. Tel: 001 608 274 4330 Fax: 001 608 277 2516 URL: http://www.promega.com Qiagen UK Ltd., Boundary Court, Gatwick Road, Crawley, West Sussex RH10 2AX, UK. Tel: 01293 422911 Fax: 01293 422922 URL: http://www.qiagen.com Qiagen Inc., 28159 Avenue Stanford, Valencia, CA 91355, USA. Tel: 001 800 426 8157 Fax: 001 800 718 2056 URL: http://www.qiagen.com Raymond A. Lamb, 6 Sunbeam Road, London NW10 6JL, UK. Roche Diagnostics Ltd., Bell Lane, Lewes, East Sussex BN7 1LG, UK. Tel: 01273 484644 Fax: 01273 480266 URL: http://www.roche.com

206

Roche Diagnostics Corp., 9115 Hague Road, PO Box 50457, Indianapolis, IN 46256, USA. Tel: 001 317 845 2358 Fax: 001 317 576 2126 URL: http://www.roche.com Roche Diagnostics GmbH, Sandhoferstrasse 116, 68305 Mannheim, Germany. Tel: 0049 621 759 4747 Fax: 0049 621 759 4002 URL: http://www.roche.com Santa Cruz Biotechnology, Autogen Bioclear UK Ltd., Holly Ditch Farm, Mile Elm, Calne, Wiltshire SN11 OPY, UK. Santa Cruz Biotechnology, 2161 Delaware Avenue, Santa Cruz, CA 95060, USA.

Schleicher and Schuell Inc., Keene, NH 03431A, USA. Tel: 001 603 357 2398 Serotec, 22 Bankside, Station Approach, Kidlington, Oxford OX5 1JE, UK. Serotec, NCSU Centennial Campus, Partners 1,1017 Main Campus Drive, Suite 2450, Raleigh, NC 27606, USA.

Shandon Scientific Ltd., 93-96 Chadwick Road, Astmoor, Runcorn, Cheshire WA7 1PR, UK. Tel: 01928 566611 URL: http://www.shandon.com

Sigma-Aldrlch Co. Ltd., The Old Brickyard, New Road, Gillingham, Dorset XP8 4XT, UK. Tel: 01747 822211 Fax: 01747 823779 URL: http://www.sigma-aldrich.com Sigma-Aldrich Co. Ltd., Fancy Road, Poole, Dorset BH12 4QH, UK. Tel: 01202 722114 Fax: 01202 715460 URL: http://www.sigma-aldrich.com Sigma Chemical Co., PO Box 14508, St Louis, MO 63178, USA. Tel: 001 314 771 5765 Fax: 001 314 771 5757 URL: http://www.sigma-aldrich.com

LIST OF SUPPLIERS

Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel: 001 858 535 5400 URL: http://www.stratagene.com Stratagene Europe, Gebouw California, Hogehilweg 15, 1101 CB Amsterdam Zuidoost, The Netherlands. Tel: 00800 9100 9100 URL: http://www.stratagene.com Tetko, 333 S Highland Avenue, Briarcliff Manor, NY 10510, USA.

United States Biochemical, PO Box 22400, Cleveland, OH 44122, USA. Tel: 001 216 464 9277 Vector Laboratories, 16 Wulfric Square, Bretton, Peterborough PE3 8RF, UK. Vector Laboratories, 30 Ingold Road, Burlingame, CA 94010, USA. Worthlngton Biochemical Corp., 730 Vassar Avenue, Lakewood, OK 74005-0788, USA.

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Index

adherence 32-5 and gene expression 33-5 and macrophage function 35 adherence-based purification 32, 35-44 collagen matrices 39-40,43, 44 detachment methods 40-2, 44, 66-7 gelatin coatings 36-7 glass 36, 43 macrophage morphology and function 43-4, 56 microexudate coatings 38-9, 43 plastic 10, 36, 43, 44 alveolar macrophage isolation 7-10 lung biopsy 7, 8, 9 lung lavage 7, 8 whole lung 9-10 antigen processing/presentation 93-113 antigen dose 105-6, 107 antigen presentation analysis 110-12 antigen transfer 109-10 cell biology 110 class I-restricted processing 103-5 class II antigen induction 98-100 class II-restricted processing 100-3 defined 93-4 microbe uptake assessment 108 T cell choice 94-5 antigen pulsing 100-2, 106-7 antigen transfer 109-10 anti-microbial activity 142-53 Candida cdbicans 147

extracellular killing, colorimetric assay 151-3 intracellular killing 149-51 phagocytosis 147-9 superoxide anion susceptibility 143-4 Leishmania spp. 144-7 oxygen dependent/ independent 143-4 anti-tumour activity 130-42 radioactive label incorporation 140-1 radioisotopic release assays 133-9 target cell sensitivity 141-2 tumour cell counting assay 130-2

Bio-Gel polyacrylamide beads 5-6 blood monocytes isolation 6-7 purification 55 bone marrow macrophages (BMM0) 10-14, 96-8, 103-5 class II antigen induction 98-100 Boyden chamber 116, 117-18 broncheolar lavage 7, 8

Candida dlbicans 147 extracellular killing, colorimetric assay 151-3 intracellular killing 149-51 phagocytosis 147-9 superoxide anion susceptibility 143-4 carrageenan 158

checkerboard titrations 100 chemotaxis 116-18 clodronate-liposomes 162-4 spectrophotometric determination 164-5 collagen adherence 39-40,43, 44 colony counting 150-1 colorimetric assay 151-3 corneal grafts 170 co-stimulation assay 111-12 counterflow centrifugal elutriation 32, 45, 50-3 cross-linking fixatives 68 soluble receptors 124-5 51 Cr release assay 119-20, 133-5 cryostats 75 cytokine receptors, soluble 122-4 cross-linking 124-5 cytolysis assessment 133-9 51 Cr release assay 119-20, 133-5 indium release assay 137-9 iododeoxyuridine release assay 135-7 thymidine release assay 135-7 cytostasis of tumour cells 140-1

DEAE dextran-mediated transfection 189-90 dendritic cells 95-6 detachment methods EDTA 41-2, 44 EDTA/lidocaine 67 lignocaine 42, 44 mechanical 40-1 physiological effects 44 dextran sulphate 158 209

INDEX

EDTA detachment 41-2, 44 EDTA/lidocaine detachment 67 electroporation transfection 191-2 elutriation 32, 45, 50-3 EMSA (electrophoretic mobility shift assay) 195-6, 199-201 probe preparation 198-9 endocytosis 78-89 quantitation 84-6 tracers 83 endosome markers 80

Ficoll-Hypaque gradient 10, 46-7 fixatives 67-8 flow cytometry 70-1 fluorescent microscopy 71 fluorochromes 65

gadolinium chloride 158-9 gelatin adherence 36-7 gene expression 173-201 and adherence 33-5 RNA detection by run-on assay 177-81 RNA preparation 174-7 RNA quantification Northern hybridization 181-2 RNase protection assay 182 RT-PCR 182-4 gene transfer 185-93 clodronate-liposome use 168-9 stable 185,192-3 transient 185, 186-92 DEAE dextran-mediated 189-90 by electroporation 191-2 lipophilic reagents 190-1 glass adherence 36, 43 gradient purification 32,45-50 acceleration/deceleration control 49 clumping 50 continuous/discontinuous gradients 50 Ficoll-Hypaque gradient 10, 46-7 gradient capacity 49-50

210

osmolarity 50 Percoll gradient 48-9 and temperature 49 wall effect 50 graft rejection 170-1 granuloma macrophage isolation 25-6 gut-associated macrophages 22-3 immunochemical labelling 62-78 detection techniques 70-1 direct 64 dual/triple 64 fixatives 67-8 fluorochromes 65 indirect 64, 68-70 macrophage preparation 65-70 markers 62 staining 76-8 tissue preparation 72-6 immunodeficient mice 169 immunofluorescence microscopy 71 indium release assay 137-9 infection-associated macrophages 25-6 inflammation suppression 170 intestinal macrophages 22-3 iododeoxyuridine release assay 135-7 isopycnic centrifugation 32, 45-50 Kupffer cell isolation 17-19 from liver wedge biopsies 19-20 lamina propria macrophages 22-3 Langerhans cell isolation 23-5 latex beads 87 Leishmania spp. 144-7 leukocyte chemotaxis 116-18 leukocyte transmigration 118-21 reverse 120, 121-2 ligands 81-2, 87 lignocaine detachment 42, 44 lipophilic reagent transfection 190-1

liposome-encapsulated clodronate 162-4 spectrophotometric determination 164-5 liposome-mediated macrophage suicide 160-8 administration 166, 167 applications 168-71 dose 165 duration 167-8 principles 160-1 selectivity 166-7 liposomes 160, 161-2 LPS contamination 56 lung 9-10 biopsy 7, 8, 9 lavage 7, 8 lysosome markers 80 lytic function analysis 127-55 anti-tumour activity 130-42 microbicidal activity 142-53

macrophage heterogeneity 1-2, 54-5 markers 62 macropinocytosis 79 malaria research 169 mannose and derivatives 159 microbe uptake, direct staining 108 microbicidal activity 142-53 Candida albicans 147 extracellular killing, colorimetric assay 151-3 intracllular killing 149-51 phagocytosis 147-9 superoxide anion susceptibility 143-4 Leishmania spp. 144-7 oxygen dependent/ independent 143-4 microexudate adherence 38-9, 43 microglial cell isolation 21-2 migration assessment 116-22 Boyden chamber 116, 117-18 by radioisotopes 119-20 MTT colorimetric assay 151-3

Northern hybridization 181-2 nuclear extract preparation 196-7 nuclear run-on assay 177-81

INDEX

oligonucleotide probe preparation 198-9 opsonization 87 organic solvents 67-8 osmotic lysis of pinosomes 104 osteoclast isolation 20-1

paraformaldehyde preparation 68 periodate-lysine 73-4 Percoll gradient 48-9 perfused tissue 72, 73-5 periodate-lysine paraformaldehyde 73-4 peritoneal macrophage isolation 2-6 Bio-Gel polyacrylamide beads 5-6 thioglycollate 4-5 purification, 55 permeabilization 64-5 phagocytic assay 86, 148-9 phagocytosis 79-80, 86-9 blocking 157-60 phagosome markers 80 pinocytosis 79 pinosomes, osmotic lysis 104 plasmid DNA purification 187-9 Plasmodium falaparum 169 plastic adherence 10, 36, 43, 44 polyacrylamide beads 5-6 protein binding 193-201 EMSA 195-6, 199-201 probe preparation 198-9 purification 31-60 adherence 32, 35-44 collagen matrices 39-40, 43, 44 detachment methods 40-2, 44, 66-7 gelatin coatings 36-7 glass 36, 43 macrophage morphology and function 43-4, 56 microexudate coatings 38-9, 43 plastic 10, 36, 43, 44 comparison of methods 32, 55

elutriation 32, 45, 50-3 by gradients 32, 45-50 acceleration/deceleration control 49 clumping 50 continuous/discontinuous gradients 50 Ficoll-Hypaque gradient 10, 46-7 gradient capacity 49-50 osmolarity 50 Percoll gradient 48-9 and temperature 49 wall effect 50 isopycnic centrifugation 32, 45-50 IPS contamination 56 principles 31 velocity sedimentation 32, 45-6

radioisotopic release assays 133-9 51 Cr 119-20, 133-5 indium 137-9 iododeoxyuridine 135-7 thymidine 135-7 radiolabelling cytostasis assessment 140-1 soluble cytokine receptor identification 124-5 receptors 81, 87 antagonists 159 reverse transcription-polymeras e chain reaction (RT-PCR) 182-4 reverse transmigration assay 120, 121-3 RNA detection by run-on assay 177-81 preparation 174-7 quantification Northern hybridization 181-2 RNase protection assay 182 RT-PCR 182-4 RNase protection assay 182 run-on assay 177-81

SCID mice 169 secondary antibodies 65 secretion 115-26 modes 115 silica particles 157-8 skin-associated macrophages 23-5 soluble receptors 122-4 cross-linking 124-5 spectrophotometry 164-5 splenic macrophage isolation 14-15, 16-17

T cell choice 94-5 thioglycollate 4-5 thymidine release assay 135-7 tissue dissociation enzymatic 16, 17-25 mechanical 15-16 TNFa 141-2 transendothelial migration 118-21 radioisotopic detection 119-20 reverse 120, 121-2 tumour-associated macrophage isolation 25-6 enzymatic method 27-8 mechanical dissociation 27 tumour cell counting assay 130-2 tumour cell cytotoxicity 130-42 radioactive label incorporation 140-1 radioisotopic release assays 133-9 target cell sensitivity 141-2 tumour cell counting assay 130-2

velocity sedimentation 32, 45-6

wall effect 50

zymosan 87

211

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