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 TM
ME T H O D S IN MO L E C U L A R BI O L O G Y

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to

www.springer.com/series/7651
 T-Cell Trafficking
Methods and Protocols

Edited by

Federica M. Marelli-Berg
Department of Immunology, Division of Medicine, Imperial College London, London, UK

Sussan Nourshargh
William Harvey Research Institute, Barts and the London School of Medicine & Dentistry, Queen Mary
University of London, London, UK
Editors
Federica M. Marelli-Berg Sussan Nourshargh
Department of Immunology William Harvey Research Institute
Division of Medicine Barts and the London School
Imperial College London of Medicine and Dentistry
Hammersmith Hospital Queen Mary, University of London
Du Cane Road Charterhouse Square
London London
UK W12 0NN UK EC1M 6BQ
[email protected] [email protected]

Additional material for this book can be downloaded from http://extras.springer.com

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-60761-460-9 e-ISBN 978-1-60761-461-6
DOI 10.1007/978-1-60761-461-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2010921222

© Springer Science+Business Media, LLC 2010

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
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Printed on acid-free paper

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Preface
Both the development of a functional immune system and the establishment of effective
immunity are orchestrated by a series of complex and coordinated migratory events. In
the last decade a large number of major discoveries have shed light on the molecular
mechanisms of lymphocyte migration and the anatomy of immune responses, including
the development of organized lymphoid tissue, the compartmentalized recirculation of
lymphocyte subsets, and their targeted access to inflammatory antigenic sites. This has
been made possible by the development and increasing availability of novel techniques,
particularly in the field of real-time imaging and genetic manipulation.
Unlike many other research areas where the use of standard techniques and off-the-
shelf kits is often sufficient and satisfactory, the study of lymphocyte trafficking requires
considerable expertise. The methods compiled in this volume are contributed by interna-
tionally recognized experts in their respective fields who have many years of experience
not only in using these techniques but also in troubleshooting and perfecting them. Each
chapter contains a step-by-step description of the method and, more importantly, it pro-
vides invaluable tips and tricks to safeguard against potential mishaps and pitfalls. The
volume is introduced by an excellent comprehensive review of the current knowledge of
lymphocyte trafficking, accessible to the non-specialist. The methods in these chapters are
divided into three parts. The first part covers state-of-the-art protocols to study lympho-
cyte migration and T-cell–endothelial cell interactions in vitro. The second part covers
various approaches used for the direct visualization of the development of the lymphoid
system, lymphocyte recirculation, and effector responses in experimental models in vivo.
Finally, a third section is dedicated to the study of lymphocyte migration and inflamma-
tion in the human system and how such investigations can lead to the identification of
prognostic markers and the identification of novel therapeutic approaches. We are confi-
dent that this book will be an essential manual for newcomers to this ever-expanding and
exciting area of research as well as a valuable addition to more specialized laboratories.
We would like to express our gratitude to all the authors who have made this book
possible and thank Professor John Walker, the Series Editor, for his guidance in editing this
volume. We would also like to express our appreciation to Abigail Woodfin for providing
the cover art for this volume.
F. Marelli-Berg’s lab is generously supported by the British Heart Foundation, The
Gates’ Foundation and the Medical Research Council of the UK.
S. Nourshargh’s work is supported by generous funds from The Wellcome Trust and
the British Heart Foundation.
Please note that additional material for this book can be downloaded from
http://extras.springer.com

Federica Marelli-Berg
Sussan Nourshargh

v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

SECTION I INTRODUCTORY REVIEW

1. How T Cells Find Their Way Around . . . . . . . . . . . . . . . . . . . . . . . 3
Alf Hamann

SECTION II MIGRATION OF T CELLS IN VITRO

2. Live Imaging of Leukocyte–Endothelium Interactions . . . . . . . . . . . . . . . 17
Olga Barreiro, Francisco Sánchez-Madrid, and María Yáñez-Mó
3. Leucocyte Adhesion Under Haemodynamic Flow Conditions . . . . . . . . . . . 31
Charlotte Lawson, Marlene Rose, and Sabine Wolf
4. Influence of Stromal Cells on Lymphocyte Adhesion and Migration
on Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Helen M. McGettrick, Chris D. Buckley, G. Ed Rainger,
and Gerard B. Nash
5. Discriminating Between the Paracellular and Transcellular Routes of Diapedesis . . 69
Jaime Millán, Eva Cernuda-Morollón, and Severine Gharbi
6. Monitoring RhoGTPase Activity in Lymphocytes . . . . . . . . . . . . . . . . . 83
Marouan Zarrouk, David Killock, and Aleksandar Ivetic
7. Visualisation of Signalling in Immune Cells . . . . . . . . . . . . . . . . . . . . 97
Leo M. Carlin, Konstantina Makrogianneli, Melanie Keppler,
Gilbert O. Fruhwirth, and Tony Ng
8. Methods for Quantitation of Leukocyte Chemotaxis and Fugetaxis . . . . . . . . 115
Fabrizio Vianello, Elda Righi, and Mark C. Poznansky
9. Analysis of CXCR3 and Atypical Variant Expression and Signalling
in Human T Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Anna Korniejewska, Malcolm Watson, and Stephen Ward
10. Transfection of Indoleamine 2,3 Dioxygenase in Primary Endothelial Cells . . . . 149
Petros XE Mouratidis and Andrew JT George

SECTION III MIGRATION OF T CELLS IN VIVO

11. Visualisation of Lymphoid Organ Development . . . . . . . . . . . . . . . . . . 161
Henrique Veiga-Fernandes, Katie Foster, Amisha Patel, Mark Coles,
and Dimitris Kioussis
12. Single-Cell Analysis of Cytotoxic T Cell Function by Intravital
Multiphoton Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Thorsten R. Mempel

vii
viii Contents

13. Imaging Interactions Between the Immune and Cardiovascular Systems

In Vivo by Multiphoton Microscopy . . . . . . . . . . . . . . . . . . . . . . . . 193
Owain R. Millington, James M. Brewer, Paul Garside,
and Pasquale Maffia
14. Applying an Adaptive Watershed to the Tissue Cell Quantification During
T-Cell Migration and Embryonic Development . . . . . . . . . . . . . . . . . . 207
D. Zhu, S. Jarmin, A. Ribeiro, F. Prin, S.Q. Xie, K. Sullivan,
J. Briscoe, A.P. Gould, Federica M. Marelli-Berg, and Y. Gu

SECTION IV MONITORING T-CELL MIGRATION IN HUMAN DISEASES

15. Identifying Homing Interactions in T-Cell Traffic in Human Disease . . . . . . . 231
Patricia F. Lalor, Stuart M. Curbishley, and David H. Adams
16. Tracking Antigen-Experienced Effector T Cells In Vitro and In Vivo . . . . . . . 253
Claire L. Gorman, Claudia Monaco, Enrico Ammiratti,
Anna-Chiara Vermi, Federica M. Marelli-Berg, and Andrew P. Cope
17. Preclinical Testing of Strategies for Therapeutic Targeting
of Human T-Cell Trafficking In Vivo . . . . . . . . . . . . . . . . . . . . . . . 267
Caroline Coisne and Britta Engelhardt
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Contributors
DAVID H. ADAMS • Liver Research Group, Division of Medicine, Institute of Biomed-
ical Research, MRC Centre for Immune Regulation, University of Birmingham,
Birmingham, UK
ENRICO AMMIRATTI • Clinical Cardiovascular Biology Research Centre, Vita-Salute San
Raffaele University and San Raffaele Scientific Institute, Milan, Italy
OLGA BARREIRO • Servicio de Inmunología, Hospital de la Princesa, Universidad
Autónoma de Madrid; Centro Nacional de Investigaciones Cardiovasculares, Madrid,
Spain.
JAMES M. BREWER • Centre for Biophotonics, Strathclyde Institute of Pharmacy &
Biomedical Sciences, University of Strathclyde, Glasgow, UK
JAMES BRISCOE • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
CHRIS D. BUCKLEY • Centre for Cardiovascular Sciences and Centre for Immune Regu-
lation, The Medical School, University of Birmingham, Birmingham, UK
LEO M. CARLIN • Cancer Studies Division/Randall Division of Cellular and Molecu-
lar Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s Medical School
Campus, King’s College London, London, UK
EVA CERNUDA-MOROLLÓN • Unidad de Histocompatibilidad, Hospital Universitario
Central de Asturias, Oviedo, Spain
CAROLINE COISNE • Theodor Kocher Institute, University of Bern, Bern, Switzerland
MARK COLES • Division of Molecular Immunology, MRC National Institute for Medi-
cal Research, The Ridgeway, Mill Hill, London; Centre for Immunology and Infection,
Department of Biology & HYMS, University of York, York, UK
ANDREW P. COPE • The Kennedy Institute of Rheumatology, Faculty of Medicine,
Imperial College London, UK
STUART M. CURBISHLEY • Liver Research Group, Division of Medicine, Institute of
Biomedical Research, MRC Centre for Immune Regulation, University of Birmingham,
Birmingham, UK
BRITTA ENGELHARDT • Theodor Kocher Institute, University of Bern, Bern, Switzerland
KATIE FOSTER • Division of Molecular Immunology, MRC National Institute for Medical
Research. The Ridgeway, Mill Hill, London, UK
GILBERT O. FRUHWIRTH • Cancer Studies Division/Randall Division of Cellular and
Molecular Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s Medical
School Campus, King’s College London, London, UK
PAUL GARSIDE • Centre for Biophotonics, Strathclyde Institute of Pharmacy & Biomedical
Sciences, University of Strathclyde, Glasgow, UK
ANDREW JT GEORGE • Department of Immunology, Hammersmith Campus, Imperial
College London, London, UK

ix
x Contributors

SEVERINE GHARBI • Department of Immunology and Oncology, Centro Nacional de

Biotecnología. CSIC, Cantoblanco, Madrid, Spain
CLAIRE L. GORMAN • Faculty of Medicine, Imperial College London, The Kennedy
Institute of Rheumatology, London, UK
ALEX P. GOULD • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
YAN GU • Developmental Neurobiology, National Institute for Medical Research, MRC,
London, UK
ALF HAMANN • Experimentelle Rheumatologie, CC12, Charité Universitätsmedizin
Berlin, Germany
ALEKSANDAR IVETIC • Cytoskeleton Research Group, Division of Cardiovascular Sciences,
Faculty of Medicine, Imperial College London, National Heart and Lung Institute,
Hammersmith Hospital Campus, DuCane Road, London, UK
SARAH JARMIN • Department of Immunology, Faculty of Medicine, Imperial College
London, Hammersmith Hospital Campus, London, UK
MELANIE KEPPLER • Cancer Studies Division/Randall Division of Cellular and Molecu-
lar Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s Medical School
Campus, King’s College London, London, UK
DAVID KILLOCK • Cytoskeleton Research Group, Division of Cardiovascular Sciences,
Faculty of Medicine, Imperial College London, National Heart and Lung Institute,
Hammersmith Hospital Campus, DuCane Road, London, UK
DIMITRIS KIOUSSIS • Division of Molecular Immunology, MRC National Institute for
Medical Research, The Ridgeway, Mill Hill, London, UK
ANNA KORNIEJEWSKA • Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath, Slough, UK
PATRICIA F. LALOR • Liver Research Group, Division of Medicine, Institute of Biomed-
ical Research, MRC Centre for Immune Regulation, University of Birmingham,
Birmingham, UK
CHARLOTTE LAWSON • Veterinary Basic Sciences, Royal Veterinary College, London, UK
PASQUALE MAFFIA • Strathclyde Institute of Pharmacy & Biomedical Sciences, Univer-
sity of Strathclyde, Glasgow, UK; Department of Experimental Pharmacology, School of
Biotechnological Sciences, University of Naples Federico II, Naples, Italy
KONSTANTINA MAKROGIANNELI • Cancer Studies Division/Randall Division of Cellu-
lar and Molecular Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s
Medical School Campus, King’s College London, London, UK
FEDERICA M. MARELLI-BERG • Division of Medicine, Department of Immunology,
Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du
Cane Road, London, UK
HELEN M. MCGETTRICK • Centre for Cardiovascular Sciences and Centre for Immune
Regulation, The Medical School, University of Birmingham, Birmingham, UK
THORSTEN R. MEMPEL • Center for Immunology and Inflammatory Diseases and Cen-
ter for Systems Biology, Massachusetts General Hospital and Harvard Medical School,
Charlestown, MA, USA
JAIME MILLÁN • Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco,
Madrid, Spain
 Contributors xi

OWAIN R. MILLINGTON • Centre for Biophotonics, Strathclyde Institute of Pharmacy &

Biomedical Sciences, University of Strathclyde, Glasgow, UK
CLAUDIA MONACO • Faculty of Medicine, Imperial College London, The Kennedy
Institute of Rheumatology, London, UK
PETROS XE MOURATIDIS • Department of Immunology, Hammersmith Campus, Impe-
rial College London, London, UK
GERARD B. NASH • Centre for Cardiovascular Sciences and Centre for Immune Regula-
tion, The Medical School, University of Birmingham, Birmingham, UK
TONY NG • Cancer Studies Division/Randall Division of Cellular and Molecular
Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s Medical School
Campus, King’s College London, London, UK
AMISHA PATEL • Division of Molecular Immunology, MRC National Institute for Medical
Research, Mill Hill, London, UK
MARK C. POZNANSKY • Infectious Diseases Unit and DFCI/Harvard Cancer Center,
Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA.
FABRICE PRIN • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
G. ED RAINGER • Centre for Cardiovascular Sciences and Centre for Immune Regulation,
The Medical School, University of Birmingham, Birmingham, UK
ANA RIBEIRO • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
ELDA RIGHI • Infectious Diseases Unit and DFCI/Harvard Cancer Center, Harvard
Medical School, Massachusetts General Hospital, Charlestown, MA, USA.
MARLENE ROSE • Veterinary Basic Sciences, Royal Veterinary College, London NW1 0TU;
National Heart and Lung Institute, Imperial College London, Harefield Hospital &
Heart Science Centre, Hill End Road, Harefield, Middlesex, UK
FRANCISCO SÁNCHEZ-MADRID • Servicio de Inmunología, Hospital de la Princesa, Uni-
versidad Autónoma de Madrid; Centro Nacional de Investigaciones Cardiovasculares,
Madrid, Spain
KATE SULLIVAN • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
HENRIQUE VEIGA-FERNANDES • Division of Molecular Immunology. MRC National
Institute for Medical Research, Mill Hill, London, UK; Immunobiology Unit., Faculdade
de Medicina de Lisboa, Instituto de Medicina Molecular, Lisboa, Portugal.
ANNA-CHIARA VERMI • Clinical Cardiovascular Biology Research Centre, Vita-Salute
San Raffaele University and San Raffaele Scientific Institute, Milan, Italy
FABRIZIO VIANELLO • Department of Haematology, Imperial College of Medicine,
Hammersmith Hospital, London, UK
STEPHEN WARD • Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath; Bath Road, Slough, UK
MALCOLM WATSON • Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath; Bath Road, Slough, UK
SABINE WOLF • Veterinary Basic Sciences, Royal Veterinary College, Royal College Street,
London, UK
xii Contributors

SHEILA Q. XIE • MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College
London, Hammersmith Hospital Campus, London, UK
MARÍA YÁNEZ-MÓ • Servicio de Inmunología, Hospital de la Princesa, Universidad
Autónoma de Madrid; Centro Nacional de Investigaciones Cardiovasculares, Madrid,
Spain.
MAROUAN ZARROUK • Cytoskeleton Research Group, Division of Cardiovascular Sciences,
Faculty of Medicine, Imperial College London, National Heart and Lung Institute,
Hammersmith Hospital Campus, DuCane Road, London, UK
DAAN ZHU • Developmental Neurobiology, National Institute for Medical Research,
MRC, London, UK
 Section I

Introductory Review
 Chapter 1

How T Cells Find Their Way Around

Alf Hamann

Abstract
Among diverse cellular systems of the body, the immune system is unique in representing a network of
interacting cells of enormous complexity yet based on single cells travelling around. Only the advanced
visualization technologies of the recent years have brought to everybody’s attention the fact that what
we see is usually a snapshot of a dynamic system, where the majority of players are highly motile and
become coordinated by diverse signals provided by their environment. This introductory chapter touches
a selection of aspects that address predominantly the functioning of the system as such. It attempts to
provide a framework of how migratory mechanisms are regulated to ensure that various cell populations
reach their destination and that the appropriate interaction partners find each other.

Key words: T cells, recirculation, tissue entry, tissue exit, homing, inflammation, adhesion
molecules, chemokines, chemokine receptors, antigen, α4 β7 integrin, memory, epigenetics,
imprinting.

1. Recirculation:
Come and See
Fifty years ago J. Gowans discovered that lymphocytes possess
the unique property to recirculate continuously between blood,
lymphoid tissues and lymph (1). In contrast to myeloid cells,
which, by and large, are only known to travel unidirectionally, the
recirculation of naive lymphocytes ensures that antigen presented
locally is seen by as many as possible cells carrying the enormous
diverse repertoire of T and B cell receptors.
Two major travelling routes through lymphoid tissues mutu-
ally complement each other that are regulated by different migra-
tory mechanisms: the circulation through the blood with a
stopover in the spleen on the one side and through both the

F.M. Marelli-Berg, S. Nourshargh (eds.), T-Cell Trafficking, Methods in Molecular Biology 616,
DOI 10.1007/978-1-60761-461-6_1, © Springer Science+Business Media, LLC 2010

3
4 Hamann

blood and the lymphatic system via lymph nodes, Peyer’s patches
and tertiary lymphoid tissue arising in chronically inflamed tissues
as gateways.
Much research has focussed on the receptors guiding lym-
phocyte entry into lymph nodes and Peyer’s patches. This process
is mediated by a well-known set of adhesion molecules assisted by
the integrin-activating function of chemokine receptors that are
triggered by chemokines presented on the endothelial surface. In
fact, L-selectin, which makes the first contacts for naive lympho-
cytes with the high endothelial venules of (predominantly, but not
exclusively peripheral) lymph nodes, was the first homing-related
molecule to be identified (2). Subsequent research discovered the
integrins LFA-1 and α4 -integrins as indispensable components of
the more complex process of transmigration that also contribute
to organ-specific properties of migration. L-selectin was found
to direct especially naïve lymphocytes into lymph nodes, whereas
α4 β7 guides cells into mucosa-associated lymphoid tissues and
also into the gut wall itself. LFA-1 is less selective and contributes
to transmigration in most tissues including inflamed sites. The
sequential operation of these molecules during migration from
blood to tissue had led to the proposal of the multi-step model of
transmigration (3), which is now part of every textbook.
In contrast to the lymphoid tissues named above, entry into
the spleen is not dependent on homing or chemokine receptors;
all evidence so far available suggests that the entry is solely driven
by mechanics of blood flow and cell motility (4, 5). Yet, local-
ization within the tissue and different compartments therein are
regulated by chemotaxis and likely also by interactions with stro-
mal cells or extracellular matrix.

2. Within Tissue:
Come and Go
(or One Comes,
Rather recent is the insight that not only the entry into tissue
One Leaves)
but also the exit requires defined molecular systems. As a simple
matter of fact, the frequency of cells disposed of in a tissue can
efficiently be regulated not only by the rate of entry but also by
modulating the exit rate. Findings of the last years have shown
that exit from the tissue is an active process controlled by chemo-
tactic mechanisms. The chemokine receptor CCR7 was shown
to be required for T cell, including Treg, exit from inflamed
peripheral tissue (6, 7). Another chemotactic agent sphingosine-
1-phosphate (S1P) and its receptors are required for the exit from
lymph nodes, a finding emerging from studies with the drug FTY
720 which displays immunosuppressive effects. Both CCR7 and
S1P receptors are modulated in the course of T cell activation and
 How T Cells Find Their Way Around 5

thereby might cause the transient retention of recently activated

T cells in the lymph node (8). When CCR7 is knocked out, the
number of T cells retained in an inflamed tissue doubles, confirm-
ing its importance for continuous circulation (9).
By technical reasons, quantification of exit rates for specific
subsets of cells and specific tissues is more difficult. However, a
variety of data are available from early studies applying cannula-
tion of the thoracic duct or even single lymph nodes, which pro-
vided clear evidence that not only naive cells entering a lymph
node via the high endothelium pass the tissue within half a day
and exit it but also that large numbers of effector/memory cells
attracted to an inflamed tissue or generated by local prolifera-
tion exit the tissue via the efferent lymph (10). It is conceiv-
able that the process of emigration underlies a variety of regu-
latory influences, too; T cell activation upon antigen encounter
within the tissue might be one factor, but also an influence
of inflammation-generated mediators such as prostaglandins has
been described (11).

3. Disposal
of Combatants
Infiltration of leucocytes into sites of an immune reaction is the
hallmark of inflammation. It can be assumed that the evolution
of leucocyte trafficking in the context of innate cellular reactions
started well before the emergence of lymphocytes. In contrast,
the specific variant of recirculation of T and B cells is a rather late
invention.
Various mechanisms contribute to the rapid delivery of
defence cells and their accumulation at sites of risk. Enhanced
expression of adhesion molecules, such as MAdCAM-1 in
mucosal sites or ICAM-1 in many tissues, as well as induction
of additional adhesion molecules such as P- and E-selectin or
VCAM-1 on endothelium under conditions of inflammation, are
the starting points for a massive adhesion and transmigration of
leucocytes from blood into the inflamed tissue, assisted by chemo-
tactic signals.
But who tells the endothelium to become activated? Most
endothelia do respond to TLR signals such as LPS, but for most
inflammatory processes it is more likely that other cells receive the
danger signals. Resident leucocytes such as mast cells and den-
dritic cells are highly sensitive sentinels for stress signals, tissue
cells such as fibroblasts or epithelial cells respond to pathogen-
derived signals, and, in later stages, antigen-specific T cells trans-
late recognition into local conditioning for high-rate recruitment
of effector leucocytes. Both innate receptors for pathogen struc-
tures and recognition of antigen by T cells lead to activation and
6 Hamann

secretion of a variety of mediators, notably cytokines such as TNF,

IFNγ and a large variety of chemokines, that are crucial within the
multi-step transmigration process and subsequently guide immi-
grated leucocytes into the centre of inflammation. In addition,
the complement system, activated by Ig-dependent or alternative
pathways, contributes significantly to the stimulation of recruit-
ment by production of the chemotactic and activating cleavage
products C3a and C5a.
The entire process shows properties of a positive feedback
loop; the first signals lead to the attraction of a few pioneer cells
(45), which, by release of activating cytokines and chemokines,
greatly accelerate the recruitment of large numbers of further
effector cells. The endothelium represents the interface between
inflammatory environment and circulation: its sensitive reaction
towards activating cytokines results in the expression of adhe-
sion molecules on the surface. Moreover, its capacity to trans-
port chemokines from the abluminal tissue side to the vessel sur-
face, where they become fixed on glycosaminoglycans, generates
tags from the inflammatory environment detectable for circulat-
ing leucocytes.
It should be mentioned that the immune system also provide
mechanisms to shut off the proinflammatory response in order
to avoid inappropriate damage and immunopathology. Different
types of regulatory T cells, including Foxp3+ Tregs and IL-10
producing Tr1 cells, appear to be generated or expanded during
the immune reaction and contribute to limit the response after
the acute reaction (12, 13). In addition, mediators such as IFNγ,
while fuelling the inflammation at the beginning, induce feed-
back mechanisms negatively regulating the response and activate
inhibitory reactions in myeloid cells such as production of NO,
IDO or others (14). Whether these negative feedback mecha-
nisms also directly down-regulate the recruitment machinery, or
whether recruitment and activation merely fades away when the
effector cells become silenced, remains to be seen. So far, there is
little evidence that, e.g., Tregs affect directly the migratory pro-
cess (Doebis et al., unpublished data).

4. Are T Cells
Attracted by
Antigen?
As mentioned above, antigen-driven activation of immigrated T
effector cells is a major factor in the establishment of a full inflam-
matory condition. It is a longstanding question to what extent the
accumulation of lymphocytes is directly related to their capacity
to recognize the antigen. It has been repeatedly reported that
antigen-reactive T and B cells become concentrated within a
tissue offering the cognate antigen (antigen-induced trapping),
 How T Cells Find Their Way Around 7

which can even lead to the complete disappearance of the reactive

cells from the circulation (15, 16). On the other side, convincing
data were provided excluding an influence of antigen on the entry
of lymphocytes into a given tissue (17), although, under certain
conditions, endothelium might also present antigen. Rather, con-
tact with the antigen and consequent activation might lead to an
alteration of so far not identified cellular properties in the cog-
nate lymphocytes that result in altered exit rates and a prolonged
residence of the cells in the respective environment. Evidence for
antigen-specific trapping has been presented for lymphoid tissue
(15, 16), for the liver (18, 19) and for peripheral tissue (20).
In our studies, using a transgenic DTH model, we found an
enhanced recruitment of both antigen-specific and non-specific
effector T cells into the inflamed cutaneous tissue upon preced-
ing encounter of the specific T cells with cognate antigen, but no
selective trapping (45). However, the specific T cells that arrived
in the site started to proliferate locally after a few days, resulting
in a cellular infiltrate that is strongly enriched for cognate T cells
(Doebis et al., submitted).

5. The Paradigm
of Organ-Specific
Homing
Already in the pioneer years of cellular immunology, the capac-
ity of distinct subpopulations of T or B cells to travel back selec-
tively into compartments of initial antigen contact was recognized
and referred to as “homing” (21). Selective homing to the gut
mucosa was observed for activated (and later also for memory)
cells (blasts) in the blood (21, 22), which, in healthy animals,
predominantly originate from the gut environment continuously
exposed to a huge burden of bacterial antigen. Application of the
famous “Stamper-Woodruff” assay, a crude approach using frozen
tissue sections to test for selective adhesion of lymphocytes to the
remnants of endothelium (23), was surprisingly effective and led
to the detection of L-selectin as major determinant of peripheral
lymph node homing (2) and of the integrin α4 β7 as a mucosal
homing receptor (24). Albeit this work gave rise to the assump-
tion that distinct homing receptors guide lymphocytes into differ-
ent tissues, the population of (predominantly) naive lymphocytes
used in that studies is – ironically – just the one population that
does not show organ-specific homing, apart from the fact that
naive lymphocytes are specialized to recirculate through any lym-
phoid tissue, but cannot enter other types of tissues.
It is therefore important to consider that lymphocytes gain
organ-specific homing properties only upon activation and dif-
ferentiation into effector/memory cells. This differentiation step
is associated with a major change in the molecular equipment
8 Hamann

required for trafficking: L-selectin as well as the chemokine

receptor CCR7, also important for lymph node entry, is down-
regulated with differentiation into end-stage memory cells, lead-
ing to a non-recirculating population with a high capacity to
enter inflamed, peripheral tissues. Down-regulation of molecules
important for recirculation, acquisition of organ-specific homing
receptors and inflammation-seeking receptors and functional dif-
ferentiation might be, but are not necessarily, synchronized. The
widely cited distinction of “central” memory cells from “effector-
memory” T cells is misleading and rather picks out two of several
possible combinations of functional (cytokine) and homing prop-
erties. For example, both, recirculating, CCR7+ T cells and ter-
minally differentiated CCR7-effector/memory cells which local-
ize exclusively in peripheral tissue are ready to produce effector
cytokines and can be simultaneously detected in vivo (25, 26).
On the other side, upregulation of organ-specific homing recep-
tors starts already with the first divisions upon antigen encounter
in vivo (27).
Early expectations were to discover a series of homing recep-
tors, each specific for a distinct organ or type of tissue. On the
level of adhesion molecules, this only holds true for the interac-
tion partner α4 β7 -integrin and MAdCAM, which guide lympho-
cytes almost exclusively into gastrointestinal (“mucosal”) sites.
The sister integrin α4 β1 was found to be a dominant mediator of
T cell migration into the brain, a fact allowing the use of anti α4
antibodies (natalizumab) as the most efficient drug against mul-
tiple sclerosis today. However, it is not excluded, that the α4 β1
integrin, which binds predominantly to its ligands VCAM-1 or
fibronectin, is also involved in the recruitment of cells to other
sites of the body, one definitely being the bone marrow (28, 29),
and possibly other sites of inflammation. Similar might apply for
the adhesion pair E-selectin (expressed on cutaneous endothe-
lium) and CLA (“cutaneous lymphocyte antigen”, a carbohy-
drate epitope, expressed on lymphocytes) being considered as a
“skin” specific system, at least in humans. However, E-selectin
is also frequently expressed in other tissues upon inflammation.
Together with P-selectin, with which it shares some common
ligands (glycosylated PSGL-1) and acts in a largely redundant
way, E-selectin might rather be considered as an inflammation-
specific system with some preference for the skin (30). Additional
adhesion molecules, such as VAP-1 (31), CD44 (32) and oth-
ers, might contribute to a significant diversity of potential address
codes, but, after all, selectins, α4 -integrins and β2 -integrins and
their respective ligands appear to be the working horses of
recognition and adhesion to endothelium, with differential, but
also widely overlapping use in various destinations of lympho-
cyte trafficking. As discussed above, the chemokine system pro-
 How T Cells Find Their Way Around 9

vides a greater degree of selectivity, although true organ-specific

chemokine receptors also might not exist.

6. Sniffing
the Way
After their discovery 30 years ago, the chemokine family has
attracted great interest as being major players in the determina-
tion of migratory pathways. The large number of family mem-
bers of these cytokine-like mediators (more than 40 chemokines
in humans) is multiplying the degree of variation provided by
adhesion molecules. Interestingly, this diversity has evolved rather
recently (with vertebrates) and appears to be driven along the
evolution of the adaptive immune system (33, 34), requesting for
sophisticated mechanisms to direct the multiple cell types, dif-
ferentiation and activation stages to appropriate sites and con-
ditions of immune reactivity within the body. Apart from the
structural homologies, the chemokine family is characterized by
its preponderant reactivity with a – also very diverse – subgroup
of receptors (almost 20 receptors in humans) belonging to the
huge superfamily of G-protein-coupled receptors. Interestingly,
within this superfamily, chemokine receptors share a subtree with
the olfactory receptors, the largest subfamily among G-protein-
linked receptors (almost 500 members in man; 33) that allows us
to orient ourselves within a complex diversity of chemical signals
of the external environment.

7. Topographical
Memories
For a long period, the paradigm of organ-specific homing
included the assumption that T cell priming within a specific tis-
sue environment led to an imprinting of the expression of spe-
cific homing receptors. Albeit even recent Nature papers use the
term imprinting, what they refer to is only induction of certain
homing receptors (35) by cells and mediators. In fact, the same
group provided experimental data challenging the concept of per-
manent imprinting and favouring the assumption of flexibility in
the expression of homing receptors (36). Indeed, organ-specific
homing could also be explained by continuing selection or re-
induction of a given receptor upon recirculation through selected
tissues providing antigen exposure and re-stimulatory capacity
associated with additional, organ-specific co-signals (37).
Studies to proof the stability of differentially expressed hom-
ing receptors in vivo were largely lacking until recently. We
10 Hamann

analysed the stability of ligands for E/P-selectins that serve as

homing receptors for inflamed tissue, notably inflamed skin. We
could provide clear evidence that recently induced selectin lig-
ands are stable only on a subfraction of T cells. However, upon
repeated stimulation under ligand-inducing conditions (presence
of IL-12) the fraction of cells stably expressing selectin ligands
for at least several weeks in vivo increased and ex vivo isolated
selectin ligand-positive effector/memory cells turned out to be
almost completely stable (38). This shows that imprinting of a sta-
ble homing phenotype appears possible, but requires repeated res-
timulation under permissive conditions, similar to what has been
found for the imprinting of a cytokine memory in T cells (39).
The above-mentioned studies on the mucosal homing recep-
tor α4 β7 in CD8+ T cells suggested that expression of this recep-
tor is not permanent after initial induction (36). We are presently
investigating this issue in CD4+ T cells. Studies not yet com-
pleted suggest that, as for selectin ligands, repeated stimulations
in the presence of retinoic acid are required to achieve expression
of α4 β7 which persist upon restimulation (Szilagyi et al., unpub-
lished). In contrast to the selectin ligands, a continuous drop of
the expression of α4 β7 is, however, observed on all adoptively
transferred cells in vivo, suggesting that stability subsists only for
a limited period, unless appropriate restimulation re-establishes
the imprinted phenotype.
For the chemokine receptor CCR9, which is also induced
(on CD8+ cells) by retinoic acid and considered to contribute to
mucosal homing (35), we could not observe a stable expression
phenotype (Szilagyi et al., unpublished).
These data suggest that variable degrees of a topographical
memory might exist, depending on the respective receptors: com-
plete stability, as in case of selectin ligands established after appro-
priate instructive differentiation of the memory cells; partial sta-
bility, which slowly fades away in the absence of permissive signals
as in case of α4 β7 ; and lack of stability, as for CCR9 in CD4+
T cells.
However, it has to be considered that, even in the absence of
a stable expression, an imprinted memory might exist. The term
“commitment” is used, for example, for T effector cells, when
polarized Th1 or Th2 cells are predestined to secrete the respec-
tive typical cytokines, IFNγ or IL-4, but require restimulation to
do so. Importantly, a committed cell only needs a reduced set of
stimulatory signals to re-acquire the specific phenotype. Further
investigations have to show whether this type of memory also
exists in case of homing receptors, as some data suggest (38).
What mechanisms could allow imprinting of a certain pheno-
type or commitment for facilitated re-expression? For the cytokine
memory, proposed mechanisms include the establishment of a
metastable signalling condition, involving positive feedback loops
 How T Cells Find Their Way Around 11

in the induction and action of key transcription factors such as

GATA-3 (39) and imprinting of information by epigenetic modi-
fication in histones (“histone code”) or especially in DNA by dif-
ferential methylation of CpGs (40). Epigenetic modification leads
to alterations in chromatin structure resulting in different degrees
of accessibility of a given gene locus. Especially DNA methylation
is reproduced upon synthesis of new DNA strains during mito-
sis and therefore allows inheritance of acquired properties. This
mechanism appears to have a key role in the imprinting of devel-
opmental changes and determination of lineage decisions (41–
43). So far, only indirect evidence for a role of epigenetic mech-
anisms in the imprinting of homing receptors has been published
(44). However, it is evident that epigenetic imprinting would be
ideally suited to match requirements for the acquisition of stable
homing phenotypes.

8. Concluding
Remarks
Every decade of immunological research appears to uncover novel
functional subsets of T cells; the latest ones being, e.g., the Th17
cells or the diverse types of regulatory T cells. How this increas-
ing universe of specialists becomes coordinated and appropriately
targeted to the hot spots of immunoreactivity would remain a
mystery if not, at the same time, our knowledge about the mech-
anisms of cell trafficking would have greatly expanded. Coop-
erating adhesion molecules and chemokine receptors equip the
migrating cells with an almost unlimited combinatorial diversity
to recognize signatures defining tissues and compartments, to dis-
tinguish inflammatory processes of multiple flavours that might
depend on the kind of triggers, site of inflammation or involved
cell populations and so on. That chemotaxis, haptotaxis and cell
contacts not only are important to regulate the macroscopic dis-
tribution of cells within the body but are equally important to
guide cells through the jungle of a tissue environment – and even
might support the marriage of individual cell partners destined to
interact in a given environment and functional stage – that insight
was greatly nourished by recent findings. The present book might
provide a number of wonderful pieces of knowledge from this
field (46).

Acknowledgements

Special thanks to present and past coworkers of my group

who contributed to ideas and findings discussed above:
12 Hamann

G. Debes, C. Doebis, S. Floess, S. Ghani, J. Huehn, S. Jennrich,

A. Menning, B. Ratsch, K. Siegmund, C. Siewert, U. Syrbe,
and B. Szilagyi. Our work was continuously supported by the
Deutsche Forschungsgemeinschaft.

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 Section II

Migration of T Cells In Vitro

 Chapter 2

Live Imaging of Leukocyte–Endothelium Interactions

Olga Barreiro, Francisco Sánchez-Madrid, and María Yáñez-Mó

Abstract
Leukocyte extravasation is a highly dynamic, interactive, and coordinated process that plays a central role
during the inflammatory response of innate immunity. The interaction of leukocytes with the activated
endothelium under shear forces is comprised of many sequential events, each involving specific leuko-
cyte and endothelial receptors, as well as chemokines and adaptor and signaling molecules. Because of
its complexity, researchers studying leukocyte extravasation at the subcellular level have been forced to
search for appropriate in vitro models that mimic pathophysiological conditions at sites of inflamma-
tion. We report methods for direct visualization of cellular and molecular processes of critical importance
to spatiotemporally dissect the different steps in the adhesion cascade. These methodologies include
techniques for the study of the dynamics of individual molecules involved in a discrete part of the pro-
cess, as well as simple procedures to label molecules and cells in order to observe the extravasation
process.

Key words: Endothelial cells, leukocytes, adhesion, flow, fluorescent proteins, fluorescent probes,
confocal microscopy.

1. Introduction

Leukocyte transendothelial migration is dependent on the

productive interaction of leukocytes with the activated endothe-
lial monolayer of the vasculature supplying the inflamed tissue.
This interaction takes place through a series of sequential steps,
which confer selectivity to the extravasation process (1). The
first step is the interaction of selectins with their carbohydrate-
based ligands (2) and allows the leukocyte to roll on the
endothelial cell wall. Leukocyte rolling increases the chances that

F.M. Marelli-Berg, S. Nourshargh (eds.), T-Cell Trafficking, Methods in Molecular Biology 616,
DOI 10.1007/978-1-60761-461-6_2, © Springer Science+Business Media, LLC 2010

17
18 Barreiro, Sánchez-Madrid, and Yáñez-Mó

leukocytes will encounter chemokines presented on the apical

endothelial surface. These cytokines activate leukocyte integrins
(3) and in cooperation with integrin-dependent signals induce
the polarization of the leukocyte (4). Polarized leukocytes firmly
adhere to the endothelial monolayer, mainly through the inte-
grin receptor–counterreceptor pairs LFA1/ICAM-1,2 and VLA-
4/VCAM-1 (5). In this firm adhesion step, binding to endothe-
lial adhesion receptors initiates intracellular signaling cascades that
lead to the cytoskeletal rearrangements necessary for the forma-
tion of a three-dimensional docking structure that concentrates
both ICAM-1 and VCAM-1 and virtually surrounds the adhered
leukocyte, preventing its detachment under flow conditions (6).
After the adhesion steps, leukocytes cross to the interstitial space
either by diapedesis through endothelial lateral junctions or by
transcellular migration (7).
Leukocyte extravasation involves the coordinated action of
multiple receptors, cytoskeletal adaptors, and signaling molecules
and results in drastic morphological changes in both leukocytes
and endothelial cells. Direct visualization of cellular and molecu-
lar dynamics is therefore of critical importance for understanding
this process. This report summarizes several approaches to the
spatiotemporal analysis of the different steps of the adhesion cas-
cade. These approaches include techniques for the study of indi-
vidual molecules involved in specific steps as well as simple pro-
cedures for labeling molecules and cells to allow observation of
the extravasation process in conditions as close to physiological as
possible.

2. Materials

2.1. Cell Models 1. For HUVEC culture, 199 medium is used supplemented
with 10% FBS, antibiotics, heparin (100 μg/ml), and ECGF
(50 μg/ml). Cells are routinely grown in culture flasks
coated with 0.5% gelatin
2. Recombinant human TNF-α (R&D Systems)
3. Ficoll-Hypaque high-density medium (Sigma)
4. PHA-L (Sigma) and IL-2, provided by the National Insti-
tutes of Health AIDS Research and Reference Reagent pro-
gram, Division of AIDS. RPMI 1640 (Gibco) supplemented
with 10% FBS
5. RPMI 1640 supplemented with 10% FBS, G418 (Cal-
biochem), and MnCl2
 Live Imaging of Leukocyte–Endothelium Interactions 19

2.2. Static Adhesion 1. BCECF-AM (Invitrogen)

Measurements and
2. HBSS (BioWhittaker) supplemented with 1% BSA
Staining
3. Plastic seal
2.2.1. Static Adhesion 4. 0.1% SDS in 50 mM Tris–HCl pH 8.5
Measurements

2.2.2. Static Adhesion 1. FN (20 μg/ml) (Sigma)

Staining
2. 4% paraformaldehyde in PBS supplemented with 2%
sacarose
3. Tris-buffered saline (TBS): 50 mM Tris–HCl pH 7.5,
150 mM NaCl

2.3. Detachment 1. A parallel flow chamber (Glycotech)

Experiments 2. Glass-bottomed Petri dishes (WillCo Wells)
3. A programmable syringe pump (Harvard Apparatus)
4. HBSS supplemented with 2% FBS at 37◦ C
5. Real-time monochrome camera coupled to a video recorder
system
6. Water bath

2.4. Leukocyte– 1. A parallel flow chamber (Glycotech)

Endothelium 2. A programmable syringe pump (Harvard Apparatus)
Interactions Under
Flow Conditions 3. Glass-bottomed Petri dishes (WillCo Wells)
4. HBSS supplemented with 2% FBS
5. Water bath

2.5. Transfection 1. 199 culture medium supplemented with growth factors

Procedures for 2. 1.5 M NaCl
Fluorescently Tagged
3. DNA plasmids
Fusion Proteins
4. Electroporator suitable for mammalian cells Gene Pulser
2.5.1. Transfection of Xcell (Bio-Rad Laboratories)
Endothelial Cells
– Electroporation cuvettes (4 mm) (Bio-Rad)
– Coated coverslips

2.5.2. Transfection of 1. Opti-MEM

R
medium (Life Technologies)
Leukocytes
2. DNA plasmids
3. Electroporator suitable for mammalian cells Gene Pulser
Xcell (Bio-Rad Laboratories)
4. Electroporation cuvettes (4 mm) (Bio-Rad)
5. Ficoll-Hypaque high-density medium (Sigma)
6. RPMI 1640 (Gibco) supplemented with 10% FBS
20 Barreiro, Sánchez-Madrid, and Yáñez-Mó

2.6. Staining of 1. A kit to stably couple antibodies with fluorescent tags (Invit-
Living Cells with rogen)
Fluorescently 2. Alternatively use a Zenon kit to directly stain primary anti-
Labeled Primary body supernatant noncovalently (Invitrogen)
Antibodies or
Fluorescent Probes
2.6.1. Fluorescently
Labeled Antibodies
2.6.2. Cell Labeling with 1. CMAC (blue), BCECF (green), CMTMR (red), or another
Fluorescent Probes permeable fluorescent probe with an ester bond that sta-
bilizes the probe in the cytoplasm (nonspecific probes
or organelle-specific ones such as mitotracker, lysotracker)
(Invitrogen)
2. HBSS supplemented with 0.5% BSA

2.7. Time-Lapse 1. Glass-bottomed Petri dishes

Fluorescence 2. Laser scanning confocal fluorescence microscope or wide-
Microscopy field epifluorescence microscope equipped with a piezoelec-
tric focusing system that allows z-axis sectioning
3. Incubation system (La-con GBr Pe-con GmbH)
4. A parallel flow chamber (Glycotech)
5. A programmable syringe pump (Harvard Apparatus)
6. Glass-bottomed Petri dishes (WillCo Wells)
7. Phenol red-free medium

3. Methods

Methods for visualizing interactions between leukocyte and the

endothelium at the subcellular level aim to mimic as far as pos-
sible the in vivo situation. The molecular behavior observed in
the in vitro models is very much dependent on the flow rate
used, the receptor expression profile and migratory capability of
the particular leukocyte subset, and the activation state of the
endothelium. Primary cells are the best choice since immortal-
ized cell lines do not usually upregulate adhesion receptors to the
same levels as primary cells and may become partially dediffer-
entiated, losing some specific endothelial or leukocyte markers.
However, to investigate a specific event (rolling, adhesion, loco-
motion, transmigration), transfected cell lines can be a valuable
tool. In addition, static adhesion experiments can give informa-
tion on the molecular dynamics or signaling cascades triggered
 Live Imaging of Leukocyte–Endothelium Interactions 21

by intercellular contact; however, details on the reorganization

of some receptors, relevance of avidity processes, etc., are only
unveiled under shear flow conditions.

3.1. Cell Models 1. HUVEC are most commonly used as a source of human pri-
mary macrovascular endothelial cells. Alternatively, primary
cultures can be obtained of microvascular, lymphatic, blood–
brain barrier, and high endothelial venule cells.
2. HUVEC are activated by changing to medium 199 10% FBS
supplemented with 20 ng/ml TNF-α for 20 h (see Note 1).
3. PBMCs are obtained from peripheral blood donated by
healthy volunteers. The PBMCs are isolated by centrifuging
the blood for 30 min at 1,800 rpm on a Ficoll gradient. The
isolated cells are then washed thoroughly and maintained in
RPMI 1640 medium 10% FBS. For a crude preparation of
PBLs, the PBMC population is depleted of monocytes by
adhesion to a plastic flask for 30 min at 37ºC.
4. T lymphoblasts are derived from PBLs by activation for 48 h
with PHA-L (1 μg/ml). After extensive washing, T cells are
cultured for 7–14 days in RPMI 1640, 10% FBS containing
50 U/ml rhIL-2.
5. K562 transfectants (expressing α4β1 or αLβ2 integrins for
VCAM-1 or ICAM-1 independent binding, respectively, see
Note 2) are grown in RPMI 1640, 10% FBS, 1 mM G418.

3.2. Static Adhesion 1. To quantify leukocyte adhesion to an endothelial monolayer

Measurements and under static conditions, HUVEC are grown to confluence
Staining in 96-well plates and treated with TNF-α (for activation, see
3.2.1. Static Adhesion Note 1) in combination with the inhibitors to be tested (see
Measurements Note 3).
2. Leukocytes are labeled with a fluorescent probe (usually
BCECF-AM) as described in Section 3.6.2. Cells are
usually pretreated for 15 min with blocking antibodies
(approximately 10 μg/ml). For experiments with chemi-
cal inhibitors, the appropriate incubation times and doses
should be determined in each case (see Note 4).
3. 105 cells/well in HBSS+1% BSA are placed in each
HUVEC-coated well and incubated for 15–30 min at 37ºC
(see Note 5).
– Wells are filled with HBSS, sealed with an adhesive plastic
seal, and maintained in an inverted position for 20 min.
– Buffer is removed and cells lysed in 0.1% SDS, Tris 50 mM
pH 8.5. Fluorescence is measured in a fluorescent microplate
reader. (If the fluorescent probe is BCECF-AM, the excita-
tion and emission wavelengths are 488 and 520 nm, respec-
tively, see Note 6.)
22 Barreiro, Sánchez-Madrid, and Yáñez-Mó

3.2.2. Static Adhesion – For staining of leukocyte interactions with endothelial cells
Staining under static conditions, a confluent monolayer of HUVEC
is grown on a gelatin- or FN-coated coverslip (see Notes 7
and 8) and activated with TNF-α (Note 1) (Fig. 2.1).

Fig. 2.1. Visualization of leukocyte–endothelial interactions under static conditions by

confocal microscopy. HUVEC were transfected with a fluorescent membrane protein and
activated with 20 ng/ml TNF-α for 20 h. α4 integrin K562 transfectants were allowed
to adhere to the endothelial monolayer under static conditions. Samples were fixed and
analyzed by confocal microscopy. Endothelial cells extend filopodial projections around
the adherent leukocyte in what has been called docking structure.6 Confocal optical
sections can be used to create three-dimensional reconstructions and rotations of the
complete cell volume, as shown in the side-view image.

– Leukocytes are allowed to adhere for different times in com-

plete medium at 37ºC (see Note 5).
– Medium is removed and samples are fixed with 2%
paraformaldehyde in PBS for 5 min and extensively washed
with TBS (see Note 9).
– Samples are then stained with the appropriate specific anti-
bodies.

3.3. Detachment Experiments to investigate the detachment of leukocytes from

Experiments the apical surface of endothelial monolayers are performed
in a parallel flow chamber. The parallel-plate flow cham-
ber used for leukocyte adhesion and transmigration under
defined laminar flow is described in detail on the Glycotech
web site (http://www.glycotech.com/apparatus/parallel.html).
These assays measure the resistance of leukocyte–endothelial
adhesion to increasing flow stresses.
– HUVEC are grown in an FN-coated glass-bottomed petri
dish and activated with TNF-α (see Notes 1, 8, 10,
and 11).
– Leukocytes (PBL or K562 transfectants) are allowed to
adhere under static conditions, in complete medium, for
15 min at 37ºC (see Note 5).
 Live Imaging of Leukocyte–Endothelium Interactions 23

– The flow chamber is carefully mounted in the petri dish (see

Note 12). HBSS+2% FBS (37ºC) is pulled through the flow
chamber with a programmable syringe pump at an initial flow
rate of 1 dyn/cm2 , which is increased up to 30 dyn/cm2 at
30 s or 1 min intervals (see Note 13). In the final seconds
of each flow rate interval, six to eight 20× fields of view are
recorded (see Note 14). Cell detachment is measured from
the differences in the numbers of adherent cells after each
flow rate interval (see Note 15).

3.4. Leukocyte– The study of leukocyte–endothelium interactions under shear

Endothelial stress allows quantification of several functional parameters, such
Interactions Under as rolling velocity, locomotion rate or percentage of rolling,
Flow Conditions detachment, adhesion, and transmigration (Fig. 2.2 and Supple-
mental Video 1).

Fig. 2.2. Leukocyte tracking under flow conditions by time-lapse confocal microscopy. A HUVEC monolayer was activated
with 20 ng/ml TNF-α for 20 h. PBLs were perfused at 1.8 dyn/cm2 for 3–4 min and then cell-free HBSS buffer containing
2% FBS was perfused for the rest of the experiment. Bright-field images were acquired every 30 s over a period of 16 min
(see Supplemental Video 1). The figure shows four representative frames from the video sequence. Each leukocyte was
assigned a letter code denoting its migratory state: R for rolling, A for adhesion, L for locomotion, D for detachment.
No cells transmigrated during this experiment. Cellular traks depicting the path followed by each cell during the whole
experiment are overlayed in the last image.

1. PBLs (106 per ml) in HBSS 2% FBS at 37◦ C are drawn

across TNF-α-activated confluent monolayers (see Note 1)
at an estimated wall shear stress of 1.8 dyn/cm2 (see Note
16) for perfusion times from 30 s to 10 min (see Note 17).
2. Lymphocyte rolling on the endothelium is easily visual-
ized because the adhered cells travel more slowly than free-
flowing cells (Fig. 2.2R). Rolling velocity, frequency, and
accumulation can be calculated after the experiment with the
use of dedicated software.
3. Lymphocytes are considered to be adherent after 20 s of
stable contact with the monolayer (Fig. 2.2A).
4. To track leukocytes that move on the apical endothelial sur-
face (locomotion, Fig. 2.2L) in search of a suitable site for
transmigration, time-lapse recording can be more illustrative
of the process (8).
24 Barreiro, Sánchez-Madrid, and Yáñez-Mó

5. Transmigrating lymphocytes can be distinguished because of

their polarized morphology and changes in brightness (see
Note 18).
6. Transmigrated lymphocytes are detected beneath the
endothelial monolayer.
7. Lymphocytes are considered to be detached when they have
returned to the free-flowing state after having been com-
pletely arrested on endothelium (Fig. 2.2D).
8. The number of rolling, adhered, transmigrating, transmi-
grated, and detached cells is quantified by direct visualiza-
tion of four different fields (20× phase contrast objective)
for each time point of every independent experiment (9)
(see Note 19). When a specific parameter is to be calcu-
lated (rolling velocity, locomotion distance, etc.), the use of a
higher magnification objective is recommended (40×–60×).

3.5. Transfection To visualize molecular dynamics, a common approach is to trans-

Procedures for fect cells with proteins labeled with fluorescent tags (EGFP, YFP,
Fluorescently Tagged CFP, RFP, etc.) (Figs. 2.1 and 2.3). It is important to confirm
Fusion Proteins that the fusion proteins have the same subcellular localization and
function as the endogenous protein (see Note 20). The relatively
easy protocols described below are considered to provide a rapid
transient expression in a reasonable percentage of cells. Alterna-
tives such as nucleofection or viral vector transduction can also be
used.

3.5.1. Transfection of 1. HUVEC are trypsinized and resuspended in complete 199

Endothelial Cells medium to a concentration of 1.5 × 106 cells in a final vol-
ume of 200 μl. Cells are placed in a electroporation cuvette
(4 mm)
2. 5 μl of 1.5 M NaCl is added (see Note 21)

Fig. 2.3. Study of molecular dynamics during leukocyte–endothelium interactions under shear flow by time-lapse fluo-
rescence confocal microscopy. The T-lymphoblastic cell line CEM was transfected with a fluorescent cytoplasmic marker,
while HUVEC were co-transfected with a fluorescent membrane protein and a intracellular marker. Endothelial cells were
treated with 20 ng/ml TNF-α for 20 h before microscopy observation. Lymphocytes were perfused at 1.8 dyn/cm2 for
3–4 min and then cell-free HBSS buffer containing 2% FBS was perfused for the rest of the experiment. Fluorescent
signals and bright-field images were acquired sequentially through a z-stack. A representative time point is shown, and
each frame shows the maximal projection of the whole fluorescence stack for the channel. The best focused bright-field
image was selected for the corresponding time point. (see Supplemental Video 2).
 Live Imaging of Leukocyte–Endothelium Interactions 25

3. 20 μg of plasmid DNA is added (see Notes 22 and 23)

4. Cells are electroporated at 200 V and 975 μF using an elec-
troporator suitable for mammalian cells
5. The cuvette is filled with 800 μl fresh complete medium 199
(see Note 24)
6. Cells are seeded as droplets onto several glass-bottomed
Petri dishes previously coated with 20 μg/ml FN (Notes
8 and 11)
7. Cells are allowed to adhere before filling the Petri dishes with
complete medium supplemented with growth factors
8. Cells are grown for 24–48 h (see Notes 25 and 26)

3.5.2. Transfection of 1. Lymphoid cell lines are transfected in serum-free medium

Leukocytes (Opti-MEM). Resuspend 10 × 106 cells in 400 μl Opti-
MEM
2. 20 μg plasmid DNA is added (see Notes 22 and 23)
3. Electroporation is performed at 250–280 V, 1,200 μF using
an electroporator suitable for mammalian cells (see Note 24)
4. Cells are incubated in a final volume of 5 ml for 12 h, and
dead cells are removed by centrifugation on a Ficoll gradient
(see Note 26).

3.6. Staining of An alternative to transfection with fluorescent protein constructs

Living Cells with is staining of endothelial cells or leukocytes, either with neutral
Fluorescently primary antibodies (see Note 27) directly coupled to fluorescent
Labeled Primary tags or with intracellular fluorescent probes.
Antibodies or
Fluorescent Probes

3.6.1. Fluorescently 1. The antibodies can be stably coupled to fluorescent tags

Labeled Antibodies using an appropriate kit (for example, Invitrogen) following
the manufacturer’s instructions.
2. Alternatively, antibodies can be transiently tagged with a sec-
ondary Fab antibody already fluorescently labeled (Zenon
kit, Invitrogen). This labeling procedure is very easy, rapid,
and quite useful for short-term experiments in living cells
(see Note 28).
3. Briefly, purified antibody or supernatant is incubated with
the fluorescent Fab anti-mouse.
4. The reaction is stopped by adding an excess of mouse
immunoglobulins.
5. The mixture is incubated with cells for 5–10 min and then
washed to remove all free mouse Ig.

3.6.2. Cell Labeling with If the aim is not to label specific molecules but instead simply
Fluorescent Probes to label cells, cells can be loaded with intracellular fluorescent
26 Barreiro, Sánchez-Madrid, and Yáñez-Mó

probes immediately before microscopy observation. Appropriate

cell probes, with characteristic excitation and emission frequen-
cies, can be combined with fluorescent proteins or antibodies.
Usually these permeable probes contain an ester modification that
is cleaved once they traverse the cell plasma membrane so that
they are retained intracellularly. There are also probes that specif-
ically label mitochondria, lysosomes, etc.
1. Leukocytes are washed with serum-free medium and resus-
pended at 5–10 × 106 cells/ml in serum-free medium con-
taining 1 μM fluorescent probe.
2. Cells are incubated for 15 min at 37ºC, centrifuged, and
washed to remove excess fluorescent probe (see Note 29).
3. For EC labeling, probes can be dissolved in serum-free
medium or HBSS+0.5% BSA and added directly to the con-
fluent monolayer.

3.7. Time-Lapse 1. HUVEC transfected or not with fluorescent fusion pro-

Fluorescence teins and/or labeled with antibodies or probes are grown
Microscopy to confluence on glass-bottomed dishes precoated with FN
(20 μg/ml) (see Note 24) (Fig. 2.3 and Supplemental
Video 2). Cells are then activated with TNF-α for the appro-
priate time (see Notes 1 and 11), transferred to phenol
red-free medium, and placed on the microscope stage (see
Note 30).
2. For experiments under static conditions, K562 transfectants
or leukocytes resuspended in 500 μl of complete medium
199 are added. Labeling with fluorescent probes can facili-
tate observation of intercellular contacts with the endothelial
monolayer.
3. Plates are maintained at 37◦ C in a 5% CO2 atmosphere using
an incubation system. Alternatively, the parallel flow cham-
ber can be coupled to the microscope stage for experiments
under flow conditions.
4. Series of transmitted light and confocal or widefield fluores-
cence images, distanced 0.4–1 μm in the z-axis, are continu-
ously obtained using a 40× or a 63× oil immersion objective
(see Note 31). Images can be processed and assembled into
movies using dedicated software (see Note 32).

4. Notes

1. Standard TNF-α treatment is 20 ng/ml for 20 h because

the expression of ICAM-1 and VCAM-1 is maximal at
this time. However, for maximal expression of E-selectin,
 Live Imaging of Leukocyte–Endothelium Interactions 27

treatment with 20 ng/ml TNF-α for 4 h would be optimal.

Alternatively, leukocyte capture under flow can be achieved
with a combination of a suboptimal TNF-α dose and the
addition of exogenous chemokines such as SDF-1, which
get immobilized at the apical glycosaminoglycans of the
endothelium (10). Other proinflammatory cytokines, such
as IL-1β, are also commonly employed.
2. Adhesion of K562 LFA-1 transfectants needs to be done
in the presence of 1 mM Mn2+ to achieve full integrin
activation.
3. If inhibitors of HUVEC function are to be tested, it is
essential to measure ICAM-1 and VCAM-1 induction by
flow cytometry in a parallel sample.
4. The use of allosteric inhibitors to specifically inhibit either
VLA-4 (BIO5192 (Biogen Idec; Cambridge, MA)) or
LFA-1 (BIRT377 (Boehringer-Ingelheim Pharmaceuticals;
Ridgefield, CT)) is explained as an example. Integrin
inhibitors (BIO5192 (10 μg/ml) or BIRT377 (10 μM))
are added to leukocytes 5 min before adhesion assay under
static or flow conditions.
5. Adhesion times are very dependent on the leukocyte trans-
migratory capacity. For example, for PBLs, average adhe-
sion times might be approximately 10 min, whereas K562
can be allowed to interact with the endothelial monolayer
for more extended periods, since they do not transmigrate
across the EC monolayer.
6. To provide the 100% adhesion reference, a separate well
is loaded with the total input of labeled leukocytes and
directly lysed.
7. 24-well plates and 12-mm coverslips are suitable for routine
staining.
8. Coverslips can be coated with 1% gelatin, 20 μg/ml FN,
or other ECM preparations. With gelatin coating, better
confluence is achieved if the gelatin is fixed with 0.5% glu-
taraldehyde and extensively washed with TBS before seed-
ing the EC.
9. Wash aldehydes with Tris-containing saline buffer to block
the fixation reaction with an excess of amine groups. Alter-
natively, glycine solutions can be used.
10. The results are more easily quantified if the transmigra-
tion rate is low: use K562 transfectants, which do not
transmigrate, or PBLs, rather than T lymphoblasts or
neutrophils.
11. Since the observation area is usually limited (the center
of the coverslip in the flow gasket or just a few fields
in a motorized confocal time-lapse microscope), it is not
28 Barreiro, Sánchez-Madrid, and Yáñez-Mó

necessary to seed large area with HUVEC. HUVEC can be

conveniently seeded as a droplet, which also favors imme-
diate confluence. Once HUVEC have spread, cells are acti-
vated with TNF-α.
12. It is important to ensure that the coverslip does not dry at
any moment and to avoid bubbles.
13. An initial wash at 1 dyn/cm2 for 1 min can be performed
to remove unbound cells. Alternatively, adhered cells are
counted only after the first 2 dyn/cm2 interval.
14. It is convenient to record the same fields each interval,
especially if initial adhesion is not homogeneous on the
preparation.
15. If the incidence of transmigration is high during the exper-
iment, transmigrated cells in each field have to be counted
and subtracted from the total count to yield the number of
detached cells.
16. The flow rate of 1.8 dyn/cm2 is similar to the shear force
generated in the human postcapillary venules.
17. When using extracellular blockers (such as antibodies or
peptides) under flow conditions, you should assess that the
perfusion with buffer does not wash them away signifi-
cantly.
18. A phase contrast objective is useful for direct observa-
tion of changes in leukocyte morphology during extrava-
sation because the initially bright round cells on top of the
endothelium spread and darken while migrating across and
beneath the monolayer.
19. Coverslips can be immediately fixed under flow with 4%
PFA at room temperature for 10 min and then washed with
TBS and stained for markers of interest.
20. If antibodies against the protein of interest are available,
it is convenient to confirm that they recognize the fusion
protein. The molecular weight of the expressed fusion pro-
tein should also be assessed by Western blot to exclude the
presence of partial degradation products, which might give
false subcellular localizations.
21. Efficiency of electroporation has been shown to increase
with higher osmotic strength.
22. Try to use DNA constructs at as high a concentration as
possible to reduce the volume to be added to the cell sus-
pension.
23. In co-transfection experiments, the amount of total DNA
should not exceed 20 μg/ml; in making adjustments,
 Live Imaging of Leukocyte–Endothelium Interactions 29

decrease the amount of plasmid encoding the protein that

is more efficiently expressed.
24. After electroporation, allow cells to recover from electric
shock and close micropores prior to seeding.
25. Allow cells to grow after electroporation, but ideally for
no more than 24 h; this will ensure the highest possible
fluorescent protein content in the culture.
26. Transfection efficiency can be routinely quantified by direct
flow cytometry.
27. The fluorescently tagged primary antibodies should be
functionally neutral in order not to interfere with cell func-
tions.
28. The Zenon kit is not advisable for long-term staining since
the tag is not covalently bound to the primary antibody and
can become detached with time, decreasing specific signal
and increasing the background signal.
29. Usually cell labeling is evident since the cell pellet will be
colored.
30. The use of medium without phenol red is important to
avoid autofluorescence.
31. Specific acquisition conditions (in terms of scanning veloc-
ity, zoom, photomultiplier gain, offset, number of series,
time-lapse parameters, etc.) need to be adjusted according
to the fluorescence intensity of the samples, the cell types,
and cell processes being studied. Photobleaching and pho-
totoxicity also need to be minimized in each experimental
setup.
32. Images acquired using a confocal microscope do not need
further processing, but deconvolution is required prior to
analysis of images acquired with a widefield fluorescence
microscope.

Acknowledgments

This work was supported by grants BFU2005-08435/BMC from

the Ministerio de Educación y Ciencia, and European Net-
work MAIN LSHG-CT-2003-502935 to FSM, by Contrato-
Investigador FIS 0019 from Instituto de Salud Carlos III to
MY-M. The authors thank Giulia Morlino and Francesc Baixauli
for providing samples for the time-lapse experiments and Simon
Bartlett for editing the manuscript.
30 Barreiro, Sánchez-Madrid, and Yáñez-Mó

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1. Ley K, Laudanna C, Cybulsky MI, 6. Barreiro O, Yáñez-Mó M, Serrador JM, et al.

Nourshargh S. (2007) Getting to the site (2002) Dynamic interaction of VCAM-1 and
of inflammation: the leukocyte adhesion ICAM-1 with moesin and ezrin in a novel
cascade updated. Nat Rev Immunol 7, endothelial docking structure for adherent
678–89. leukocytes. J Cell Biol 157, 1233–45.
2. Ley K, Kansas GS. (2004) Selectins in T-cell 7. Vestweber D. (2007) Adhesion and signaling
recruitment to non-lymphoid tissues and molecules controlling the transmigration of
sites of inflammation. Nat Rev Immunol 4, leukocytes through endothelium. Immunol
325–35. Rev 218, 178–96.
3. Laudanna C, Alon R. Right on the 8. Shulman Z, Pasvolsky R, Woolf E, et al.
spot. (2006) Chemokine triggering of (2006) DOCK2 regulates chemokine-
integrin-mediated arrest of rolling leuko- triggered lateral lymphocyte motility but
cytes. Thromb Haemost 95, 5–11. not transendothelial migration. Blood 108,
4. Sanchez-Madrid F, del Pozo MA. (1999) 2150–8.
Leukocyte polarization in cell migration 9. Goetz DJ, Greif DM, Shen J, Luscinskas FW.
and immune interactions. EMBO J 18, (1999) Cell-cell adhesive interactions in an in
501–11. vitro flow chamber. Methods Mol Biol 96,
5. Barreiro O, de la Fuente H, Mittelbrunn 137–45.
M, Sanchez-Madrid F. (2007) Functional 10. Cinamon G, Alon R. (2004) Real-time in
insights on the polarized redistribution of vitro assay for studying chemoattractant-
leukocyte integrins and their ligands dur- triggered leukocyte transendothelial migra-
ing leukocyte migration and immune inter- tion under physiological flow conditions.
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 Chapter 3

Leucocyte Adhesion Under Haemodynamic Flow Conditions

Charlotte Lawson, Marlene Rose, and Sabine Wolf

Abstract
Vascular endothelial cells (EC) line the luminal side of all blood vessels and act as a selective barrier
between blood and tissue. EC are constantly exposed to biochemical and biomechanical stimuli from the
blood and underlying tissue. Fluid shear stress acts in parallel to the vessel wall, resulting from friction
of blood against EC. Despite the importance of flow on normal EC function, much of the information
regarding EC function and dysfunction has been derived from cells harvested, grown and studied in
static culture. In order to study the effects of shear stress on EC function, a number of in vitro models
have been developed. This chapter provides methodology for use of a system which enables recirculation
of leucocytes and cell culture medium over the endothelium for a period of several minutes to days
and enables investigation of the effects of prolonged leucocyte co-culture on both the endothelial and
leucocyte populations.

Key words: Endothelium, shear stress, parallel-plate flow chamber.

1. Introduction

1.1. The Endothelium Vascular endothelial cells (EC) line the luminal side of all blood
vessels and act as a selective barrier between blood and tissue. EC
are constantly exposed to biochemical and biomechanical stimuli
from the blood and underlying tissue. It is well established that
maintenance of a quiescent endothelium is vital to prevent coagu-
lation and control vascular permeability as well as regulating vas-
cular tone through production of nitric oxide. In addition, EC
contribute to maintenance of the quiescence of circulating leuco-
cytes (reviewed in (1)). Conversely, failure to control vascular per-
meability and coagulation, an increase in vascular tone and loss of

F.M. Marelli-Berg, S. Nourshargh (eds.), T-Cell Trafficking, Methods in Molecular Biology 616,
DOI 10.1007/978-1-60761-461-6_3, © Springer Science+Business Media, LLC 2010

31
32 Lawson, Rose, and Wolf

leucocyte quiescence can all contribute to EC dysfunction. Thus

EC pathology contributes to many conditions such as atheroscle-
rosis, hypertension, thrombosis, stroke, vasospastic disorders and
diabetic microangiopathy, as well as the increase in mortality and
morbidity associated with chronic inflammation and autoimmune
disease (review in, e.g., (2)).
There is increasing evidence that endothelial cells are also
bona fide antigen presenting cells (APC). In vitro, they present
antigen to B7-independent memory T cells inducing prolifera-
tion and IL-2 production. In vivo, human endothelium is con-
stitutively positive for major histocompatibility (MHC) class II
and vascular structures can be identified by HLA-DR staining in
normal tissue sections (3–6).

1.2. Forces on the Blood vessels are constantly exposed to haemodynamic forces in
Endothelium: Shear the form of cyclic stretch, fluid shear stress and hydrostatic pres-
Stress sures. Shear stress is the major haemodynamic force EC respond
to, whereas vascular SMC responses are more influenced by cyclic
stretch (7, 8).
Fluid shear stress acts in parallel to the vessel wall. It results
from the friction of blood against the inner lining of the blood
vessel wall and is principally sensed by EC (Fig. 3.1; (9)). In
“linear”, unbranched areas of the vasculature, blood flows in uni-
form, laminar patterns and EC experience a mean positive shear
stress, around 10–40 dyn/cm2 in the arterial network and 1–20
dyn/cm2 in the venous microcirculation (see Fig. 3.1). In areas

A Laminar Flow

Blood viscosity, η
R

Volumetric flow rate, Q

B Disturbed Flow

Fig. 3.1. Diagram showing flow patterns for laminar flow (a) and disturbed flow (b)
(adapted from (9)).
 Leucocyte Adhesion Under Haemodynamic Flow Conditions 33

with abrupt curvations or bifurcations, the steady laminar flow

pattern is disrupted by regions of separated blood flow creating
recirculating sites. Shear stress in these regions varies from neg-
ative, zero and positive values (Fig. 3.1; (10, 11)). Parts of the
vasculature exposed to steady laminar flow with high shear stress
are atheroprotective, whereas areas of turbulent, disturbed flow
and low fluid shear stress are prone to develop atherosclerotic
lesions (12, 13).

1.3. Atherosclerosis It is well established that when EC are subjected to disrupted

and the Endothelium flow, they take on an activated pro-inflammatory phenotype that
supports leucocyte transendothelial migration in vitro and in
vivo. Atherosclerotic lesions form at branch points in arteries
where flow is not laminar and leucocyte accumulation is observed
even in very early lesions, with accumulation of T cells as well
as monocytes being well documented in humans (15–17) and
in the ApoE–/– or LDL-R–/– mouse models of atherosclerosis
(18, 19). The potential importance of T cells for progression
of the lesions has been demonstrated using ApoE–/– /Rag-1 or
LDL-R–/– /Rag-1 mice which are defective in both T and B cells,
but not monocytes. Early lesion development in the Rag-1 mice,
compared to wild types, was significantly diminished after 8 weeks
on a Western-type diet (WTD), suggesting that lymphocytes play
an active role in early lesion development (19).
During chronic allograft vasculopathy (CAV), lesions are seen
which are not dissimilar to those seen in atherosclerosis, although
there are several features that are different (reviewed in detail
elsewhere; (20)). As with native atherosclerosis, large accumu-
lations of fibro-fatty deposits have been observed in the sub-
endothelial space as well as proliferating smooth muscle cells
that have migrated from the media of blood vessel wall. These
VSMC secrete inflammatory cytokines and extracellular matrix
proteins, all of which contribute to the progression of the lesion.
As with “native” atherosclerosis, elevated numbers of leucocytes
have been observed adhering to and transmigrating into the sub-
endothelial space in both human and animal models including
increased numbers of CD4 T cells even in the presence of an intact
endothelium in non-branching parts of the vasculature where
shear stress is high (21, 22).

1.4. Use Much of the information regarding EC function and dysfunc-

of Endothelial Cells tion has been derived from cells harvested, grown and studied
In Vitro in culture. EC have been isolated from many different vascular
beds and various species including humans. The most common
approach for obtaining EC is by enzymatic digestion of cells from
large blood vessels, which provides a good yield of high-purity
cells. These can be further purified with magnetic bead separation
34 Lawson, Rose, and Wolf

and use of selective media (23). Human umbilical vein endothe-

lial cells (HUVEC) are probably the most widely used model for
human endothelia (24).

1.5. Use A disadvantage of using cultured cells is the difficulty in recapit-

of a Parallel-Plate ulating the forces that EC are exposed to in vivo, in a culture
Flow Chamber dish. To some extent, this can be overcome by use of two-
dimensional “flow chambers”. A number of different apparatus
have been described including the parallel-plate flow chamber
(25) and the cone and plate viscometer (26), both of which
have been shown to mimic the flows seen in vivo using a two-
dimensional/single-cell monolayer setting to enable molecular
dissection of the responses due to EC alone. Here, we outline
protocols using a parallel-plate flow chamber for long-term expo-
sure of cultured EC to arterial flow conditions, in the presence of
purified T-cell populations.

2. Materials

2.1. HUVEC Culture 1. HUVEC culture medium: Medium 199 with HEPES (PAA)
on Glass Slides supplemented with 20% foetal bovine serum (BioSera) and
L -glutamine (sigma) and penicillin/streptomycin (PAA).
2. HUVEC flow medium: M199 with HEPES supplemented
with 10% FCS; L-glutamine, penicillin/streptomycin;
amphotericin B (PAA).
3. Sterile 1x PBS (10x PBS; for 1 L add 2 g KCl, 2 g KH2 PO4 ,
80 g NaCl, 11.5 g Na2 HPO4, dilute to 1x with ddH2 O and
autoclave before use).
4. 1x trypsin/EDTA (PAA).
5. Glass microscope slides (76 × 38 mm; Fisher Life Sciences)
[sterilise by autoclaving before use].
6. Sterile 9 cm Petri dishes.
7. Human fibronectin (Sigma) diluted to 50 μg/ml in 1x PBS.
8. Haemocytometer (e.g. Fisher LifeSciences).
9. Trypan Blue (Sigma).

2.2. T-Cell 1. 15% EDTA for blood collection.

Purification 2. 1x PBS supplemented with 2% FBS and 1 mM EDTA.
3. RosetteSep human CD4 T-cell-negative selection cocktail
(Stemcell Technologies Inc.). Store at 4◦ C.
4. Histopaque 1,077 cell separation gradient (Sigma).
5. Sterile pastettes (Greiner Bio-One).
 Leucocyte Adhesion Under Haemodynamic Flow Conditions 35

6. RPMI (PAA) supplemented with penicillin/streptomycin,

L -glutamine, 10% FCS (T-cell medium).
7. Haemocytometer.

2.3. Flow Loop 1. Flow chamber and apparatus for recirculating flow loop from
Cytodyne Inc. (www.cytodyne.net).
2. HUVEC flow medium.
3. For total RNA extraction; TRIzol reagent (Invitrogen) (this
will require further reagents including chloroform, iso-
propanol, 70% ethanol, RNase-free pipette tips and tubes,
RNase-free water).
4. For protein extraction; RIPA buffer (20 mM MOPS, pH
7.0; 150 mM NaCl; 1 mM EDTA; 1% NP40; 1% Na deoxy-
cholate; 0.1% SDS), protease and phosphatase inhibitor
cocktails (Sigma P2714, P5726), tray containing ice, 1 ml
syringes and 21-G needles, microcentrifuge (ideally cooled).

2.4. 1. Ice-cold acetone. (Ensure that acetone is only stored in

Immunohistochemistry spark-proof freezers. If this is not available, pre-cool on ice
before use.)
2. 100-ml beakers to hold oversized glass slides. (Slides used in
the Cytodyne setup described below do not fit in standard
Coplin/staining jars.)
3. 1x PBS.
4. Primary antibodies as appropriate (e.g. against CD31;
DAKO).
5. Fluorescently conjugated secondary antibodies (e.g. goat-
anti-mouse-Ig-Alexa 594; Invitrogen).
6. Phalloidin-Alexa 488 (Invitrogen).
7. VectaMount with DAPI (VectorLab).
8. 22 × 50 mm coverslips (e.g. VWR).

2.5. T-Cell 1. RPMI T-cell medium.

Alloproliferation 2. Second HUVEC isolate.
Assay
3. 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester
(CFSE; Sigma) diluted to 1 μM.
4. Flat-bottomed 24-well tissue culture plate.

2.6. PHA Proliferation 1. RPMI T-cell medium (27).

Assay 2. Lectin from Phaseolus vulgaris (phytohaemagglutinin; PHA;
Sigma) diluted to 2 μg/ml.
3. γ-Irradiation source to prevent division of antigen present-
ing cells. (It is possible to use 60 μg/ml mitomycin C
for 25 min [Sigma] if a suitable radioactive source is not
available.)
36 Lawson, Rose, and Wolf

4. [3 H] thymidine ([3 H] TdR) (GEC or Sigma).

5. V-bottomed 96-well tissue culture plate.
6. Cell harvester apparatus and β-counter.

3. Methods

3.1. Cell Culture HUVEC may be obtained from commercial sources (e.g. Promo-
cell, Oxford, UK) or may be isolated from umbilical cords col-
3.1.1. HUVEC Culture lected with appropriate ethical permission and informed consent
from a local maternity unit, according to methods described in
detail elsewhere (e.g. (23)) (see Notes 1 and 2).
1. Place 76 × 38 mm glass slides in 90 mm sterile Petri dishes
and pretreat with 0.5 ml 50 μg/ml human fibronectin for
45 min at room temperature in a Class II safety cabinet.
Then remove excess fibronectin using a sterile pipette.
2. Passage confluent HUVEC cultures following well-
described protocols using trypsin/EDTA or the supplier’s
recommended protocol.
3. Count live cells by Trypan Blue exclusion using a haemocy-
tometer
4. Seed fibronectin-coated slides with approximately 2 × 106
per ml HUVEC in 1 ml of HUVEC medium onto each glass
slide (see Note 3).
5. Incubate slides in a 37◦ C/5% CO2 incubator for at least 4 h
to allow HUVEC to adhere and then flood slides with 12 ml
of flow medium (M199 supplemented with L-glutamine,
penicillin (100 units), streptomycin (0.1 mg/ml), 10% FBS
and 1/200-dilution amphotericin B) and incubate overnight
in 37◦ C/5% CO2 incubator.

3.1.2. T-Cell Purification There are many protocols for purification of human CD4+ T cells
from peripheral blood (28). Protocols employing negative selec-
tion are preferred to minimise activation of the T-cell population
under examination.
1. Collect peripheral blood by venepuncture into tubes con-
taining 15% EDTA (1 ml for every 50 ml blood collected).
2. Purify CD4+ T cells using method of choice (e.g. Rosette-
Sep negative selection cocktail (Stemcell Technologies Inc.)
followed by gradient separation on Histopaque 1,077 and
collection of the buffy coat layer using sterile pastettes).
3. Wash purified T cells twice in 1x PBS/2% FBS/1 mM
EDTA.
 Leucocyte Adhesion Under Haemodynamic Flow Conditions 37

4. Count purified T cells using haemocytometer and Trypan

Blue exclusion to distinguish viable cells.
5. Verify purity by FACS analysis of a sample of cells after stain-
ing with fluorochrome-conjugated anti-CD3 and anti-CD4
antibodies.
6. For enumeration of adhered T cells, they may be labelled
with CFSE by addition of 1 μl of 1 μM CFSE to 5 × 106
T cells in a volume of 5 ml; incubate in the dark for 5 min,
then quench with 5 ml FBS and incubate for a further 5 min.
Dilute to 50 ml with 1x PBS; pellet cells and resuspend as
appropriate. Uptake of CFSE should be verified by fluores-
cent microscopy or FACS.

3.2. Use of A parallel-plate recirculating flow loop system as first described

Parallel-Plate Flow by Frangos (25) may be used for shear stress experiments carried
Chamber for Laminar out over a longer period of time to the traditional setups utilising
Flow Experiments a syringe pump to draw fluid over the parallel-plate flow cham-
with HUVEC ber. The system consists of two reservoirs connected with a flow
chamber (Fig. 3.2) to enable recirculation of the flow media and
therefore the opportunity to acclimatise EC to flow conditions.

Fig. 3.2. Diagram of flow loop apparatus showing the flow chamber, silicon gasket and
the glass slide with the attached confluent monolayer of endothelial cells, which are
held together by a vacuum pump at the periphery of the chamber complex. The flow
chamber has two slits through which flow medium enters and exits the channel. The
(arterial) flow rate is controlled by the peristaltic pump. The medium is recirculated from
the reservoir to the inlet tubing onto the flow chamber and back into the reservoir.
38 Lawson, Rose, and Wolf

The parallel-plate flow chamber consists of the flow chamber,

a gasket and glass slide seeded with EC (Fig. 3.3), all of which
are held in place by a vacuum pump. Additionally, the use of a vac-
uum pump ensures a uniform channel depth (d = 220 μm) across
the flow chamber area (a = 16 cm2 ). Flow media is pumped by
a peristaltic pump from the lower reservoir to the upper reser-
voir at a constant rate. Overflow of excess media drains down the
glass tube and is collected into the lower reservoir where it can be
recirculated. The design of two reservoirs prevents the entry of air
bubbles into the primary flow section upstream of the flow cham-
ber and allows the maintenance of a constant hydrostatic pres-
sure head between the upper and the lower reservoir. Flow media
enters the flow chamber via the entry port. It passes through the
entry slit, over the channel where cells are located, into the exit slit
and leaves the flow chamber via the exit port (Fig. 3.3). The flow
media is then returned to the lower reservoir for recirculation.

E C
G

F
D

A
Fig. 3.3. Cartoon showing parallel flow chamber. When assembling the flow chamber,
the gasket (b) is carefully placed onto the flow chamber (c). The glass slide (a), which is
coated with HUVEC, is added on top of the gasket with the cells facing towards the flow
chamber. A vacuum pump is attached onto the flow chamber (d) to hold glass slides,
gasket and flow chamber in place. Media (grey arrows) enters the flow chamber via the
entry port (e), runs through the slit (f) over the channel back into the slit (g) and exits the
flow chamber through exit port (h). When aligning the gasket, great care is required not
to cover the entry (f) and exit slit (g) which would prevent flow of the culture medium.
 Leucocyte Adhesion Under Haemodynamic Flow Conditions 39

The use of a recirculating system allows for longer term cul-

ture of EC under laminar flow conditions (up to 96 h in our
laboratory) without use of large volumes of cell culture media,
and inclusion of a septum port in the lower reservoir enables
collection of samples of flow media for analysis of soluble fac-
tors released by EC at different time points using appropriate
assays.

3.2.1. To Set Up the 1. Sterilise parallel-plate flow loop system using ethylene
Apparatus oxide.
2. Pre-warm sterile flow media and warm the 37◦ C chamber
and apparatus for at least 1 h before intended use
3. Assemble parallel-plate flow chamber and flow loop accord-
ing to Fig. 3.2 inside a class II safety cabinet to maintain
sterility (see Note 4)
4. Ensure that the connectors on tubing (e.g. Masterflex tub-
ing and connectors from Cole Parmer, London, UK) and
on glassware are firmly attached and close the flow loop
5. Add pre-warmed flow media to the bottom reservoir via
the three-way tap.
6. Align the sterile gasket (Fig. 3.3b) onto the flow chamber
(Fig. 3.3c) in the tissue culture hood being careful not to
cover the channel and slits (Fig. 3.3f, g) on the parallel-
plate flow chamber.
7. The slide (Fig. 3.3a), seeded with a confluent monolayer of
HUVEC, can then be mounted onto the gasket on the flow
chamber and attached immediately to the vacuum pump
(Fig. 3.3d) to hold the flow chamber together. Ensure
great care is taken not to move the gasket and glass slide
on the flow chamber out of place during the process in
order to avoid leakage.
8. After attaching the inlet (Fig. 3.3e) and outlet tubing
(Fig. 3.3h) to the flow chamber, carefully move the flow
loop apparatus to a pre-warmed 37◦ C incubator (Note 5).
9. Place tubing onto the peristaltic pump and observe sys-
tem for signs of leakage. During assembly of the flow
loop, tubing and/or flow chamber itself should be adjusted
to ensure the optimal flow loop conditions, i.e. no hin-
drance of flow and no air bubbles are trapped in the flow
chamber. Air bubbles can be removed using a needle and
syringe placed in the septum port located at the exit of the
chamber.
10. Level of flow media remaining in the reservoir should be
observed to ensure that no leakage has occurred.
40 Lawson, Rose, and Wolf

11. After switching on the peristaltic pump, HUVEC on the

slide are immediately exposed to a laminar shear stress of
>10 dyn/cm2 for 1–96 h.
12. Before co-culture of T lymphocytes with HUVEC, allow
HUVEC to become accustomed to the presence of lam-
inar shear stress for at least 18 h (see Note 6). Position-
ing of a phase contrast microscope within the incubator
before commencement of flow will allow visualisation of
slides whilst they are being subjected to flow to ensure the
presence of an intact monolayer, with cells aligned to the
direction of the flow.
13. If desired, carefully wash the HUVEC monolayer whilst
maintaining flow by removal of excess flow medium in the
lower reservoir and replacement with fresh flow medium
(via the septum port).
14. For prolonged co-culture (up to 4 h) of purified CD4+
T cells and HUVEC, resuspend T cells in HUVEC flow
media at 1 × 107 per ml and inject 2–5 × 106 into the flow
loop via the septum port located in the lower reservoir.
15. At the termination of flow, slides should be quickly
removed from the chamber and processed for analysis
using immunohistochemistry (whole slides), flow cytom-
etry (intact cells), Western blotting (cell lysates) or PCR
(mRNA), and flow media can be collected for measurement
of soluble factors.
16. To harvest cells for flow cytometry, rinse slides briefly in 1x
PBS and place in a clean dry Petri dish (see Note 7). Add a
1 ml “drop” of Accutase (PAA L11-007) to the top of the
slide and incubate for 2–3 min at room temperature. Care-
fully remove Accutase containing disaggregated cells and
place in a 15 ml conical tube containing 0.5 ml FBS. Wash
the slide carefully with 2 ml PBS to collect any remaining
cells. Pellet and process for flow cytometry.
17. To harvest cells for collection of total RNA, rinse slides
briefly in 1x PBS and place in a clean dry Petri dish. Place
0.5 ml TRIzol reagent (Invitrogen) to the top of the slide
and incubate 5 min at room temperature. Carefully collect
the lysate into a clean 1.5 ml Eppendorf tube and follow
manufacturer’s instructions for purification of total RNA.
18. To harvest cells for collection of protein lysates, rinse slides
briefly in 1x PBS and place in a clean dry Petri dish. Place
the Petri dish in a tray of ice and add 0.5 ml RIPA buffer
to the top of the slide. Incubate 10 min on ice, then care-
fully scrape the lysate to loosen cellular material. Collect the
lysate into a clean Eppendorf tube. Push the lysate through
a syringe and small bore needle × 10, then centrifuge at
 Leucocyte Adhesion Under Haemodynamic Flow Conditions 41

13,000 rpm for 10 min to pellet nuclear debris. Keep super-

natant and store at –80◦ C.
19. Use cells exposed to static culture conditions as controls
for all experiments.

3.2.2. 1. At termination of flow, remove glass slides from the flow

Immunohistochemistry loop or take out of static culture and quickly rinse in PBS.
2. HUVEC are fixed by submerging slides in ice-cold acetone
for 5 min followed by 3 × 5 min washes in 1x PBS.
3. During the washes, prepare the primary antibody solution
at the appropriate dilution and aliquot 250 μl onto a clean
dry Petri dish. The glass slide is carefully placed on top of
the antibody solution ensuring HUVEC on the slide are in
contact with the diluted antibody.
4. Incubate for 30 min at room temperature, then remove
slides from Petri dishes, rinse and wash three times with
1x PBS.
5. During the washes, prepare the secondary antibody conju-
gate (e.g. Alexa Fluor 594 goat anti-mouse IgG, Molec-
ular Probe/Invitrogen, UK) at the appropriate dilution
together with fluorescently conjugated phalloidin to stain
F-actin, if appropriate (e.g. Alexa 488 conjugate, Molecu-
lar Probes/Invitrogen, UK).
6. Incubate the slides HUVEC side down in the antibody solu-
tion (250 μl) for 30 min at RT.
7. Rinse slides, then wash three times in PBS before care-
fully adding two drops of mounting medium containing the
nuclear counterstain 4,6 diamidino-2-phenylindole (DAPI)
for visualisation of cell nuclei.
8. Place long cover slips (Fisher Scientific) on top of the
mounting media and store slides at 4◦ C in the dark under
humid conditions to avoid drying out before visualisa-
tion by fluorescent/confocal microscopy (Supplementary
Fig. 3.1).

3.2.3. Analysis of T-Cell 1. Seed a 24-well tissue culture plate with 1 × 105
Functionality After HUVEC/well in triplicate or quadruplicate using a separate
Co-culture with EC isolate to the one used for co-culture under laminar flow
Under Laminar Flow conditions, 24 h before the flow co-culture.
Conditions:
Alloproliferation to 2. During laminar flow co-culture (Section 3.2.1) remove
Third-Party HUVEC medium from HUVEC in the 96-well plate and treat with
60 μg/ml mitomycin C for 25 min to prevent HUVEC pro-
liferation followed by three washes with 1x PBS to remove all
traces of mitomycin C. Replace medium with 250 μl T-cell
medium/well.
Another random document with
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would cheer him up so, wouldn't it? I'm sure we ought to try to make him happy—poor
Tom! He says nobody cares for him!"

Mrs. Triggs had not paid much attention to the conversation, but she now turned her head
sharply round.

"Is that my Tom you be speakin' of? His mother cares for him more'n all the world! He was
such a handsome baby—took arter his father—who were a fine, upstandin' man, but with a
taste for the beer. Tom be made arter the same pattern. An' I says if God and Natur' made
him so, why blame the poor lad? An' he never have given his mother an unkind word!"

"I like Tom very much," Harebell answered her eagerly. "And when Aunt Diana comes back
I'll beg her with tears to let me go and see him, and I'll find him a wife as quick as ever I
can!"

"A wife?" screamed the old woman. "You let my Tom be! What do he want a wife for? He
have a good home, and there isn't a girl in this village who'd do him aught but harm. Idle
worthless hussies they be! Go on with you! A wife, indeed!"

Harebell looked frightened. She said good-bye and slipped away. Miss Triggs said in a
whisper to her:

"Never you mind mother, dear. She don't mean to be rude, but she don't take kindly to a
wife for Tom, and I can't say he ought to have one, unless his heart gets changed, and his
life too!"

Harebell went back to the Rectory slowly and thoughtfully, but when she found Peter and
Nan had put up a swing in the orchard, and were enjoying themselves upon it, she joined
them gleefully. They forgot their squabble, and she was a happy light-hearted child again.

The return of her aunt was the next event. Mrs. Garland kept her till after the arrival, and
when Harebell went home the next day, the whole house seemed to have altered its ways.

There was a man's coat and hat in the hall. A strange man-servant was sitting in the
pantry talking to Andy. A little cheerful bustle pervaded the house. There was a smell of
tobacco in the morning-room. Two or three newspapers and pipes lay on the table.

Mrs. Keith came out of the drawing-room to greet Harebell. The child was so startled at the
difference in her aunt's face that all fear of her vanished. Putting up her slim little arms,
she clasped her round the neck.

"Your ice has gone!" she exclaimed. "Oh, I'm so very glad!"

And Mrs. Keith did not stare, or frown, or reprove her coldly for such words. She looked
tired, but very happy, and there was a light and softness in her eyes that had never been
there before. "Would you like to see your uncle? You must be very quiet, as he is quite an
invalid at present. But I have told him about you. Come this way."

Harebell trod on tip-toe, with eager eyes and a beating heart. On a couch near the open
window was a grey-haired man propped up with pillows. His hands looked white and thin;
his face was lined with pain; he had a hooked nose, and thick bushy eyebrows, but when
he saw Harebell, both his lips and eyes smiled.

"The little niece! Come and welcome a poor old sick soldier, who isn't worth the trouble he
gives."
"I'm so very glad you're here," said Harebell standing before him with clasped hands. "Me
and God have talked you over often, and God seemed to tell me He would send you back
soon."

If Colonel Keith was surprised at such a welcome, he did not show it. He looked at his wife,
and his eyes grew soft and tender. Then he spoke to Harebell.

"Life deals hardly with those who quarrel with her. Don't you let your passions ever get
mastery of your love, little woman."

"I don't understand a bit what you mean."

"And there is no need that you should," said her aunt a little sharply.

Colonel Keith put his hand on his wife's arm, as she stood by his couch. Her voice softened
at once.

"Come and sit down and talk to your Uncle Herbert; I must go away for a little. I have
letters to write."

So Harebell took a chair by the couch, and when her aunt had left the room, her tongue
began to move, and she poured into her uncle's ear a flood of talk. She told him of her
home in India, of Chris, of the Rectory children, of Tom Triggs and his sister and his
mother, of Fanny Crake and her mother, and the little cottage. But she did not talk much of
her aunt, and Colonel Keith noticed the omission. Harebell found him as good a listener as
Mr. Graham. She ended up by saying impulsively:

"I do like you so much, Uncle Herbert! You quite understand what I mean. I haven't to
keep explaining; Andy thinks me quite easy and understandable, he says, but Goody is
always saying I amaze her. I've always said I like men better than I do women."

"But you can't and must not like me better than your aunt!" expostulated Colonel Keith.
"You don't know what trouble I brought upon her by my hot temper and wicked pride! She
has suffered, and yet now has no reproach upon her lips. I'm a bad lot, and she's a saint!"

Harebell did not answer for a minute; then she said solemnly:

"I'll try and like you both the same."

Certainly she found life much gayer now. Her aunt and uncle were much together, and she
was left more than ever to her own devices; but when she was with them at meals, her
busy tongue was no longer repressed. Her uncle encouraged her to talk, and liked to hear
all about her lessons and play. Her aunt's voice was getting softer, her smiles were more
frequent. And as for Andy, his old face was radiant with happiness.

"Ah! The good old times have come back," he said to Harebell. "The days of mourning are
over for this old house."

The little girl nodded.

"I haven't to hush about the house any more, I can almost make as much noise as they do
at the Rectory."

It was not very long before she begged permission from her aunt to go and see Tom Triggs
again. Mrs. Keith did not actually refuse her; but she said she must wait. And then one day
at the Rectory, Nan informed her that Tom was very nearly well, and was going away from
the village altogether. Harebell was much surprised, and rather uneasy.

"Why is he going away? How can he leave his mother? Oh, I must see him, and ask him all
about it."

It happened to be a Saturday, and every Saturday, Harebell dined at the Rectory and spent
her half-holiday there.

"We'll go and see him this afternoon," suggested Nan. "Peter wants to see him, don't you,
Peter? Tom is making him a box with lock and key to keep his birds' eggs in. He's out of
hospital, and living with his mother."

"That will be lovely!" exclaimed Harebell. It was only when she was actually starting with
them, that she remembered her aunt had not given her permission to do it. With a little
hesitation, she told Nan and Peter that perhaps she had better not go.

"Go home and ask your aunt," said Peter; but Nan vehemently opposed this suggestion.

"We have no time. It's such a long way off; and if we go to the village, we can go on to the
woods and have some fun."

Harebell hesitated.

"I'll go," cried Peter, "on my bicycle. I'll go, and catch you up before you get to the village."

Peter had only lately owned a bicycle, so he liked to use it on every occasion.

Harebell brightened up.

"That will be jolly! And I don't believe Aunt Diana will say 'no' to you."

He rode off at full speed, and the little girls walked in the direction of the village. They had
barely reached it before he overtook them.

"Can I go?" Harebell asked him eagerly.

"Yes," he said.

She skipped for joy. Tom's future held a big place in her thoughts, and she was delighted
to see him again.

"Oh, do come on," she besought the others when the village sweet shop brought them to a
standstill.

"I want some bull's eyes," said Peter.

He wheeled his cycle up to the shop, leant it against the wall, and then disappeared inside.
Nan followed him.

Harebell stamped with impatience: then determined not to wait for them, and walked on
quickly to Mrs. Triggs' cottage.

She had one more check.

Colonel Keith was coming out of the post office and met her. He was rapidly getting
stronger, and now got about in a low pony trap, which for the present, he hired.
"Hulloa!" he said. "Where are you going?"

"I'm with Peter and Nan. We're going to see Tom Triggs. He's going away."

"Oh, that's your friend, is it? And your aunt knows?"

Colonel Keith knew all about the forbidden visits; for Harebell had besought him to help
her, and he had been doing his best in that way.

"Oh yes," Harebell said with assurance; "she has given me leave to-day."

"And is it to be a wife, or work, or a cottage?" Harebell laughed, and ran on. She was
breathless when she stood at the cottage door.

Tom himself came to open it, and smiled all over his face when he saw who it was.

"Why, I thought you and me was friends no longer!" he said.

Harebell seized hold of his hand.

"Oh, Tom, dear Tom, don't go away! Do stay and have a little cottage here. I don't want
you to go."

He led her into the little parlour.

"Hessie be out to-day, and mother and me be mindin' each other."

"And how's your leg?"

Tom swung it slowly to and fro.

"Near as good as ever 'twas! You see, missy, I be what you call going on the tack. And I
have an offer of work in a town firm. 'Tis a contrac' for some big house, ten mile or so
away. 'Twill be a change and a beginning! But I ain't goin' so very far arter all!"

Harebell smiled.

"Did you get any message about Fanny? That's what she said—the drinking to be given up
first, and then the work and then the wife?"

Tom's eyes twinkled.

"That there Fanny be too forward. Her must wait till her is axed!"

"Oh, but I asked her; I besoughted her; I begged her with all my heart, to marry you just
as you were, and very quickly too! She was a great disappointment to me; I did hope she
would have married you directly you came out of hospital!"

Tom threw his head back, and laughed aloud. There was a clearer look in his eyes, and he
held his head higher than he had ever done before.

"I shan't sit down and cry, if her don't want me," he said. "I can't keep a wife just at
present. The girls be too expensive in these days."

Harebell was silent. This seemed quite a new Tom; a man who could scorn a wife was
beyond her comprehension.
"And you're never going to a nasty public-house again?"

"Ay, well, there be no tellin'; but I ain't visitin' the 'Black Swan' just now."

"Tom," said Harebell looking up at him with solemn eyes, "are you through?"

His eyes met hers rather gravely.

"Through? How d'ye mean?"

"Through the Door? You know I almost think you are. And I believe that's the first thing of
all to be done. I wonder if you did it first."

"I wonder," said Tom, in a low grave voice, looking over Harebell's head as he spoke.

"I wish you'd tell me. Because we'd be on the same side, then. I ask God every day to
keep me on the right side, the inside you know, and not to let me run out."

"Hi! Tom! Where's my box?"

Peter's shrill voice coming up the garden-path interrupted them. There was no more
opportunity for serious talk. Tom took the children to the backyard where he was working,
and for half an hour they stayed there chattering and watching him complete Peter's egg-
box. Then they left him, and went on to the woods, where they had a very happy time.

Coming home, Harebell said:

"Haven't we had a jolly afternoon? And isn't Tom Triggs nice? Quite different to when he
was drinking!"

Peter edged up to her.

"I want to tell you a secret. Go on, Nan; it isn't for you."

Nan laughed.

"I'm not a bit curious. You never have interesting secrets, Peter."

She obligingly crossed the road. Peter sank his voice to a whisper.

"You needn't think your aunt gave you leave to go and see Tom, for she didn't. You'd
better keep quiet about it, and not let her know you went."

"Oh, Peter, what do you mean?"

"Don't shout, you stupid! I did go to ask her; but she was out, so I couldn't!"

"But you told a lie! You said she had given me leave."

"I didn't!" said Peter, a little sullenly. "You asked me if you could go, and I said, 'Yes.' I
didn't say anything about your aunt!"

Harebell stopped still in the road. He dragged her on by the arm.

"There's nothing to make a fuss about! I didn't tell a lie. You needn't say a word about
going to Tom. Tell her you went to the woods, if she asks you."
"But I met Uncle Herbert, and told him I was going to Tom, and I told him aunt had given
me leave to go!"

"You were a little fool."

Then he changed his tone.

"Look here, Harebell, don't you get me into a row. Don't split, will you? You aren't a sneak,
and it would be awfully mean to tell tales. You see, your aunt and uncle are coming to
dinner to-night at our house, and they'd make a row over it. I only wanted you to have a
good time. I needn't have interfered at all, and it wasn't a lie, and of course they'd think it
was, they'd never understand. I'll never forgive you if you split!"

"Oh, I won't say a word about you! You needn't be afraid."

Harebell's voice was scornful.

Peter got rather red in the face.

"Such a fuss about nothing!" he muttered. "I don't expect your aunt will care where you've
been. You can tell her you had to come with us; you couldn't help yourself."

Harebell did not speak. Then she said slowly:

"I have told lies myself in India, but not since I've been in England. I couldn't have done it,
as you did!"

"I didn't tell a lie."

Peter left her and joined Nan. They were rather a constrained trio for the rest of their walk.
Nan remarked—

"You and Peter don't seem to have enjoyed the secret."

And Harebell said quickly:

"I shouldn't think so!"

When they reached the Rectory, Harebell said good-bye. She kissed Nan, but turned her
back on Peter.

"Remember!" he called after her threateningly.

"You needn't be afraid!" she retorted.

But she entered her aunt's house with a sinking heart.

CHAPTER IX
IN DISGRACE
HAREBELL had her tea in the schoolroom alone, as she very often did. Andy waited upon
her.

"There be visitors in the drawin'-room," he said. "'Tis like old times, gentlemen a-comin'
here! For years we've had nothing but ladies, and a few on them. Sir Robert Ferguson and
his lady have been to tea, and the Colonel be quite spry. What have you been a-doin' to-
day? Somethin' to get a scoldin' for! Mistress says to me, 'Tell Miss Harebell to go to her
bedroom after she has finished her tea, and stay there till I come to her.'

"Then she knows," said poor Harebell with a deep sigh. "Did she look very angry, Andy?"

"Very cold and quiet," said Andy. "What have you been doin'?"

But Harebell for a wonder would not tell him.

"Mayn't I go and see Chris?" she asked.

"Best not. I've given you the message exack'ly as it were given me!"

Harebell's tea almost choked her. She left it unfinished and went upstairs.

"It's no good," she said to herself as she sat down disconsolately in her little chair by the
window, "to say I'm not frightened of Aunt Diana, because I am; and she'll say I've
disobeyed her, and so I did. And I never, on my word and honour, meant to be naughty to-
day. God knows about me; that is one comfort. He knows I didn't mean to be naughty. And
as for Peter, he's the wickedest, meanest boy I ever knew, and I don't think I shall ever be
friends with him again!"

When she heard her aunt's step at last, she stood up with a beating heart.

To her aunt, as she came into the room, Harebell looked the picture of guilt.

Mrs. Keith's face was very hard and stern. "I have come," she said, "to have some
explanation from you of your conduct this afternoon. You not only directly disobeyed me,
and went off to see that drunken man, but you told your uncle a lie, and said that you had
my permission to do so. Do you remember what I told you when you first came here about
lies?"

"Yes," said Harebell miserably. "I remember quite well, but I haven't told a lie, I really
haven't."

"Don't try to cover up one lie with another; that is only making matters worse."

Harebell was silent. What could she say?

"Have you anything to say for yourself?"

"I don't know," faltered Harebell; "it was—was a mistake. I—I thought you'd given me
leave."

"How can you have the face to say such a thing to me? You know I did not."

"I didn't tell a lie," Harebell murmured.

Her aunt looked at her with an expression of disgust. "I suppose I was foolish to think that
you were a truthful child. My eyes are open now. If you had only frankly confessed, I might
have regarded it more leniently. However, I keep my word, I shall send you to school after
the summer holidays. Never will I have a child in my house who deceives, or tries to
deceive me."

Harebell began to cry.

"Oh," she sobbed in the depths of her despair, "if you were God, you'd understand!"

"Don't add hypocrisy to lies," said her aunt sharply. "You are not to come downstairs to-
night. Go to bed, and remember that I might have forgiven your disobedience—but I will
never forgive lying!"

She left the room.

Harebell flung herself on the floor.

"I shall never, never be happy again! I'm not a liar, I'm not even disobedient; it's all a
muddling mistake, and it's Peter, and not me, who ought to be punished!"

She began to feel justly angry with Peter.

"He'll go on living and people will think him a good boy, and I shall be thought a liar for
ever and ever! And school is a prison, and—oh, I never thought of it! I shall have to leave
my darling Chris! My heart will be broken. I wish I could die!"

She lay there sobbing her heart out, and Goody, entering the room later, was much
astonished and alarmed.

Harebell raised a white tear-stained face to her.

"I ought to be in bed," she said slowly. "Aunt Diana said I was to go. She thinks I've told a
lie, and I haven't, Goody, and I'm to be sent to school in disgrace."

"Dearie me! What an upset! You must get hold of the Colonel. He'll put things right."

A gleam of hope stole into Harebell's eyes; then it died away.

"He thinks I told him a lie. He won't help me. I'm what you call doomed, Goody."

She began to undress. She would give no explanation to Goody, for fear of inculpating
Peter.

She heard a carriage come to take her uncle and aunt to dinner at the Rectory.

She wondered if her aunt would tell them all there of her wickedness; and if so, how Peter
would feel when he heard it. She began to hope that perhaps his conscience would compel
him to confess and to clear her. But she remembered that Nan said once that Peter never
owned himself to be in the wrong.

Goody went away at last, and she was left alone in bed.

It was hours before she slept, and when she did, she dreamt that a school-mistress with
flaming red curls and bony hands was pushing her down some steep steps into a dark
cellar!

When the morning came, she wondered at first what awful thing had happened to her. The
birds were singing. It was a lovely sunshiny morning in June, and when she remembered
the trouble in which she was, she felt that some help would come to her.
"Aunt Diana won't really send me away. Peter will be sorry and tell."

Yet as she dressed, fear overcame hope. She ran softly downstairs and made her way to
the stable. Chris neighed in delight when he heard her step, and rubbed her all over with
his nose. Of course he was told all, and Harebell clasped him passionately round the neck.

"If they send me away from you, I shall die," she assured him.

Then the prayer bell rang, and she slowly went into the house. Her uncle did not come
down to breakfast, but had it in his room. He was still quite an invalid. Mrs. Keith hardly
spoke to her, but as she was leaving the breakfast-table, she said:

"Are you ready to confess the lie you told? Are you sorry?"

Harebell looked at her aunt nervously.

"I feel," she said, "if I said I had told a lie, that would be a lie."

"You will be in disgrace till you do confess," said her aunt shortly.

Harebell went to the Rectory with a heavy heart.

She could hardly say "Good Morning" to Peter. Nan asked her at once what was the matter,
and Harebell looked Peter straight in the face as she said:

"I'm in disgrace. Aunt Diana says I've told a lie, when I haven't. I'm going to be sent away
to school, and I shall never come back again!"

"Oh yes, you will," said Peter fast and eagerly, whilst his cheeks got hot and red. "School is
awfully jolly; and you always come home in the holidays. I wish I could get sent to school.
No such luck for me."

"School is enchanting," said Nan. "A girl in the next village goes to a boarding-school, and
she loves it. I don't pity you, if you go to school, Harebell."

"And how can I part with Chris?"

"You'll have him in the holidays," said Peter; "and p'raps dad will keep him for you when
you're away, and we'll exercise him for you!"

This was too much for Harebell. She turned upon Peter in a blazing fury.

"I hate you! I'd like Chris to kick you off and tread on you, if you ever dare to ride him. He
knows all about you. I've told him. And I've told God, too, and I'll never play with you
again, and I won't speak to you, and if you leave any of your birds' eggs about, I will
smash them in bits!"

"My dear child!"

Mrs. Garland had come into the room unnoticed.

Harebell's fury was stayed. She hung her head.

Nan was looking quite frightened; Peter red and uncomfortable.

"What has Peter done to provoke such an outburst?" Mrs. Garland said.
Harebell flung herself into her arms.

"I can't say, but I never tell lies, do I? Do I? Aunt Diana says I do."

"And does Peter say you do?"

Mrs. Garland looked at her small son very keenly.

"No—no!" he stammered. "I never said she did. It isn't my fault!"

"She's going to be sent to school, and she doesn't like it," said Nan. "Her aunt is angry
with her."

Mrs. Garland tried to discover what had happened, but neither Peter nor Harebell would
tell her, and Nan was as much in the dark as she was.

Miss Forster interrupted them, and lessons began. Harebell naturally did hers very badly,
but Miss Forster saw she was much upset and made allowances. When twelve o'clock
came, they went into the garden to play. Harebell left the others, and wandered round the
paths in the shrubbery, feeling very miserable.

"I'm not a bit like a child who is inside the Door," she told herself. "I've been in a temper
with Peter, and I'm sure I oughtn't to be. Jesus Christ wasn't angry when He was ill-
treated, and I know He doesn't want me to be. But it's very hard not to call Peter names.
He is the meanest—sneakiest—oh, I mustn't! But how can I love him when it's all his fault,
and not mine at all!"

It was a hard struggle with Harebell. Her sense of justice was great, and her punishment
she knew was not deserved. But before she left the Rectory she went up to Peter.

"I'm sorry I said I would like to smash your eggs. I won't. I'll try and forgive you. But
you're making me awfully miserable, and you know you are."

Peter walked away from her.

"You're making a fuss about nothing," he said; "you chose to think I meant what I didn't
mean. It was only a mistake."

He was feeling miserable too, but he would not allow it, and tried to make excuses for
himself.

"Such a fuss!" he repeated to himself. "It isn't worth thinking about. I'm sure Mrs. Keith
won't really send her to school. She'll forget all about it in a few days."

When Harebell went home she found her uncle pacing the garden paths. He called her to
him cheerily, and wished her "Good morning" as usual.

Harebell looked up at him wistfully. She longed to confide in him.

"Well," he said, "how have the lessons gone?"

"Very badly," said Harebell, shaking her head. "I've an extra lesson to learn for not
attending; but my soul was in such a state, that I couldn't work at sums, so they got
jumbled up."

Her uncle sat down on the garden seat and drew her to him.
"Tell me about it, little woman."

Harebell worked her fingers in and out of his coat buttonhole nervously.

"Do you think I told you a lie yesterday? I didn't. It was a mistake, not a lie, and Aunt
Diana won't believe me."

"How was it?"

Harebell was silent.

"I can't explain myself—but I'm telling true. And if—"

Here she got excited and waved her hands about.

"If Aunt Diana was to burn me, or flog me, or drown me, I couldn't say anything but that I
didn't tell a lie!"

"Try and explain," said her uncle gently. "Your aunt has such a horror of deceit and lying
that perhaps she did not give you time to speak."

"I can't tell her. She won't believe me. But oh, Uncle Herbert, I can't live without Chris. If
she sends me away from him, I shall die. I shall never live to come back. Please don't let
her send me away to school."

"I hope that will not be necessary."

Mrs. Keith came up to them.

"Harebell, go into the house. Until you confess your fault you are in disgrace."

Harebell turned disconsolately away. Colonel Keith said something to her aunt, which she
could not hear, but she heard her aunt's clear cold voice reply:

"It is her mother over again! I warned her when she came to me. There is no mistake. She
disobeyed, told a lie, and sticks to it. I will not undertake the charge of her any more. I
shall send her to a strict school, for I will not be responsible for her training."

With despair in her heart, Harebell crept indoors.

The following days were very unhappy ones. She grew very quiet, moped about the house,
lost her appetite, did not sleep at nights, and got a peaked white look upon her face. But
as time passed, she grew accustomed to her aunt's cold displeasure, and as no more was
said, began to hope that perhaps she would not carry out her threat.

The summer holidays came. The Rectory children went away to the seaside with their
parents.

For over a month, Harebell had not been allowed to ride out on Chris; but now, owing to
her uncle's intercession, she was permitted to begin her rides again.

Mrs. Keith hardly ever took any notice of her, but at last one day she called her to her.

"I have made all arrangements about school, and you will go next Monday. Goody will take
you. The school is at Eastbourne."

Harebell looked at her aunt with frightened miserable eyes.

Then her aunt said in a gentler tone:

"You have still four days before you. If you will frankly confess, and express real sorrow for
the lie you told, I may be induced to forgive you. Your uncle has made me promise that I
will."

Harebell's lips quivered, but she said nothing. She knew there was no hope now. Peter was
away, and was not coming home till after Monday. Unhappy as she was, the thought never
crossed her mind that she might break her promise to Peter.

The four days gradually slipped away.

She watched Goody pack her clothes; Miss Triggs had come round to make her some new
frocks, but she, as well as Andy and Goody, considered that going to school was nothing so
very bad after all. The only comfort that came to her was hearing from Miss Triggs that
Tom was getting on splendidly; he had signed the pledge and was keeping it.

"He's a first-class workman, Tom is, when he's sober, and we've heard his master thinks no
end of him."

Harebell was nearly desperate when Sunday came, and when she laid her head on her
pillow in the evening a tempting plan came into her head.

This was to get up very early on Monday morning, saddle Chris, and ride off with him out
of reach of all the people who were taking part in sending her to school.

"I shall go along and get my food in farmhouses where they make nice hot bread and have
cream with their porridge. I have five shillings of my own, and that will last a long time. I
will get lost where no one can find me. And then Peter will be sorry and confess what he
did, and aunt will be sorry too!"

The more she thought about this the more easy and delightful it seemed to be.

"Aunt Diana wants to get rid of me, and, if I go away, she'll be glad!"

Then, after a good deal more thinking, she fell asleep.

CHAPTER X
A LITTLE RUNAWAY

IT was a lovely summer morning. Harebell woke up a little before five o'clock. With a set
determined face she got up and dressed herself, stepping about her room as quietly as
possible. She tied up a nightgown and brush and comb and toothbrush in a bundle. Then
she began to think that she might want more clothes than that. She took a few things out
of her drawers, and put them into a red cotton bag which she tied round her waist.

Then on tip-toe, she stole downstairs, and softly unbolted the back-door. It was easy then
to find her way to the stable. Andy had taught her how to saddle Chris, and in about half
an hour's time she had got free of the house, and was cantering along the country lanes.
Then she remembered that she had not said her prayers. Her conscience began to trouble
her. Was this like a child of the Kingdom? Harebell refused to let herself think. In a
whisper, she gabbled over her prayers; for she felt that she wanted God to take care of
her, though she did not mean to mention her plan in her prayers to Him.

The fresh air and the birds' singing did not seem as enjoyable to her, as she expected they
would be. She passed through the village as quickly as she could, and took the road that
the signpost said led to London.

"Everybody goes to London," she said. "But I will stop before I get there. I'll find a nice
pretty farm, with apples in the garden, and they'll give me some breakfast."

But as time passed she began to feel hungry, and no pretty farm came in sight. The
country was singularly desolate. She came upon two or three small cottages by the
wayside, and an inn; but none of these seemed to her attractive enough for breakfast. At
last she turned up a leafy lane.

"I must try and lose myself thoroughly, Chris," she said; "so that nobody can possibly find
me and take me back. I feel quite frightened now, when I think of Aunt Diana finding me
gone. How very angry she'll be!"

Childlike, she was living entirely in the present. Her future never troubled her. The lane
wound about in a wonderful way, then suddenly ended. A white gate appeared and a high
wall on either side of it.

"This must be a house," said Harebell to herself.

She found the gate open and rode up a neglected drive; nettles and rank grass flourished
on either side of a mossy road. Overgrown shrubs and thick trees lined the way.

Her heart began to beat excitedly.

"It's like the palace grounds of the Sleeping Beauty. I wish I could have a real adventure."

The drive seemed an interminable distance to her, but at last, to her great delight, she saw
a big grey house in the distance. It looked still and deserted.

When she came up to the big flight of steps leading up to the front door she persuaded
herself that it was, indeed, the Sleeping Palace. Slipping off Chris, she let him turn aside to
munch at the long grass on the lawn, and then mounted the steps with eager expectancy.
Would the door open at her touch? Would she go in and find the remains of feasting in the
great hall, and the servants all asleep at their posts?

Alas! the door was fast shut and barred, the windows were shuttered, and through a small
peephole in a broken shutter, she saw that the inside of the house was empty and
unfurnished.

Slowly and reluctantly she turned away; then, seeing a side path near the house, she ran
along it, wondering if the back of the house would prove more cheerful than the front. She
found a side door, and to her joy, as she turned the handle and pushed, it yielded to her
touch. The next moment, she was inside a long wide passage. It was light, and looking up,
she saw there was a big glass dome high up in the centre. Rather fearfully she made her
way along, till she reached the centre hall. A great staircase wound up to a gallery round
it. She was just mounting the stairs, when she suddenly heard a man's laugh.
Now she was frightened. Into her brain rushed stories of ogres, giants, burglars, and
criminals. Panic seized her; she fled back along the passage, missed her way, got into
another part of the house, and could not find an open door anywhere. Then she screamed.
It seemed like some hideous nightmare. She beat and kicked against a door with her
hands and feet. The horrible thought came to her that she had been purposely locked in,
and that some wicked man would come and kill her.

Suddenly, from behind, a big hand laid hold of her shoulder. She screamed louder than
ever in real terror, and then she turned to confront Tom Triggs, and to hear him say with a
little gasp of bewilderment:

"Why, I'm blest if it ain't little missy!"

She clung to him in a tempest of sobs.

"Oh, take me out! Dear Tom, save me! Where am I?"

The next moment a door was opened and she was in the fresh air, with the sun shining and
the birds singing, and Chris still calmly munching the grass a little distance off. It took
some minutes to soothe and calm her, but Tom did it. He was in his working clothes, with
his carpenter's apron on, and looked strangely out of place in this great empty house.

"It's the funniest thing out that you should have come straight to the very house I'm
workin' at. Me and my mates were havin' our breakfast in the back yard. We are doin'
repairs to the stables, and all on a sudden we heard a scream, and it seemed to come
from inside the house, an' I come along to find out whether it be a ghost or a h'owl, and
then I catches sight of you a-beatin' your fists against a door. Now, do you just tell me
what has brought you here. Did you come to find out whether good-for-nothing Tom were
keeping off the drink?"

Harebell smiled through her tears, but she kept a tight clutch of Tom's hand.

"I didn't think of you. I didn't know you were here. I was a dreadful coward, but I felt I
was lost a good deal more than I meant to be. And generally when I'm frightened, I ask
God to take care of me; but I couldn't, and I felt He was a million miles away from me,
and wouldn't dream of coming near me. And then I knew it was because I must have run
outside the Door, and wasn't safe any more!"

She spoke with feverish intensity. Tom looked at her and then at Chris in a puzzled sort of
way. Then he sat her down on the broad balustrade at the bottom of the steps.

"Take yer time, missy. Tell me just how you come to be so far away from home this
morning!"

Then Harebell poured it all out, every bit of her trouble. She felt that she could even tell
Tom about Peter's deceit, after making him promise that he would not tell any one. And
Tom listened and rubbed his head, and then delivered his verdict.

"You must go back, missy; there's no help for it. You must get you back!"

Harebell began to cry. She was tired and hungry. She began to wonder how she had dared
to run away in such a fashion.

"Aunt Diana will be so very, very angry."

"But she'll be in a terrible state about you now. You can't bide alone in the world, trampin'
the roads without food and money. It be a stoopid thing to do—"

"I s'pose you haven't got a cottage yet? Couldn't you take me somewhere? I'm afraid of
Aunt Diana now. She'll never forgive me!"

"You must get you back," Tom repeated with conviction. "It be bad you're comin' off in that
fashion, but every hour you stay away, it be badder!"

Harebell looked up at him beseechingly.

"I don't know what to do. I can't go back."

"Oh yes, you can! I'll come a bit of the way with you, and if you trot your pony pretty fast,
you'll get home not so very late for breakfast after all. Would you like a sip of hot tea? You
wait here a minute."

He disappeared, but soon came back with a hot tin of tea, and some bread and cheese.

"'Tis mos' remarkable you comin' away in a straight line to the house which I be workin'
on! How did you do it now?"

Harebell drank the tea thirstily, then she looked at Tom gravely.

"I s'pose God brought me to you, so that you would tell me how wicked I was, and send
me back. I used to think when I first knew about you, Tom, that you were much wickeder
than I was. Now it's me that is wicked, and you're trying to make me good. It's dreadfully
wicked to run away, isn't it? Almost as bad as telling a lie."

"It's a poor thing to do—to run away," said Tom slowly; "but I don't know that I ain't just
done it myself! You see, I knowed how my old pals would be gettin' over me, so I come
away twelve mile off to make a fresh start where I couldn't be baited!"

"But you didn't run away from home, Tom. Your mother and sister knew you were coming."

"Bless their hearts, that they did! And I be gettin' along fine. And some day I hopes I'll
come back and be able to look my fellow-creatures straight in the face. For I shan't be
feared then o' nobody. An' I do allow 'tis a happy thing to feel inside the Kingdom's Door,
missy. I humbly 'ope I've crawled through, and the Lord be holdin' my feet straight, and
my mouth from even wanting the accursed stuff; and He have got me by His hand, so I
just steps behind Him, and He goes first."

Harebell smiled for the first time.

"Oh, Tom dear, I'm so very glad. I always did know you would get through soon. When did
you do it?"

"Well, I can't rightly say as to day an' hour—but I had a try in hospital, and then agen at
home—an' it seemed to me as one day I was for goin' in, an' the next for comin' out, an' I
didn't get much forrarder, so at last I gets down on my knees and tells the Lord He must
please do it all Hisself, for I were come to the Door an' He must do the rest. Bless His
name, He seemed to stoop right down an' get hold of me—a reg'lar safe grip—and there I
be—very afeared of myself, but very sure o' Him!"

"And do you think I've been naughty and so He's put me outside? Oh, Tom, do you think
you're inside now and I'm outside?"
Harebell's lips were quivering.

"I ain't no scholar, missy, but there be one chapter in the Bible I reads over and over and
over! 'Tis the one you mentioned first about the Door. If we be inside the Door, I take it we
be in the sheepfold; and if we be in the sheepfold, we be the sheep; and if we be the
Lord's sheep, He has us safe, sure enough, for it says, 'Neither shall any man pluck them
out of My hand.' You be right enough—just a slip—and you're a-goin' back now to say
you're sorry—an' I'm a-comin' a bit o' the way with ye!"

"I haven't said my prayers properly this morning," Harebell confessed with shame. "I
gabbled them through. I'll just speak to God here, if you go away—and tell Him I'm sorry."

Tom moved away, rolled up his apron, then caught Chris, and by the time he joined
Harebell again, the cloud was off her face.

She mounted Chris, and Tom walked by her side till they reached the high-road.

"There!" he said. "Now 'tis a straight road home, and you can't miss it. Good-bye, little
missy; and just put up a prayer for good-for-nothing Tom, will you?"

"I will," promised Harebell, "but p'r'aps You'll never see me again. I'm goin' to be sent to
school, you know."

She conquered a rising sob.

Tom looked at her thoughtfully.

"Ay, 'twas through me, you be in this trouble! Well, p'r'aps I can help of 'ee out."

"It will be too late. I shall be gone," said Harebell.

Tom rubbed his head.

The little girl added, "And you mustn't tell about Peter, you promised not to; and I don't
mean to tell."

"Well, you be doin' a fine thing, a-bearin' his fault."

Harebell rode away, waving her hand to her friend.

He looked after her in perplexity. "If I were a scholar now! But I'll venture on it!"

He returned to his work with a plan in his head.

And Harebell rode on home, feeling more and more frightened and unhappy as she drew
nearer the village.

"It all seems as bad as it can be, and when I say I've seen Tom, Aunt Diana will think I
went to him on purpose, and it will make her angrier still!"

Presently she met Andy at the entrance to the village. He threw up his hands.

"Ay! You naughty child, we've all turned out to catch ye! To think of your going off for a
ride this very morning when you're to go to school."

"I'm sorry, Andy. I'm coming back!"

"Comin' back! High time, too. The missis an' the master be in a terrible way. What did you
go to do it for?"

Harebell did not answer. Even Andy, her friend, was scolding her.

The house was reached. Andy took her pony, and when Harebell reached the front door,
her aunt met her in the hall.

"Come in here," she said. "Where have you been? Did you not know a cab was coming at
ten o'clock to take you to the station? It is now nearly eleven."

She drew her into the morning-room. Colonel Keith was not there. Harebell's heart sank
within her. She looked up at her aunt. Somehow or other, Mrs. Keith was not looking as
angry as she had expected.

"I am very sorry, Aunt Diana, but I meant to run away and never come back again; I quite
meant to. And—and—I met Tom—I didn't mean to meet him—he and me think God
managed it, and—and—he made me come home again."

There was silence.

"Where did you meet him?"

"At an old, old house far away. I found it by accident and—" here Harebell's love of
romance seized her, and she forgot she was in disgrace—"do you know it was exactly like
the palace of the Sleeping Beauty. It was still and silent, and the weeds were enormous;
and I quite hoped to see everybody asleep, and all that was left of the feast. And then I
got into the house and found it empty and dark, and I was dreadfully frightened, and I
couldn't find my way out, and I thought I was locked in; and then I screamed and
screamed, and Tom heard me and came to me, and he's the carpenter who's mending the
stable there!"

She paused for breath.

Her aunt was silent for a moment. She seemed to be turning over things in her mind.

Harebell put her arm out timidly and touched her aunt's arm.

"Do please forgive me, Aunt Diana. I know it was wicked of me to run away. I knew when I
did it that it was, and that made it worse, didn't it!"

"What made you come back?" her aunt asked sharply.

"It was Tom. I wasn't even going to do it for him, but when he told me he was inside the
Door of the Kingdom and would never drink any more, I was so glad, it made me—well, it
made something different in my heart—and I knew I must come back if you—if you
whipped me to death!"

She ended her sentence with desperate emphasis.

"I have never yet raised my hand against you," her aunt said gravely.

"No, but I thought you might," Harebell replied quickly. "I thought of such a lot of things
you could do to me; but, you know, it was God and Tom who made me come back. I had
to."
"It was exceedingly naughty of you to think of running away. If you had gone on, you
might have met with accidents. We should, of course, have followed you and brought you
back before the day was over. And nothing then would have prevented my sending you to
school to-morrow. A little girl who acts like that wants a great deal more discipline than I
can give her. But as you turned back of your own accord, I am going to forgive you. I have
received a letter from Mrs. Garland this morning. If you had been here at breakfast-time,
you would have heard about it. Of course, the letter has explained what you ought to have
explained to me long ago—"

Harebell's eyes were open wide.

"What?" she gasped.

"It seems that Peter has been unhappy a long time, and confessed to his mother yesterday
that he was the cause of your disobeying me. Why did not you tell me so before?"

"I—he—I promised him I wouldn't tell," faltered Harebell.

"You had no right to promise such a thing. It was not being frank with me, and led me to
think what was not true—"

"I—I told you it was a mistake I made, and not a lie," said Harebell. "I couldn't explain
properly; I really couldn't, Aunt Diana."

Tears came into her eyes. She was relieved that she was cleared of untruthfulness, but she
still seemed to be in disgrace.

Then Mrs. Keith spoke more gently:

"I want to be fair with you, Harebell. I am deeply thankful to find that you did not tell me a
lie, and to think that I can still trust your word. And for the present, I shall not send you to
the school I intended for you. As I told you just now, if you had not come back of your own
accord, I should still have done it. But as it is, Miss Forster will still continue to teach you. I
am sorry to think that there is so little confidence between us that all this trouble has been
the result. You ought to have told Peter at once that you could not withhold truth from me.
You did not tell me an untruth, but you withheld the truth, and both are wrong. Do you
understand me?"

"Not quite," said Harebell; "isn't it wrong to tell tales?"

"Not if it helps to deceive. Your not telling about Peter helped to deceive me; and I acted
wrongly because of it. I want you to remember this, for people have made themselves and
others very miserable because of it. If shielding one person makes another act unjustly, it
is wrong. Now I shall say no more—you had better have some breakfast."

She stooped and kissed Harebell, then led her into the schoolroom, where some food was
awaiting her.

Harebell began to feel much happier, and when her uncle came in presently, and told her
how glad he was to hear that the mystery was all cleared up, she heaved a deep sigh and
said:

"I feel as if a heavy weight has lifted out of my chest. And now that aunt has forgiven me,
and I'm not going to school, may I tell you about Tom?"
 CHAPTER XI
TOM'S LAST EFFORT

THAT very same evening, Mrs. Keith received an ill-written letter from Tom:

"MADAM,—This is to say as little Miss to my sertain nowledge have not

toled a lie. There be anuther party in the bisness wich if you cud
discover wud be rather near home but I am pleged to say nothing. They
that lives most with her knows and is hidinge the truth.

"Your obedient servant

"TOM TRIGGS."

Mrs. Keith showed this to her husband. As Harebell was cleared, they did not tell her
anything about it, but Tom was written to and thanked for his intervention.

And very soon the Rectory party returned from the seaside. Peter and Harebell had a very
solemn interview. He was made by his mother to come up and tell Mrs. Keith exactly what
he had said; and then he apologised to Harebell.

She took his shamefaced apology very gravely. But when she began to relate to him her
runaway ride, he brightened up and was most interested.

"It's just like a story! What a pity you came back. I should have gone to sea!"

"I couldn't have. I couldn't have taken Chris with me. It was him I didn't like leaving."

"Girls never keep things up. They always get frightened and stop in the middle."

"What would you have felt like if I had never come back?"

Peter reflected.

"I think I should have told people it was my fault, and then I should have felt obliged to
run away after you to find you. That would have been good fun! I should have gone on the
donkey, and you bet I should have caught you up!" His eyes gleamed at the idea.

"I'm very glad I didn't go on. It's horrid if you feel you're quite alone in the world. I felt
when I was in that empty house, as if I had lost my friends and my home—and the most
awful thing of all—that I had lost God, and didn't belong to Him any more."

It was Peter's turn to look grave.

"I'm glad I'm not you, without a mother. Mother is ripping. She wasn't a bit angry when I
told her, only very sorry—and—well—loving. I was rather a cad, and, of course it was a lie
I told. I'm never going to tell another as long as I live. If I die for it, I won't!"

Peter clenched his fists determinedly.

His sin, and the burden of it, and then the confession, had made him a different boy.
Harebell felt he was a much nicer Peter afterwards, and other people felt so too.

Lessons began again, and life went on with cheerful regularity, varied by picnics on half-
holidays, and later on by blackberrying and nutting expeditions. Harebell grew into a
strong rosy girl. Her aunt was fast losing her cold indifferent manner towards her, and
Harebell now chatted to her as freely and unconstrainedly as to any one else. Then the
winter came: but Harebell enjoyed the cold, and welcomed the frost and snow with delight
and interest.

Christmas was a most enjoyable time, and she and the Rectory children were inseparable
during the holidays.

When the New Year came, there was a good deal of sickness in the village, and many of
the old people died. Amongst them was Mrs. Crake and Mrs. Triggs. Tom came to his
mother's funeral, and the village hardly knew him. He had gone steadily on in the right
way, and was now a respectable sober hardworking man.

Mrs. Keith no longer objected to Harebell's interest in him, and one day she was allowed to
go to tea with Tom and his sister. She walked off very proudly, and was soon sitting up at a
well-spread table in the little front parlour.

Miss Triggs was in deep mourning and was rather sad. Harebell was too excited to be so.
She had never much cared for old Mrs. Triggs, who did not welcome her as a visitor, and
made no secret of her dislike to children. And now she could not pretend to mourn for her.

"Do you know, Miss Harebell, I may be going away?" said Miss Triggs with a sigh.

"You'll have to get somebody else to make your frocks for you. I've had an offer from an
uncle of ours, who is a big draper in Swansea. He wants me to go there and be one of the
skirt hands, and Tom and me have been talking it over, and I think I'd like to go."

"Oh, dear! This is dreadful news," said Harebell in dismay. "And who will live in your
cottage?"

"I'll give it up. I have only to give a month's notice."

"But won't Tom want it? You'll come back and live here again, won't you, Tom? I do want
you so much."

"Not yet awhile, missy. I'm feelin' a bit unsettled like. True, the Squire talked about wantin'
an estate carpenter now his head man be off to Canada: but that be too good a billet for
me. There be a small cottage, too, for the lucky chap who gets it—"

"Oh, but what a lovely thing for you! And then you'd get a wife. Do say you would, Tom.
And Fanny isn't married to anybody else, and now she's all alone, it would be just right,
wouldn't it, Miss Triggs? She has lost her mother as well as you."

Miss Triggs smiled, but Tom did not. He got rather red.

"Eh, dear? But you do go on about a wife; I ain't ready for one yet."

"I'll help you to get ready," said Harebell earnestly; "there seem a lot of cottages ready for
your wife. This one, and Fanny's, and the Squire's."
"Have one of these little cakes, dear," said Miss Triggs, who wished to change the
conversation.

Harebell ate the cake and thought hard. At length she said:

"You see, Tom, you've given up the beer, and you've got work, and now you've got
cottages all round you, and the wife is the last thing and the best of all."

"'Tis to be hoped she will be," said Tom, half to himself.

"Well," said Miss Triggs, "I wouldn't go off and leave him, Miss Harebell, if he'd make his
home with me. But he won't. 'Tisn't disagreeableness; the fact is, he have kept on sendin'
his money home to mother, and he hasn't enough just now to start a house on his own,
and he have got proud, and won't let me pay the rent and such like."

"You have spent too much on me all these years," muttered Tom. "Now we'll let my affairs
bide for a bit, missy. Do you think you could find room in your home for a little chair I've
been making in my odd moments? I brought it back with me when I come—"

He went into the back kitchen and brought out a beautiful little rocking-chair, which he
presented to Harebell. Then he showed her some letters carved on the back:

F.T.T.
A.B.S.
I.T.D.

Harebell screamed with delight over the chair, but puzzled over the letters.

Tom at length enlightened her.

"I'm not a very good scholar, and my tools be not fine enough to carve words, but I've put
the first letter, see?"

"From Tom Triggs

A black sheep
Inside the Door."

"That do describe me, by God's help."

"Oh, it's beautiful!" exclaimed Harebell. "And so clever! I am sure Aunt Diana will let me
have it, and I do adore a thing that rocks! It will be much nicer than a rocking-horse. I
used to have one of those in India."

Tom promised to bring it up that night; and Harebell was overwhelming in her thanks.

At last she had to leave her friends, and she trotted home. It seemed to her that it was not
chance that made her meet the Squire of the neighbourhood riding home from a hunt,
with two other gentlemen with him. It was too good an opportunity to be lost.

Harebell had often seen the Squire in church, and had spoken to him once when he had
been calling upon her uncle. She now stepped into the middle of the road and held up her
hand authoritatively.
The Squire reined up his horse with a good-natured laugh.

"Well, little maid, are you a suffragette? Or a policeman? What do you want?"

Harebell was nothing if not direct.

"I want you to stop to listen to me. Tom Triggs is a very good man, and a friend of mine,
and he can make beautiful rocking-chairs fit for a queen. Please take him as your
carpenter and give him a cottage."

"Upon my word! Tom Triggs! Who is he? Surely not that drunken loafer who spends his
days over his beer-pot!"

"Oh, that was long, long ago; he's quite a changed man; everybody says so. At least, he's
really the same, only much, much nicer."

"I know him," said one of the Squire's companions. "He's doing up the house I'm buying.
Tom Triggs—I know the chap. He isn't a bad workman. My foreman of works says he's the
most dependable of the whole lot out there."

The Squire looked down upon Harebell with laughing eyes.

"Now may I ask why you and he have chummed up together? An odd companion for you, I
should say."

Harebell looked up with big earnest eyes.

"I liked him when he was wicked, because I'd never seen a wicked man before. And then—
well, you know I've been trying to get him to do things, and he's done them all except a
cottage and a wife. I could get the wife if you would give the cottage."

The Squire laughed heartily.

"This is most entertaining. So you can get wives for people. Could you get one for me? I
haven't one, you know."

Harebell shook her head.

"I don't know you well enough. Tom is a very great friend of mine. We talk over things,
specially since we're together inside the Door. That's the best thing of all he has done. He
has got right through and is quite safe."

"What door?"

"It's the Door of the Kingdom. He likes to call it the sheepfold, but it means the same. It
tells you all about it in the Bible."

There was a minute's pause; then the Squire said genially:

"I really must see this wonderful carpenter. Send him up to see me—not to-day. To-
morrow morning about ten. I shall be in, and I'll have a talk with him."

He nodded to her, then rode on; and Harebell danced along the road for joy.

"He'll get that cottage, and then Fanny must marry him!"
She told her aunt all about it when she got home. Mrs. Keith told her she must not speak
to gentlemen whom she hardly knew when they were riding.

"It makes them think you're a most forward little girl," she said.

"I was so full of Tom, I didn't stop to think," said Harebell; "and he's made me the most
beautiful rocking-chair, Aunt Diana, and he's going to bring it round to-night. You'll let me
take it, won't you? And may I tell him the Squire wants to see him to-morrow?"

Permission was given. When Tom arrived, he looked quite confused when Harebell gave
him the message.

"Upon my word, missy, you don't let the grass grow under your feet! Why, you be just
wonderful with your tongue."

"And you'll tell me if you get the cottage, Tom? You'll tell me directly it's settled."

"There be not much chance of that, missy. The Squire have knowed me in the old days."

However, Tom had his interview with the Squire, and was taken on trial for a month, to his
unbounded pride and delight. Harebell went straight off to see Fanny Crake when she
heard of it.

Unfortunately, Fanny was away from home. But Harebell was not easily daunted. She came
home and procured a piece of paper. In large copper-plate writing she wrote on it:

"Please Fanny, Tom Triggs may have the Squire's cottage. Get ready
for his wife. From Harebell."

And then the next day she took the paper and slipped it underneath the door.

But she heard nothing from Fanny. And Tom was too busy at work to come near her.

Lessons and games and talks with everybody who came across her path now occupied her
time.

Mrs. Keith said to her husband one night:

"I really dread the development of that child. I don't know which is busiest—her brain or
tongue. I hope she won't grow into a chattering woman. I found her having a long
dissertation to-day on the back doorstep with a wandering pedlar. She knows his history,
and all the names and ages of all his relations, for I heard her repeating it all to Andy in
the pantry."

"Oh, she's all right!" said Colonel Keith kindly. "She is interested in her fellow-creatures. It
is better to have one's interest circle round them than round oneself."

And then one day Harebell got a letter, and it was a letter that filled her small heart with
joy and satisfaction. It was from Tom Triggs:

"MY DEAR MISSY,—I have not yet wrote you a letter but I do so now
hoping this will find you well as it leaves me. I write to say the Squire,
he have give me the job and I thank you down to the ground for arsking
 of him to do it. I have the cottage in the wood, and all is going on as you
said it ought. Nex April I hopes to get it, and mother's bits will stock it
fine. And Fanny Crake and me are walking out, for on looking round I
felt as how I ought to oblige you, and there seems no other to soot me,
and Fanny says your heart be terrible set on to it. So we hopes if things
go on well, to be husband and wife in April. Wishing you well.

"Your obedient servant,

"TOM TRIGGS."

"P.S. The best which has come to me is what I have not the learning to
speak on. But I am still I humbly trust, I. T. D."

She carried this letter all day about with her and slept with it under her pillow.

Her aunt found her one evening spelling it out to herself by the light of a candle.

"You are very fond of Tom Triggs," she said.

"He's such a good kind of friend," said Harebell in her old-fashioned way, as she folded up
her letter and tucked it under her pillow. "He has done every single thing I wanted him to.
And not many friends do that, do they?"

"Perhaps not."

"It's just five things he has done, and the wife is the last and best of all."

"What things?"

"Well, first he gave up drinking, and then he got some work, and then he got his cottage,
and now he has got his wife."

"That is four."

"I s'pose the other thing is the really best thing," said Harebell, slowly and thoughtfully.
"He has put it last in his letter, but I really believe it came first of all."

"And what is that?"

"He came to the Door of the sheep and got through it into the sheepfold."

Harebell's eyes were shining and earnest as she spoke.

Mrs. Keith stooped and kissed her.

"Yes, that is the only thing that really matters," she murmured.

And then when she left the room, Harebell added to herself:

"And in April, I mean to go to tea with them in their cottage. I said from the very first I
would do it."

She hugged her letter as she spoke, then she fell asleep.
 THE END

PRINTED BY WILLIAM CLOWES AND SONS, LIMITED, LONDON AND BECCLES.

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