Direct Labelling Presents New Opportunities to Study Blood Leukocyte Migration

Bill Ristevski

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Depanment of Laboratory Medicine and Pathobiology University of Toronto, 2000

O Copyright by Bill Ristevski 2000

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Direct LabelLing Presents New Opportunities to Study Blood Leukocyte Migration

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Laboratory Medicine and Pathobiology University of Toronto, 2000

Abstract

Leukocyte trafic in the blood compartment is a dynamic and complex process.

There is a constant influx of leukocytes fiom primary lymphoid organs and recirculating

lymphocytes via the lymphatic system, which is counterbalanced by monocyte and

lymphocyte emigration fiom the blood and leukocyte death. These ongoing processcs

working in coordination to maintain blood leukocyte homeostasis produce characteristic

half-times of leukocytes within the blood compartment. The half-times of fluorescently

labelled monocytes, neutrophils, and lymphocytes were investigated in the blood of

sheep. The results obtained represent the most detailed and technically advanced half-

times to date. In addition a methodology was established to directly label the entire blood

leukocyte pool in situ, and the functional ability of lymphocytes to recirculate was

assessed post labelling. This method establishes the framework for many novel

experiments to be performed investigating low fiequency blood leukocyte populations

that were not previously possible.

Ac knowledgements

I would Iike to express my deep gratitude to Dr. Jack Hay for providing the

opportunity for me to work in his laboratory under his guidance. Interacting with Dr. Hay

has expanded me both on a scientific and personal level. His enthusiasm in al1 aspects life

is contagious, and because of this, I will always remember my introduction into research

as one of my rnost positive expenences.

I would dso like to thank Dr. Miles Johnston and Dr. Myron Cybulsky, rny

cornmittee rnem ben, for their inval uable input and advice.

Over the past two yean I've had the fortunate oppomuiity to work with many

fantastic people in the laboratory. My fhends Dr. Binh A U Dr. Jodi Dickstein and Dr.

Tim Seabrook have ail provided hours of surgical assistance which I am gateful for.

Many others made this work possible. Cheryl Smith trained me to use the flow

cytometer, and Frank, Ranier, Angela, Wendy, Parn and many othen in the animai

facility spent a great deal of time taking care of the anirnals that were used in these

investigations.

1 am also thankful for my family whose encouragement, support and

understanding is boundless and unconditional.

Table Of Contents

CHAPTER 1

7 ...................................................................................................................... 1.1 THE lMMUNE SYSTEM - ............................................................................................ 1.2 THE RECIRCULATION OF LYMPHOCYTES 3

............................... 1.3 LEUKOCVTE RECRUITMENT FROM THE BLOOD VIA EMX)THELIAL INERACTIONS 3

...................................................................................................... 1.4 SECONDARY L YMPHOID ORGWS 7

......................................................................................... 1.5 CELLULAR HO~OSTASIS [N THE BLOOD 8

....................................... 1.6 TISSUE SPECFIC MIGRATION AND LWCIOCYTE SUBSET REASSORTMENT 13

1.7 SIZE OF THE BLOOD POOL VERSUS THE RECIRCULATING POOL . VERSUS

........................................................................................................ TOTAL BODY LYMPHWYTES 19

....................................................................................................... I.8 BLOOD LEUKOCYTE DYNAMICS 20

..................................................................................................................................... 1.9 RATIONALE 20

CHAPTER 3

97 ............................................................................................. GENERAL MATERIALS AND METHODS

2.1 ANtMtUs .................................................................................... ................................................. 23

........................................................................................................................................ 2.2 SURGERY 23

.................................................................................................... 2.3 C m LABELING AND D ~ C T ~ O N 24

2.4 ~MUNOF~.UORESCENCE STAIN[NG FOR FLOW CMUMEIWC ANALYSIS OF CD14 EVRESSION ......... 25

............................................... 2.5 CELLULAR SUSPENSIONS FROM Som TISSUES ... ..................... 26

.................................................................................. 2.6 REAGENTS ...................... ., ... .. 27

2.6. I Dulbeccos ' Phosphate BMered Safine IOX... ........................................................................... 27

........................................................................................................... 2.6.2 Anticwgulant So/utio~ts 28

2.6. 3 Par@ormakiéW Solution ...................................................................................................... 29 . . .................................................................................................... 2.6.4 Heparrntzed .iàl ine Solutions 29

CHAPTER 3

THE HALF-TIMES OF FLUORESCENTLY LABELLED MONOCYTES. GRWL'LOCYTES AM)

.............................................................................. LYMPHOCYTES IN THE BLOOD .............. .... 30

................................................................................................................. 3.1 AEWRACT ............... .. 31

................................................................................................................................ 3.2 ~NTRODU~ON 32

............................................................................................................. 3.3 MATERWLS AND ~&TCIODS 33

.................................................................................................................................... 3.3. I Animais 33

............................................................................... 3.3.1 Leuk~yrr Labelhg with CFSE and FITC 33

.................................................................................................................. 3.3.3 Ana/ysis of Sam pies 34

....................................................................................................... 3.3.4 Càia~lariots and Statistics 35

......................................................................................................................................... 3 .4 RESLJLTS 37

................................................................................................................ 3.4. i CFSE C d La6e f iing 37

....... .................................... 3.42 Rrrtucrion of LubeiIed C.dIsfiom the B f d Cornpartment ...... W

3.4 3 FIK* C'ri1 Labrilit~g and Thwr Reassarmrrrt .......... .. ............ .. ................ 59

.................................................................................................................................... 3 -5 DISCUSSION 59

..................................................................................... IN VIVO BLOOD LELKOCYTE LABELLMG 84

4.1 Assmc-r ...................................................................................................................................... 85

.............................................................................................................................. 4.2 hmo~ucrro~ 56

............................................................................................................. 4.3 MATERIALS AM) WODS 87

.................................................................................................................................. 4.3.1 Animais.. 87

........................................................................................................................... 43.2 CeU labelhg 87

.................................................................................................................. 4.3.3 At~u/ysis of sarnpfes 87

....................................................................................................... 4.3.4 Caicuhriom anci statistim 8 7

......................................................................................................................................... 4.4 RESULTS 88

1.4. i CeIlhbefIing .......................................................................................................................... 88

4.5 DISCUSSION .................................................................................................................................... 99

1.5.1 Disamion of Dota in Appe radac. .. ........,................................ ................................................ I I I

CHAPTER 5

..................................................................................................................... GENERAL DISCUSSION 114

............................ .............................. 5.1 LABELLING METHODOLOG~ES FOR CELL ~ C K I N G ..... 115

........................................................ 5.3 REDUC~ON OF ~ B E L L E D LYMPHOCYTES FROM THE BLOOD 115

...................................... 5 -3 F U N ~ O N A L ASPECTS OF HALF-TMEs IN THE BLOOD COMPARTMENT .... 120

.................................................................................... 5.4 TISSE REASSORTMENT OF L Y M P H O C ~ S 124

.................................................................................................................. 5.5 F m - EXPERMENTS 126

................................... 5.5. i Equriments ru Further Characterizr the In 1 ïvo LubeIfing Procechrre 126

.... 5.5.2 Erperiments to Re-Ewnine the Rehction of Labef/ed CelfsfLom rhr B l d Cornpurmient 127

......... 5.53 Eqwerirnrtit to Ercmine Rrcovery Kimtics of Laoelied Lymphq~es i11 E.erent Lymph 128

............................................................................................................ 5.6 SWY ................ .... 128

..................................................................................................................................... REFERENCES 130

List of Figure

.................... Figure 1.1 The Lymphocyte lnput and Output of a Single Node.. 1 O

......................... Figure 3.1 Differential Gating of Sheep Blood Leukocytes.. -38

Figure 3.2 Fluorescence Intensity of Unstained, CFSE Labelled

......................... (In Vitro) and Post Infusion Blood Leukocytes.. ..40

Figure 3.3 Detection of CFSE Labelled Mononuclear Cells 12

...................................... Days Post infusion of Labelled Cells.. .44

.......... Figure 3.4 Detection of CD 14 Expression on Sheep Blood Leukocytes.. -46

Figure 3.5 Composite Graph of the Reduction of CFSE Labelled Monocytes.

.............................. Neutrophils, and Lymphocytes in the Blood ..J9

Figure 3.6 Reduction of CFSE Labelled Monocytes fiom the

.......................................................... B lood Cornpartment .5 1

Figure 3.7 Reduction of CFSE Labelled Neutrophils From the

......................................................... Blood Corn partment.. .53

Figure 3.8 Reduction of CFSE Labelled Lymphocytes from the

........................................................ Biood Cornpartment.. - 3 5

Figure 3.9 Fluorescence Intensity of Unstained and FITC Labelled

.................................................................. BIood Cells.. -60

Figure 3.10 Tissue Reassortment of FïïC Labelled Lymphocytes. ................ .61

Figure 3.1 1 Diagrammatic Representation of the Reassortment of

Labelled Lymphocytes Afier Infusion into the Blood. ................. .69

Figure 4.1 Differential Gating of Sheep Bload Leukocytes for

........................................... In Vivo LabelLing Experiments.. ..89

Figure 4.2 Fluorescence Intensity of Undneci, and In Vivo

......................................... CFSE Labelled Blood Leukocytes.. .9 1

Figure 4.3 Fluorescence Intensity of Efferent Lyrnph Derived Lymphocytes

Before and Mer In Vivo Blood Labelling ................................. .95

Figure 4.4 Graph S howing the Percent of Labelled Lymphocytes Recovered

from the Efferent Lymph of a Single Sheep ................................. 97

Figure 4.5 Reduction of CFSE Labelled Neutrophils and Smdl Lymphocytes

.................................................. From the Blood Cornpartment 100

Figure 4.6 Fluorescence Intensity of Unstained, and Ln Vivo CFSE

............................................ Labelled Rat Blood Leukocytes.. .103

Figure 4.7 Tissue Reassortment of Labelled Mononuclear cells (RI)

..................... 10 Minutes Post In Vivo Blood Labelling in the Rat.. 107

Figure 5.1 Hypothetical Reduction of Labelled Lymphocytes ftom the

........................................ Blood Based on Thoracic Duct Input. 1 17

Figure 5.2 Hypothetical Reduction Cuve for Labelled Lymphocytes in the Blood

in the Absence of the Blood Resident Lymphocyte Population.. ......... 12 1

Figure A. 1 Graphs Showing the Percent of Labelled Lymphocytes

Recovered From the Efferent Lymph of Two Sheep ....................... 137

Figure A.2 Tissue Reassortment of In Vivo CFSE Labelled Lymphocytes.. ....... 139

Figure A-3 Tissue Reassortment of Leukocytes 1 Hou Post

In Vivo Blood Labelling ........................................................ 14 1

List of Tables

Table 1 . 1 Key Endothelial and Leukocyte CAMs Involved in the Multi-Step Paradigm. ........................................................... 5

Table 1 .2 Distibution of Lymphocyte Subsets in Blood Afferent Lymph. Efferent Lymph and Subcutaneous Nodes.. ................................. 16

Table 3.1 Sumrnary of the Reduction of Labelled Leukocytes Based on Linear Regressional Anaiysis.. ........................................... -57

............... Table 3.7 Hal f-Times of Leukocytes Obtained fiom Earlier S tudies. .79

List of Abbreviations

Y r

Ab

M C

BLC

CAM

CCR7

CD

CFSE

CXCRS

DF;?P

EDTA

ESL- 1

FITC

GlyCAM-I

HEV

Lm.

1.v.

ICAM- 1

1s

LFA- I

rnAb

C hrorniurn

anti body

allophycocyanin

B-lymphocyte c hemoattractant

ceIlular adhesion molecule

chemokine receptor 7

cluster of differentiation antigen

carboxy ff uorescein diacetate succinimidyl ester

chemokine receptor 5

di-isopropy l fl uorophosphate

ethylenediamine tetraacetic acid

E-selrctin ligand- 1

fluorescein isothiocyanate

glycosylation-dependent ce11 adhesion rnoiecule- 1

high endothelia1 venules

intramuscuiar

intravenous

interceIIular adhesion molecuie- 1

leukocyte fiuiction antigen-1

monoclonal anribody

Mac- l

MadCAM

MCP- I

MZP

PAF

PBS

PECAM- 1

PSGL- 1

SDF- 1

SLC

vcm- 1

macrophage- 1 antigen

mucosal addressin ceIl adhesion molecde

monocyte chernotactic protein- 1

macrophage inflarnmatory protein

platelet activating factor

phosphate buffered saline

platelet endothelial ceIl adhesion rnolecule- 1

P-selectin giycoprotein ligand- 1

revolutions per minute

stroma1 cell-derived factor

secondaxy lymphoid tissue chemokine

vascular ce11 adhesion molecule- l

Chapter 1

General Introduction

1.1 The Immune System

The immune system of mammals is organized in such a manner that form helps

dictate fiinction. The pnmary or central Iyrnphoid organs are the thymus and bone

marrow, which are the sites of immune ce11 generation. The secondaiy or peripheral

Iymphoid organs are lymph nodes. spleen. tonds. adenoids. appendix. Peyer's patches.

and branchial-associated lymphoid tissues, which are the sites of initiation of the adaptive

immune response. This form of compartmentalization of immune ce11 genesis versus sites

of initiation of adaptive imrnunity makes immune ceIl traffic and reassortrnent a requisite

for proper fùnction of the immune systern. In particular the continuous recirculation of

naïve and memory lymphocytes throughout the body via the vascular and Iymphatic

systems provides the opportunities for lymphocytes to traverse lymph nodes and other

peripheral lymphoid organs. This dynamic lymphocyte trafic throughout the entire body

is the basis of immune surveillance and coordination of immune responses. One of main

actions of this traffic is to increase the probability that a naïve lymphocyte will encounter

and have the opportunity to respond to its specific antigen, which are collected into

lymph nodes. The collection of antigen into lymph nodes or other peripheral lymphoid

organs is accomplished by drainage of extracellular fiuid containing the antigen via

afferent lymphatics, or by uptake by specialized celIs such as Langerhans' cells which

have the ability to migrate to local lymph nodes after engulfing antigen fiom the

perip hery.

At the level of the node, interdigitaihg dendritic cells and macrophages can

process and present this antigen in a major histocompab'bility cornplex-restricted manner

to begin an adaptive immune response. Another function of this trafic is the

dissemination of clonally expanded lymphocytes via the efferent lymphatic(s) of the

infected node to allow reassortment of these cells to their effector sites. Once the ensuing

immune response is completed antigenic rnemory is retained by a selected group of

lymphocytes. These lymphocytes preferentially migrate through peripheral tissues where

it is more likely that they will encounter a future challenge of the same antigen, which

would elicit a much more rapid secondary immune response. It should be noted that this

thesis focuses on the migration and recirculation of naive and memory lymphocytes and

other leukocytes at steady state. The migration and recirculation of activated cells

participating in an immune response is considerably different, and can not be inferred

from steady state kinetics. Since leukocyte trafic is intimately linked with every major

facet of the immune response, investigating trafic and recirculation patterns of immune

cells is of considerable importance.

1.2 The Recirculation of Lymphocytes

Pioneering work by James Gowans and colleagues lead to the development of the

tenn "recirculation" referring to the circulatory migration patterns of lymphocytes fiom

blood to lymph. Although it was known that cells were present in lymphatics it was

specdated that these were recently formed cells that were not traficking fkom the blood

Gowan's classical experiments performed in rats demonstrated that chronic diversion of

lymph fiom the thoracic duct resuited in a Iymphopenia in the thoracic duct lymph and

blood (Gowans 1959).The loss of lymphocytes fiom the thoracic duct lyrnph could be

conected by returning the diverted cells back to the animal by intravenous ( i x ) injection.

Gowans dso established îhat cells collected from the thoracic duct and Iabelled with

radioactive tracen couid be recovered in the thoracic duct lymph &er i.v. infusion,

demonstrating blood to lymph migration (Gowans and Knight 1964). It was found that

the main route of migration of lymphocytes from blood to lymph occurred at the level of

the node. Bede Morris and colleagues expanded on this research by using methods to

carnulate efferent lymphatics of a single lyrnph node in larger animals (Lascelles and

Morris 1961; Hall and Moms 1962; Hall and Morris 1965). Sheep offered particular

advantages because of their size, and docile nature. Unlike hurnans sheep do not have

nodes that often occur in chains where the efferent lymphatic of one node becomes the

afTerent lymphatic of another. In fact most nodes have a single efferent lymphatic that

eventually drains into the thoracic duct.

Although many technological advances have been made in terms of cell tracking

compounds and anaiysis techniques, the generai procedures developed by these scientists

are still used to study this phenornenon and are used in these studies.

1.3 Leukocyte Recruitment from the Wood via Endothelial Interactions

The multi-step paradigrn for leukocyte recruitment from the blood along

postcapillary venule endothelium can be divided into four general categories: tethering

and rolling, activation through G-protein Iinked receptoa, activation dependent arrest and

diapedesis (Butcher, Williams et al. 1999). Cellular adhesion moiecuie (CAM)

interactions are involved in al1 of the steps, and at the least the process is partially

regdated by chemoattractants (DeVries, Ran et al. 1999). Although many of the studies

are centered on investigating this process during inflammation, adhesion is very

important for leukocyte emigration during steady state. Table 1.1 lists the stages in the

Table 1.1 Key Endothelial and Leukocyte CAMs Invotved in the Multi-Step Pa rad igm

Table shows the stage of the multi-step paradi-m for leukocyte extravasation and the key

CAMs involved in each of the steps.

Table 1 . I

i STAGE 1

Key Endothelial Molecules

i Tethering and Rolling E-selectin P-se lectin GlyCAM- 1

: Transendothelial 1 VCAM-1 Migration (Diapedesis) ' [CAM- I 1 PECAM-I

I i Activation l

Key Leukocyte Molecules 1 I

C hemokine presentation CSa, PAF, MCP- 1, SDF- 1

ESL- 1 I PSGL- 1 1 L-selectin

1

1 i

1 MIP, SLC Stable Adhesion 1 VCAM-I

1 [CAM-I

Speci fic G-protein linked ! ! receptors j

1

a 4 integrins LFA- 1 , Mac- l PECAM-I i

References for Tabie 1 . 1 : (Butcher and Picker 1996; Butcher, Williams et al. 1999;

Kulidjian, Inman et al. 1999)

multi-step paradigm along with the key endothelid and leukocyte molecules involved in

the respective step. Although this thesis doesn't directly center on the molecular

mechanisms of leukocyte extravasation it is a relevant theme, as any of the Ieukocytes

that emigrate out of the blood vasculature wil1 do so by the general process described

above.

1.4 Secondary Lyrnphoid Organs

Lymph nodes are ofien thought of as antigenic and celluhr filters along

lymphatics. Their counterpart in the blood is the spleen. For this reason some

investigaton have referred to passage of ce1 1s through the spleen as '-reci rculation".

However, in the spleen the majority of lymphocyte emigration occurs via the blood

sinusoids in the marginal zone of the spleen, an ''open" system (Girard and Springer

1995). Therefore in this thesis the term recirculation will be reserved for its traditionai

meaning of blood to lymph migration.

The spleen is made of two areas red pulp which is the site of senescent red blood

ceIl destruction intenpersed with lymphoid white pulp. Upon entry into the spleen 90%

of lymphocytes will end up in the red pulp, with a transit time of 5 minutes, and 10% of

the cells will migrate into the white pulp, with an average stay of 4 - 5 hours (Kraal,

Rodngues et al. 1989). At the level of the white pulp, recirculation of lymphocytes in the

traditionai sense can occur as there are deep splenic lymphatic vessels, and migrating

lymphocytes cm exit white pulp via these Iymphatic vessels (Pellas and Weiss 1990).

Unfomuiately, there is a paucity of data on the control of cellular organization

within the spleen and other lymphoid organs. However, an interesting observation is the

disorganization of lymphoid organs in CXCRS or CCR7 deficient anirnals (Forster,

Mattis et al. 1996; Forster, Schubel et al. 1999), which supports the idea that chemokines

play a crucial role in creating segregated microenvironments. However, binding of

CXCRS on B cells to its ligand BLC expressed in B ce11 follicles is insufficient to target

these celIs on its own (Campbell and Butcher 2000). Therefore this organization is most

likely due to a complex interplay of CCR7 binding its ligands SLC and MIP-3B to direct

T cells, combined with CXCRS directing B ceII organization and likely additional

chemokines not yet identified. Additionally in vivo there is a large overlap of chernotactic

fields and this combined with c hem0 kine receptor redwidancy suggests extremely

complex mechanisms involved in microenvironmental homing in lyrnphoid organs.

Microenvironmental horning in lymphoid tissues is likely very important in ternis of

detemining the transit times of cells through these tissues.

1.5 Cellular Homeostasis in the Blood

Although the majority of experiments contained in ths thesis deal with leukocytes

in the blood cornpartment, undentanding lymphocyte migration through lymph nodes is

essential as this traffic plays a major role in blood cell homeostasis. The term steady state

applied to the blood is misleadhg At steady state the blood is nevertheless a very

dynamic comparmient. The blood cornparmient has a continuous loss of cells due to

lymphocyte recircuiation and monocyte emigration as well as normal leukocyte death.

There is also a continuous input of leukocytes fiom primary Iymphoid organs and an

input due to the retum of recircuiation competent lymphocytes via the thoracic duct,

prescapdar and c e ~ d efferent lymphatics. As efferent lymphatics in the sheep foilow a

path to the thoracic duct it is helpful to study an individual node to understand the origins

of thoracic duct lymphocyte input back to the blood.

Early sheep experiments showed significant differences in ce11 concentration and

composition between afferent and efferent lymph. Concentration of cells in afferent

lymph is approximately 106 cells/ml while efferent lymph is typically at least IO-fold

higher (Smith, McIntosh et al. 1970; Mackay, Kimpton et al. 1988). The composition of

afferent iymph is also different as compared to efferent lymph. Approximately 10-20% of

afferent derived cells are macrophages or dendritic cells that are CDI positive and

express high levels of class II MHC, while the remainder of cells are lymphocytes (Miller

and Adams 1977; Knight 1984; Bujdoso, Hopkins et al. 1989). Efferent lyrnph ceils are a

very pure population of lymphocytes with the exception of the rare macrophage precursor

(Pugh and MacPherson 1982). This ability of the node to filter macrophages and dendntic

cells from rntering the efferent lymph is the cause of the abrogated recirculation

experienced by these cells, as opposed to lymphocytes which have the ability to traverse

the node.

Ce11 outputs also differ in afferent versus efferent lyrnph. A single afferent

Iymphatic supplies about 1 x 106 cells per hour to a 1 gram subcutaneous lymph node

with the typical node having 6-12 afkrent lymphatics (= 8 x 106 cells/hr) (Hall and

Morris 1965). in contrast to afTerent output, efferent output from a subcutaneous lymph

node is approximately 30 x 106 cells per hour per gram of lymph node, and only 10% of

these cells (1 3 x 1 o6 cells/hour/grarn of node) are actually derived fiorn cells that entered

the node via afEerent lymphatics. (Hall and Morris 1962). Figure 1.1 shows the

quantitative input and output of lymphocytes to a 1 gram subcutaneous lymph node.

Figure 1.1 The Lymphocyte Input and Output of a Single Node

Diagram shows the lymphocyte input via the afferent lymphatics and blood supply as

well as the lymphocyte output Ma the efferent l ymphatic.

Figure 1.1

Bl00d A 1 gram node receives approximately 1 2x1 o8 lymphocytes/hour, 1 in 4 will cross HEVs and enter the riode

Arterial Blood Supply

Lymph Node 1 gram node = a1 o9 C~I IS

---+ Lymph Flow Lyrnph Flow

Afferent Lymph Efferent Lymph 6-1 2 afferent lyrnphatics per node Flows at 3x1 o7 cells/hour Each flows at approximately 1 XI o6 œllslhr for a 1 gram node 10-1 5% macrophage-like cells (Some Dend Aic) 1 00% lymphocytes 85.90% lymphocytes 5% recently produœd

within node 10% derived from afferent

h'mph 85% recently ernigraîed

from blood

Adapted fioom: (Young 1999)

In addition experiments using tritiated thymidine to label lymph node cells in situ found

that no more than 5% of the cells in efferent lymph were recently produced within the

node itself (Hall and Momk 1965). Approximately 85%, the remainder of cells present in

efferent lymph are extracted fiom the arterial blood passing through the node by HEVs.

Later experiments demonstrated that the blood flow to a node was about 0.012% of the

cardiac output per gram of node. This translates to roughly 1.2 X 10' cells per hour

flowing through the blood vasculature of a 1 gram node (Hay and Hobbs 1977). In order

to accommodate the 85% of lymphocytes in efferent lymph that are not accounted for by

afferent input and proliferation, 1 in 4 lymphocytes passing dong the HEVs in the node

mut extravasate (Hay and Hobbs 1977; Bjerknes, Cheng et al. 1986). This extravasation

eficiency is remarkable given the load that a single node accommodates. This is even

more surprising given that recently different laboratones have demonstrated that as rnuch

as 40% of lymphocytes in the blood are not actively recirculating in vivo (Young,

Marston et al. 1997; Andrade, Johnston et al. 1998; Gupta, McConnell et al. 1998). This

would mean that about 1 in 2 of recirculating cornpetent lymphocytes cross HEVs and

enter efferent lymph. Consolidating a11 of this experimental information shows that ongin

of efferent lymph lymphocytes can be divided into three general categorïes:

1. Approximately 10% of these efferent lymph lymphocytes are derived h m afferent

lymph input.

2. Approximately 5% of the efferent lyrnph lymphocytes are recently produced within

îhe node.

3. Approximately 85% of efferent lymph lymphocytes have recently emigrated fiom the

blood across HEVs of the node.

in addition it should be noted that approxirnately 70% of the efferent lymph derived

lymphocytes are T cells while the remaining 30% is comprised of B celIs (Young 1999).

These data refer to subcutaneous lymph nodes which may not represent the physiolagy of

lymphocyte recirculation through mesentenc lymph nodes or other secondary lymphoid

tissues. However, it is important to consider the ongins of cells in efferent lymph as it

provides the bulk of lymphocytes rehiming to the blood cornpartment.

1.6 Tissue Specific Migration and lymphocyte Subset Reassortment

The presence of a non-recirculating pool of lymphocytes in the blood venus a

recirculation comptent pool shows that lymphocytes difler in their ability to recirculate.

Experiments have shown that certain lymphocytes have a tropism to recirculate

preferentially through certain tissues of the body. It must be stressed that none of these

preferentiai migration patterns are absolute. The interpretation of these patterns is that

there is a greater likelihood of a group of lymphocytes to migrate to a specific area of the

body (but not excluding other areas) with a higher efficiency than other lymphocytes.

Eariy experiments demonstrated tissue homing of lymphoblasts to gut tissue

(Gowans and Knight 1964; Hall and Smith 1970), aithough activated cells IikeIy migrate

differently than midl resting lymphocytes. However, later e.yperiments showed that midl

resting lymphocytes were capable of tissue specific migration. The initial observations

made were that lymphocytes collected from efferent mesenterïc lymph, radiolabelled and

retunied intravenously, would retum to mesenterie lymph in the approxirnate proportion

of 2: I versus subcutaneous efferent lymph collections. Vice versa, cells collected from

subcutaneous efferent lymph showed a tropism to r e m to subcutaneous lymph. Thus it

was shown that there was two distinct recirculating pools that tended to return to their

cornpartment of origin with a higher degree than other cornpariments (Cahill, Poskitt et

al. 1977; Chin and Hay 1980). Non-random lymphocyte recirculation has also been

demonstrated through normal and inflamed skin in sheep, suggestive of a third pool (Chin

and Hay 1980; Issekutz, Chin et al. 1980). In mice cells obtained by making cellular

suspensions from Peyer's patches or peripheral nodes demonstrated preferentiaf

migration back to the tissue of origin (Butcher, Scollay et al. 1980). This is contrast to the

observation in sheep where cells teased fiom lymph nodes did not show the same

recirculation preferences (Reynolds, Heron et al. 1982).

Tissue specitic homing is suspected to be accomplished by differential expression

of CAMs on the various cells allowing these cells to interact with a greater or lesser

avidity with the different microenvironments that exist throughout the body. For instance

L-selectin binding with GIyCAM-I or CD34 on endothelium is thought to home

lymphocytes to peripheral lymph nodes (Butcher and Picker 1996). While a4P7

interaction with MadCAM- I is thought to be the main regulator of lymphocyte homing to

the gut (Butcher and Picker 1996). However, the cornplex interplay of leukocytes with

endothelium shows that these general des need much more investigation. For instance

knock outs of the L-selectin gene or infusion of anti-L-selectin antibodies can prevent

localization of lymphocytes within peripheral lymph nodes (Gallatin, Weissman et al.

1983; Arbones, Ord et al. 1994; Kuiidjian, h a n et al. 1999). However, granulocytes

also express L-selectin and have been shown to bind HEVs of frozen lymph node

sections (Hallman, Jutila et al. 199 1 ; Jutila, Kishimoto et al. 199 1 ), yet these cells are not

found in efferent lymph during steady state conditions. In fact monocytes and neutrophils

express to varying degrees, al1 of the adhesion molecules found on lymphocytes with the

exception of However, these different ce11 types expenence very different migration

patterns from the blood. Granulocytes under steady state conditions are not thought to

exit the blood vasculature. Monocytes have the ability to exit the blood vasculature and

are found in afferent lymphatics however, they don't have the ability to enter efferent

lymphatics. Thus monocytes have an abrogated recirculation because lymph nodes act as

a "filter" to these cells. The majority of lymphocytes however, have the ability to exit the

blood cornpartment traverse nodes and r e m to the blood. This implies that subtle

differentiai expression of CAMs may produce large changes in tünctional migration

capabilities. Altematively, this may mean that other factors such as chemokines have a

large ro le in regulating these migratory di fferences.

Lymphocyte subset specific migration through van'ous compartments has also

been well docurnented. The use of multi-colour flow cytometry has made it possible to

track specific lymphocyte subsets. Table 1.7 shows the percentage of T and B ce11 subsets

in blood, afferent lymph, efferent lymph and subcutaneous nodes. Some of the major

fin& include that approximately 40% of lymphocytes in the blood do not recirculate and

the majority (85%) of these cells are CD2 1 ' B cells (Young, Marston et al. 1997). This

shows that the recirculating lymphocyte pool is quite dîfferent than the blood when

relative subset concentrations are compared Additional interesting finds with regards to

subset-specific migration are:

Table 1.2 Distribution of Lymphocyte Subsets in Blood, Afferent Lymph, Efferent Lymph and Subcutaneous Nodes

Table shows the percent of lymphocyte subsets found in the blood, afferent lymph,

e fferent lymph and subcutaneous nodes.

Table 1-2

Adapted from: (Young 1999)

Efferent 1 Node Btood Afferent

1 1

Lymph i Lymph 1 1 Non-recirculating Ce11 Recirculating

1. y 6 T cells have a tropism for subcutaneous lymph nodes (Washington, Katerelos et

al. 1994).

2. C D ~ + T cells appear to recirculate well through intestinal Iymph nodes (Washington,

Katerelos et al. 1994).

3. CM' T cells show a preference to migrate back to their tissue of origin (Abemethy,

Hay et al. 1990; Abernethy, Hay et al. 1991; Mackay, Andrew et al. 1996).

4. The rnajority of C D ~ ' cells in afferent lymph express a memory phenotype while

rnost of the CD@ cells in efferent lymph are naïve (Mackay, Manton et al. 1990).

However, in tems of actual number of mernory lymphocytes recirculating per hour,

there is a greater number of memory cells in efferent lymph as compared to afFerent

bmph.

Although both tissue-specific and subset-specific migration in vivo are very

reproducible and most likely have a functional significance it is not the focus of this

thesis. The central goal was to investigate the overall monocyte, neutrophil and

lymphocyte kinetics in the blood cornpartment. However, the overall kinetics of

lymphocytes in the blood is due to the cumulative effect of al1 the intncate tissue and

subset-specific migration patterns. It is not known if different lymphocyte subsets or

populations with tropisms for certain tissues experience different kinetics in the blood, or

have di fferent li fe spans. Although these differences may exist, reductionism was avoided

in this study, and Iymphocytes as a single group were investigated. However it is

important to note that these different populations of subset specific, tissue specific and

non-recirculating lymphocytes may experience different kinetics in the blood

cornpartment. With new developrnents in ce11 tracking, such as the results presented in

Chapter 4, such issues cm now be experirnentally tested.

1.7 Sire of the Blood Pool versus the Recirculating Pool versus Total Body Lymphocytes

The blood cornparmient houses 1 2 % of the total lymphocytes found in a sheep at

any given time. This is based on approximate calcuiations using the total mass of

lymphoid tissue found in a sheep, which is approximately 1

lymphocytes weigh 1 gram the estirnate of total lymphocytes is 10"

1985). Howevet, it seems that not al1 of the lymphocytes in the

kilogram. Since lo9

(Chin, Pearson et al.

body are capable of

recirculating. Experirnents that involved diversion of thoracic duct lymph showed a sharp

drop in the output of lymphocyies over 3 days at which point a low baseline output was

obtained. Over these 3 days about 10" lymphocytes were collected indicating that only

10% of total lymphocytes were actively recirculating (Schnappauf and Schnappauf

1968). Further evidence cornes from experirnents where efferent lymph cells were

fluorescentiy labeled ex Mvo and infused into the blood comparement When 109

lymphocytes were infùsed and allowed to equilibrate, only 1% of the lymphocytes in the

efferent lymph were labelled (Young and Hay 1995). This shows that these labelled

lymphocytes equilibrated with a pool that is a 100 times larger, which again suggests that

only 10" lymphocytes are in the recirculating lymphocyte pool. These are rough

approximations at best and must be considered to have substantial margins for error,

however, such measurements fiom sheep represent the best estimates available currently.

1.8 Blood Leukocyte Dynamics

Many processes contribute to the dynamics of leukocyte tiaffic in the blood.

These processes discussed above include new leukocyte formation, lymphocyte

recirculation, leukocyte emigration from the blood, leukocyte death, transient leukocyte

compartmentalization, subset specific migration, tissue specific migration, rnargination,

and the size of leukocyte pools present in the various compartments of the body.

Although much of this thesis centen on the blood compartment it is crucial to understand

the processes both within and outside of the blood that contribute to the dynarnic

leukocyte aaffic in the blood-

.9 Rationale

The blood compartment is very important in tems of leukocyte migration and

lymphocyte recircdation for many reasons. It is the entq point for newly formed cells

From prïmary lymphoid organs and is also the retuming point for recirculating

Iymphocytes. Although the blood contains only 1.2% of the lymphocytes in the body at

any time (Chin, Pearson et al. 1985) its dynamic nature and its ability to disseminate

lymphocytes to various parts of the body indicates the centrai role it performs in this

phenornenon These factors combined with the clinical practice of using the blood to

gauge immune status makes understanding the dynarnics of ce11 ttaffic through the blood

compartment very important.

The main objective of this thesis was to determine the steady state half-tirnes of

fluorescently labelleci Ieukocytes in the blood compartment. Monocytes, lymphocytes,

and granulocytes were simultaneousIy investigated Determining the half-times of these

leukocytes in the blood gives information on the basic biology of these different ce11

types-

Chapter 3 of this thesis documents the reduction of fluorescently labelled

leukocytes in blood from which the half-tirnes of these labelled leukocytes were

determined mathematically. Chapter 3 also shows the reassortment of fiuorescently

labelled lymphocytes in lymphoid compartments afler recirculation equilibration was

reac hed,

Chapter 4 outlines a new rnethodology for in situ labelling of al1 Ieukocytes in the

blood cornpartment using the intmcellular fluorescent dye carboxy fluorescein diacetate

succinimidyl ester (CFSE).

blood and aIso demonstrate

vivo.

These

a new

studies give insight

powemil method to

into immune ce11 trafic in the

study blood leukocyte trafic in

Chapter 2

General Materials and Methods

2.1 Animals

Outbred ewes weighing 30-35 kilograms were used for al1 experiments with the

exception of three experiments in Chapter 4 that used pregnant Wistar rats. Ewes were

obtained either fiom Boxwood Farms (London, Ontario) or Renwick Farrns (Oshawa,

Ontario). The animals had unrestricted access to hay and water and in addition were

given pellets of alfalfa once daily. Sheep were ailowed a minimum of three days to

acclimatize to the animal facility at the University of Toronto. Al1 experiments were doue

in accordance with the Canadian Council on Animai Care, the Animais for Research Act

of Ontario, and by the Animal Care Cornmittee at the University of Toronto.

Animals were initially anaesthetized with pentothal sodium 15-25 mgkg i.v.

(Boehringer hgelheim, Burlington, Ontario). An endotracheai tube was used to intubate

the animai, and a surgical plane of anesthesia was maintained with 2% isofluorine in 0,.

Mer ligation of either the right or left jugular vein a catheter with a 3-way stopcock was

surgicaily placed in the vein through an incision downstream of the ligation site. The

catheter was sutured in place and the stopcock was exteriorized Depending on the

expriment perfmed (See Chapters 3 and 4) prescapular, and/or prefemoral efferent

lymphatics were cannulated The method for this surgical winulation are described

elsewhere in detail (Young, Hein et al. 1997). Briefly, a short stretch (a 2cm) of the

lymphatic vesse1 of interest was exposed and ligated downstream of the site of

cannulation. Forceps were used to carefully remove fascia away fiom the lymphatic until

the vessel wall of the lymphatic was clearly exposed. A small incision was made in the

lymphatic vessel wail large enough to allow insertion of the cannula. Polyvinyl chloride

cannulas (SV45, Dural Plastics and Engineering, Dural, Australia) with an outer diameter

of 0.6 - 1.2rnrn were Bushed with sterile heparinized saline, and inserted into the

lymphatic. The cannula was sutured in place and extenorized to the outside of the body.

The entrance used to gain surgical access was sutured and the cannda was diverted into

stenle bottles that contained hepannized saline (0.5rnl containing 500 units of heparin;

Hepalean, Organon Teknika, Toronto, Ontario). Animals were given O.OOSmg/kg i.m.

buprenorphine HCI analgesic (Temgesic, Reckitî and Colman, Hull, England)

immediately afler surgery was cornpleted. Animais were allowed at least one &y after

surgeiy to recover before experiments were continued.

2.3 Cell Labeling and Detection

Ex vivo labelling of cells (Chapter 3) was accomplished using one of two

fluorescent ce11 tracking dyes, fluorescein isothiocyanate (FITC) (Sigma, Oakville,

Ontario), and CFSE (Molecular Probes, Eugene, Oregon). S ince di fferent protoco 1s were

used for the various dyes, the methodologies will be discussed in each of the chapten. A

newly developed method for in vivo labelling of blood leukocytes will be discussed in

Chapter 4.

Flow cytometric analysis of blood and lymph sarnples was accomplished using a

Becton Dickinson FACScalibur flow cytometer equipped with a 488nm argon laser and

530n.m band-pass filter. Lymph samples were washed twice ( 1400rprn, for 10 minutes

4°C) with phosphate bufYered saline (PBS), then fked with 500pI of 1%

pamformaidehyde before flow cytornetric analysis. For blood samples, red blood cells

were lysed using distilled H20 in a 4: 1 ratio to blood, incubated on ice for 15 seconds

before king m h e d (1400rpm, for 10 minutes 4OC) with PBS. The procedure was

repeated with just PBS unless upon visual inspection red blood cells were still present in

the pellet, in which case water lysis was performed again The sample was then fixed

with 500pl of I% paraformaldehyde, before flow cytometric analysis. Whenever possible

both iymph and blood sarnples were kept on ice.

2.4 lmmunofluorescence Staining for Flow Cytometric Analysis of CD14 Expression

Samples of blood and lymph were washed as described above in section 2.3. The

cellular pellet was resuspended in PBS containing magnesium and calcium, so that a

150~1 aliquot would contain 2x1 o6 cells. 150pI (2x1 o6 cells) was aliquoted per well of a

96-well, U-bottom microtitre plate (Becton Dickinson, Lincoln Park, New Jersey). 40p1

of the prhary Ab (CD14, MCA 920 Cederlane, Homby, Ontario, 1 :20 dilution; Ab:PBS

containing magnesium and calcium) was added to the well and mixed thoroughly. The

plate was incubated for 10 minutes on ice. The plate was centnfuged (1400rpm,

IOminutes at 4OC) and the cells in each well were washed using 200jd of PBS containing

magnesiurn and calcium (1400rpm, 1Ominutes at 4°C). The cellular pellet was

resuspended in 150pi of PBS containing magnesium and calcium and 40pi of the

secondary Ab (1:200 dilution; Ab:PBS containing magnesium and calcium) conjugated to

allophycocyanin (MC, Cederlane, Hornby, Ontario) was added and the plate was

incubated on ice in the dark for 10 minutes. The plate was centrifiiged (1400rprn,

lominutes at 4OC) and the cells in each well were washed using 200pl of PBS containing

magnesium and calcium (1400rpm, lominutes at 4OC). The pellet was resuspended in

200~1 of PBS containing magnesium and calcium and 500pl of I% paraformaldehyde

was used to fix each sample. Samples were stored at 4OC and were analyzed by flow

cytornetry within a maximum of 48 hours. Control wells included a well with unstained

cells, cells incubated with secondary Ab alone, and cells incubated with non-specific

mouse igG (undiluted) followed by secondary Ab. Secondary Ab done did not have

background staining due to non specific binding. The mouse IgG antibody followed by

secondary antibody gave a background level of staining that was consistently around 1%.

2.5 Cellular Suspensions From Solid Tissues

In order to determine the percentage of labelled lymphocytes in solid tissues the

following procedure was used Immediately after the animal was sacrificed biopsies of

solid lymphoid tissues (refer to Chapter 3) were collected in 50 ml conical tubes (VWR

Canada, Mississauga) that contained Iscove's Modified Duibecco's Media (Gibco Life

Technologies, Burlingtoo, Ontario) with approximately 200 units of heparin. Samples

were kept on ice whenever possible. The contents of the 50ml tube were deposited into a

petri dish and kept on ice. A scalpel and forceps were used to gently tease apart solid

tissues in order to release cells From the tissue. A 0.22p.m cellular filter (VWR CanaQ

Mississauga) was used to strain the sample and the cells were washed with PBS

( 1400rpm, lOminutes at 4OC). If any aggregates formed during washing the sample was

filtered again using a 0 . 2 2 ~ cellular Nter (VWR Canada, Mississauga). Samples were

fixed using 500~1 of 1% pdomaldehyde, and were andyzed by flow cytometry within

48 hours.

2.6 Reagents

2.6.1 Dulbecco$ Phosphate Buffered Saline 10X

For al1 ceil washes excluding immunostaining procedures Dulbecco's phosphate

buffiered saline was used (PBS). The PBS ( IOX) was made using the following protocol:

NaCl (sodium chloride) 400.0g

KCl (potassium c hioride) 10.0g

Na2HP04 (sodium di-phosphate) 57.58

KHzPOr (potassium bi-phosphate) 10.0g

Reagents were added to 4 Iiten of distilled H20 and stirred until completely dissolved

The PBS was dispensed in 500ml aliquots and autoclaved. As needed 10X PBS was

diluted 1: 10 with distilled H20 in order to make 1X PBS which was used for cellular

washes.

Dulbeccos' phosphate buffered saline with calcium and rnagnesium was used for

resuspending cells and Abs in immunofluorescence staining procedures. PBS with

calcium and magnesium was made with the following protocol:

SoIution E

NaCl (sodium chloride) 40.0g

KCI (potassium chloride) 1 .Og

NazHPOJ (sodium di-phosphate) 5.758

K&POs (potassium bi-phosphate) 0.2g

Reagents were completely dissolved in 4 liters of distilled H20, dispensed in 400rnl

aliquots and autoclaved.

Solution II

CaCI? (calcium chloride) 0.5g

Reagent was completely dissolved in 500mI of distilled H20, dispensed in 50ml aliquots

and autoclaved.

Solution III

MgCIi (magnesium chloride) 0.5g

Reagent was completely dissolved in 500mI of distilled H20, dispensed in 50ml aliquots

and autoclaved.

As needed an individual aliquot of solution 1, II, and III were mixed together to make the

completed form of PBS with calcium and magnesium.

2.6.2 Anticoagulant Solutions

Heparin 1000units/ml (Hepalean, Organon Teknika, Toronto, Ontario), acid

citrate dextrose (ACD) and ethylenediamine tetraacetic acid (EDTA) were al1 used for the

various experiments performed (see Chapters 3 and 4). ACD was prepared using the

following protocol :

C&N&O7 (Trisodium citrate) 2.5g

C&Na (Citric acid) 1.5g

Ca&) (Dextrose) 2.0g

Reagents were dissolved in 1 OOrnl of dHzO autoclaved ACD was used as a blood

anticoagulant in the ratio 1:6, (ACD:blood).

A 7.5% solution of EDTA by weight, in 0.9% saline was also used as an

anticoagulant. 2.5 ml of 7.5% EDTA was used for every 60 ml of blood.

2.6.3 Parafowaldehyde Solution

1 Oh parafomaldehyde solution was made usine the followine protocol:

1OOml of PBS was heated to 56OC and 1 gram of paraformaldehyde was added The

mixture was stirred at 56°C until the paraformaldehyde was completely dissolved. The

solution was left at room temperature to cool. The 1% parafomaldehyde solution was

fi Itered through a 0.2 micron filter (VWR Canada, Mississauga) to remove precipitated

particles. The 1% paraformaldehyde solution was stored at -20°C until required.

2.6.4 Heparinized Saline Solutions

Low dose heparin/saline solutions were made by adding lOm1 of heparin

( 1000uniWml, Hepalean, Organon Teknika, Toronto, Ontario) to a 1 O O m i bag of sterile

0.9% saline. Low dose hepa.rin/saline solution was used to flush indwelling jugular vein

catheters. High dose heparinfsaline solutions were made by adding 2ml of heparin

(10000units/mi, Hepalean, Organon Teknika, Toronto, Ontario) to 18 ml of sterile 0.9%

saline. High dose heparinlsaiine solution was used as an anticoagulant for chronic iymph

collections.

Chapter 3

The Half-Times of Fluorescently Labelled Monocytes,

Granulocytes and Lymphocytes in the Blood

Leukocyte migration and recircuiation along with hansient tissue distribution is

thought to be the basis of immune surveillance. In order to study the physiological basis

of this phenomenon both Buorescein-5-isothiocyanate ( FITC ), and (5-(and-6))

carbo-fluorescein diacetate succinimidyl ester (CFSEI. were used to mark blood

leukocytes. The labelled blood leukocyies were returned via the venous circulation of

sheep and tracked in the blood, lymph, peripheral lymph nodes and spleen. The

emigration kinetics out of blood were studied over a 12 day period o b s e ~ h g the initial

leukocyte reassortment and Iater recirculationdependent reduction of labelled blood

lymphocytes. The half-times obtained for these leukocy-tes in the blood cornparmient

were 12.8 hours, 9.8 hours, and 406.0 hours for monocytes, neutrophils, and lymphocytes

respectively. Tissue reassortment of labelled lymphocytes in lyrnphoid compartrnents was

also obtained indicating that the overwhelming majority of lymphocytes in a resting

lymph node are part of the recirculaiing lymphocyte pool and are "in transit" rather than

residential.

3.2 Introduction

Cellular traffic through the blood compartment is very dynamic and complex. The

blood cornpartment although it houses oniy 1.2% of the bodies total lymphocytes (Chin,

Pearson et al. 1985) is the center of recircdation as it is the entry point for newly formed

cells h m prirnaty iymphoid oreans. and the retum point for recirculating lymphocytes.

Lymphocyte input via the thoracic duct has been determined to be approxirnately 1 x lo9

cells per hour (Heath, Lascelles et al. 1962). This input of lymphocytes from the thoracic

duct equals the total number of lymphocytes in the blood every 10 hours. In addition to

thoracic duct input, the blood receives recirculating lymphocytes from prescapular and

cervical efferent Iymphatics, and newly formed leukocytes from primary lyrnphoid

organs. Homeostatic balance is maintained through lymphocyte and monocyte emigration

out of the blood and ce11 death. Superimposition of all of these processes results in the

characteristic half-times of leukocytes in the blood compartment which were studied

here.

Sheep offered particular advantages in performing these experirnents as they

ailowed many sequential blood samples to be taken, accurately mapping the reduction of

fluorescently labelled leukocytes from the blood. This was accomplished while not

comprising the physiology of the animal due excessive blood Ioss. The use of sheep also

allowed sampling of the efferent lymph which is not possible is smaller animals (Hein

1995).

In addition, after allowihg five days for equilibration of lymphocytes through the

body it was possible to obtain the percent of fluorescently labelled lymphocytes in the

spleen, lympb nodes, mesenteric efferent lymph and blood

3.3 Materials and Methods

3.3.1 Animais

A total of ten sheep were used for these studies. Five sheep were used in

experiments to obtain the reduction of CFSE labelled leukocytes from the blood Five

sheep were used in experirnents to analyze the tissue reassortment of FITC positive blood

lymphocytes. The surgical procedure for cannulation of the jugula vein were described

in Chapter 2.

3.3.2 Leukocyte Labelling with CFSE and FITC

For al1 labelling protocols stede equipment and techniques were used throughout

the procedure.

CFSE labelling protocol

340-360ml of blood was colleaed via the jugular vein catheter into 60ml sphges

contain 2.5mi of 7.5% EDTA (Sigma, Oakville, Ontario). Blood was placed in two 250ml

plastic bonles and spun at 1400rprn for 20 minutes with the centrifuge brake off. The

plasma supernatant was removed by vacuum pipetting using a lOml pipette, being

extremely careful not to disturb the bu@ coat The bIood cells were resuspended in cold

PBS (kept on ice) and equaiiy divided into four 250rnl bottles which were each filled

with PBS to a total volume of 200mI. The blood was centrifbged as above, and again the

supernatant was removed The wash was repeated one more time. The cells were

resuspended in 8pM CFSE with a volume equal to the original blood volume (ie. IOOml

of 8pM CFSE was added to the ceUs obtained from lOOd of blood). 8p.M CFSE was

made fiom a 500pM stock solution of CFSE in dimethyl sulphoxide @MSO, Sigma,

Oakville, Ontario), diluted with the appropriate volume of PBS to obtain a 8pM

concentration. The cells were incubated at 37°C for 15 minutes and then centrifüged at

1400rpm for 20 minutes with the centrifuge brake off. The supernatant was removed and

cells were washed in PBS as above two additional rimes. Approximately 40ml of 0.9%

saline was added to the labelled cells before infusion via the jugular vein catheter.

FITC labelling protocol

The above CFSE labelling procedure was followed to label blood cells with FITC

with a few exceptions (Andrade, Johston et al. 1996). First, blood was collected in ACD

indead of 7.5% EDTA (Sigma, Oakville, Ontario). Secondly, instead of resuspending

cells in CFSE, cells were resuspended in a total of 4OOrnl of saturated FITC solution and

incubated for 30 minutes at 4OC. Saturated FITC solution was made by adding 0.05g of

FITC in 500mI of PBS and stirring the solution overnight at 4OC. The saturated FITC

solution was filtered using a 0 . 2 2 ~ vacuum filter unit (VWR Canada, Mississauga)

before use.

3.3.3 Analysis of Samples

Blood and lymph samples were collected using heparin or EDTA as an

anticoagulant For blood samples a 2 - 3ml sample of blood was taken and discarded

before the officia1 sample was taken, to ensure that blood that may have been present in

the catheter wasn't sampled The samples were prepared for flow cytometric anaiysis as

descriid in Chapter 2. Samples were always analyzed twice and whenever possible

1x10~ cells were analyzed each time. Cells were distinguished on their characteristic

forward and 90" side scatter of light For the experiments investigatiog the half-times of

leukocytes in the blood, a CD14 rnAb was used for immunofluorescence staining of

blood leukocytes (procedure in Chapter 2). Gating on mononuclear cells based on their

forward and side scatter, CD14 positive cells were distinguished by their fluorescence as

the CD14 antibody was detected with a goat anti-mouse secondaxy Ab conjugated to

APC. Cells that were CD14 positive within the mononuclear gate were considered to be

monocytes, while cells that were CD14 negative within the mononuclear gate were

considered to be lymphocytes. Granulocytes were distinguished based on their forward

and side scatîer of light Using the above method to distinguish between the ce11 types,

the percentage of fluorescently label led ce1 1s wi thin that ce1 1 population was determined

based on CFSE fluorescence intensity.

in experiments investigating the tissue reassortment of fluorescently labelled

lymphocytes, blood, mesenteric efferent I p p h and secondary lymphoid tissue samples

were prepared as outlined in Chapter 2. Sarnples were anaiyzed twice and whenever

possible at least l x l ~ ' cells were analyzed each time. For al1 sarnples small lymphocytes

were detected and gated on based on îheir characteristic forward and 90' side scatter of

light. The percent of FITC positive lymphocytes was determined in each sample based on

fluorescence intensity.

3.3.4 Calculations and Statistics

Ushg sheep in these experiments was advantagrnus as many sequential blood

sarnples couid be taken in each of the experiments, documenthg the initiai and long term

reduction of labelled leukocytes. The reduction curves were plotted as percent of labelled

cells (log scale) versus time in h o m (linear scale). Since monocytes were identified

using a CD14 mAb, the background level of fluorescence was subtraaed from the

obtained value before a semi-log plot was completed. A line of best fit was obtained in

the form of log y = bix + bo, where bi = dope and bo = y-intercept, using linear

regressional analysis. Half-times were calculated fiom this equation by calculating the

time that was required for the initial percent of labelled cells (at time = O) to half

Calculations for bi and bo were as follows:

The sample estirnate (s') of the population deviation o' from the line of best fit

was obtained by summing the squares of the residuals as follows:

The standard errors for the estimated regression coefficients were calculated as

follows:

The correlation coefficient r was cdculated as follows:

r = d(+).

Tissue reassortment data was collected as a percent of labelled lymphocytes found

in the various tissues anatyzed The percent of labelled lymphocytes found in the various

tissues were averaged from the different animais tested and the standard deviation and

standard error of the mean were calculated. Analysis of variance was used to determine if

there was a sipifkant difference between compartments.

3.4 Results

3.4.1 CFSE CeII Labelling

Figure 3.1 shows a dot-plot of forward scatter versus side scatter of light

generated by flow cytometric analysis of a unstained blood sample. Gates 1, 2 and 3 are

shown which remained constant throughout this series of experiments. Gate I (RI)

surrounds mononuclear cells, gate 2 (R2) surrounds granulocytes, and gate 3 (R3)

surrounds mail lymphocytes. Figure 3.2 shows botb negative (unstained) blood

leukocytes, pre-infusion blood leukocytes afler in vitro labelling with CFSE, and

leukocytes fiom a blood sample taken 5 minutes pst infusion of labeiled leukocytes for

al1 three gates. The labelling of blood leukocytes was well demarcated as compared to

unstained cells with 100% of cells king Iabelled CFSE is a long lived fluorescent dye,

therefore cells were readily distinguishable at &y 12 (Figure 3.3) and beyond Figure 3.4

Figure 3.1 Differeotial Gating of Sheep Blood Leu kocytes

Representative flow cytometric analysis of sheep blood leukocytes. The dotplot shows

forward light scatter (linear scale) venus side Iight scatter (log scale). Gates were

constnicted as shown in the dotplot Gate 1 (RI ) encompasses mononuclear cells, gate 2

(R2) encompasses granulocytes, and gate 3 (R3) encompasses smali Iymphocytes. The

gates were kept constant throughout this series of experiments and are refened to in text

as R 1, R2, or R3 or as mononuclear cells (R 1 ), _mulocytes (neutrophils, R), and small

lymphocytes (R3) respectively.

Figure 3.1

Figure 32 Fluorescence Intensity of Unstained, CFSE Labelled (ln Vitro) and Post Infusion Blood Leukocytes

Representative histograms generated by flow cytometric anaiysis of unstained leukocytes,

CFSE labelled leukocytes before infusion and a blood sample taken 5 minutes p s t

infusion of labelled leukocytes. Al1 histograms shown are ceil count versus CFSE

intensity (FL 1, log scale). Panel A, B and C are unstained blood leukocytes, stained blood

leukocytes, and blood leukocytes five minutes p s t infusion fiom the R1 gate

respectively. Panel D, E and F are unstained blood leukocytes, stained blood leukocytes,

and blood leukocytes five minutes pst infusion from the R2 gate respectively. Panel G,

H and 1 are unstained blood leukocytes, stained blood leukocytes, and blood leukocytes

five minutes poa infusion from the R3 gate respectively.

Figure 3.2

Panel A, unstained mononuclear cells R 1 gate

Panel B, CFSE labelled mononuclear cells R1 gate

Panel C, 5 minutes p s t infusion of labelleci cells RI gate

Figure 3.2

Panel D, unstained granulocytes R2 gate

Panel E, CFSE labelled granulocytes R2 gate

Panel F, 5 minutes p s t infusion of labelled cells R2 gate

Figure 3.2

Panel G, unstained mononuclear cells R3 gate

Panel A, CFSE labelled mononuclear cells R3 gate

Panel ï, 5 minutes p s t infusion of labelled cells R3 gate

Figure 3 3 Detection of CFSE Labelled Mononuclear Cells 12 Days Post Infusion of La belled CeIb

Representative histograrn generated by flow cytometric analysis of blood mononuclear

cells (R 1 ), 12 days pst infusion of labelled cells. Histogram shown is ceIl count versus

CFSE intensity (FL 1, log scale).

Figure 3.3

Figure 3.4 Deteetion of CD14 Expression on Sheep Blood Leu kocytes

Representative flow cytometric analysis of sheep blood leukocytes. Dot plots show CFSE

Intensity (FL 1 ) versus APC intensity (FL4, CD 14 expression). Al1 dot plots were

renerated with cells withm the R 1 gate. Panel A, B, C and D show a blood sample of c.

unstained mononuclear cells, unstained mononuclear cells checked for CD 14 expression,

CFSE labelled mononuclear cells (pre-infusion) checked for CD 14 expression, and

mononuclear cells collected 15 minutes p s t infusion of labelled leukocytes checked for

CD 14 expression, respectively .

Figure 3.4

Panel A Panel B

Panel C Panel D

shows dot-plots of CFSE intensity venus APC intensity. Cells were tested for CD14

expression, detected by a secondary Ab conjugated to M C . The dot-plots were generated

using cells within the mononuclear ce11 gate (Rl). Four dot-plots are shown in this figure.

Panel A is unstained mononuclear cells (R 1), that were not c hec ked for CD 14 expression.

The other panels B, C, and D show CFSE venus CD14 expression on unstained

mononuclear cells (RI), CFSE positive pre-infusion mononuclear cells ( R I ) and dso a

blood sample taken 15 minutes after infusion of CFSE positive cells (Rl). 100% of

monocytes labelled in vitro, and in the dotplot shown 15 minutes p s t infusion, 1.8% of

monocytes were CFSE positive.

3.4.2 Reduction of Labelled Cells from the Blood Cornpartment

Figures 3.5 - 3.8 show the reduction from the blood compartment of labelled

Ieukocytes for each of the three leukocyte types investigated. Figure 3.5 is a composite

graph showing the reduction of dl three leukocyte types on the same time sa le for

comparative rasons. Figures 3.6 - 3.8 show the reduction From the blood compartment

of al1 three leukocyte types individually. Each reduction cuve is shown twice, first on a

linear scale of percent of labelled cells venus time and than a semi-log scde showing log

of percent of labelled cells versus time. Neutrophils and monocytes demonstrated a linear

fit as shown by the semi-log plots, indicating the reduction of these cells from the blood

compartment closely followed an exponentiai c w e . Lymphocytes however showed a

biphasic response with an initial sharp reduction from O to 8 hours followed by a second

more gradual reduction Eoom 10 to 276 hours. These distinct phases each represent an

exponential reduction and were andyzed independently. Table 3.1 shows the caiculated

Figure 3.5 Composite Graph of the Reduetion of CFSE Labelleci Monocytes, Neutrophils, and Lymphocytes in the Biood

Graph shows percent of labelled cells venus time in hours. The y-axis and error bars

have been omitted for clarity and are shown in additional tigures below.

Figure 3.5

--

+ MONOCYTES + NEUTROPHILS +- LYMPHOCYTES

Time (Hours)

Figure 3.6 Reduction of CFSE Labelled Monocytes from the Blood Cornpartment

Graph A shows percent of labelled monocytes (linear scale) venus time in houn. Error

bars were generated using standard error of the mean. Graph B shows percent of labelled

monocytes (log scale) venus time in hom. (n = 4)

Figure 3.6

Graph A

t MONOCYTES

Tirne (Hours)

Graph B

I O - -+ MONOCYTES

0.1 O 20 40 60 80 100

Time (Hours)

Figure 3.7 Reductioo of CFSE Lebelled Neutrophils from the Blood Cornpartment

Graph A shows percent of labelled neutrophils (linear scale) venus time in houn. Error

bars were generated using standard error of the mean. Graph B shows percent of labelled

neutrophils (log scale) venus time in hours. (n = 5 )

Figure 3.7

Graph A

NEUTROPHILS

Graph B

-

-c NEUTROPHlLS

40 60

Time (Hours)

Figure 3.8 Reductioo of CFSE Labelled Lymphocytes from the Blood Cornpartment

Graph A shows percent of labelled lymphocytes (linear scale) venus time in hours. Error

bars were generated using standard error of the mean. Graph B shows percent of labelled

lymphocytes (log scale) venus time in hours. (n = 5 )

Figure 3.8

Graph A

.c LYMPHOCYTES

Time (Hours)

Graph B

-e LYMPHOCYTES

Time (Hours)

Table 3.1 Summary of the Reduction of La belled Leukocytes Based on Linear Regressional Analysis

Shown in the table are the linear equations (log y = bix + bo) defining the reduction

cwes, calculated half-times correlation coefficient (r), sample estimate of the

population a (s), standard error of the dope (sbl), and standard error of the y-intercept

(sbO)? based on linear regressional analysis of logarithmic reduction plots.

Table 3.1

Sb0

1 Monocytes O - 8 hours

Cell

I i ) Lymphocytes -0.0723~ + 0.5344 4.2 0.91 0.1033 0.0 177 0.0606

r

-0.06 17x + O. 1578

1 0 - 8 hours I

I I Lymphocytes

10 - 276 hours

I

I 1

s 1 sbl

1

Equation

! i

I -O.OMx - 0.3547 1 19.9 1 0.69

1 ! l

1 Monocyes -û.O?%x - 0.0200 ' i 0.87 ' 0.2605 l O -60 houn i i2-8 I I i

Tm

4.9

10 - 60 hours

-0.00074~ +O. 1 109

(logy=)

1 i

0.98 0.0004 0.03 13

1 I I

I / I

(Hrs.)

Neutraphilr , -0.0307~ + 0.55 13 ! I

l 9.8 , 0.99 0.0810 1 0.0011 , 0.0274 1 0-60 houn

406.0 0.94 0.0252 7.1 x 10" 0.0 1 O3

line of regression determined from the semi-log plots for each of the ceIl types dong with

the associated errors and calculated half-tirnes. Although monocytes had a strong linear

correlation coefficient on the semi-log plot, this c w e shows a slight biphasic response,

so in addition to a single linear analysis, each of these phases was analyzed independently

as well (See Table 3.1 ).

3.4.3 FITC Cell Labelling and Tissue Reassortment

FITC was employed to label blood leukocytes in a series of experiments aimed at

investigating the tissue reassortment of labelled blood lymphocytes. The tissue

reassortment experiments were completed pnor to the use of CFSE in our laboratory.

CFSE could have been used in these experiments and an example of tissue reassortment

of lymphocytes using CFSE is shown in the appendix (Figure A.?). Figure 3.9 shows

unstained mononuclear cells, mononuclear cells labelled in vitro with FITC (pre-

infusion) and rnononuclear cells from a blood sample taken 3 days after infusion of

labelled cells.

Figure 3.10 shows the average percent of RTC labelled lymphocytes found in the

various tissues tested The graph was generated by flow cytornetric anaiysis of small

lymphocytes (R3) for al1 samples tested.

3.5 Discussion

The two main fluorescent dyes used in these studies CFSE and FITC label cells in

different ways. CFSE is the acronym that is used to describe the dye CFDA-SE,

carboxyfiuorescein diacetate succinimidyl ester. This molecule is highly membrane

Figure 3.9 Fluorescence Intensity of Unstained and F'ITC Labelleci Blood Cells

Representative histograrns generated by flow cytomenic analysis of unstained cells. FITC

labelled cells before infusion and 3 days pst infusion via the jugular vein catheter. Ail

histograms shown are ce11 count versus FITC intensity (FL 1 , log scale). Panel A, B and C

are unstained blood cells, stained blood cells, and blood cells collected 3 days pst

infusion of labelled cells, fiom the RI gate respectively.

Figure 3.9

Panel A

Panel B

Panel C

Figure 3.10 Tissue Reassortment of FlTC Labelled Lymphocytes

Graph shows the percent of labelled small lymphocytes (R3) in the blood, spleen,

mesentenc 1 ymph, mesenteric 1 ym ph node, pre femo rai 1 ym ph node and prescapuiar

lymph node five days pst infusion of Iabeiled blood cells. Error bars were generated

using standard error of the mean. (n = 4)

Figure 3.10

Blood Spire n Masanbric Memnteric Pmhrnoml Pnscapular L P P h Noda Node Node

Tissue

permeant and enters and leaves cells quickly. However, during the short stay in the ceil

the esterases in the leukocytes can cleave the two acetate groups off the molecule which

considerably slows the exit of the molecule out of the cell. This leaves enough time for

the succinimidyl moiety to make stable amide bonds to intracelldar proteins (Parish

1999). Many of the proteins are short lived, so there is a significant loss of dye within 24

hours. After this initial loss of label, subsequent loss is very slow allowing in vivo

tracking of CFSE positive non-dividing lymphocytes for weeks to 6 months (Parish

1999). FITC works by reacting and forming a covalent bonds with extracellular proteins

contauiing lysine residues. Fluorescence is also seen in the cytoplasm and is most likeiy

due to membrane protein turnover. Both dyes can be toxic to ceils at high levels,

however, since lymphocyte extravasation is an active process (Springer 1994),

recircdating cells must be alive and active. In addition viability of cells labelled in vitro

was tested by trypan biue exclusion and typically >95% of the cells were viable pnor to

retuming the cells to the animais blood.

The reduction of labelled cells in the blood cornpartment is a very complex

process and it is furthet convoluted by the tenninology used in fields like this that

investigate very dynamic processes. The tenn half-time in the blood cornpartment has

been used above which refen to the time it takes for the percent of labelled ceils in the

blood cornpartment to half However, this half-time in the blood does not necessariiy

reflect the cells haif-life which is the time it takes for half the cells in a population to die

or divide. These terms are adapted meanings because the term half-life was onginally

used to describe the time it takes the radioactivity of a substance to fail to half its original

value (Hawkins 1988). This involves a single parameter of radioactivity, while in vivo

cell tracking involves two parameters, time spent by cells in a particuiar cornpartment of

the body, and life span of the cells in question. Hence, half-time and half-life will be used

to describe these two panuneters respectively.

There are many reasons why the half-tirne in the blood rnay not be equivdent to

the half-life of the cells. The processes involved in the reduction of labelled cells fiom the

blood are varied and complex and their cumulative effect on blood dynarnics is what

gives the characteristics half-times of different ce11 populations in the blood. One process

that causes a reduction in the percent of labelled leukocytes in the blood is the input of

newly formed leukocytes from primary lymphoid organs. An additional input to the

blood includes recirculating lymphocytes entering the biood via the thoracic duct,

prescapular and cervical efferent lymphatics. In tems of output fiom the blood

cornpartment there is ernigration of cells out of the blood system by extravasation, a

process that monocytes and lymphocytes can undergo at steady state, as wetl as

leukocytes death. Although both monocytes and lymphocytes share the ability to

ernigrate out of the blood, they don? have the same capacity to return to the blood. This

is due to the ability of lymphocytes to traverse nodes and enter efferent lymphatics, while

monocytes can not, and thus do not return to the blood via efferent lymphatics (Pugh and

Macpherson 1982).

In addition leukocytes may be capable of marginating in the blood vasculature.

Although this is not a reduction of labelled cells from the biood cornpartment it does

represent a loss in the ability to sample these marginated cells by an i.v. blood sampling.

Therefore, this is seen as a reduction of labelled cells from the blood. The spleen also

represents a complex transit location dong the blood system where again if labelled cells

enter the spleen the observation is a reduction of labelled cells as determined by blood

sampling via the jugular vein Finally, there are large microvascular beds present in

locations such as the lung and liver that leukocytes must migrate through at a retarded

rate as compared to larger veins and arteries. Once again entry of labelled cells into these

microvascular beds results in the observation of a reduction of labelled cells as gauged by

jugular vein sampling.

The three processes mentioned above margination, migration through the spleen,

and migration through large microvascular beds are of particular importance when

considering what initially happens to a bolus of labelled leukocytes that are infused into

the blood. Within 2 - 3 minutes the bolus of labelled leukocytes will equilibrate in terms

of concentration of labelled leukocytes in large arteries and large veins (unpublished

observations, Hay, Cahill). This is important to note because this means that teduction

kinetics observed in the blood after a few minutes are not due to labelled cells being

dihted in the blood

Another important consideration for initial reduction kinetics in terms of

lymphocytes is that in the first two hours there are no labelled lymphocytes retuming to

blood due to recirculation. The recirculation process in ongoing and in tems of the

labelled lymphocytes begins as soon as the labelled cells are infused into the blood.

However, there is a lag time before lymphocytes that have emigrated from the blood can

retu..cn to the blood This lag time is approxhately 2 hours at which point labelled

lymphocytes begin to migrate back to the blood (Seabrook, Au et ai. 1999). M e r 2 hours

there is a steady increase in the concentration of labelfed lymphocytes retuming to blood

with peak concentration k ing obtained between 21 and 24 houn (Abemethy and Hay

1992). The important point about this is that the initial reùuction of lymphocytes occurs

before biologicd equilibrium of recirculation is established with respect to labelled

lymphocytes. Therefore, the initial reduction of lymphocytes is not counteracted by the

retum of label led recirculating lymphocytes. In addition during the initiai reduction

kinetics there is the chance for ce11 death to occur which would contribute to the

reduction of Iabelled leukocytes. One expects ce11 death to be minimal for labelled

lymphocytes during the fint 8 hours, as the half-life of these cells was determined to be

406.0 hours. In contrast ce11 death for monocytes and neutrophils in the fim 8 hours may

be substantial. However, the potential ability of cells to marginate, combined with

migration through the spleen and microvascular beds, is most likely the major cause of

the initiai leukocyte reduction observed in the blood. There is likely a very large input of

labelled leukocytes into these areas before a biological equilibriurn is established for

labelled leukocytes in tems of relation of output to input. It is important to note that a

biological equilibrium in tems of labelled leukocytes does not necessady mean output

of labelled leukocytes is nurnerîcally balanced by the input. It refen to the individual

system in question reaching a state of output that is in accordance with steady state

dynarnics to its input with respect to labelled leukocytes. Essentially the spleen and

rnicrovascular beds need to be "primeci" with labelled leukocytes before a biological

equilibrium can be established with the labelled leukocytes. Therefore, just as there is a

lag time with recircdation, there wiIl be an initial state where there is an entry of labelled

leukocytes into these systems with no output of labellecl leukocytes. However, by i.v.

sampling as done in this experiment this priming represents a reduction of labelled ceils

fiom the blood To complicate the matter the rates of migration through the spleen, and

microvascular beds are most likely different leading to different biological equilibration

times for these individual areas.

For al1 of the reasons discussed above the initial half-times obtained for

leukocytes may not reflect their half-lives. Therefore the haif-time in the blood is a

measure of emigration, margmation, reduction due to lag time before biological

equilibrium is establisheâ, recirculation, ce11 death, and new ce11 formation, superirnposed

on one another and therefore rnay not initially reflect the half-life of labelled cells.

As tirne goes on biological equilibrium is established with respect to the Iabelled

leukocytes in the different compartments. This is well documented for labelled

recirculating lymphocytes that return to the blood via thoracic duct, prescapular and

ceMcal lymphatics as mentioned above. Labelled lymphocytes c m be detected in these

lymphatics at approxirnately 2 hours pst infusion and the concentration of labelled

lymphocytes increases steadily with maximal concentration occurring between 2 1 to 24

houn, More it begins to decline and level off as biological equilibrium is established

(Abemethy and Hay 1992). Figure 3.1 1 below shows the different stages that wiil occur

afler a bolus of Buorescently labelled leukocytes are infused into the blood. AAer

biological equilibration is obtained the half-times in the blood represent a measure of

different processes than the initial kinetics. This will be discussed in detail below.

Once ail of the compartments have equilibrated with respect to the labelled cells

the subsequent half-time may actually reflect the half-life of that ce11 population. For this

discussion it is imperative to look at neutrophils, monocytes and lymphocytes separately.

The semi-log plot of neutrophils versus tirne in Figure 3.7 shows a very linear plot with

correlation coefficient of 0.99. Therefore, it closely approximates an exponential curve.

Figure 3.11 Diagrammatic Representation of the Reassortment of Labelleci Lymphocytes After Infusion into the Blood

Each successive diagram shows the reassortment of lymphocytes as time passes. The

time points shown are just estimates and represent a single time point in an ongoing

dynarnic process, illustrating how different compartments in the body can reach

Iriological equilibriurn at difkrent times with respect to labelled lymphocytes. D i a m A

shows that within minutes the bolus of labelled lymphocytes has already been equally

distributed in large artenes and veins of the blood system. Diagram B shows that afler 15

minutes lymphocytes have already begun to migrate to the different compartments

shown. Diagram C shows that afler 1 hour a noticeable amount labelled lymphocytes

have entered the various compartments and in the case of microvascular beds labelled

lymphocytes are already retuming to the blood at their biological equilibration rate. There

is also labelled lymphocytes retuniing to the blood via the spleen. By one hou there is

already a decrease in the concentration of labelled lymphocytes in the blood as the other

compartments are receiving these lymphocytes. By 8 hours (Diagram D) the spleen has

reached biological equilibrium, there is an even greater decrease of labelled cells in the

blood, and some labelled recircdating lymphocytes are returning to the blood via the

lymphatic system. By 36 hours (Diagram E) recircuiation through Iymph nodes and non-

lymphoid tissues has also reached biologicai equilibrium. Note that the concentration of

labelled lymphocytes in the differed compartments is not equal even though shown as

the same shade of grey. This process is similar for neutrophils and monocytes with the

exception of emigrating out of the blood into tissues and retuming to the blood via

efferent lymphatics respectively.

5 MINUTES POST BLOOD LABELLING

Wood Circuiatow Svstem

LYMPH NODE

u

Bfood Microvascular Bed Non-fymphoid

Tissue

15 MINUTES POST BLOOD LABELLING

SPL

C

Non-fymphoid Blood Microvascular Bed Tissue

1 HOUR POST BLOOD LABELLING

Blood Microvascular Bed Non-lyrnphoid

Tissue

8 HOURS POST BLOOD LABELLlNG

Biood Circuiatorv Svstem

SPLEEN

Bidogical Equilibration Established

Bidog ical Equilibrium

LYMPH NODE

Blood Microvascular 8ed Non-lymphoid

Tissue

36 HOURS POST BLOOD LABELLING

SPLEEN

P Biological

Equilibrium

Biological Equilibrium

Biolog ica l

Blood Microvascular Bed Non-lymphoid

Tissue

in this case the half-time of 9.8 hours may achially reflect the half-life of neutrophils.

This is due to rnany reasons, one is that neutrophils will not extravasate out of the blood

vasculahire unless an immune or inflammatory response is ongoing. Therefore, the

reduction of neutrophils is due to ce11 death, margination, and entry into microvascular

beds and/or the spleen. However, when the reduction curve reaches O%, at no point after

that can CFSE positive neutrophils be detected in the blood. This suggests that there is

not a delayed migration of neutrophils through the spleen or microvascular beds that

would result in labelled neutrophils appearing in the blood after the reduction c w e

reaches 0%. This implies that biological equilibrium is obtained quickly for labelled

neutrophils, or that the output of labelled cells from sites such as the spleen or

microvascular beds is very srnall. Therefore, the half-time obtained would reflect the

half-life of neutrophils. However, this conclusion does not mle out the possibility that

neutrophils are sequestered in areas such as the spleen, liver and microvascular beds

waiting to die. This residency time would not be accounted for by the half-time obtained

in these experiments. Therefore, if this residency is prolonged the experimental half-tirne

obtained would not reflect the half-life of these cells, as they would still be dive but

sequestered, and not detected in the b l d

Figure 3.6 shows a semi-log plot of percentage of labelled monocytes versus time

which aiso has a linear fit with a correlation coefficient of 0.87. The same scenario above

for neutrophils applies to monocytes however there is an additional complication.

Monocytes can extravasate under normal steady state conditions and it is unknown what

their life span is in various tissues, or what their life-span is afler migrating to lymph

nodes via afZerent lyrnphatics. Therefore the half-time in the blood of 12.8 houn obtained

for monocytes likely reflects a combination of cell de& in the blood, spleen, liver and

emigration out of the blood system. However, their half-life can be rnuch larger than the

half-time obtained in the blood because these cells may sw ive in tissues and nodes for a

substantial period, and this would not be determineci by blmd sampling. Chapter 4

outlines a new in vivo method for labelling al1 blood leukocytes. Using this method it is

now possible to track dendritic cells derived fiom blood monocytes into afferent lymph.

This will give insight into the haif-lives and tissue residency times of blood derived

monocytes (unpublished, Young).

Lymphocytes show a biphasic response on a semi-log plot of percent of labelled

lymphocytes venus time. The correlation coefficient of the initiai phase was 0.91 with a

haIf-time of 4.2 hours. The second phase had a correlation coefficient of 0.94 and a half-

time of 406.0 hours. The half-times obtained for these two phases represent different

phenonmena The initial phase is likely the reduction kinetics of this bolus of labelled

lymphocytes to the various areas of the body such as the spleen, large rnicrovascular

beds, in combination with emigration of these lymphocytes out of the blood. This is

therefore the half-time experience by lymphocytes that enter the blood h m the thoracic

duct or primary Iyrnphoid organs. The haif-time obtained for the second phase likely

represents the half-life of lymphocytes. This is due to many reasons. Although there is a

reduction occurring through emigration out of the blood system this reduction has

reached biological equilibrium with the input of labelled lymphocytes back into the blood

via the lymphatic system. This combined with the fact that these are restiag lymphocytes

with no ensuing immune response, suggests that very little celi division is taking place.

Therefore, the reduction of labeiied lymphocytes in the blood would arguably be due to

ce11 death. This half-life of 406.0 hours for the second phase of the lymphocytes cuve

does not mean that the lymphocytes are trafficking through the blood at slower rate than

the initial half-time of 4.2 hours observed in the first phase of the lymphocyte curve.

Labelled lymphocytes are most li kely still Ieaving the blood with a hal f-time of 4.2 hours

during the second phase of lymphocyte curve, however this reduction is counteracted by

the large-scale retum of labelled recirculating lymphocytes back to the blood at the

biological equilibration rate. Therefore the first phase of the lymphocyte curve is a

measure of lymphocyte traffic kinetics in blood while the second phase is a measure of

the half-life or Iife span of blood derived lymphocytes.

Although the monocyte plot has a strong correlation coefficient of 0.87, upon

closer examination it shows a slight biphasic response with phase one being from O to 8

hours and phase two from 10 to 60 hours. When analyzed in this manner phase one has a

half-time of 4.9 hours and phase two has a half-time of 19.9 hom. Therefore the initiai

half-time of 4.2 hours for lymphocytes (O to 8 hours) is very similar to the initial half-

time of 4.9 hours for monocytes (O to 8 houn). Neutrophils actually show a half-time of

18.8 houn between O to 8 hours. This is an interesting cornparison especially when

considering monocytes and 1 ym p hocytes s hue the ability to extravasate under steady

state conditions, while neutrophils do not. The additional route of reduction via

extravasation for lymphocytes and monocytes as compared to neutrophils allows these

cells to t raac through the blood more quickly. The similarity in initial half-time for

monocytes and lymphocytes is more startiing when the main emiption route of

lymphocytes is considered The majority of lymphocytes exit the blood via HEVs in

lyrnph nodes. In order for these half-times to be equal it suggests that monocytes wodd

also exit the blood and enter nodes via HEVs at a ratio to lymphocytes equivaient to the

ratio of monocytes to lymphocytes in the blood (approximately 1 : 10). However, this type

of concIusion treats cells as if they were inert particles in the blood that have the sarne

migratory kinetics through the spleen and microvasculature. This may not be the case. If

monocytes are not crossing HEVs in lymphoid tissues than in order to have the same

half-time as lymphocytes they would have to behave differently. Monocytes would

therefore have an enhanced ability to extravasate into non-lymphoid tissues or possess a

slower migration rate through microvasculature and the spleen, or increased death rate

due to a shorter half-life than lymphocytes, or a combination of these possibilities. These

interpretations and possibilities are now experimentally testable, see Chapter 5.

The half-tirnes of leukocytes have been tested in humans, rats and other animals

using different methods. Table 3.2 A, B, and C show the half-times obtained by different

researchen in previous experiments, and the pmicular method used to determine the

half-iime for monocytes, neutrophils and lymphocytes respectively. Most of the earlier

experiments suffered from serious drawbacks, with respect to cell identification and

labelling. This was pointed out in a study that examined the half-tirnes of granulocytes in

the blooâ cornpartment using 5 1 ~ r and D F ~ ~ F . A half-time of 16.1 houn was obtained

when j'cr was used, and a half-time of 5.4 houn was obtained using DF~'P, even though

the same experimental procedures were used (Dresch, Najean et ai. 1 974). m e r studies

used injections of tritiated thymidine which setects for cycling cells. This is a drawback

as cycling cells rnay not have the same half-time as resting cells, and the overwhelming

majority of blood leukocytes are not cycling. Some studies used peroxidase or Lysoqme

activity to iden* monocytes. However, a large proportion of peroxidase-positive

Table 3.2 Balf-Times of Leu kocytes Obtained from Earlier Studies

Table 3.3 A, B, and C show a number of previous studies that have investigated half-

times of monocytes, neutrophils and lymphocytes in the blood cornpartment respectively.

Shown is the year the study was completed, the primary author, species used, rnethod of

leukocyte labelling, and the half-time (Tic in hours) obtained.

Sm1 = Smail Int. = Intemediate Lrg. = Large Virg. = Very Large

Table A, Monocytes

YEAR

1 I 1 1968 Van Furth Human

1 1979 1 van FU& 1 Human 1 Tritiated Thymidine / 104 I

Primary Author

1972

Ttitiated Thymidine

1 1984 1 Panvaresch 1 Rat 1 Tritiated Thymidine 1 1

I

Species

36 1 l

1

1 1972 1 Whitelaw 1 Rat l Tritiated Thymidine / 14 i

Meuret 1 Human 1

l 1974 i Syren

Method of Labelling

'H-DFP / 8.4 t

1 Rat 1 Tritiated Thymidine 1 12- 13

l

1986

Tic (Hom) i

N o m m 1 Guinea Pig 1

Tritiated Thymidine / Sml. Monocyte 10.8 Int. monocyte 18.2 Lrg. Monocyte 5.7 Vlrg. Monocyte 36.5 Estimated composite Half-time 10

Table 3.2

Table B, Neutrophils

1 YEAR 1 Pnmary Species 1 Method of Labelling 1 TIC (Hours) 1 Author 1

1972 Meuret Hurnan 4.4

1

1975 Dresch Human ' ' ~ r 16.1 ~ ~ j 2 - p 5.4 1

Table C, Lymphocytes

1961

1972

Athens

Whitelaw

References for Table 3.2: (Athens, Haab et al. 196 1 ; van Furth and Cohn 1968; Meuret

1972; Whitelaw and Batho 1972; Meuret and Hoffmann 1973; Dale, Fauci et ai. 1974;

Dresch, Najean et al. 1974; Syren 1974; van Furth, Raeburn et al. 1979; Issekutz,

Issekutz et al. 198 1 ; Panvaresch and Wacker 1984; Normann and Noga 1986; Young and

Hay 1995)

YEAR

1995

1973

Hurnan

Rat

Method of Labelling Primary 1 Species Author 1

I

DF"-P i 6b6 Tritiated Thymidine ( 4.4

I

Tic (Hom)

Young

Whitelaw

S heep I

19.2 I

Rat Tritiated Thymidine

mononuclear blood cells in the rat lack the lysozyme activity seen in 96% of the

rnorphologicdly typical monocytes (Syren and Raeste 1971). The present study has

eliminated these problems by identifjnng monocytes using a mAb directed against CD 14,

and by using a intracellular fluorescent dye. Another advantage included the use of flow

cytometry, whch examines individual cells rather then populations of cells. In addition

flow cytometry routinely pemitted anaiysis of 10' - 106 events, which increased the

number of observations made by 2 - 4 orden of magnitude as compared to earlier studies.

This increased the number of cells analyzed and eliminated human error due to the

manual counting of cells. Finally, the use of the sheep allowed many sequential blood

sarnples to be taken in each animal tested. This pemitted data on the long term kinetics

of fluorescently labelled lymphocytes to be obtained This is a major improvement as

most of the previous studies observed kinetics for oniy 24 to 48 hours.

Another point of interest was to detenine the subsequent lymphoid tissue

reassortment of fluorescently labelled blood lymphocytes, show in Figure 3.10. As

expected the blood had the highest percent of labelled lymphocytes, as there is a blood

resident lymphocyte pool. This difference was significant between blood and al1 other

compartrnents pc0.001 (for al! compartments) as previously demonstrated (Andrade

1996; Young, Marston et al. 1997; Andrade, Johnston et al. 1998). However, there was

no signifiant difference between mesenteric nodes and mesenteric lymph p = 0.428. This

observation indicates that there is no measurable retention of labelled lymphocytes in the

node that would increase the percentage of IabelIed lymphocytes in the node as compared

to the lymphatic that is dmining the node. This shows that the majority of cells within a

node are in transit through the node and are not residentid.

These data collectively show the dynamics of leukocyte traffïc through the blood

and reassortment of lymphocytes in secondary lymphoid tissues. These &ta also indicate

that naffic through the blood should be detenined From the initial reduction kinetics

observeci, before biological equilibrium is reached as this represents a true reduction from

the blood without being buffered by the retum of labelled cells from different areas back

to the blood.

Chapter 4

In Vivo Blood Leukocyte Labelling

4.1 Abstract

A new method has been devised to label the entire blood leukocyte pool in vivo

with the fluorescent dye carboxyfluorescein diacetate succinimidy l ester, CFSE. Previous

methods of labelhg blood cells have involved whole blood labelling ex vivo, or

purification steps where leukocytes are separated From the rest of the cornponents of

btood by density gradient centrifugation These methods have allowed snidies of blood

ce11 migration, however, they often involve a great ioss of cells and hours of ex vivo

manipulation of the blood cells. This new method involves a single i.v. injection that

labels the entire blood leukocyte pool in minutes. In this study this in vivo method was

used to label leukocytes in both sheep and rats. In addition the functional capability of

lymphocytes to recircdate after king labelled was assessed by making chronic efferent

lymph colleciions and tracking the kinetics of blood labelled lymphocytes into efferent

lymph. This new development now permits a variety of new experiments on the tracking

of minor populations of leukocytes in animals and, perhaps, in the future will lead to new

data on leukocyte migration in humans.

4.2 Introduction

Leukocyte trafic is imperative for proper function of the immune system. In

particular the continuous reassortment and recirculation of lymphocytes is fundamental to

immune surveillance (Mackay, Marston et ai. 1990), dissemination of the effector cells

(Smith, Cunningham et al. 1970; Chin and Cahill l984), many pathologies and infectious

diseases (Rosenberg and Janossy 1999). Fluorescent cell-tracking cornpounds employed

in conjunction with Bow cytometry has been a powerfbl technique to investigate

lymphocyte traffic in experimental animais. Investigations in sheep venus smaller

animals have been of major importance as sheep allow sequential samples to be taken

from the blood and many lymphatic comparûnents via chronic sampling and drainage

cannulae. Furthemore, this physiological system has been crucial is validating cellular

labels as fiinctiond capacity can be investigated, since dead or darnaged lymphocytes

cannot recirculate behveen blood and lymph (Springer 1994; Andrade 1996). Here is

descriied a new in situ whole blood labeling method which uses CFSE to mark

essentially the entire blood leukocyte pool in minutes. It is docurnented how this now

facilitates novel experiments into the migratory characteristics of low frequency su&

populations of cells such as dendritic cells. In addition, the potential now exists to

perform a wide varies- of new experiments in many different areas of physiology, cell-

biology, and pathology.

4.3 Materials and Metfiods

4.3.1 Animais

For this set of experiments 3 sheep (Renwick Farms, Oshawa) 30 - 35kg each and

3 Wistar pregnant rats (&y 12-14, Charles River, Quebec) approximately 300 g r a s each

were used. The surgical procedures used for cannulating prescapular and prefemoral

efferent lymphatics and the jugular vein were outlined in Chapter 2. The protocol for

making cellular suspensions fiom solid tissues was aiso described in Chapter 2.

4.3.2 Cell labelling

For sheep a dose of 25-37 mg of CFSE dissolved in 4-6 ml of DMSO and 3 - 4

drops of heparin (1000 units/rnl, dispensed fkom a 20 gauge needle) was prepared This

injection was given via the jugular vein, as a single bolus. For rats a greater dose of CFSE

per body weight was given compared to sheep. Two rats received a tail vein injection of

0.80 mg of CFSE dissolved in 200ul of DMSO. One rat received a tail vein injection of

3.8 mg of CFSE dissolved in 150ul of DMSO.

4.3.3 Analysis of samples

Samples were prepared for flow cytornetric analysis as described in Chapter 2.

4.3.4 Calculations and statistics

Standard error of the mean was calculated for the percent of labelled mononuclear

cells found in various secondary lymphoid tissues examined fiorn rats.

4.4 Results

4.4.1 Cell Labelling

Figure 4.1 shows a dot plot of forward light scatter venus 90" side light scatter

generated by flow cytomrtric anaiysis of shsep blood leukocj.tes. This dotplot shotts the

designated gates used to analyze the cells. R1 gates on mononuclear cells while R2 gates

on granulocytes, and R3 gates on srnall lymphocytes. Identical gates were constmcted for

rat blood leukocytes. Figure 4.2 shows representative histograms of cell count venus

CFSE fluorescence intensity for sheep blood leukocytes. Blood samples before and 15

minutes post i.v. administration of CFSE are shown for RI , R2 and R3.Leukocytes in al1

three gates had well demarcated labelling in vivo above their respective controls. in these

examples shown >98% of dl leukocytes fluoresced with an intensity greater than the

negative control. As with in vitro labelling, granulocytes had a more hornogeneous

intensity of CFSE staining as compared with mononuclear cells. This resulted in 100% of

granulocytes fi uorescing with an intensity greater than unstained granulocytes.

Granulocytes also stained more brightly than mononuclear cells. The peak channel in the

R 2 gate was 1382 versus 1 197 for the R I gate in the example s h o w

Figure 4.3 A and B are representative histograms of ce11 count versus CFSE

fluorescence intensity generated from analysis of prefemoral efferent lymphocytes using

the R3 gate, at time O and a collection of lympti made 18 to 20 hours pst CFSE blood

labelling respectively (5.7% labelled lymphocytes). These histognims demonstrate that

labeiled blood lymphocytes rnaintained their functional capabitity of recirculating &er in

vivo IabelIing, as labelled lymphocytes were present in efferent lympà Figure 4.4 is a

Figure 4.1 Differentiai Gating of Sheep Blood Leukocytes for In Vivo Lsbelling Experimeats

Representative flow cytometric analysis of sheep blood leukocytes. The dotplot shows

fonvard Iight scatter (linear scaie) vesus side light scatter (log scale). Gates were

constructed as shown in the dotplot Gate 1 (R 1 ) encompasses mononuclear cells, gate 2

(R2) encompasses granulocytes, and gate 3 (R3) encompasses small lymphocytes. The

gates were kept constant throughout this series of experiments and are referred to in text

as R 1, R2, or R3 or as mononuclear cells (R 1 ), granulocytes (neutrophils, R2), and small

lymphocytes (R3) respectively.

Figure 4.2 Fluorescence Intensity of Unstained, and In Vivo CFSE Labelid Blood Leu kocytes

Representative histograms generated by flow cytometric anaiysis of unstained

leukocytes, and 15 minutes p s t i.v. infusion of CFSE. All histograms show are ce11

count venus CFSE intensity (FL 1, log scale). Panels A and B show unstained blood cells,

and blood cells 15 minutes post labelling for the R 1 gate respectively. Panels C and D

show unstained blood cells, and blood cells 15 minutes pst labelling fiom the R2 gate

respectively. Panels E and F show unstained blood cells and blood cells 15 minutes post

labelling fiom the EG gate respectively.

Figure 4.2

Panel A

Panel B

Figure 4.2

Panel C

Panel D

Figure 4.2

Panel E

Panel F

Figure 4.3 Fluorescence Latensity of Efferent Lymph Derived Lymphocytes Before and After In Vivo Blood Labelliag

Panel A is a representative histogmm showing the fluorescent intensity of efferent lymph

derived lymphocytes before in vivo blood labelling. Panel B is a representative histogmm

showing the fluorescent intensity of lymphocytes derived from the same efferent

lymphatic fiorn a collection made 18 to 20 houn pst blood labelling. Both histograms

are ce11 count venus CFSE intensity (FL 1 ) gating on a11 the cells found in efferent lymph.

Figure 4.3

Panel A

Panel B

Figure 4.4 Graph Showing the Percent of Labelled Lymphocytes Recovered from the Efferent Lymph of a Single Sheep

Continuous prefernoral efferent lymph collections were made during the first 40 hours

after in vivo blood labelling. The percent of labelled lymphocytes found in each sample

was detemined by fiow cytomeby and plotted over the time the sample was collected.

Figure 4.4

graph of percent of labelled efferent lymphocytes versus time for one sheep. Continuous

sampling of prefemoral eflerent Iymph afier blood leukocyte labelling was done to obtain

the data for this graph. The purpose of this was to assess the arnount of labelled

lymphocytes that could be recovered and also the kinetics of this recovery. (Figure A. 1 in

the appendix shows data from two other sheep, where catheten flowed for 7 and 1 1 hours

pst blood labelling). Figure 4.5 shows the reduction of labelled srnail lymphocytes and

neutrophils fiom the blood cornpartment afier in vivo blood labelling (n = 3). Figure 4.6

shows representative histograrns of ce11 count venus CFSE intensity generated fiom rat

blood sarnples before and 10 minutes pst CFSE blood labelling, for the RI, R2, and R3

gates respectively. In these examples shown >98% of al1 leukocytes in blood labelled

with a p a t e r intensity than unstained blood sample. Figure 4.7 shows ce11 count versus

CFSE intensity generated fiom cellular suspensions made from various rat lyrnphoid

organs 20 minutes pst blood labelling.

4.5 Discussion

This study extends previous methods of blood labelling by demonstrating it is

possible to label essentially al1 of the leukocytes found in both sheep and rat blood in situ.

The functional capability of the labelled lymphocyte to recirculate was assessed by

tracking the migration of labelled lymphocytes into efferent lymphatics as shown in

Figure 4.3. Figure 4.4 shows the percentage of labelled efferent lymphocytes collected

venus time for one sheep. There are three interesting points to note about this graph. One

is the actuaI percent of IabeIIed cells obtained in the collections. The peak percentage

obîained was 5.7%, which is approximately 5 times higher than the percentage that is

obtained with ex vivo labelling procedures where a unit of blood is labelled

Figure 4.5 Reduction of CFSE Labelled Neutrophiis and Small Lymphocytes from the Blood Corn partment

Graph A and B show the percent of labelfed small lymphocytes (R3) venus time in hours

on a linear and log scale respectively. Graph C and D show the percent of labelled

neutrophils venus time in hours on a linear and log scale respectively. Error bars were

generated using standard error of the mean. (n = 3)

Figure 4.5

Graph A

+ LYMPHOCYTES

20 40 60 80 Time (Hours)

Graph B

+ LYMPHOCYTES

40 60 Time (Hours)

Percent of Labelled Neutrophils (Log Scale) ,

a O O O

Percent of Labelled Neutrophils

a h ) I P I Q ) œ o

O 0 0 0 0 0

Figure 4.6 Fluorescence Intensity of Unstained, and In Vivo CFSE Labelled Rat BIood Leukocytes

Representative histograms generated by flow cytometric analysis of unstained leukocytes,

and leukocytes 10 minutes p s t blood labelling. Ail histograms shown are ce11 count

venus CFSE intensity (FL l , log scale). Panels A and B are unstained blood ieukocytes

and blood leukocyies collected 10 minutes p s t blood labelling from the R1 gate

respectively. Panels C and D are unstained blood Ieukocytes and blood leukocytes

collected 10 minutes p s t blood labelling from the EU gate respectively. Panels E and F

are unstained blood leukocytes and blood leukocytes collected 10 minutes p s t blood

labelling fiorn the R3 gate respectively.

Figure 4.6

Panel A

Panel B

Figure 4.6

Panel C

Panel D

Figure 4.6

Panel E

Panel F

Figure 4.7 Tissue Reassortment of Labelled Mononuclear cells (RI) 20 Minutes Post In Vivo Blood Labelling in the Rat

Histograms are ceIl count versus CFSE intensity (FL 1. log sale). Panel A shows axillary

node cells from a rat labelled with 0.8 mg of CFSE i.v.. Panel B shows mesenteric node

cells From a rat labelled with 3.8 mg of CFSE i.v.. Panel C shows the spleen From a rat

labelled with 3.8 mg of CFSE i-v..

Figure 4.7

Panel A

Panel B

Panel C

(Abernethy and Hay 1992; Andrade 1996). This demonstrates the signifiant increase in

recirculating labelled lymphocytes, due to the increased amount of cells labelled with the

in vivo procedure. The other two sheep tested showed comparable but higher percentages

of labelled lymphocytes in the efferent lymph (Figure A. 1, Appendix). The second point

is that the highest concentration of labelled cells was obtained in the collection made

between 7 and 18 hours, which is considerably earlier than the recovery obtained using

ex vivo labelling, where the highest concentration of labelled cells is recovered between

2 1 to 24 hours(Abemethy and Hay 1992; Andrade 1996). This is not a function of CFSE

as blood lymphocytes IabeIled ex vivo with CFSE have followed the traditional kinetics

of al1 other ex vivo labelled cells (personal communication, Young, 2000). This leaves

two explamtions to describe this potentiai increase in recirculation rate. One is that ex

vivo labelling due to plasma loss, time ex vivo, temperature changes, centrifugation,

contact with lab equiprnent etc., is altering cells leading to a slower recirculation time of

lymphocytes. The second possibility is that the DMSO used to dissolve the CFSE is

causing a systemic increase in ce11 diapedesis leading to increased lymphocyte

recirculation kinetics. An experiment using ex vivo CFSE labelled blood cells re-infused

with the same dose of DMSO used in the in vivo Iabelling procedure may be able to

support or eliminate the possibility of increased cell extravasation due to DMSO. This

experiment is currently king performed The third point is the recovery of labelled

lymphocytes in lymph 40 hours pst injection per 109 celIs is 0.67%, which is consistent

with ex vivo labelling procedures where typically 0.5% is recovered (Abemethy and Hay

1 992; Andrade 1996).

The reduction c w e s in the blood compartment for neutrophils and small

lymphocytes were obtained using the in vivo technique to label cells. The initial half-time

obtained for reduction of labelled lymphocytes (O to 7 hours) was 4.3 hours which is in

agreement with curves generated using cells labelled ex vivo (4.2 hours). The half-time

obtained from 9 to 96 houn was 293.4 hours for lymphocytes. Neutrophils had a haif-

t h e of 15.1 houn which is larger than the 9.8 hours obtained previously. However, more

experiments are needed to confin these prelirninary results as they are only based on

three sheep.

The leukocytes in the rat blood, iike the sheep, had well demarcated labelling with

99 + 1% of the cells having a fluorescence intensity greater than unstained leukocytes

(n=3). Pregnant rats were used to test if fetaI blood could be labelled by giving the

mother a tail vein injection of the fluorescent dye. At the concentrations used it was

found that fetal biood was not labelled (data not shown). This may be a question of

dosage, or it rnay be that the rat placenta doesn't allow the passage of CFSE. Rats were

sacrificed 20 minutes p s t injection of CFSE and lymphoid tissues were tested for the

presence of labelled cells. The cellular suspensions made from rat spleens showed

significant ce11 labelling. In addition, the rat that received the higher dose of 3.8 mg of

CFSE showed that approximately 30% of its mesenteric node cells were slightly labelled

(Figure 4.7, Panel B). It is important to note that in order to cause this very low level of

labelling in nodes a concentration of CFSE 10 hmes that used in the sheep per body

weight had to be administered The peak c b e l of this population of labelled cells was

25 as compared with 1596 in the b l o d in addition a considerable amount of CFSE

intensity is Iost over a 24 hour period after labelling, which would make this low level of

labelling in the nodes indistïnguishable From negative node cells. In the spleen 40 k 3%

of cells were labelled, however these cells were labelled with a lower intensity than cells

in the b l o d The peak channel of labelled cells in the spleen was 583, compared to 1596

in the blood for the representative example shown.

4.5.1 Discussion of Data in Appendix

Appended to this thesis are preliminary results that were obtained from a single

animal or repeated only two or three tirnes using the in vivo blood labdling technique. As

show above in Figure 4.4 the recovery of labelled lymphocytes in efferent lyrnph was

assessed in a single sheep. Figure A.1 in the appendix contains plots showing two

additional experirnents that examined this recovery in efferent lymph. Unfortunately the

catheten remained intact only for 7 and 1 1 hours respectively. However, these plots still

dernonstrate the significant increase in percent of labelled lymphocytes recovered in

efferent lyrnph in cornparison to ex vivo labelling techniques. In addition Graph B of

Figure A. 1 shows that the recovery of labelled lymphocytes was beg i~ ing to plateau for

the 1st collection of efferent lyrnph made between 9 and 1 I hom. This supports the idea

of faster recovery kinetics of labelled lymphocytes into efferent lymph using the in vivo

labelling method, discussed above and show in Figure 4.4.

Figure A.2 shows the percent of labelled small lymphocytes found in different

compartments five days pst in vivo blood labelling for a single sheep. This represents a

12-14 fold increase in the percent of labelled lymphocytes found in these tissues as

compared to ex vivo Iabelling techniques, with the exception of the spleen which

experienced an 8 fold increase. This also illustrates the significant increase of labelled

cells available for tnicking using the in vivo procedure. This is a tremendous advantage

when attempting to track low frequency blood ce11 populations, see Chapter 5.

As show in Figure 4.7 with the concentration of CFSE given to rats it was

possible to label approximately 40% of the spleen cells, albeit at a much lower intensity

than the blood leukocytes. Sheep received a much lower concentration of CFSE per body

weight and Figure A.3 in the appendix shows tissue distribution of labelled mononuclear

cells (Rl) 1 hour d e r in vivo blood labelling in a single sheep. It should be noted that

this animal was previously used for in vivo blood labelling experiment approximately I

month prior to this additionai experiment, therefore a residual amount of labelled

lymphocytes was present in this sheep from the first expriment. Figure A.3 shows that 1

hour after in vivo blood labelling there was no sign of labelled lymphocytes in the

mesenteric node (Panel D) and the spleen showed 9% of labelled mononuclear cells (RI,

Panel C). This is considerably smaller than the 40% obtained for rats, and is likely due to

blood to spleen migration of labelled cells during the hour afler blood labelling. This is

preliminary evidence that the dose of CFSE is extremely important and will most likely

determine if spleen cells will or will not be labelled during the procedure.

Collectively, the experiments contained in this chapter show that it is possible to

perform this in vivo labelling of blood leukocytes, and lymphocytes stained are

fiui&onal ly capable of recirculation. The preliminary data O btained suggests that

lymphocytes have faster recovery kinetics into efferent lymph using this method and

there is likely a dose dependent relationship that determines what cells will be labelled In

addition, although aot directly tested, it seems plausible that pIatelets and vascular

endothelium may also be stained using this procedure. Supplementary experiments to this

thesis are in progress to enhance the data shom in the appendix, and to prepare for

publication.

Chapter 5

General Discussion

5.1 Labelling Methodologies for Cell Tacking

The method of labeiling blood leukocytes for tracking is an important tod for

investigating in vivo cellular WIC (Andrade, Johnston et ai. 1996). A new in vivo

labelling procedure using CFSE for blood leukocytes has be dernonstrated and applied in

this thesis. The âdvantages of this dye are numerous due to the speed at which it labels

cells, the longevity of the dye and that the dye labels intracellular proteins (Parish 1999).

M e r labels have different rnechanisms to label cells, such as PKH26 which is a

membrane intercalating dye or FITC which binds extracellular proteins. One criticism is

that these dyes may cause stenc interference with CAMs, however, use of many different

dyes over the years have yielded the same recircuiation kinetics for lymphocytes, even

though the dyes label cells by different rnechanisms (Abernethy and Hay 1992; Young

and Hay 1995; Andrade 1996; Andrade, Johnston et al. 1998). The second criticism of in

vivo cell tracking experiments is that the cells are usually labelled ex vivo, leaving the

potential for cellular damage, loss, and up/down regdation of CAMs. The ability to

address this criticism has b e n dificult as there were no effective alternative ways to

label ceils. This new method now gives the opportunity to address the criticism of ex vivo

labelling.

5.2 Reduction of Labelled Lymphocytes from the Blood

Loolcing at the initial blood reduction kinetics fiom O to 8 hours both in vivo and

ex vivo labelhg methods gave a half-tirne of approxhately 4 hom in the blood

cornpartment for lymphocytes. This is an important find as this is redly the half-time that

a lymphocytes entering the blood via thoracic duct or a primary lymphoid organ will

experience. This half-time is probably correct for monocytes as well, although monocyte

kinetics in the blood were only investigated using ex vivo labelling techniques (initial

halctime 4.9 hours). Neutrophils that are not normally capable of emigrating out of the

blood experience a much higher initial half-time. This half-time was t 8.8 houn for ex

vivo labelling and 9.3 hours for in vivo labelling.

Some perspective can be gained on ths initial half-time of 4 hours when the

hypothetical situation of reduction of labelled blood lymphocytes from the blood

compartment based solely on thoracic duct input is considered Figure 5.1 A and B show

the hypothetical reduction of labelled blood lymphocytes fiom the blood compartment

based on a thoracic duct input of 1x10~ lymphocytes per h o u on a linear and log scale

respectively (Heath, Lascelles et al. 1962). This graph assumes that lymphocytes are not

recirculating back to the blood and that al1 lymphocytes have the sarne ability to emigrate

out of the blood This assumption that cells don3 recirculate back to the blood is done to

mimic the initial reduction of cells fiom the blood as the return of labelled lymphocytes

to the blood is protracted in time (Abernethy and Hay 1992; Andrade 1996). The half-

time of this hypothetical curve is 6.6 hours. However, this curve ody considers input

from the thoracic duct, and ignores input to the b l d fiom prescapular and cemkal

efferent lymphatics, fiom newly forrned lymphocytes entering the blood, and the

reduction of lymphocytes due to ce11 death. These additional processes would decrease

this hypothetical haif-the based solely on thoracic duct input. Factoring in these

additional inputs to the blood would arguably show that the initial reduction of labelled

lymphocytes obtained experïmentally folIows the theoreticai resuit

Figure 5.1 Bypothetical Reduction of Labelled Lymphocytes from the Blood Based on Thoraeic Duct Input

The curve shown assumes a thoracic duct input of 1 x 10' cells/hour, that al1 lymphocytes

in the blood have an equai ability to migrate and extravasate, and that there is no retum of

labelled lymphocytes via the lymphatic system back to the blood. Graph A and B show

the percent of labelled lymphocytes in the blood on a linear and log scale respectively.

Figure 5.1

Graph A

- Lymphocytes

20 40 Time (Hours)

O 20 40 60 Time (Hours)

Additional insight is gained by comparing a hypothetical reduction cuve with a

half-the of 4 hours to the curve that is obtained expximentally. At 24 hours the

hypothetical cuve would have 1.6% labelled lymphocytes in the blood The experimental

in vivo labelling results show 28.8% labelled lymphocytes in the blood. It is clear that

although the curves share an initial half-time of about 4 hours they clearly deviate from

each other as time passes. An obvious answer for this deviation would be the r e m of

recircdating labelled lymphocytes to the blood via the lymphatic system. The 24 hour

average output obtained experirnentaily for the prefemoral efferent lymphatic was 4.0%.

Taking this result and projecting it to the thoracic duct would mean that the thoracic duct

would contain an average of 4.0% labelled lymphocytes over 34 houn. Assurning a

thoracic duct output of 1x10~ lymphocytes per hour this would arnount to 9 . 6 ~ 108

Iabelled lymphocytes entering the blood. This represent 9.6% of the blood pool, leaving

17.6% of labelled lymphocytes in the experimental resdt unaccounted for. Other sheep

showed a higher recovery of labelled cells in the efferent lymph (Figure A. 1, Appendix),

however, even doubling the amount of labelled cells returning to the blood via the

thoracic duct would still leave an excess of 8% labelled lymphocytes in the blood. This

discrepancy can be reconciled due to an interesting finding that discovered that as much

as 40% of lymphocytes in the blood are part of population of non-recirculatùig

lymphocytes, a blood resident population (Young, Marston et al. 1997; Andrade,

Johnston et al. 1998; Gupta, McCome!I et al. 1998). However, this doesn't mean that this

population of ceUs can't experience a reduction nom the blood cornpartment, as they

most likely have the ability to migrate into the spleen, and in addition these celIs may

have a different haif-life from lymphocytes capable of recirculating-

The blood resident

the reduction curves. The

population also

observation is

explains another interesting point seen with

that in both in vivo and ex vivo labelling

experiments the second phase of the lymphocyte curve begins between 8 and 10 hours. In

theory this seems quite early given that the rnajority of recirculating lymphocytes don?

retum to the blood until a time around 24 hours. Ths retwn of labelled lymphocytes may

be quicker for the in vivo labelling technique, but not fast enough to assume that the

recirculating lymphocytes retuming to the blood are causing the second phase of the

lymphocyte reduction cuve to begin. in addition the fact that this phase begins at the

same time for ex vivo labelhg experiments dismisses the chance of faster recirculation

times being the cause of initiation of the second phase. Thus another group of labelled

lymphocytes must be in the blood to cause this sustained elevation of labelled

lymphocytes begiming at the second phase of the c w e .

Therefore, the recovery kinetics of labelled lymphocytes in efferent lyrnph along

with the early transition to phase two in the lymphocytes reduction c w e support the idea

of a blood resident population of lymphocytes. The predicted lymphocyte reduction curve

in the blood if the blood resident population didn't exist is show in Figure 5.2. In this

curve there is no blood resident population of lymphocytes to effectively buffer the

reduction of lymphocytes nom blood before the r e m of labelled recirculating

lymphocytes.

5.3 Functional Aspects of HaIf-Times in the Blood Cornpartment

The blood represents a widely used cornpartment for clinical anaiysis. Immune

status is ofien gauged by analysis of b l d samples. In ternis of lymphocytes, the blood

Figure 5.2 Hypothetieal Reductioo Curve for Labelleci Lymphocytes in the Blood in the Absence of the Blood Resident Lymphocyte Population

The graph shows the predickd percent of labelled lymphocytes in the blood versus time

in hours.

Figure 5.2

- Lymphocytes

Time (Hours)

only houses 1 - 2% of the total lymphocytes in the body, at any given time (Chin, Pearson

et al. 1985). This alone demonstrates the shortcomings of using the blood to infer

immune status. The danger of doing this is fbrther illustrated in pathologies such as SIV

and HIV where there is a marked depletion of CW' T cells in the blood (Rosenberg and

Janossy 1999). However, recent investigations have shown that there is not the sarne

depletion in lymphoid organs (Pabst and Rosenberg 1998; Rosenberg and Janossy 1999).

The functional significance of this has yet to be determined, but it provides a good

example of the extrapolation error that can occur by using the blood to decipher the

immune status of the entire body.

Assuming the experimental half-time of lymphocytes obtained in the blood

cornpartment is 4.3 hours this would mean that lymphocytes would be reduced from the

blood cornpartment at a rate of approximately 1 .2xlo9 cells per hour. By definition at

steady state the same amount of lymphocytes would enter the blood via recirculation and

new lymphocytes from primary lymphoid organs. This lymphocyte entry rate would

equal the number of lymphocytes found in the blood in just 8.3 hours. Howevcr, this

doesn't mean that al1 lymphocytes in the blood get replaced by incoming lymphocytes

every 8.3 hours. An exarnple to illustrate this would be cornparhg two independent blood

compartments with an equivalent number of lymphocytes. One compartment does not

have a blood resident population of lymphocytes (A). The second compartment has a

lymphocyte blood resident population of 50% (E3). Thus ody half of the lymphocytes in

population B would have the ability to recirculate. Assuming an equal lymphocyte

recirculation speed for both cornpartments, the entry rate of lymphocytes via the

lyrnphatic system into blood cornpartment B would be half the entry rate into blood

cornpartment A. These compartments couid have the sarne lymphocyte entry rate due to

recirculation, if the recirculating lymphocytes in population B recircuiated twke as fast

as the lymphocytes in population A However, ody half of the lymphocytes would be

involved in the recirculation process in cornpartment B, whereas d l of the lymphocytes

would be involved in the recirculation process for cornpartment A This type of

knowledge in combination with leukocyte traffic kinetics in the blood is crucial when

using the blood to determine immune status of the rest of the body.

5.4 Tissue Reassortment of Lymphocytes

The data obtained on tissue reassortment of lymphocytes demonstrated that the

mesenteric nodes and mesenteric efferent lymph contained approximately the sarne

percentage of labelled lymphocytes, showing that there wasn't a measurable

accumulation of labelled lymphocytes in the node. An interesting point can be made

when the percentage of labelled lymphocytes in mesenteric lymph is considered against

the percentage of labelled lymphocytes in blood Previous experiments have show that

approximately 1 in 4 lymphocytes in the blood will cross KEVs in the node and enter

ef5erent lymph, assuming dl lymphocytes have the same ability to cross HEVs (Hay and

Hobbs 1977). At biologicai equilibrium this ratio of 1:4 lymphocytes crossing KEVs and

entering efferent lymph would predict that for every 3 labelled lymphocytes in the blood

there would be 1 labelled lymphocyte in the efferent iymph Based on the tissue

reassortment data obtained using ex vivo labeiling techniques (Fig 3.10) the percentage

of labelied mesenteric efferent lymphocytes was 0.35% compared with 1.52% labelled

lymphocytes in the blood. Previous experiments have aiso demonstrated that 85% of

efferent lymph cells are derived frorn blood lymphocytes that cross HEVs and enter

efferent lymphatics (Hay and Hobbs 1977). Given that afferent lymph contains

approximately the same percentage of labelled lymphocytes as efferent lyrnph (data not

shown), and that 10% of the cells in efferent lymph are derived fiom afferent lyrnph cells

(Hall and Momk 1962), it would be a reasonable assumption that 10% of the labelled

lymphocytes in efferent lymph would be derived fiom afferent lymph. Given the

experimental resuit of 0.35% labelled lymphocytes in mesenteric efferent lymph it would

be expected that 10% of these labelled cells (0.035%) would be denved fiom the afferent

lymph. The remaining percent of labelled cells in the efferent lymph 0.315% would

theoretically be derived fiom blood lymphocytes migrating across node E-EVs. Since the

predicted value is three labelled lymphocytes in the blood for every labelled lymphocyte

in efferent lymph the predicted percent of labelled cells in the blood wouid be 0.95%.

This is significantly less than the 1.52% of labelled lymphocytes found in the blood

experimentally (Figure 3.10). In fact this shows that 38% of the labelled lymphocytes in

blood are not accounted for using this method. This wodd suggest that 38% of the cells

in the blood do not recirculate into efferent lymph, which is in agreement with recent

discoveries that have found that 40% of lymphocytes do not recirculate (Young, Marston

et al. 1997; Andrade, Iohnston et al. 1998; Gupta, McConnell et al. 1998). When this

analysis is applied to the tissue reassortment data obtained using the in vivo technique to

label blood, 40% of the labelled lymphocytes in the blood were not accounted for.

However, ody one animai was tested using the in vivo technique, whereas four animais

were tested using ex vivo labelling techniques.

5.5 Future Experiments

The new method for in vivo blood leukocyte labelling provides opportunities for

many novel experiments to be performed that were not possible previously. The three

major advantapes to this method that provide opprtunities for new experiments are: 1 )

the entire blood pool is labelled arnplifjmg the numben of cells that cm be tracked; 2)

the speed of this labelling process; 3) the labelling is in vivo and avoids the complications

of ex vivo labelling. Below are some suggestions for htme experiments using this

method.

5.5.1 Experiments to Further Characterize the In Vivo Labelling Procedure

Since this process is in its infancy many experiments remain to be perfonned to

charactenze this labelling procedure. One immediate question is to determine the

percentage of spleen cells that are labelled at different i-v. doses of the label. These

experiments would involve a fairly large number of animals as multiple anhnals would

have to be used at each concentration tested, therefore it wouid favourabie to use mice or

rats for these experiments. Furthemore, valuable data on labelling of cells in nodes at

very high concentrations of CFSE would simultaneously be gained.

Secondly, it may be that platelets and/or vascular endothelid cells are also being

labelled with CFSE using this method If this is tnie it may extend the applications of this

process. This can be investigated by andyzing platelets by flow cytornetry after in vivo

labeliing. Different techniques can be used to check for fluorescence in endothelial cells,

one way would be using confocal microscopy.

5.5.2 Experiments to Re-Examine the Reduction of Labelled Cells from the Wood Cornpartment

Additional experirnents are necessary to obtain the reduction cuves of leukocytes

in the blood using the in vivo labelling m e t h d In addition to examining different

leukocyte populations, comparative data between different subsets of lymphocytes could

be obtained by performing immunoEiuorescence staining to detect B cells, CD4, CD8,

and y6 T cells. Since it has been shown that the blood resident population of B cells could

be detected by coordinated expression of CD2 1 and L-selectin (Young, Marston et al.

1997), it would be interesting to see if there is a e ~ c h m n t of these cells in blood during

the lag time before the majority of recirculating lymphocytes retum to the blood.

Experiments have shown in rats that there is an enrichment of B cells in the blood afler

the diversion of thoracic duct lymph (Westermann, Matyas et al. 1994). This experiment

can be perfonned in sheep after in vivo labelling to obtain the reduction kinetics of

lymphocytes without reac hing recirculation equi li brium.

Another expenrnent could be to test the reduction of labelled ceils in the blood

cornpartment of splenectomized animals. The hypothesis would be that the initial half-

times experïenced by leukocytes would increase because there would be no traffic

through the spleen.

5.5.3 Expriment to Examine Recovery Kinetics of Labelled Lymphocytes in Efferent Lymph

The experiments performed in this thesis suggest that the recovery kinetics of in

vivo labelled blood lymphocytes into efferent lymph are faster than previous methods of

labelling. Sorne additional experiments are needed to confimi if this preliminary result is

correct As lymphocytes labetlcd uith CFSE or iiw h v c the same rccovcr). kinetics, of

lymphocytes stained ex vivo with other labels (personal communication, Young, 2000),

this quicker recovery is particularly interesting. A control experiment using leukocytes

labelled ex vivo with CFSE and re-infused with an equivalent amount of DMSO used in

the in vivo procedure, will need to be completed to rule out the possibility of DMSO

causing this potential increase in recovery kinetics.

Additional tracking expenments are already underway tracking blood monocyte

derived dendritic cells into afferent and pseudo-afferent lymphatics (Young,

unpublished). This experiment has previously proved to be very difficult due to the low

numbers of labelled dendritic cells recovered using previous labelling methods.

Experiments such as these combined with the knowledge of half-times in the blood

cornpartment will hopefully be the beginning of dissecting tissue residency times of

blood derived monocytes.

5.6 Surnrnary

These experiments have used the most ment meîhods avail able to investigate

leukocyte half-times in the blood The experimeats have demonstrated the similarity in

initial reduction kinetics for monocytes and lymphocytes versus neutrophils and have

also determined the half-lives of neutrophils and blood derived lymphocytes. Also shown

is a rnethod for in vivo blood leukocyte labelling extending the capabilities of blood

leukocyte tracking experiments.

and relate migratory kinetics of

tems of in vivo immunity.

Future experiments are needed to expand on this work

leukocytes in the blood with their functional ability in

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Appendix

Figure A.l Graphs Showing the Percent of Labelled Lymphocytes Recovered from the Efferent Lymph of Two Sbeep

Continuous prefemoral efferent lyrnph sampling was made during the fint 7 or I 1 hours

after in vivo blood labelling. The percent of labelled lymphocytes found in each sarnple

was determined by flow cytometry and plotted over the tirne the sample was collected

Figure A. l

Graph A

-% of Labelled Lymphocytes

O I O 20 30 4C Time (Hours)

Graph B

- % of La belled Lymphocytes

O 10 20 30 40 Time (Hours)

Figure A2 Tissue Reassortment of In Vivo CFSE La belled Lymphocytes

Graph shows the percent of labelled small lymphocytes (R3) in the bfood, spleen,

rnesenteric lymph, prefemoral lyrnph node and popliteal lymph node five days pst in

vivo Iabelling of a single sheep.

Figure A.2

Bloood Spleen Mesenteric Prefemoral Popfiteal L Y ~ P ~ Node Node Tissue

Figure A 3 Tissue Reassortment of Leukocytes 1 Bour Post In Vivo Blood Labelliog

AI1 histograms are ce11 count versus CFSE intensity. Panel A shows mononuclear cells

(RI ) of an unstained sample of blood. Panel B shows a blood sample taken 10 minutes

p s t in vivo blood labelling (Rl). Panel C shows a spleen sample taken 1 hour p s t in

vivo blood labelling. Panel D shows a sample of mesenteric node taken 1 hour ps t in

vivo blood labelling.

Figure A3

Panel A Panel B

Panel C Panel D