University of Calgary

PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2013-01-25

Activation of Epiplexus Cells by ATP

Maslieieva, Valentyna

Maslieieva, V. (2013). Activation of Epiplexus Cells by ATP (Unpublished master's thesis).

University of Calgary, Calgary, AB. doi:10.11575/PRISM/24689

http://hdl.handle.net/11023/504

master thesis

University of Calgary graduate students retain copyright ownership and moral rights for their

thesis. You may use this material in any way that is permitted by the Copyright Act or through

licensing that has been assigned to the document. For uses that are not allowable under

copyright legislation or licensing, you are required to seek permission.

Downloaded from PRISM: https://prism.ucalgary.ca

UNIVERSITY OF CALGARY

Activation of Epiplexus Cells by ATP

by

Valentyna Maslieieva

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF NEUROSCIENCE

CALGARY, ALBERTA

JANUARY, 2013

© Valentyna Maslieieva 2013

ii

Abstract

Epiplexus cells represent a population of innate immune cells on the surface of the

choroid plexus (CP) in the brain ventricles. I hypothesized that the epiplexus cells are involved in

immune responses of the CP via sensitivity to immune mediators such as ATP. A novel

technique for the isolation of live and intact rat CP was developed that allowed, for the first time,

observation and quantification of physiological responses of epiplexus cells in situ. Bath

application of ATP, as well as ADP or UTP, activated movement (chemokinesis) of epiplexus

cells. This was not dependent upon P2X7 and P2X4 receptors, but may involve epiplexus cell

P2X1, P2X5, and / or P2Y13 and P2Y2 receptors and epithelial cell pannexin-1. Therefore,

epiplexus cells respond to the immune mediator, ATP, and may receive signals directly from the

epithelium on which they reside to mediate localized immune responses in the ventricle.

iii

Acknowledgements

I would like to sincerely thank all the people and institutions that helped me throughout

my study. This thesis would not have been possible without patient guidance, enthusiastic

encouragement, and constructive criticism from my supervisor Dr. Roger Thompson and my

committee members Dr. Patrick Whelan, Dr. Grant Gordon, and Dr. Michael Antle. I also would

like to express my gratitude to the University of Calgary and Hotckiss Brain Institute for the

support and opportunities they provided. I am grateful to the labs of Dr. Shalina Ousman, Dr.

Keith Sharkey, Dr. Quentin Pittman and Dr. Jaideep Bains for sharing some of the materials used

in experiments for this thesis. I am obliged to many of my colleagues for helpful advice and a

great work environment, especially Evelyn Ma, Peter Tang, Nickolas Weilinger, Jennifer

Bialecki, Wataru Inoue, Cheryl Sank, Dinara Baimoukhametova, Mio Tsutsui, and other

members of Thompson’s, Bains’, Teskey’s and Pittman’s labs, as well as everyone else I had a

pleasure of meeting. I am truly thankful to my boyfriend, family, friends, and mentors from both

Canada and Ukraine for supporting, inspiring and believing in me.

iv

Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgements ............................................................................................................ iii

Table of Contents ............................................................................................................... iv

List of Tables ..................................................................................................................... vi

List of Figures and Illustrations ........................................................................................ vii

List of Symbols, Abbreviations and Nomenclature ......................................................... viii

CHAPTER ONE: INTRODUCTION ..................................................................................1

The choroid plexus ...........................................................................................................1

The immune cells of the CP .............................................................................................3

Origin of the native immune cells in the CP ....................................................................4

The epiplexus cells and the CP in normal and pathological conditions ..........................6

ATP as an extracellular messenger for immune cells ......................................................9

Purinergic signalling in innate immune cells .................................................................11

Migration of immune cells and ATP .............................................................................13

Panx1 as a source of extracellular ATP .........................................................................15

CHAPTER TWO: MATERIALS AND METHODS ........................................................19

The isolated and intact CP preparation ..........................................................................19

Live cell fluorescent imaging ........................................................................................19

Data analysis and statistics ............................................................................................21

Immunohistochemistry ..................................................................................................22

CHAPTER THREE: RESULTS ........................................................................................24

v

Identification of epiplexus cells as immune cell ............................................................24

Epiplexus cells are activated by extracellular ATP .......................................................26

P2X7 receptors are not involved in chemokinesis of epiplexus cells ............................29

P2X4 receptors are not involved in epiplexus cells chemokinesis by exogenous ATP 29

P2Y2 receptors contribute to ATP-induced chemokinesis of epiplexus cells ...............32

The effects of other purines on chemokinesis of epiplexus cells ..................................37

Panx1 channels contribute to epiplexus cell’s activation ..............................................41

LPS and Poly(I:C) do not trigger increased motility .....................................................41

CHAPTER FOUR: DISCUSSION ....................................................................................46

Immune origin of epiplexus cells ...................................................................................47

Chemokinesis of epiplexus cells in intact isolated CP ..................................................48

Investigation of molecular mechanisms ........................................................................49

Additional findings ........................................................................................................53

Potential limitations of the new approach of studying epiplexus cells and solutions ....53

Conclusions ....................................................................................................................54

REFERENCES ..................................................................................................................57

vi

List of Tables

Table 1. Changes in the number of epiplexus cells and the expression of various molecules

on these cells in infection, injury and auto-immune disease MS. ........................................... 7

Table 2. A list of all known purinergic receptors, and those expressed on monocytes,

macrophages, microglia, and dendritic cells. ........................................................................ 12

vii

List of Figures and Illustrations

Figure 1. The schematic representation of a hypothesised cascade. ............................................ 18

Figure 2. Epiplexus cells are labelled with IB4 and Iba1. ........................................................... 25

Figure 3. Representation of the tracked paths superimposed on the original image ................... 27

Figure 4. Distance travelled by epiplexus cells in control and in the presence of ATP. ............. 28

Figure 5. Immunofluorescent staining for P2X7 receptors in epiplexus cells. ............................ 30

Figure 6. P2X7 receptors are not involved in epiplexus cells activation by extracellular ATP .. 31

Figure 7. P2X4 receptors are not involved in epiplexus cells activation by exogenous ATP. .... 33

Figure 8. Blockage of P2X1, P2X2, P2X3, P2X5, P2Y1 and/or P2Y13 together with P2X7

receptors by PPADS and BBG decreased epiplexus cell's motility. ..................................... 35

Figure 9. Immunofluorescent staining for P2Y2 receptors in epiplexus cells ............................. 36

Figure 10. Tangeretin (blocks P2Y2) significantly decreased chemokinesis, and in

combination with PPADS (blocks P2X1, P2X2, P2X3, P2X5, P2Y1, and P2Y13)

abolished it ............................................................................................................................ 38

Figure 11. ADP (activates P2Y1, P2Y12, P2Y13) and UTP (activates P2Y2, P2Y4, P2Y6),

but not adenosine (activates A1, A2A, A2B, A3 receptors) triggered chemokinesis ............... 40

Figure 12. Immunofluorescent staining for Panx1 in the CP ....................................................... 42

Figure 13. Blockage of Panx1 channels by probenecid gradually decreased chemokinesis ....... 43

Figure 14. LPS and Poly(I:C) do not trigger chemokinesis. ........................................................ 45

Figure 15. Model of the findings from this thesis ........................................................................ 56

viii

List of Symbols, Abbreviations and Nomenclature

Symbol Definition

µM micromolar

µm micrometer

aCSF artificial cerebro-spinal fluid

AD Alzheimer’s disease

ADP adenosine-5'-diphosphate

ANOVA one-way analysis of variance

ATP adenosine-5'-triphosphate

Aβ amyloid beta peptide

BBB blood-brain barrier

BBG brilliant blue G

BCSFB blood-cerebrospinal fluid barrier

BSA bovine serum albumin

BV blood vessel

bzATP 3'-Benzoylbenzoyl adenosine 5´-triphosphate

C5a complement component 5a

Ca2+

calcium ion

CaCl2 calcium chloride

cAMP 3'-5'-cyclic adenosine monophosphate

CCL chemokine (C-C motif) ligand

CD cluster of differentiation

CNS central nervous system

ix

CP choroid plexus

CR3 complement receptor 3

CSF cerebrospinal fluid

CXCL10 C-X-C motif chemokine 10

DAMPs damage associated molecular patterns

DAPI 4',6-diamidino-2-phenylindole

DMSO dimethyl sulfoxide

EC epiplexus cell

EC50 half maximal effective concentration

EDTA ethylenediaminetetraacetate

EM electron microscopy

EP choroidal epithelium

Gi/o / Gq/11 G protein subunits

HBSS Hank’s balanced salt solution

HLA-DR human leukocyte antigen-D related

HRP horseradish peroxidise

IB4 isolectin B4

IFN-γ interferon gamma

IgG immunoglobulin G

IL interleukin

IP intraperitoneal

IP3 inositol 1,4,5-trisphosphate

IV intravenous

x

KCl potassium chloride

kDa kilodalton

LCA leukocyte common antigen

LED light emitting diode

LPS lipopolysaccharide

LV lumen of the lateral ventricle

MgCl2 magnesium chloride

MgSO4 magnesium sulphate

MHC major histocompatibility complex

min minute

ml millilitre

mM millimolar

MS multiple sclerosis

mV millivolt

n sample size

NA numerical aperture

NaCl sodium chloride

NaH2PO4 monosodium phosphate

NaHCO3 sodium bicarbonate

ng nanogram

NLRP3 Nacht Domain-, Leucine Rich Repeat-, and PYD-containig Protein 3

nM nanomolar

nm nanometer

xi

NO nitric oxide

NOS nitric oxide synthase

P postnatal

p probability

PAMPs pathogen associated molecular patterns

Panx1 pannexin-1

PBS phosphate buffered saline

PFA paraformaldehyde

PLC-β phosphoinositide phospholipase C β

Poly(I:C) polyinosinic:polycytidylic acid

PPADS pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid

SEM error of the mean

shRNA small hairpin ribonucleic acid

TBI traumatic brain injury

TLR toll-like receptor

TNF-α tumor necrosis factor-alpha

UDP uridine-5'- diphosphate

UTP uridine-5'-triphosphate

VCAM-1 vascular cell adhesion molecule-1

1

CHAPTER ONE: INTRODUCTION

The immune system is a combination of structures, specialized cells and processes that

have co-ordinated functioning to protect the organism from disease due to infection. There are

innate and adaptive immune systems that can function physiologically by reacting to toxins,

foreign organisms and injuries, or pathologically resulting in autoimmune diseases such as

multiple sclerosis (MS). Innate immune cells function by utilizing their pattern recognition

receptors to recognise molecules on pathogenic microorganisms or signals released from an

injured or distressed cell. In contrast, adaptive immune cells react specifically to certain

pathogens whose signature antigen is presented by innate immune cells.

The choroid plexus

The choroid plexus (CP) is located in the brain ventricles and forms important structural

and immune barriers between the cerebrospinal fluid (CSF) and the blood. The CP has a

lobulated structure and its appearance is sometimes compared to a bunch of grapes. Overall, the

total apical surface of the epithelium is large compared to its volume because of microvilli and it

is estimated to be approximately half of the size of the blood-brain barrier (BBB) (75 cm2 for 30-

day-old rats versus 155 cm2) (1). Although it is derived from and connected to the ependymal

cells that line the ventricles and face the ventricular lumen, the CP has unique structure and

functions (2). The most well-known function of the CP is CSF production. This involves

transport of sodium, chloride and bicarbonate to the ventricles from the blood to create an

osmotic gradient for water movement into the ventricles through aquaporin channels (3, 4). In

adults the CSF secretion rate is very high (0.4 ml/minute), leading to a complete turnover of the

CSF six times per day (5, 6).

2

In addition to CSF production and turnover, the CP may also produce a nutritive

“cocktail” of neuropeptides, growth factors and cytokines and play an integral role in distributing

them to distant brain targets. This may be facilitated by the expression of a broad array of

receptors on epithelial cells (for example, to some leptins and growth factors) as well as apically

and basally located transporters. Taken together, the CP appears to be fundamental for the

bidirectional movement of substances in and out of the brain, into the CSF or blood (7). The

highly regulated environment of the brain requires this facilitated filtering at the blood CSF

interfaces, thus a blood-cerebrospinal fluid barrier (BCSFB) exists.

The BCSFB is located on the apical face of the CP that is in contact with the CSF and it

is comprised of a single-layer of polarised cuboidal epithelial cells with basal nuclei and apically

located microvilli (8). Tight junctions between the epithelial cells restrict the flow of substances

across the BCSFB (7). The epithelium rests on the basal lamina, which in turn is underlined by a

dense vascular bed. Blood vessels in the BCSFB of the CP, unlike those constituting the BBB,

are thin-walled and fenestrated, indicating that they are permeable to ions and cells. In addition

to the physical barrier of the epithelial cells, there is also a population of resident native immune

cells resting on the surface of the epithelial cells. These are called epiplexus cells, which are also

referred as Kolmer cells or intraventricular macrophages (9). Together the BCSFB and the

epiplexus cells constitute the local immune system of the CP.

It is now becoming clear that inflammation and/or pathological changes occur in the CP

of the brain in individuals affected by infection, MS, Alzheimer’s disease, hydrocephalus,

hypoxia and injury (7, 9–11). Clarification of the immunological cascades in the CP will give a

new perspective into these pathological conditions. Indeed, CP dysfunction may be involved in

many diseases, suggesting that the epiplexus cells are a potential therapeutic target for treating

3

infection and neuro-immune disorders. This thesis is devoted to understanding the functional

activation of epiplexus cells.

The immune cells of the CP

Along with microglia, epiplexus cells represent a resident group of native immune cells

in the central nervous system (CNS). Epiplexus cells were first described by Kolmer in 1921,

and have since been extensively imaged with electron microscopy (EM) or

immunohistochemistry (12–16). Native immune cells can be found on the CP surface (epiplexus

or Kolmer cells) or in the stroma (connective tissue between epithelial and endothelial cels),

which we refer to as intraplexus cells. In addition, two more ventricular locations for immune

cells have been described: on the surface of the ventricular lining (supraependymal) and free-

floating in the CSF. They all share similar ultrastructural features and are often collectively

referred to as intraventricular macrophages (9).

Enzyme expression by epiplexus cells corresponds to the monocyte and macrophage

profiles, including activity for nonspecific esterases, acid phosphatase, beta-glucuronidase, and

weak activity for naphthol-AS-D-chloroacetate-esterase. However, epiplexus cells do not have

peroxidise activity, while monocytes and macrophages usually do (17–20). In most reports, both

intraplexus and epiplexus cells are referred to as macrophages, though there is some evidence

that this pool consists of both dendritic cells and macrophages. Dendritic cells are innate immune

cells whose main function is antigen presentation. They were named based on their long surface

projections. Dendritic cells in the CP were distinguished by a few factors, such as their

dendriform morphology, expression of MHC II, immunostaining for integrin alpha E2, and

absence of cluster of differentiation (CD) 68/CD163 (ED-1/ED-2) markers (21–23).

4

Macrophages and microglia share most of the known biochemical markers that have been

investigated for immune cells (18). Epiplexus cells are located on the CNS side of the barrier,

which poses the question whether epiplexus cells are microglia, macrophages, dendritic cells, or

a unique cell type. A few notable differences between epiplexus cells and microglia have been

described. Unlike microglia, innate immune cells of the CP express CD14 (co-receptor with toll-

like receptor 4 (TLR4), recognizes pathogen associated molecular patterns (PAMPs) such as

lipopolysaccharides (LPS)), CD200R (also called OX2, conducts immunosuppressive signals),

and CD206 (mannose receptor (MR) involved in phagocytosis) markers. Also, expression of

CD16, CD32 and CD64 (various affinities Fc (fragment crystallisable) receptors that bind to Fc

domain on IgG antibodies, mediate antibody-dependent responses, such as cytotoxicity and

phagocytosis), by epiplexus cells is noticeably higher (24). Additionally, the epiplexus cells

express low levels of various antigens that are often upregulated during infection, injury and

autoimmune disease, suggesting that they are bona fide immune cells. Examples of such antigens

are complement type 3 (CR3, CD11b) receptors, which are involved in phagocytosis and

adhesion, and the leukocyte common antigen (LCA; CD45), major histocompatibility complex

(MHC) I and II, which are required for antigen presentation to cells of adaptive immunity (9).

Therefore, while epiplexus cells express features of macrophages, dendritic cells and microglia,

there appear to be enough differences to justify classification as unique immune cells that are

resident in the CP.

Origin of the native immune cells in the CP

It is thought that epiplexus cells are resident immune cells in the CP, although there is an

outstanding question regarding from where they arise and how they get to their place of

residence. There are different hypotheses about how the epiplexus cells arise in the CP. A

5

generally accepted theory suggests that blood-derived immune cells travel from the blood to the

ventricle (9, 15). This is based on experiments where intravenous injection with India ink tracer

(labelled carbon) gave a delayed labelling of immune cells in the CP. On the 1st-4th days after

injection, the tracer could be found in circulating monocytes and intraplexus cells, but not in the

epiplexus cells, where it appeared 5-6 days after the injection (15). This suggests that labelled

monocytes were moving from the blood into the CP and then to the apical surface to become

epiplexus cells. An alternative theory suggested that microglia might migrate from the brain to

the CP during pathological conditions, such as induced hydrocephalus. In this model, the number

of intraventricular macrophages was increased and immunoreactive cells were observed crossing

the ependymal lining, while the corpus callosum microglial pool above the ventricle was

depleted (25).

The adhesion of monocytes to endothelial cells, the first step of transmigration of

leucocytes from the blood to the tissue through the blood vessel, called diapedesis, has been

observed at the EM level in the CP (26). Also, cells of round and elongated shapes were found in

the stroma of the CP, with some of them extending their processes to capillaries, and some

sending them to and in between epithelial cells, supporting the circulation origin theory of

immune cells in the CP because this resembles the shape of immune cell during movement.

However, these studies only provided snapshots of precursors of epiplexus cell’s migration in the

CP and need to be supported by time lapse imaging experiments.

There were some studies that investigated how monocytes migrate through the CP to the

CFS-facing surface in order to become epiplexus cells. Using either scanning or transmission

EM, monocytes have been observed emerging on the apical side of epithelial cell or even inside

the cell itself (8, 26). Authors suggested two hypotheses to explain this phenomenon. One states

6

that monocytes travel between epithelial cells of the CP (8, 26). The other says that the stromal

cells get to their final destination by the process of emperipolesis, travelling through the other

cell, which in this case is epithelial (8, 26).

These experiments demonstrate that precursors of epiplexus cells are motile, while for the

resident epiplexus cells, their ability to move is currently unknown. Regardless of how the

epiplexus cells arrive at the surface of the CP, they likely have specific functions that include

detection of antigens presented by epithelial cells, or acting as a ready pool of immune cells in

case of infection in the ventricle (10).

The epiplexus cells and the CP in normal and pathological conditions

The epiplexus cells appear to share hallmark features, and function similarly, to innate

immune cells such as tissue macrophages and microglia because they react to infection and

injury, and are involved in autoimmune CNS diseases, such as MS (Table 1). Phagocytic

properties have been described for epiplexus cells, as evident by their ability to endocytose

intraperitonealy (IP) or intravenously (IV) injected horseradish peroxidise (HRP) (27–29). The

immune responses of epiplexus cells have been studied for several models of disease, and in

disease states which all strongly support an immunological role for epiplexus cells.

In models of infection, such as IP injection of LPS or interferon gamma (IFN-γ), an

immune response was elicited in the epiplexus cells that included increased expression of CR3,

LCA, MHC I and II, and ED1 (Table 1), plus an increase in the total number of epiplexus cells

(30, 31). Additionally, increased transferrin expression was also observed, which may be

explained by the iron withholding hypothesis: bacterial survival is impeded when the level of

free iron in the environment drops as the result of transferrin chelation of iron (30, 32).

7

Nitric oxide (NO) plays various functions, but in the case of infection, it is implicated in

vasodilatation and immune defence and can be used as an indicator of immune cell activation.

Interestingly, there is no detectable nitric oxide synthase (NOS) expression under normal

conditions, but NOS increases in both the epiplexus cells and epithelial cells of the CP after LPS

injection (33). All of these indicate an inflammation process and response to infection by

epiplexus cells.

Table 1. Changes in the number of epiplexus cells and the expression of various molecules on

these cells in infection, injury and auto-immune disease MS (10, 25, 30–43).

Number

of cells

CR3 MHC I MHC II LCA

CD68/

ED1

NOS

Trans-

ferrin

Control moderate weak weak weak weak no weak

LPS increase strong strong strong strong strong moderate strong

IFN-γ moderate moderate moderate weak weak moderate

Viral en-

cephalitis

increase increase increase

TBI increase increase increase

Hydro-

cephalus

increase increase increase

Hypoxia increase increase increase increase increase

MS increase increase increase

8

The response of epiplexus cells to brain injury has been modelled in four ways: by non-

penetrating traumatic brain injury (TBI), hydrocephalus, hypoxia, and the crotoxin complex

(phospholipase A2) injection (for oxidative stress and neuroinflammation) (Table 1). Increased

levels of CR3, MHC I and II in epiplexus cells were observed during these models, as well as an

elevated number of epiplexus cells during hydrocephalus and increased expression of NOS and

CD68 in hypoxia. Emperipolesis is a very rare observation in normal conditions, while its

occurrences after TBI were more frequent, suggesting this is an important mode for recruitment

of new cells to the CP (25, 34–44). In the hydrocephalus model, the epiplexus cells were

phagocytising erythrocytes that normally are not present in the ventricles (25). Within two hours

after cisternal injection of the crotoxin complex, the epiplexus cells have spread their processes

over the epithelial cells (37). Beads covered with crotoxin complex and injected into ventricles

were surrounded by epiplexus cells (36). Hypoxia caused weakening of the BCSFB,

demonstrated by the higher rates of HRP penetration through the barrier (34, 35).

In the case of actual disease, viral encephalitis, there was an increase in HLA-DR (MHC

II) and CD 68 expression, as well as higher numbers of epiplexus cells (10). In patients with MS,

an autoimmune disease, the CP had no visible morphologic changes, but showed signs of

inflammation. In particular, intraplexus and epiplexus cells had increased immunoreactivity to

HLA-DR (MHC II), CD 68 and CD 3 (T-lymphocyte marker). The number of epiplexus cells

was also increased (10). In the CSF the number of T cells and CD4/CD8 positive cells were also

elevated (45). Endothelial cells of the CP had high levels of VCAM-1 (vascular cell adhesion

molecule-1) expression, while in normal controls it is not expressed at all (10). Taken together,

these studies suggest that epiplexus cells become activated during immune challenges and

autoimmune diseases.

9

In patients with Alzheimer’s disease (AD) pathological changes in the CP are pronounced.

Defective production of the amyloid beta peptide (Aβ), one of the main players in AD in the

brain, can be neurotoxic (46, 47). Accumulation of Aβ in the CP is detrimental as well. There it

results in increased production of NO, mitochondrial dysfunction, cell death and probably

dysfunction of the BCSFB. Inefficient clearance of Aβ can also be an underlying cause of AD

development, in particular due to abnormal clearance by the CP (11).

To summarise, the epiplexus cells show hallmark features of innate immune cells, such as

phagocytosis, scavenging, promotion of infection, antigen presentation, accumulation of iron,

and production of NO. There is a hypothesis that the immune response of epiplexus cells,

probably the subpopulation of dendritic cells, involves migration out of the CNS and recruitment

of T cells to the ventricle (7, 10, 22, 48). Together with the CP stroma and epithelium, epiplexus

cells create not only a functional barrier between the blood and the CSF, but also a system

involved in routine immune surveillance and responses to infection via release of inflammatory

molecules.

ATP as an extracellular messenger for immune cells

It is not known how epiplexus cells detect infection and injury. Are they sensing changes

by themselves or receiving signals from the damaged epithelium, or both? It is now becoming

clear that, to be fully activated during infection, cells of innate immunity should not only detect

PAMPs released or present on a foreign microorganism, but also recognise damage associated

molecular patterns (DAMPs) (49–51).

The purine nucleotide, adenosine-5'-triphosphate (ATP), has several characteristics that

let it serve as a DAMP. Firstly, the physiological concentrations of ATP in the extracellular

space and blood plasma are within the nanomolar range (400-700nM, some sources stating 1-

10

10nM), compared to cytosolic concentrations of 1-10mM (52–56), allowing it to be rapidly

released along a concentration gradient so that even small quantities can be read as a strong

signal. Secondly, ATP is water-soluble and can easily diffuse in the extracellular environment.

Thirdly, purinergic signalling is plastic due to the large family of purinergic receptors with

various expression patterns on different types of cells. Lastly, there are many extracellular

ATPases that quickly inactivate the signal and lead to spatial and temporal restrictions (57, 58).

There are multiple mechanisms proposed for the ATP release pathway from cells during

both physiological and pathological conditions. These include, rupture of the cellular membrane

under pathological conditions, conductance by connexin and pannexin channels, exocytosis,

transporters, and P2X7 receptors (59–69). An example that may be relevant to the intact CP is

that ATP release is augmented under inflammatory conditions; much more ATP is released by

non-damaged endothelial cells when stimulated by a combination of LPS and shear stress,

compared to shear stress only (70). Released ATP and its catabolic products can then initiate

numerous cellular responses, depending upon the types of purinergic receptors that are

expressed.

There are two large families of purinergic receptors, P1 and P2, that have expression in

virtually all cells (71). The P1 receptors, A1, A2A, A2B, and A3 are all G-protein coupled

metabotropic receptors that use adenosine, an ATP degradation product, as their ligand. The P2

receptors on the other hand have two groups of receptors that are metabotropic (P2Y) or

ionotropic (P2X). The P2Y group has eight G-protein coupled receptors that are activated by a

variety of nucleotides (preferred agonist in brackets): P2Y1 (adenosine-5’-diphosphate (ADP)),

P2Y2 (ATP and uridine-5'-triphosphate (UTP)), P2Y4 (UTP, less potently by ATP, uridine-5'-

diphosphate (UDP)), P2Y6 (UDP, less potently by UTP), P2Y11 (ATP), P2Y12 (ADP), P2Y13

11

(ADP, ATP), and P2Y14 (UDP-glucose). The other group, P2X, includes seven ligand-binding

ion channels (P2X1-7) that are all opened by ATP. Agonist binding affinities (EC50) of

purinergic receptors are typically less than 10µM, with the exception of the P2X7 receptor,

which is sensitive to higher ATP concentrations (>100µM) (55, 56).

As noted above, a subset of purinergic receptors can be activated by UTP and its

derivative UDP. For a long time it was believed that UTP participates in the intracellular

processes only, such as being a substrate for the synthesis of ribonucleic acid (RNA), an

activator of substrates in metabolic reaction and a source of energy. Now UTP and UDP are

known to participate in extracellular P2Y receptor-mediated signaling as well (72–74). A

concerted effort has been applied exploring the roles of purinergic receptors in immune cells

because ATP and other nucleotides can critically function as DAMPs (49–51).

Purinergic signalling in innate immune cells

Purinergic receptors are expressed on monocytes, macrophages, microglia, dendritic cells

and other immune cell types (Table 2) (55, 56, 75–83). Interestingly, expression patterns are not

always consistent in the literature for a certain cell type, but likely reflect the source of the cells

and their maturation stage. For example, Franke et al found that microglia in their basal state

expressed only one P2 receptor, P2Y1. However, after brain injury, microglial cells had a much

more diverse purinergic receptor profile, expressing P2X1, 2, 4, 7 and P2Y1, 2, 4, 6, 12 (77). It is

also important to mention that P2Y11 receptor is present in humans, but is not expressed by

rodents (84). To date, there is no information whether epiplexus cells express purinergic

receptors, but considering their clear immune cell origin (see above), they likely express one or

multiple subtypes.

12

Table 2. A list of all known purinergic receptors, and those expressed on monocytes,

macrophages, microglia, and dendritic cells (55, 56, 75–83).

Where expressed Ionotropic P2X Metabotropic P2Y Metabotropic P1

Systemic 1, 2, 3, 4, 5, 6, 7 1, 2, 4, 6, 11, 12, 13, 14 A1, A2a, A2b, A3

Monocytes 1, 4, 5, 7 1, 2, 4, 6, 11, 12, 13 A1, A2a, A2b, A3

Macrophages 1, 4, 5, 7 1, 2, 4, 6, 11, 12 A1, A2a, A2b, A3

Microglia 1, 2, 4, 7 1, 2, 4, 6, 12 A1, A2a, A2b, A3

Dendritic cells 1, 4, 5, 7 1, 2, 4, 6, 11, 13, 14 A1, A2a, A2b, A3

Extracellular ATP, as well as other nucleotides, can modulate responses of innate and

adaptive immune cells to injury and infection in both a facilitating and an attenuating manner.

Extracellular ATP acting through the P2X7 receptor causes increases in intracellular Ca2+

in

most innate immune cells (56, 79, 80, 85). This is often followed by cellular activation as evident

by assembly of the NLRP3 inflammasome (Nacht Domain-, Leucine Rich Repeat-, and PYD-

containig Protein 3), a component of innate immune system and a multiprotein oligomer that

induces cleavage and maturation of IL-1β and IL-18, and release of these inflammatory

cytokines (86–88). Inflammatory responses may also be enhanced by P2X2, P2X4, P2X7

receptors, and pannexin-1 (Panx1) channels mediated activation of effector T-cells, selective

P2X7-mediated depletion of Treg cells, and P2X7-mediated enhancement of neutrofil

microbicidal activity (89–96). On the other hand, extracellular ATP can also inhibit the LPS-

triggered secretion of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1, IL-12,

CCl2, CCL3, CCl5 and CXCL10, by dendritic cells (97–99). Thus, innate and adaptive immunity

13

can be differentially regulated by ATP. It is important to note that the outcome of these types of

experiments depends upon the concentration and species of extracellular nucleotides, the stage of

the immune response, the types of purinergic receptors expressed, and the types of immune cells

being investigated. While it has been demonstrated that ATP modulates the activity of

macrophages, microglia and dendritic cells (55, 56), the regulation of epiplexus cells by ATP

remains unexplored. Here, I hypothesize that epiplexus cells are activated by extracellular

ATP.

Migration of immune cells and ATP

How might ATP alter the activity of epiplexus cells? An important aspect of the

development of inflammation is migration of innate immune cells to the site of distress. There

are two types of cellular motility, chemotaxis and chemokinesis. Chemotaxis is the directed

movements of cells to or from some chemical signal. In the absence of a defined (or detectable)

gradient of the chemotactic signal, cellular movements may be undirected and this process is

called chemokinesis.

A concentration gradient of extracellular ATP or other nucleotides can be present in the

inflammatory site of damaged tissue (55, 56). There is some evidence that suggests that ATP,

ADP, UTP and / or adenosine can directly cause chemokinesis (100, 101) or chemotaxis of

various immune cells, such as monocytes, macrophages, immature dendritic cells, microglia,

mast cells, eosinophils and neutrophils (74, 102–110). Chemotaxis was shown to be mediated by

both G-protein coupled (A1, A3, P2Y2, and P2Y12) and ionotropic receptors, though P2X-

mediated chemotaxis was less potent (74, 104–108, 110). It is important to mention that the

results of these studies varied depending on the type of cells used in the experiments. Other

14

motile responses of microglia have been described. These include a P2Y-mediated behaviour

where microglia rapidly extend filopodia to sites of injury or ATP application (111).

Additionally, microglia also constantly survey the brain environment by extension and retraction

of filopodia. Some authors argue that extracellular nucleotides enhance chemotaxis, or together

with other molecules, such as chemokines and formyl-peptide signals, result in immune cell

migration (112–114). Thus, it appears that purine nucleotides and other DAMPS can affect

several different behaviours of immune cells.

ATP can affect immune cell’s chemotaxis in both paracrine and autocrine ways (115).

Chemokine C5a - induced migration of mouse monocytes and macrophages was found to be

translated and amplified by autocrine ATP signalling (78). In this study, combined but not

individual inhibition of P2Y2, P2Y12 and/or adenosine receptors resulted in impaired

chemotaxis. Autocrine ATP-mediated signal amplification, gradient sensing and promotion of

cell migration was also found in human neutrophils. This process was mediated by P2Y2 and A3

receptors (116). The precise mechanism of autocrine ATP signalling is currently unknown. A

potential candidate for this role, Panx-1, was found to be implicated in ATP release from non-

excitable cells (66), and facilitation of T-cells activation (89, 95, 96), but not in ATP-mediated

amplification of C5a-chemotaxis (78).

There are multiple signalling pathways regulating immune cell motility (117), though

nucleotide-triggered mechanisms are not well understood. Activation of chemotaxis-related

metabotropic receptors A1 (Gi/o), A3 (Gi/o), P2Y2 (Gq/11 and Gi/o), and P2Y12 (Gi/o) results in

inhibition of 3'-5'-cyclic adenosine monophosphate (cAMP) production, as well as

phosphoinositide phospholipase C β (PLC-β) activation and inositol 1,4,5-trisphosphate (IP3)

production that, in turn, releases Ca2+

from internal stores (84, 115). Activation of ionotropic

15

purinergic receptors also results in increased intracellular Ca2+

due to influx (79, 118, 119). Even

though a connection between rises in intracellular Ca2+

and cell motility are not fully established,

there is some evidence that supports this idea (120–123). IP3 and Ca2+

accumulates in the

chemoattractant-facing region of the cell, where polymerisation of actin is followed by

polarisation and migration of the cell (80, 115, 117, 120–123).

Thus, extracellular ATP and other nucleotides are important for paracrine and autocrine

signalling to and migration of various immune cells, including monocytes, macrophages,

microglia and dendritic cells. To date it is unknown if epiplexus cells respond to ATP, and what

that response is. Since this population is believed to consist of macrophages and dendritic cells,

epiplexus cells could have changes in their motility, such as protraction of cellular processes and

/ or cell movement; determining this is the major focus of this thesis.

Panx1 as a source of extracellular ATP

The sources of extracellular ATP can arise from infectious organisms, damaged or dying

cells and release from healthy cells during normal signalling. For example, neurons in the CNS

undergoing apoptosis can release ATP via Panx1 to call immune cells to phagocytise them (102).

Panx1 is a large conductance channel with broad tissue expression (124). One of the more

interesting features of Panx1 is the channel’s permeability to molecules that are < 1 kDa in size,

which includes amino acids and ATP (124). Panx1 opening is triggered by various physiological

and pathological conditions, like membrane depolarisations beyond −20mV, intracellular Ca2+

,

mechanical stretch, ischemia and extracellular ATP (P2X7-mediated activation). Also, it was

found that prolonged activation of NMDA receptors can open a large-conductance pore that was

sensitive to Panx1 blockers or knockdown of Panx1 by shRNA and this occurred independently

of intracellular Ca2+

(124–127).

16

Panx1-mediated ATP release, regardless of the trigger, is thought to be involved in

physiological signalling. In taste bud receptor cells, ATP from Panx1 is implicated in activation

of gustatory afferent nerves (128, 129). Panx1 is also important for controlling microcirculation

via ATP released from Panx1 expressed on red blood cells (130). Interestingly, Panx1 is

expressed on epithelial cells of the airway where is plays a role in purinergic-mediated

mucociliary clearance (131).

In the immune context, Panx1 is linked to cell death in several ways that involve high

concentrations of extracellular ATP, which acts as a danger signal (96, 125, 131). Initially, it was

shown that Panx1 is activated by P2X7 purinergic receptors and contributed to activation of a

large “death pore” (132, 133). More recently, Panx1 was proposed to be an integral part of the

cellular inflammasome that leads to activation of caspase-1 and release of IL-1β (134–138).

Thus, Panx1 can be an important source of extracellular ATP under physiological and patho-

physiological conditions. This raises the important question of whether or not Panx1 is present

on the CP epithelium and if it can contribute to activation of the epiplexus cells.

Therefore I hypothesize that epiplexus cells are activated by extracellular ATP and

that the CP epithelial cells express Panx1 channels that are involved in activation of the

epiplexus cells (Figure 1).

This will be tested in three specific aims:

1. To determine and characterize if epiplexus cells are activated by ATP.

2. To identify the purinergic receptors responsible for epiplexus cell responses to ATP.

3. To determine if the CP epithelium expresses Panx1 channels and investigate if they are

involved in ATP-mediated activation of epiplexus cells.

17

Clarification of the mechanisms of epiplexus cell activation will improve our

understanding of the immune responses cascades at the level of the BCSFB. Dysfunction of this

barrier is involved in many diseases, thus we anticipate that the epiplexus cells can be a potential

therapeutic target for neuro-immune disorders.

18

Figure 1. The schematic representation of a hypothesised cascade. Extracellular ATP binds

to the purinergic receptors on epiplexus cells, resulting in activation of these cells. The possible

source of extracellular ATP is Panx1 channel expressed on CP epithelium.

19

CHAPTER TWO: MATERIALS AND METHODS

Materials were obtained from Sigma (St. Louis, MO) unless otherwise stated. Sprague-

Dawley rats were housed according to the Canadian Council for Animal Care guidelines.

The isolated and intact CP preparation

A new technique for the isolation of live and intact CP was developed. Postnatal (P) 21-

40 day old Spague-Dawley rats were anaesthetized by inhalation of isofluorane (from Baxter) in

air. Animals were killed by decapitation and brains were quickly removed and placed in ice-cold

artificial cerebro-spinal fluid (aCSF) consisting of (in mM) NaCl (120), NaHCO3 (26), KCl

(2.5), NaH2PO4 (1.25), MgSO4 (1.3), CaCl2 (2), and glucose (10). Osmolarity was carefully

maintained at 291±2 milliosmole. The frontal and medial corticies were resected to expose the

corpus callosum and two additional incisions along the hippocampus were used to remove the

remaining cortex and expose the lateral ventricles. The CP was gently extracted with forceps and

placed into a chamber filled with aCSF at 30-33 ºC to recover for 30 minutes; this is similar to

standard brain slice procedures that are routinely used in the lab. The CP from the third and

fourth ventricles can also be easily obtained, but experiments were performed on CP from the

lateral ventricles because it tended to be flat and rested on the cover glass making it very suitable

for live cell imaging.

Live cell fluorescent imaging

Alexa Fluor 488 isolectin B4 conjugate (IB4) from Griffonia simplicifolia (from

Invitrogen) was used to label live epiplexus cells. IB4 has been shown to selectively label

immune cells, such as macrophages and brain microglia (139–141). A 1 mg/ml stock solution of

IB4 was prepared using phosphate buffered saline (PBS) (pH 7.4) and 0.5mM CaCl2. The

isolated CP was placed into a 10µg/ml solution of IB4 in modified Hank’s Balanced Salt

20

Solution (HBSS, MgCl2 (4mM), CaCl2 (1mM), pyruvic acid (1mM), kynurenic acid (1mM),

glutathione (0.005mM), pH 7.4) (142) for 15 minutes at room temperature.

After labelling in IB4, the CP was placed onto cover glass type 0 (from Fisher Scientific)

and studied under a light microscope (Zeiss Axioimager inverted microscope) equipped with

fluorescence. IB4 was excited at 470 nm with light emitting diode (LED) light sources (Zeiss

Colibri) and emission was filtered through a high efficiency GFP filter set with single band pass

550/25 nm. Baseline fluorescence was collected with a 40x air objective (NA=0.6) for 25

minutes while the CP was perfused with oxygenated (95% O2 / 5% CO2) aCSF and then

switched to the experimental solution for up to 95 minutes. The experimental solutions contained

adenosine-5'-triphosphate (ATP), other agonists (adenosine-5'-diphosphate (ADP), uridine-5'-

triphosphate (UTP), adenosine, 3'-Benzoylbenzoyl adenosine 5´-triphosphate (BzATP)) and/or

antagonists (brilliant blue G (BBG), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid

(PPADS), tangeretin) or potentiator (ivermectin) of purinergic receptors, Panx1 blocker

probenecid, TLR ligands lipopolysaccharide from Escherichia coli 055:B5 (LPS) and high

molecular weight polyinosinic:polycytidylic acid (Poly(I:C) (from InvivoGen), dissolved at the

final concentration in aCSF. If necessary, 0.1% solution of dimethyl sulfoxide (DMSO) (from

VWR) was used as a vehicle. The temperature of all solutions was maintained at 30-33 ºC with

an in-line heater placed close to the microscope chamber.

AxioVision Multidimensional Acquisition software (Zeiss) was used to acquire four

adjacent images in the xy plane (using the MosaiX module; Zeiss) and each was comprised of 4

to 6 z-stacks collected at 0.5-0.7 µm steps. Images were taken every 5 minutes. Adjacent images

were stitched using the Convert Tile Images parameter and stacks were collapsed using the

maximum projection method to create a single image that was used for analysis.

21

Data analysis and statistics

Images were processed and analyzed with ImageJ v.1.46 software (National Institutes of

Health, Bethesda, MD). The StackReg (143) plug-in was used to align the images to the original

position if the xy axes drifted during the course of the experiment. Brightness and contrast were

adjusted to an optimal level and remained consistent throughout each image series. The Manual

Tracking (144) plug-in for ImageJ was used to measure the distance travelled by cells for each

frame, and generated representations of the tracked path superimposed on the original image.

Data obtained with ImageJ was further processed in Microsoft Office Excel 2007. Cells

with missing or less than three data points in the 5 points / 25 minutes long baseline were

excluded from further analysis. Data for each time point for a given experiment were averaged

(arithmetic mean), the standard error of the mean (SEM) was calculated and these plotted as

“Raw average distance travelled”. To obtain normalised data (plotted as “Normalised distance

travelled”), the baseline of each cell was averaged and subtracted from each data (time) point for

each cell. Then, data for each time point for all cells was averaged and the SEM was calculated

and plotted. Cumulative normalised data (plotted as “Normalised cumulative distance travelled”)

for each data (time) point for each cell was calculated by adding up all previous data points to the

next data point. Finally, the average and SEM was calculated for each time point for all cells.

Bar graphs were plotted using normalised distance and, in most cases, were calculated as

follows: Firstly, all data for all time points for each cell were averaged. These means represented

average distance travelled by an individual cell. The mean distances for each cell was then

averaged and used to determine the standard error of the mean for the entire population. If cells

were travelling noticeably different distances during the experiment, these parts were analysed

separately.

22

GraphPad Prism 4 software was used for all statistical analysis. One-way analysis of

variance (ANOVA) and Bonferroni’s test for post-hoc analysis were used to compare means.

Means were considered significantly different when probability (p) was lower than 0.05. Error

bars in all plots represent the standard error of the mean (SEM). Both GraphPad Prism 4 and

Microsoft Office Excel 2007 software were used for graphic representations of the data.

Immunohistochemistry

The CP was isolated as described above and subsequently fixed overnight in 4%

paraformaldehyde (PFA) (from EMD). The samples were then cryoprotected in 30% sucrose at

4oC. The CP was washed in PBS three times for 10 minutes to remove the sucrose and then

subjected to antigen retrieval that involved submersion into a heated (80oC) sodium citrate buffer

(trisodium citrate (dihydrate) 10mM (from Fisher Scientific), 0.05% polyoxyethylene sorbitan

monolaurate (Tween 20) (from BioRad), pH 6.0) for 30 minutes. CPs were then cooled for 30

minutes and washed in bovine serum albumin (BSA) (from Roche) three times for 10 minutes.

Nonspecific binding of the antibodies was blocked by a 2 hour exposure to BSA-based blocking

solution (solution of 1% BSA, 0.2% Triton X-100, 0.5% sodium azide, 0.4% Na

ethylenediaminetetraacetate (EDTA) in PBS) at room temperature.

Primary antibodies were dissolved in blocking solution and applied to the fixed CP tissue

for 24 hours at room temperature. The CP was washed with 0.2% Triton X-100 in PBS three

times for 10 minutes. When another primary antibody was used to co-label the CP, the above

steps were repeated. All antibodies against purinergic receptors were used at 1:200 dilutions, and

antibodies against Iba1 and Panx1 were at 1:500. Subsequent incubation in a secondary antibody

cocktail (secondary antibodies at 1:100 and 100ng/ml 4',6-diamidino-2-phenylindole (DAPI)

dissolved in blocking solution) was for 2 hours. Finally, the CP was washed with 0.2% Triton X-

23

100 solution in PBS three times for 10 minutes, mounted onto chrome alum (chromium (III)

potassium sulfate)-coated superfrost white slides (from VWR) in Vectashield (from Vector Lab),

covered with a coverslip type 0 (from Fisher Scientific) and studied under the microscope. When

cells were co-labelled with IB4, immunolabelling was modified so that IB4 was applied before

PFA fixation (i.e. to live tissue) and antigen retrieval was omitted because it resulted in loss of

the fluorescent IB4 labelling.

The following primary antibodies were used – from rabbit: anti - P2X2R (APR-003),

P2X7R (APR-004), P2Y1R (APR-021), P2Y11R (APR-015) from Alomone, anti - P2X3R

(AB5895) from Millipore, anti - P2Y2R (AB10270) from Abcam, anti – Iba1 (#019-19741) from

Wako; and from mouse anti - Panx1 (#H00024145-M07) from Abnova. The following secondary

antibodies were used: Alexa Fluor (AF) 488 donkey anti-rabbit IgG (A-21206), AF donkey anti-

mouse 488 IgG (A-21202), AF donkey anti-rabbit 555 IgG (A-31572) from Invitrogen.

24

CHAPTER THREE: RESULTS

To study the activation of epiplexus cells by ATP, two approaches were taken. Firstly,

motility of the cells was observed and recorded with fluorescence imaging in the presence of

ATP and other purinergic receptors agonists and antagonists. Secondly, immunohistochemical

analysis was performed to investigate the compliment of purinergic receptors expressed on

epiplexus cells and to determine if Panx1 is expressed in the CP.

Identification of epiplexus cells as immune cell

Prior to recording epiplexus cellular physiology, I first sought to confirm they were

innate immune cells and develop a method for identifying them in situ. This was achieved using

a fluorescent analogue of IB4, a known generic marker for immune cells (Figure 2) that has been

extensively used to label macrophages and microglia (139–141). The fluorescence of most

labelled cells appeared similar, and was found to be effective for visualisation of cell bodies and

intracellular vesicles (Figure 2), but less so for fine cell processes (140). The epiplexus cells

appeared evenly distributed across the CSF-facing CP surface (apical surface). They usually had

more than one identifiable large process and were round or elongated in shape. IB4 is reported to

be effective at labelling endothelial cells (145), but in our acute, live CP preparation blood

vessels did not stain. However, staining of CPs for IB4 after fixation in 4% PFA resulted in

labelling of both the epiplexus and endothelial cells (not shown).

Co-staining with an antibody to the immune cell marker, ionized calcium binding adaptor

molecule 1 (Iba1), was performed in order to confirm that the IB4-labelled cells are of immune

origin (Figure 2B). This marker is specifically expressed on the cells of monocytic lineage,

including macrophages and microglia (146, 147). Epiplexus cell were co-labelled with IB4 and

Iba1, although there were some differences in the observed staining patterns. For example, Iba1

25

Figure 2. Epiplexus cells are labelled with IB4 and Iba1. A –an epiplexus cell resting

on the choroidal epithelium, visualised with transmitted light or by labelling with Alexa Fluor

488 isolectin B4 conjugate from Griffonia simplicifolia. EC – epiplexus cell, EP – choroidal

epithelium, BV – blood vessel, LV – lumen of the lateral ventricle. Scale bar 20 μm. B –

immunofluorescent staining for immune cell markers Iba1 and IB4 in the CP. Note the

significant co-localisation. Scale bar 50 μm.

26

was better visualised on the cellular processes and evenly distributed on the cell bodies, whereas

IB4 staining was concentrated around the nucleus (Figure 2), which likely reflects their distinct

molecular targets.

Epiplexus cells are activated by extracellular ATP

The data in Figure 2 demonstrate that the epiplexus cells are of immune origin and can be

labelled with IB4 as live cells in CP explants. To investigate their activation by ATP, images of

IB4 labelled live epiplexus cells in the intact CP were acquired at 5 minute intervals and

movements were manually tracked with the aid of ImageJ software (Figure 3 and 4). Activation

was quantified as the distance travelled by epiplexus cells in these 5 min intervals. In the absence

of ATP (i.e. baseline control), epiplexus cells resided mainly in one place within the CP tissue

(Figure 3 and 4), but sometimes showed spontaneous motility that appeared to remain consistent

throughout both the baseline and experiments (raw, not normalised distance travelled was 0.84

+/- 0.09 μm/frame in control conditions; n=124 cells from 5CPs).

To investigate the immune responsiveness of epiplexus cells, ATP was bath applied to

the intact, isolated CP. I chose to bath apply ATP because it was reasoned that unlike focal

application, bath ATP would mimic a general infection of the CSF. Application of 100µM

extracellular nucleotides was shown to be optimal for triggering a maximal rise in internal Ca2+

in human alveolar macrophages (79). In the presence of exogenous ATP (100 µM), cells began

‘crawling’ around the sheet of CP epithelial cells. Epiplexus cells travelled varying distances that

ranged from tens to hundreds of microns in an hour (Figure 3, 4 and 6). ATP application

significantly (p<0.0001) increased the mean normalised (+/-SEM) distance travelled by the

epiplexus cells from 0.05 +/- 0.15 μm/frame in control (n=124 cells from 5CPs) to 0.93 +/- 0.12

μm/frame (n=293 cells from 9CPs). Movements of the cells were clearly chemokinesis because it

27

Figure 3. Representation of the tracked paths superimposed on the original image. 0

minutes – locations of the cells at the start of the experiment, 25 minutes – end of the baseline,

120 min – end of the experiment for control conditions (A) and bath-applied 100µM ATP (B).

Bar is 50µm.

28

Figure 4. Distance travelled by epiplexus cells in control and in the presence of ATP. Each

line represents individual epiplexus cell. A, B – raw distance travelled, C, D – normalised

cumulative distance travelled.

29

lacked directionality (see Figure 3).

P2X7 receptors are not involved in chemokinesis of epiplexus cells

The P2X7 receptor has been implicated in activation of microglia by ATP during brain

injury (148–150). Epiplexus cells express P2X7 receptors (Figure 5), so I tested if P2X7

receptors are involved in ATP-induced chemokinesis of epiplexus cells by bath application of the

P2X7 receptor antagonist, BBG (1μM, as shown by Jiang et al. to effectively block rat P2X7

receptors (151)), or the agonist bzATP (100μM, same concentration as used for other

nucleotides; Figure 4). Application of BBG with ATP did not significantly alter (p=0,4602) the

mean normalised (+/-SEM) distance travelled by the epiplexus cells, 1.22 +/- 0.11μm/frame

(n=177 cells from 7CPs) compared to ATP alone 0.93 +/- 0.12 μm/frame (n=293 cells from

9CPs). The distance travelled in the presence of BBG plus ATP was significantly (p<0.0001)

further than in control 0.05 +/- 0.15 μm/frame (n=124 cells from 5CPs). In the presence of

bzATP to activate P2X7, epiplexus cells travelled significantly (p<0.0001) shorter distances

when compared to ATP alone (-0.26 +/- 0.10 μm/frame; n=150 cells from 5CPs). The mean

normalised distance travelled in the presence of bzATP was not significantly different from

control (p=0,8825). Thus, the P2X7 receptor blocker, BBG did not prevent activation of

epiplexus cells by ATP, and the P2X7 receptor agonist bzATP did not trigger an increase in

epiplexus cells activity, suggesting that P2X7 receptors were not involved in epiplexus cells

activation by extracellular ATP.

P2X4 receptors are not involved in epiplexus cells chemokinesis by exogenous ATP

The data in Figures 5 and 6 suggest that the P2X7 receptor is not linked to chemokinesis

of epiplexus cells induced by ATP. Therefore, I sought to identify other candidate purinergic

30

Figure 5. Immunofluorescent staining for P2X7 receptors in epiplexus cells.

31

Figure 6. P2X7 receptors are not involved in epiplexus cells activation by extracellular

ATP. P2X7 receptor blocker BBG did not prevent activation of epiplexus cell by ATP, and

P2X7 receptor agonist bzATP did not trigger increase in epiplexus cells activity. A – raw

average distance travelled, B – normalised distance travelled, C – normalised cumulative

distance travelled, D – normalised average distance and statistical analysis.

32

receptors. Most immune cells, including macrophages, microglia and dendritic cells, express

P2X4 receptors, that are functional (mediate ionic current) and participate in activation of

immune cells (81, 82, 152). The P2X4 receptor potentiator, ivermectin, (10μM) was used to test

whether P2X4 receptors could be involved in chemokinesis (Figure 7). Since ivermectin

potentiates the magnitude of P2X4 receptor activation to a given concentration of ATP (81, 153,

154), the changes in activity of epiplexus cells were compared at 10μM exogenous ATP, both

with and without concomitant application of ivermectin. DMSO (0.1%) alone was used as the

vehicle control.

Application of both DMSO (0.1%) and ATP (100μM) together did not significantly

(p=0,9504) affect the mean normalised distance travelled by epiplexus cells, 0.81 +/-

0.14μm/frame (n=162 cells from 5CPs), compared to ATP (100μM) alone 0.93 +/- 0.12

μm/frame (n=293 cells from 9CPs), but was significantly different (p=0,0021) from control (i.e.

no ATP) 0.05 +/- 0.15 μm/frame (n=124 cells from 5CPs). In the presence of 10μM ATP,

epiplexus cells had a mean normalised movement of 0.19 +/- 0.11 μm/frame (n=140 cells from

4CPs) that was significantly (p=0,0003) different than 100μM ATP but not significantly different

from the control rate (p=0,9623). When 10μM ivermectin was applied concomitantly with 10μM

ATP, epiplexus chemokinesis increased to 0.55 +/- 0.10 μm/frame (n=109 cells from 4CPs), but

this was not significantly different from either control (p=0,1678), 10μM ATP (p=0,4692) or

from 100μM ATP plus DMSO (p=0,7546).

P2Y2 receptors contribute to ATP-induced chemokinesis of epiplexus cells

The data reported above suggested that neither P2X4, nor P2X7 ionotropic purinergic

receptors play a role in induction of chemokinesis of epiplexus cells by ATP. Here, I sought to

33

Figure 7. P2X4 receptors are not involved in epiplexus cells activation by exogenous ATP.

Potentiation of P2X4 by ivermectin did not result in statistically significant increase in

chemokinesis. A – raw average distance travelled, B – normalised distance travelled, C –

normalised cumulative distance travelled, D – normalised average distance and statistical

analysis.

34

determine if other purinergic receptors might be involved. While the pharmacology of P2X and

P2Y receptors lack complete specificity, the combination of multiple pharmacological agents and

immunocytochemistry are viable options for narrowing down potential contributing receptors.

To this end, I applied the non-selective P2X/Y antagonist PPADS. PPADS is a well-described

(155–157) antagonist of several P2X (1-3 and 5) and P2Y (1 and 13) receptors.

Immunocytochemistry with the available purinergic receptor antibodies suggested that there is

no expression of P2X2, P2X3, and P2Y1 by epiplexus cells (data not shown), while expression

of P2X1, P2X5, and P2Y13 has not yet been investigated.

Bath application of PPADS ((50μM); Figure 8) appeared to decrease ATP-induced

activity of epiplexus cells from the control level of 0.93 +/- 0.12 μm/frame (n=293 cells from

9CPs) to 0.45 +/- 0.18μm/frame (n=115 cells from 6CPs). However, this failed to reach

statistical significance when compared (by ANOVA) to either ATP alone (0.93 +/- 0.12

μm/frame; n=293 cells from 9CPs, p=0,0976) or control (0.05 +/- 0.15 μm/frame; n=124 cells

from 5CPs, p=0,5016). Interestingly, the combination of BBG (1μM) plus PPADS (50μM) and

ATP (100μM) slowed epiplexus cells significantly (p=0,0125) 0.41 +/- 0.10μm/frame (n=189

cells from 7CPs) when compared to ATP alone, and this was not different (p=0,4928) from

control (Figure 8).

Since PPADS and BBG partially decreased activation of epiplexus cell by ATP, but did

not completely block it, I hypothesised an involvement for P2Y2 receptors, because they are

expressed in many immune cell types and participate in immune cell chemotaxis (74, 78, 116).

Immunohistochemistry confirmed that P2Y2 receptors were highly expressed on epiplexus cells

(Figure 9). The P2Y2 receptor blocker, tangeretin (30μM) (158), was used alone or in

35

Figure 8. Blockage of P2X1, P2X2, P2X3, P2X5, P2Y1 and/or P2Y13 together with P2X7

receptors by PPADS and BBG decreased epiplexus cells motility, thus, some of the listed

receptors may be involved in activation. A – raw average distance travelled, B – normalised

distance travelled, C – normalised cumulative distance travelled, D – normalised average

distance and statistical analysis.

36

Figure 9. Immunofluorescent staining for P2Y2 receptors in epiplexus cells.

37

combination with PPADS (50μM) (Figure 10). Tangeretin alone decreased motility of epiplexus

cells to 0.21 +/- 0.10 μm/frame (n=165 cells from 6CPs), which was significantly (p=0,0278)

different from vehicle (DMSO at 0.1%) and ATP (0.81 +/- 0.14 μm/frame; n=162 cells from

5CPs) but not from control 0.05 +/- 0.15 μm/frame (n=124 cells from 5CPs, p> 0,9999). It is

important to note that the activation of epiplexus cells by ATP in the presence of tangeretin

changed with time. At first the cells were actively moving, but their activity started decreasing at

~40 minutes, and then remained at a stable and low (similar to control) level for the remainder of

the experiment.

Combined application of PPADS and tangeretin completely abolished the response to

ATP (Figure 10). The mean normalised distance travelled by epiplexus cells was -0.24 +/- 0.10

μm/frame (n=168 cells from 6CPs), indicating that they spent a significant number of frames

motionless. This rate was significantly different from vehicle plus ATP (p< 0,0001) and PPADS

plus ATP (p=0,0133), but not from tangeretin plus ATP (p=0,2475).

The effects of other purines on chemokinesis of epiplexus cells

ATP activates most P2 receptors (except P2Y14) (159) making it an experimentally

useful, and physiologically important ligand, but some P2Y receptors have higher affinity to

other naturally occurring ligands. For example, P2Y1, 12 and 13 are potently activated by ADP,

whereas P2Y2, 4 and 6 can be activated by UTP. The UTP metabolite, UDP can also activate the

P2Y6 receptor (159). Furthermore, ATP can be spontaneously or enzymatically degraded to

other purine phosphates, such as ADP, AMP and also adenosine (56–58). Adenosine is the

ligand for the P1 receptors, A1, A2A, A2B and A3. Another naturally occurring extracellular

nucleotide is UTP, as well as its degradation product UDP (72–74). Chemotaxis of various

38

Figure 10. Tangeretin (blocks P2Y2) significantly decreased chemokinesis, and in

combination with PPADS (blocks P2X1, P2X2, P2X3, P2X5, P2Y1, and P2Y13) abolished

it. A – raw average distance travelled, B – normalised distance travelled, C – normalised

cumulative distance travelled, D – normalised average distance and statistical analysis.

39

immune cells was triggered or influenced by these nucleotides (74, 101, 104–109, 112, 113) and

G-coupled proteins (including P2Y and A receptors) (78, 104, 110, 116). Thus, experiments with

ADP (100μM), adenosine (100μM) and UTP (100μM) were conducted to test the hypothesis that

P2Y receptors are involved, and more specifically, P2Y2 receptors (Figure 10 and 11).

ADP induced epiplexus cell chemokinesis, increasing the rate of movement to 0.90 +/-

0.19 μm/frame (n=125 cells from 6CPs), which was similar (p>0,9999) to the level of activation

by ATP (0.93 +/- 0.12 μm/frame; n=293 cells from 9CPs; Figure 11), and significantly different

(p=0,0081) from control 0.05 +/- 0.15 μm/frame (n=124 cells from 5CPs). Interestingly, in the

presence of ADP, cellular motility increased for the first 45 minutes and then returned to

baseline. This was clearly different than the response to ATP, which resulted in a steady

elevation of epiplexus cell activity (Figure 11).

Adenosine, the final degradation product of extracellular ATP and ADP, had no effect on

epiplexus cells’ activity. The mean normalised distance travelled in this case was 0.15 +/- 0.20

μm/frame (n=123 cells from 4CPs), which was not significantly different (p>0,9999) from

control but significantly different (p=0,0024) from ATP, suggesting that adenosine receptors

were not involved.

Interestingly, UTP (100μM) had similar effects as ADP when bath applied to the isolated

and intact CP preparation. For example, the initial increase in activity was followed by a

decrease (Figure 11). Furthermore, the rate of activation by UTP appeared similar to ATP at the

beginning of an experiment and peaked by 30 minutes with a return to the baseline level by 60

minutes. The mean normalised distance travelled by epiplexus cells was 0.74 +/- 0.15 μm/frame

(n=165 cells from 4CPs) during the first 5-55 minutes and -0.21 +/- 0.13 μm/frame (n=150 cells

40

Figure 11. ADP (activates P2Y1, P2Y12, P2Y13) and UTP (activates P2Y2, P2Y4, P2Y6),

but not adenosine (activates A1, A2A, A2B, A3 receptors) triggered chemokinesis. A – raw

average distance travelled, B – normalised distance travelled, C – normalised cumulative

distance travelled, D – normalised average distance and statistical analysis.

41

from 4CPs) during the next 60-95 minutes. Results from the first 5-55 minutes were significantly

different from control (p=0,0377) and the next 60-95 minutes were not (p>0,9999), but were

significantly slower than ATP (p<0,0001).

Panx1 channels contribute to epiplexus cell’s activation

Panx1 channels are activated by both ionotropic and metabotropic purinergic receptors

(133, 134, 160), although the mechanisms are not clearly established. Panx1 was shown to

participate in the process of ATP-mediated activation of effector T-cells, monocytes and

macrophages (102, 152). Thus, I first used immunocytochemistry to investigate if Panx1 is

expressed in the CP and then blocked Panx1 channels with probenecid to investigate whether

these ATP-permeable channels (95, 161) participate in activation of epiplexus cells.

Immunohistochemical analysis clearly demonstrated that Panx1 was abundantly

expressed on the CP epithelium but was not detectable on the epiplexus cells (Figure 12).

Probenecid (500μM) gradually decreased ATP-triggered epiplexus cell motility (Figure 13).

When the chemokinesis data were divided into two parts for analysis, the mean normalised

distance travelled by epiplexus cells was 0.44 +/- 0.13 μm/frame (n=165 cells from 6CPs) during

the first 5-45 minutes, and 0.13 +/- 0.17 μm/frame (n=153 cells from 6CPs) during the rest 50-95

minutes. The first part of the experiment was not different from control (p=0,8591) or ATP plus

vehicle (DMSO) (p=0,7845), while the second part was significantly different (p=0,0156) from

ATP plus vehicle (Figure 13).

LPS and Poly(I:C) do not trigger increased motility

Immune cell migration can be activated by LPS, which is a bacteria derived coat protein

acting at the TLR-4 receptor. Poly(I:C) is a TLR-3 receptor activating synthetic analog of

double-stranded RNA that can also activate immune cells by a mechanism involving P2

42

Figure 12. Immunofluorescent staining for Panx1 in the CP.

43

Figure 13. Blockage of Panx1 channels by probenecid gradually decreased chemokinesis. A

– raw average distance travelled, B – normalised distance travelled, C – normalised cumulative

distance travelled, D – normalised average distance and statistical analysis.

44

receptors (162–169). LPS and Poly(I:C) are common tools for immune cells activation (170–

173) so they were applied to the bathing solution. Application of either LPS (10 μg/ml), or

Poly(I:C) (10 μg/ml) failed to increase the motility of epiplexus cells (Figure 14). The mean

normalised distance travelled by the epiplexus cells was 0.05 +/- 0.10 μm/frame (n=198 cells

from 6CPs) and 0.20 +/- 0.07 μm/frame (n=170 cells from 4CPs) for LPS and Poly(I:C),

respectively. Data obtained from both of the experiments were significantly different (p<0.0001)

from ATP (100μM) alone, 0.93 +/- 0.12 μm/frame (n=293 cells from 9CPs), but not from

control, 0.05 +/- 0.15 μm/frame (n=124 cells from 5CPs; p> 0,9999).

45

Figure 14. LPS and Poly(I:C) do not trigger chemokinesis. A – raw average distance

travelled, B – normalised distance travelled, C – normalised cumulative distance travelled, D –

normalised average distance and statistical analysis.

46

CHAPTER FOUR: DISCUSSION

The CP is important for brain homeostasis through production and turnover of CSF (5, 6),

production and distribution to distant brain targets of a nutritive “cocktail” of neuropeptides,

growth factors and cytokines, regulation of bidirectional movement of substances in and out of

the brain, and the constitution of a BCSFB (7). CP dysfunction may be involved in many

diseases, such as infection, injury, Alzheimer’s disease and the auto-immune disease multiple

sclerosis (7, 174). This suggests that epiplexus cells, the immune cells of the CP (9), can be a

potential therapeutic target for treating infection and neuro-immune disorders. Therefore, this

thesis was devoted to understanding the activation of epiplexus cells by the inflammatory

mediator ATP (see Figure 1).

To investigate the activation of epiplexus cells by ATP, I first developed a novel isolated

and intact CP preparation akin to acute brain slices that are commonplace in electrophysiology

and imaging. This preparation allowed me to identify epiplexus cells as members of the immune

cell family, and to label them with fluorescent IB4 for in situ live cell imaging. In the thesis I

report that epiplexus cells are potently activated by exogenous ATP, which induced clear

chemokinesis (Figure 15).

Application of a P2X7R blocker or agonist, as well as a potentiator of P2X4 showed that

these two receptors are not involved. P2X1, P2X5, and/or P2Y13 and P2Y2 receptors may be

important for increased motility since antagonists of these receptors abolished chemokinesis.

Some other receptors, like P2Y4, P2Y6, and P2Y12 could also be potentially involved, since

application of ADP and UTP triggered chemokinesis. The response was different from the

reaction to ATP though, with an initial peak and subsequent decline in motility, which points to

temporally distinct contributions of these receptors to chemokinesis. Such a pattern could be

47

explained by internalization or desensitization of receptors. For example, P2Y1, P2Y2 and

P2Y12 receptors were found to be internalized in response to prolonged agonist application

(175–178). Thus, there could be some alternative pathways of activation that are not sufficient

for triggering chemokinesis when P2X1, P2X5, and/or P2Y13 and P2Y2 receptors are blocked

(Figure 15).

Investigation of a potential role of Panx1 channels, which were expressed only in

epithelial cells, suggested that Panx1 was required for maintenance of epiplexus cell activation,

but not initiation of activation. Finally, acute application of the infection mimetics, LPS or

Poly(I:C) failed to trigger chemokinesis, suggesting that systemic immune responses may be

required to alter epiplexus cell responses. Taken together, I have characterized a novel

mechanism of activation of the innate immune cells of the CP that involves acute responses to

ATP and, possibly, sustained responses that involve Panx1, metabotropic P2Y2 receptors, and,

potentially, a few other purinergic receptors (Figure 15).

Immune origin of epiplexus cells

There are some reports suggesting that epiplexus cells may be comprised of a mixed

population of macrophages and microglia (25). In the majority of papers however, epiplexus cell

are described as a population of macrophages and dendritic cells with a very small numbers of T-

cells (21–23). Since microglia and macrophages share most of the known markers of immune

cells (179), it is difficult to rule out the presence of microglia. Nevertheless, a few differences in

biomarker expression in microglia and CP immune cells have been recently identified (24).

In our experimental model, the cells on the surface of the CP were co-labelled by the

immune cell markers, IB4 and Iba1 (Figure 2). IB4 was reported to stain endothelial cells as well

(145), but in my hands endothelial cells labelled with IB4 were detected only in the fixed CP

48

tissue following application of PFA. Since IB4 and Iba1 label both macrophages and microglia

(139–141) it was not possible to conclude if epiplexus cells belong to one cell type or another.

Nevertheless, it is clear that these IB4-labelled cells on the CSF-facing surface of the CP are

immune cells and the application of IB4 for labelling live epiplexus cells was a novel

development for this thesis.

Chemokinesis of epiplexus cells in intact isolated CP

A new technique for the isolation of live and intact CP was developed that allowed us for

the first time to observe and quantify the behaviour of epiplexus cells in situ over several hours

(Figure 3). In control experiments (CP bathed in aCSF alone), the epiplexus cells mainly resided

in one place, only rarely were translocations of their somas observed. However, their processes

were actively exploring the surrounding area (Figure 3 and 4) in a manner that may be similar to

that reported for brain microglia (111). It appears that under normal conditions epiplexus cells

are actively and effectively monitoring the state of epithelial cells and that this does not require

extensive movement of the epiplexus cell body, and this behaviour is consistent with the current

understanding of immune cell surveillance.

Myrtek et al. determined that 100µM extracellular nucleotides was optimal for triggering

a maximal rise in internal Ca2+

in human alveolar macrophages (79). To investigate if ATP could

activate epiplexus cells, I bath applied 100μM ATP, which initiated active movements of

epiplexus cells that can be best characterised as chemokinesis: the undirected movements of cells

in response to a chemical stimulus (Figure 3, 4 and 6). While this may reflect an inability of

epiplexus cells to determine a source of ATP, we chose this paradigm because it likely mimics

conditions in which there is a systemic infection or brain injury. Chemotaxis towards focally

applied ATP is also possible, but to investigate this a different approach in delivering ATP

49

should be taken. Activation by ATP was dose-dependent, with 10 μM ATP failing to induce

activity (Figure 7). In some instances, several epiplexus cells would cluster together and move

through the CP tissue as a single group, but the significance of this to CP function and defence of

the BCSFB is not yet known. Thus, for the first time epiplexus cells’ behaviour was recorded and

characterized, as well as ATP was shown to trigger chemokinesis for this type of cell.

How does the chemokinesis of epiplexus cells fit with our current knowledge of central

nervous system immune cells? The characteristic feature of brain microglial activation is that

microglial cells send their processes into the site of injury or ATP application very rapidly,

without moving the soma, to create a wall between damaged and healthy tissue (111), and only

later exhibit features of whole-cell migration (109). In contrast, macrophages move their cell

bodies towards the signal (180). Our results showed that epiplexus cells respond to ATP by the

movement of their cell bodies (Figure 3). However, it is not possible to firmly state that

epiplexus cells’ behaviour is completely macrophage-like. Firstly, there was a report about the

formation of a protective network of epiplexus cell’s protrusions on the surface of epithelial cells

in the presence of a toxic agent (37), although this was not observed in the time frame of my

experiments. Secondly, the present thesis used experiments with bath-applied ATP, whereas in

the case of microglia, the experiments used focally delivered ATP or injuries induced by a laser.

Investigation of molecular mechanisms

To investigate the molecular mechanisms underlying ATP-triggered chemokinesis of

epiplexus cells, various agonists and antagonists, as well as a potentiator of purinergic receptors

were used. Immunohistochemical analysis allowed us to investigate the expression of purinergic

receptors and Panx-1 by the CP epithelial and epiplexus cells. It was anticipated that epiplexus

cells express some of the purinergic receptors present on monocytes (P2X1, 4, 5, 7 and P2Y1, 2,

50

4, 6, 11, 12, 13), macrophages (P2X1, 4, 5, 7 and P2Y1, 2, 4, 6, 11, 12), microglia (P2X1, 2, 4, 7

and P2Y1, 2, 4, 6, 12), or dendritic cells (P2X1, 4, 5, 7 and P2Y1, 2, 4, 6, 11, 13, 14) (55, 56,

75–83). It was unclear, however, which would be functionally important.

The first target investigated was P2X7 receptors because they are extensively studied and

participate in activation of macrophages, microglia and other immune cells (86–88, 134, 181).

Surprisingly, the P2X7 receptor antagonist, BBG (151) did not prevent ATP-induced

chemokinesis. Application of the P2X7 receptor agonist, BzATP did not result in increased

motility of the cells (Figure 6). This suggests that P2X7 receptors are not involved in activation

of epiplexus cell chemokinesis, even though they are expressed as shown by

immunohistochemistry (Figure 5). It is possible, that P2X7 receptors activation acts as a “stop

signal” to migrating immune cells, since these receptors are activated by very high

concentrations of extracellular ATP, which is usually present in the center of inflammation or

injury (109). Another possibility is that these receptors participate in activation of epiplexus cells

in some alternative way that is not related to chemokinesis, such as increasing projections of fine

processes in a manner similar to that reported for microglia (111).

Since there are so many purinergic receptors, the wide-spectrum blocker, PPADS, was

used to investigate whether P2X1, P2X2, P2X3, P2X5, P2Y1, or P2Y13 receptor could be

involved in epiplexus cell activation by ATP (Figure 8). It is known that most of these receptors

are expressed by other innate immune cells (55, 56, 75–83). PPADS decreased the level of

epiplexus cell chemokinesis by ~50% when applied alone or in combination with BBG. Only the

results with the combination of these two antagonists were statistically different from ATP alone.

Taking into account that BBG alone did not cause any decrease in activation levels, and that the

results of these two experiments (PPADS alone and PPADS + BBG) were very similar (51% and

51

56% block, respectively), this suggests that some of the receptors blocked by PPADS might be

involved, but more experiments to increase n’s may confirm this. According to the

immunohistochemical analysis, P2X2, P2X3, and P2Y1 receptors are not expressed on epiplexus

cells and can be ruled out as a target for PPADS. Expression of P2X1, P2X5, and P2Y13– other

targets of PPADS – was not determined and needs to be investigated more fully in the future.

The ionotropic P2X4 receptor is expressed on most immune cells where they are

functional (mediate ionic current) and participate in cellular activation (82, 152). Since these

receptors are not blocked by PPADS, a P2X4 receptor potentiator ivermectin (82) was used to

investigate involvement of the P2X4 receptor in epiplexus cell activation by ATP (Figure 7).

Application of ivermectin together with 10μM ATP resulted in a 2.9 fold increase in normalised

distance travelled compared to 10μM ATP alone, which was 69% of the activity induced by

100μM ATP+DMSO. Nevertheless, the difference from both control and ATP experiments was

not statistically significant, suggesting that P2X4 receptors may not be involved in epiplexus cell

activation. At the same the statistically non-significant 2.9 fold change could suggest the

presence of type II statistical error. This could be clarified by increasing the sample size or

choosing less strict post-hoc test.

The P2Y2 receptor is highly expressed in immune cells and is known to participate in

immune cell chemotaxis (74, 78, 116). Indeed, application of the P2Y2 receptor blocker,

tangeretin, resulted in a significant decrease, by 74%, in the average normalised distance

travelled (Figure 10). Interestingly, epiplexus cell motility was not stable during the span of the

experiment. At first it was increasing, peaking at 40 minutes and then declining to the levels

close to control. This can be explained by temporary activation of some other purinergic

receptor(s) at the beginning of the experiment. To investigate this question further, the

52

combination of tangeretin and PPADS was applied and resulted in complete abolishment of

chemokinesis (Figure 10). Immunohistochemistry confirmed that P2Y2 receptors are widely

expressed on epiplexus cells (Figure 9). This indicates that P2X1, P2X5, and/or P2Y13 (blocked

by PPADS, but not tested for expression) and P2Y2 receptors may be necessary for epiplexus

cells activation by extracellular ATP.

Extracellular ATP degradation to ADP, AMP, and adenosine occurs naturally and is

facilitated by a wide array of enzymes over a relatively short period of time (56–58). UTP is

another naturally occurring extracellular nucleotide, as well as its degradation product UDP (72–

74). Some of the purinergic receptors more sensitive to ADP (P2Y1, P2Y12, P2Y13), adenosine

(A1, A2A, A2B and A3), UTP (P2Y2, P2Y4, P2Y6), and UDP (P2Y6), compared to ATP and / or

other naturally occurring and artificial ligands, are present on innate immune cells (56). All of

these nucleotides trigger or participate in chemotactic response of various immune cells (74, 101,

104–109, 112, 113). Also, it was shown that G-protein coupled receptors (including P2Y and A

receptors) are involved in immune cell chemotaxis (78, 104, 110, 116).

In my work, application of adenosine had no effect on epiplexus cell’s motility (Figure

11), which argues against P1 receptors in mediating chemokinesis. At the same time, ADP

increased motility of epiplexus cells to a level, similar to ATP (Figure 11), but the pattern of

activation was different with an initial peak at 45 minutes and a decline to the control levels. This

suggests that P2Y12 and/or P2Y13 could be present on epiplexus cells (there was no expression

of P2Y1 receptors), and that a portion of the observed effects of ATP may be caused by ADP.

Finally, UTP application triggered increased activity at the beginning of the experiment but,

similar to ADP, declined to the control level by 60 minutes (Figure 11). Thus, P2Y2, P2Y4

and/or P2Y6 (all activated by UTP), and P2Y12 and/or P2Y13 (activated by ADP) (56), may be

53

present on epiplexus cells and involved in the activation process at the early stages. This was

confirmed for P2Y2 by using tangeretin, but further investigation is required to determine the

potential roles of P2Y4, P2Y6, P2Y12, and P2Y13.

In the final set of experiments, the Panx1 antagonist, probenecid was bath applied. It was

demonstrated that Panx1 participates in the process of immune cells activation by ATP (102,

152). Application of probenecid, a Panx1 blocker, together with ATP resulted in a gradual

decrease of chemokinesis (Figure 13). Surprisingly, Panx1 is not present on epiplexus cells, but

is abundantly expressed on epithelial cells (Figure 12). This suggests that Panx1 contributes to

late activation of epiplexus cells and might mediate release of some signalling molecule from

epithelial cells to enhance or sustain epiplexus cell’s reaction to ATP.

Additional findings

The common tools for immune cell activation (170–173), LPS (TLR-4 ligand) and Poly(I:C)

(TLR-3 ligand), did not trigger an increase in epiplexus cells motility (Figure 14). This is

surprising, taking into account that these molecules were reported to cause immune cell

migration (162–169). A possible explanation of our results would be that insufficient time or

concentrations were used to trigger chemokinesis in this specific type of cells. Additionally, a

systemic immune response may be required to signal to the epiplexus cells. These two

possibilities represent exciting avenues for future investigations that will link epiplexus cell

physiology to the systemic immune response.

Potential limitations of the new approach of studying epiplexus cells and solutions

The CP is a very delicate and fragile tissue, which complicates the task of maintaining

choroidal epithelium health. The development of the isolated and intact CP preparation was very

challenging but we have devised a successful protocol for routinely isolating tissue that remains

54

healthy for several hours, akin to the acute brain slice preparation. This now limits the possibility

that dying epithelial cells release signals that activate the epiplexus cells.

Another potential complication is that the measurements of distance travelled by

activated epithelial cells are restricted, largely, to the x-y plane, while data from z-axis makes a

more minor contribution. Thus, if the cells move preferentially in the z-plane, our measurements

will underestimate their total movement. We have addressed this issue by taking image stacks in

the z-plane and collapsing them into a single image for each time point (i.e. a z projection) prior

to analysis. Further, the morphological structure of the epithelial cell layer is as a sheet that is

only one cell layer thick. This may have the effect of restricting movements of cells largely in

what we have defined as the x-y plane, thus reducing the error associated with z movements.

Conclusions

The current knowledge of epiplexus cells’ physiology is very limited. There have been no

reports on live imaging of motility of these cells, as well as on potential reactions to ATP and

expression of purinergic receptors. The main hypothesis of this work was based mainly on

information available about other types of innate immune cells. In summary, the main results of

this study were:

1. A new technique for isolation of intact rat CP preparation, as well as approach to visualise

with IB4 and analyse epiplexus cells’ behaviour were developed.

2. Bath application of ATP initiated movements (chemokinesis) of epiplexus cells, and the

extent of activation depended on the concentration of ATP.

3. P2X7 receptors (blocked by BBG and activated by BzATP) were not involved in

chemokinesis, but are expressed on epiplexus cells.

55

4. Potentiation of P2X4 by ivermectin did not result in increased chemokinesis, which means

that P2X4 is not involved in chemokinesis.

5. Block of P2X1, P2X2, P2X3, P2X5, P2Y1, and/or P2Y13 (by PPADS) and P2Y2

(tangeretin) attenuated chemokinesis. P2Y2 receptors are expressed on epiplexus cells, but

P2X2, P2X3 and P2Y1 are not.

6. ADP (activates P2Y1, P2Y12, P2Y13) and UTP (activates P2Y2, P2Y4, P2Y6) triggered

temporally distinct chemokinesis with initial peak and subsequent decline in cells motility.

7. Probenecid block of Panx1 channels, expressed on epithelial cells, but absent on epiplexus

cells, decreased sustained chemokinesis, but not the early response to ATP.

8. LPS and Poly(I:C), the common tools for immune cells activation, did not trigger

chemokinesis when applied directly to the CP.

Taken together, this work asserts that extracellular ATP triggers a complex chemokinesis

of epiplexus cells (Figure 15). This may be mediated by a combination of P2X1, P2X5, and/or

P2Y13 and P2Y2 receptors, even though some other receptors, like P2Y4, P2Y6, and P2Y12

could be potentially involved as well. Metabotropic receptors could be responsible for early

activation of epiplexus cells, while Panx1, expressed on epithelial cells, could mediate late

activation through release of some signals that facilitate exogenous ATP-triggered chemokinesis.

In the future, expression and functions of P2X1, P2X5, P2Y4, P2Y6, P2Y12, and P2Y13

should be investigated, as well as the role of Panx1 in the CP. Since epiplexus cells actively react

to the immune mediator ATP, this suggests that epiplexus cells, the immune cells of the CP (9),

can be a potential therapeutic target for treating infection and neuro-immune disorders.

56

Figure 15. Model of the findings from this thesis. Extracellular ATP binds to P2X1,

P2X5, and/or P2Y13 and P2Y2 receptors on epiplexus cells, resulting in chemokinesis of these

cells. Chemokinesis is fully abolished when these receptors are blocked. Extracellular UTP and

ADP also trigger chemokinesis. Panx1 channel is expressed on CP epithelium and is important

for maintaining chemokinesis by yet unknown mechanism.

57

REFERENCES

1. R. F. Keep, H. C. Jones, A morphometric study on the development of the lateral ventricle

choroid plexus, choroid plexus capillaries and ventricular ependyma in the rat., Brain research.

Developmental brain research 56, 47–53 (1990).

2. C. Ide et al., Grafting of choroid plexus ependymal cells promotes the growth of regenerating

axons in the dorsal funiculus of rat spinal cord: a preliminary report., Experimental neurology

167, 242–51 (2001).

3. K. Oshio, Y. Song, A. S. Verkman, G. T. Manley, Aquaporin-1 deletion reduces osmotic

water permeability and cerebrospinal fluid production., Acta neurochirurgica. Supplement 86,

525–8 (2003).

4. T. Speake, C. Whitwell, H. Kajita, a Majid, P. D. Brown, Mechanisms of CSF secretion by the

choroid plexus., Microscopy research and technique 52, 49–59 (2001).

5. C. May et al., Cerebrospinal fluid production is reduced in healthy aging., Neurology 40, 500–

3 (1990).

6. N. K. Tanna et al., Analysis of brain and cerebrospinal fluid volumes with MR imaging:

impact on PET data correction for atrophy. Part II. Aging and Alzheimer dementia., Radiology

178, 123–30 (1991).

7. D. F. Emerich, S. J. M. Skinner, C. V Borlongan, A. V Vasconcellos, C. G. Thanos, The

choroid plexus in the rise, fall and repair of the brain., BioEssays : news and reviews in

molecular, cellular and developmental biology 27, 262–74 (2005).

8. E. A. Ling, Ultrastructure and mode of formation of epiplexus cells in the choroid plexus in

the lateral ventricles of the monkey (Macaca fascicularis)., Journal of anatomy 133, 555–69

(1981).

58

9. E. A. Ling, C. Kaur, J. Lu, Origin, nature, and some functional considerations of

intraventricular macrophages, with special reference to the epiplexus cells., Microscopy research

and technique 41, 43–56 (1998).

10. M. Vercellino et al., Involvement of the choroid plexus in multiple sclerosis autoimmune

inflammation: a neuropathological study., Journal of neuroimmunology 199, 133–41 (2008).

11. X. Alvira-Botero, E. M. Carro, Clearance of amyloid-β peptide across the choroid plexus in

Alzheimer’s disease., Current aging science 3, 219–29 (2010).

12. D. J. Allen, Scanning electron microscopy of epiplexus macrophages (Kolmer cells) in the

dog., The Journal of comparative neurology 161, 197–213 (1975).

13. Y. Hosoya, T. Fujita, Scanning electron microscope observation of intraventricular

macrophages (Kolmer cells) in the rat brain., Archivum histologicum Japonicum = Nihon

soshikigaku kiroku 35, 133–40 (1973).

14. M. Kemali, Third ventricle suprachoroidal cells., The Anatomical record 202, 113–6 (1982).

15. E. A. Ling, Ultrastruct and origin of epiplexus cells in the telencephalic choroid plexus of

postnatal rats studied by intravenous injection of carbon particles., Journal of anatomy 129, 479–

92 (1979).

16. R. R. Sturrock, A semithin light microscopic, transmission electron microscopic and

scanning electron microscopic study of macrophages in the lateral ventricle of mice from

embryonic to adult life., Journal of anatomy 129, 31–44 (1979).

17. A. Bos, R. Wever, D. Roos, Characterization and quantification of the peroxidase in human

monocytes, Biochimica et Biophysica Acta (BBA) - Enzymology 525, 37–44 (1978).

59

18. G. J. Guillemin, B. J. Brew, Microglia, macrophages, perivascular macrophages, and

pericytes: a review of function and identification, Journal of leukocyte biology 75, 388–397

(2004).

19. E. Ulvestad et al., Phenotypic differences between human monocytes/macrophages and

microglial cells studied in situ and in vitro., Journal of neuropathology and experimental

neurology 53, 492–501 (1994).

20. E. W. Schwarze, The origin of (Kolmer’s) epiplexus cells. A combined histomorphological

and histochemical study., Histochemistry 44, 103–4 (1975).

21. P. G. McMenamin, Distribution and phenotype of dendritic cells and resident tissue

macrophages in the dura mater, leptomeninges, and choroid plexus of the rat brain as

demonstrated in wholemount preparations., The Journal of comparative neurology 405, 553–62

(1999).

22. P. G. McMenamin, R. J. Wealthall, M. Deverall, S. J. Cooper, B. Griffin, Macrophages and

dendritic cells in the rat meninges and choroid plexus: three-dimensional localisation by

environmental scanning electron microscopy and confocal microscopy., Cell and tissue research

313, 259–69 (2003).

23. A. Hanly, C. K. Petito, HLA-DR-positive dendritic cells of the normal human choroid

plexus: a potential reservoir of HIV in the central nervous system., Human pathology 29, 88–93

(1998).

24. J. Melief et al., Phenotyping primary human microglia: Tight regulation of LPS

responsiveness., Glia 60, 1506–17 (2012).

60

25. J. Lu, C. Kaur, E. a Ling, An immunohistochemical study of the intraventricular

macrophages in induced hydrocephalus in prenatal rats following a maternal injection of 6-

aminonicotinamide., Journal of anatomy 188 ( Pt 2, 491–5 (1996).

26. E. A. Ling, Scanning electron microscopic study of epiplexus cells in the lateral ventricles of

the monkey (Macaca fascicularis)., Journal of anatomy 137 ( Pt 4, 645–52 (1983).

27. S. J. Carpenter, L. E. McCarthy, H. L. Borison, Electron microscopic study of the epiplexus

(Kolmer) cells of the cat choroid plexus., Zeitschrift für Zellforschung und mikroskopische

Anatomie (Vienna, Austria : 1948) 110, 471–86 (1970).

28. J. Lu, C. Kaur, E. A. Ling, Intraventricular macrophages in the lateral ventricles with special

reference to epiplexus cells: a quantitative analysis and their uptake of fluorescent tracer injected

intraperitoneally in rats of different ages., Journal of anatomy 183 ( Pt 2, 405–14 (1993).

29. J. Lu, C. Kaur, E. A. Ling, Uptake of tracer by the epiplexus cells via the choroid plexus

epithelium following an intravenous or intraperitoneal injection of horseradish peroxidase in

rats., Journal of anatomy 183 ( Pt 3, 609–17 (1993).

30. J. Lu, C. Kaur, E. A. Ling, Immunophenotypic features of epiplexus cells and their response

to interferon gamma injected intraperitoneally in postnatal rats., Journal of anatomy 185 ( Pt 1,

75–84 (1994).

31. J. Lu, C. Kaur, E. A. Ling, Up-regulation of surface antigens on epiplexus cells in postnatal

rats following intraperitoneal injections of lipopolysaccharide., Neuroscience 63, 1169–78

(1994).

32. J. Lu, C. Kaur, E. A. Ling, Expression and upregulation of transferrin receptors and iron

uptake in the epiplexus cells of different aged rats injected with lipopolysaccharide and

interferon-gamma., Journal of anatomy 187 ( Pt 3, 603–11 (1995).

61

33. J. Lu, C. Kaur, E. A. Ling, Histochemical demonstration of nitric oxide synthase-like

immunoreactivity in epiplexus cells and choroid epithelia in the lateral ventricles of postnatal rat

brain induced by an intracerebral injection of lipopolysaccharide., Brain research 699, 275–85

(1995).

34. V. Sivakumar, J. Lu, E. A. Ling, C. Kaur, Vascular endothelial growth factor and nitric oxide

production in response to hypoxia in the choroid plexus in neonatal brain., Brain pathology

(Zurich, Switzerland) 18, 71–85 (2008).

35. D. Wang, C. Kaur, Response of epiplexus cells associated with the choroid plexus in the

lateral ventricles of adult rats to high altitude exposure., Neuroscience letters 285, 197–200

(2000).

36. C. Kaur, E. a Ling, P. Gopalakrishnakone, W. C. Wong, Response of intraventricular

macrophages to crotoxin-coated microcarrier beads injected into the lateral ventricle of postnatal

rats., Journal of anatomy 168, 63–72 (1990).

37. E. A. Ling, P. Gopalakrishnakone, C. K. Tan, Electron-microscopical study of the choroid

plexus and epiplexus cells in cats following a cisternal injection of crotoxin complex., Acta

anatomica 131, 241–8 (1988).

38. W. L. Maxwell et al., Changes in the choroid plexus, responses by intrinsic epiplexus cells

and recruitment from monocytes after experimental head acceleration injury in the non-human

primate., Acta neuropathologica 84, 78–84 (1992).

39. W. L. Maxwell, J. McGadey, Response of intraventricular macrophages after a penetrant

cerebral lesion., Journal of anatomy 160, 145–55 (1988).

62

40. C. Kaur et al., Studies of the choroid plexus and its associated epiplexus cells in the lateral

ventricles of rats following an exposure to a single non-penetrative blast., Archives of histology

and cytology 59, 239–48 (1996).

41. M. Cerdá-Nicolás, A. Peydró Olaya, [Experimental hydrocephalus. Morphological and

ultrastructural study of Kolmer cells]., Archivos de neurobiologiá 51, 81–8 (1988).

42. K. G. Go, I. Stokroos, E. H. Blaauw, F. Zuiderveen, I. Molenaar, Changes of ventricular

ependyma and choroid plexus in experimental hydrocephalus, as observed by scanning electron

microscopy., Acta neuropathologica 34, 55–64 (1976).

43. E. A. Ling, C. Y. Tseng, W. C. Wong, An electron microscopical study of epiplexus and

supraependymal cells in the prenatal rat brain following a maternal injection of 6-

aminonicotinamide., Journal of anatomy 140 ( Pt 1, 119–29 (1985).

44. M. N. Ghabriel, I. M. Zdziarski, C. Leigh, R. Vink, Changes in the blood-CSF barrier in

experimental traumatic brain injury., Acta neurochirurgica. Supplement 106, 239–45 (2010).

45. C. Oreja-Guevara, E. Sindern, M. Raulf-Heimsoth, J. P. Malin, Analysis of lymphocyte

subpopulations in cerebrospinal fluid and peripheral blood in patients with multiple sclerosis and

inflammatory diseases of the nervous system., Acta neurologica Scandinavica 98, 310–3 (1998).

46. J. Hardy, D. J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and

problems on the road to therapeutics., Science (New York, N.Y.) 297, 353–6 (2002).

47. R. E. Tanzi, R. D. Moir, S. L. Wagner, Clearance of Alzheimer’s Abeta peptide: the many

roads to perdition., Neuron 43, 605–8 (2004).

48. P. Kivisäkk et al., Human cerebrospinal fluid central memory CD4+ T cells: evidence for

trafficking through choroid plexus and meninges via P-selectin., Proceedings of the National

Academy of Sciences of the United States of America 100, 8389–94 (2003).

63

49. C. A. Janeway, R. Medzhitov, Innate immune recognition., Annual review of immunology 20,

197–216 (2002).

50. A. la Sala et al., Alerting and tuning the immune response by extracellular nucleotides.,

Journal of leukocyte biology 73, 339–43 (2003).

51. A. Rubartelli, M. T. Lotze, Inside, outside, upside down: damage-associated molecular-

pattern molecules (DAMPs) and redox., Trends in immunology 28, 429–36 (2007).

52. S. B. Coade, J. D. Pearson, Metabolism of adenine nucleotides in human blood., Circulation

research 65, 531–7 (1989).

53. L. M. Ryan, J. W. Rachow, B. A. McCarty, D. J. McCarty, Adenosine triphosphate levels in

human plasma., The Journal of rheumatology 23, 214–9 (1996).

54. L. M. Ryan, J. W. Rachow, D. J. McCarty, Synovial fluid ATP: a potential substrate for the

production of inorganic pyrophosphate., The Journal of rheumatology 18, 716–20 (1991).

55. L. Vitiello, S. Gorini, G. Rosano, A. la Sala, Immunoregulation through extracellular

nucleotides., Blood 120, 511–8 (2012).

56. M. J. L. Bours, E. L. R. Swennen, F. Di Virgilio, B. N. Cronstein, P. C. Dagnelie, Adenosine

5’-triphosphate and adenosine as endogenous signaling molecules in immunity and

inflammation., Pharmacology & therapeutics 112, 358–404 (2006).

57. M. J. L. Bours, P. C. Dagnelie, A. L. Giuliani, A. Wesselius, F. Di Virgilio, P2 receptors and

extracellular ATP: a novel homeostatic pathway in inflammation., Frontiers in bioscience

(Scholar edition) 3, 1443–56 (2011).

58. A. Rayah, J. M. Kanellopoulos, F. Di Virgilio, P2 receptors and immunity., Microbes and

infection / Institut Pasteur (2012), doi:10.1016/j.micinf.2012.07.006.

64

59. Z. Zhang et al., Regulated ATP release from astrocytes through lysosome exocytosis., Nature

cell biology 9, 945–53 (2007).

60. S. C. Schock, D. Leblanc, A. M. Hakim, C. S. Thompson, ATP release by way of connexin

36 hemichannels mediates ischemic tolerance in vitro., Biochemical and biophysical research

communications 368, 138–44 (2008).

61. M. Darby, J. B. Kuzmiski, W. Panenka, D. Feighan, B. A. MacVicar, ATP released from

astrocytes during swelling activates chloride channels., Journal of neurophysiology 89, 1870–7

(2003).

62. P. Ballerini et al., Glial cells express multiple ATP binding cassette proteins which are

involved in ATP release., Neuroreport 13, 1789–92 (2002).

63. R. T. R. Huckstepp et al., Connexin hemichannel-mediated CO2-dependent release of ATP

in the medulla oblongata contributes to central respiratory chemosensitivity., The Journal of

physiology 588, 3901–20 (2010).

64. R. Iglesias, G. Dahl, F. Qiu, D. C. Spray, E. Scemes, Pannexin 1: the molecular substrate of

astrocyte “hemichannels”., The Journal of neuroscience : the official journal of the Society for

Neuroscience 29, 7092–7 (2009).

65. R. Corriden, P. A. Insel, Basal release of ATP: an autocrine-paracrine mechanism for cell

regulation., Science signaling 3, re1 (2010).

66. H. a Praetorius, J. Leipziger, ATP release from non-excitable cells., Purinergic signalling 5,

433–46 (2009).

67. G. Burnstock, Unresolved issues and controversies in purinergic signalling., The Journal of

physiology 586, 3307–12 (2008).

65

68. P. Bodin, G. Burnstock, Purinergic signalling: ATP release., Neurochemical research 26,

959–69 (2001).

69. R. Corriden, P. Insel, Basal release of ATP: an autocrine-paracrine mechanism for cell

regulation, Science’s STKE 3, 1–25 (2010).

70. P. Bodin, G. Burnstock, Increased release of ATP from endothelial cells during acute

inflammation., Inflammation research : official journal of the European Histamine Research

Society ... [et al.] 47, 351–4 (1998).

71. G. Burnstock, G. E. Knight, Cellular distribution and functions of P2 receptor subtypes in

different systems., International review of cytology 240, 31–304 (2004).

72. B. Saiag et al., Uptake and Flow-induced Release of Uridine Nucleotides from Isolated

Vascular Endothelial Cells, (2009) (available at

http://informahealthcare.com/doi/abs/10.3109/10623329509024644).

73. E. R. Lazarowski, R. C. Boucher, UTP as an extracellular signaling molecule., News in

physiological sciences : an international journal of physiology produced jointly by the

International Union of Physiological Sciences and the American Physiological Society 16, 1–5

(2001).

74. M. R. Elliott et al., Nucleotides released by apoptotic cells act as a find-me signal to promote

phagocytic clearance., Nature 461, 282–6 (2009).

75. K. Färber, H. Kettenmann, Purinergic signaling and microglia., Pflügers Archiv : European

journal of physiology 452, 615–21 (2006).

76. C. Matute, F. Cavaliere, Neuroglial interactions mediated by purinergic signalling in the

pathophysiology of CNS disorders., Seminars in cell & developmental biology 22, 252–9 (2011).

66

77. H. Franke, C. Schepper, P. Illes, U. Krügel, Involvement of P2X and P2Y receptors in

microglial activation in vivo., Purinergic signalling 3, 435–45 (2007).

78. M. Kronlage et al., Autocrine purinergic receptor signaling is essential for macrophage

chemotaxis., Science signaling 3, ra55 (2010).

79. D. Myrtek et al., Activation of human alveolar macrophages via P2 receptors: coupling to

intracellular Ca2+ increases and cytokine secretion., Journal of immunology (Baltimore, Md. :

1950) 181, 2181–8 (2008).

80. A. del Rey et al., Knock-out mice reveal the contributions of P2Y and P2X receptors to

nucleotide-induced Ca2+ signaling in macrophages., The Journal of biological chemistry 281,

35147–55 (2006).

81. J. W. Bowler, R. J. Bailey, R. A. North, A. Surprenant, P2X4, P2Y1 and P2Y2 receptors on

rat alveolar macrophages., British journal of pharmacology 140, 567–75 (2003).

82. J. a Sim, C.-K. Park, S. B. Oh, R. J. Evans, R. a North, P2X1 and P2X4 receptor currents in

mouse macrophages., British journal of pharmacology 152, 1283–90 (2007).

83. K. Inoue, Purinergic systems in microglia., Cellular and molecular life sciences : CMLS 65,

3074–80 (2008).

84. M. Abbracchio, G. Burnstock, J. Boeynaems, International Union of Pharmacology LVIII:

update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and

pathophysiology to therapy, Pharmacological 58, 281–341 (2006).

85. S. D. Skaper, P. Debetto, P. Giusti, The P2X7 purinergic receptor: from physiology to

neurological disorders., The FASEB journal : official publication of the Federation of American

Societies for Experimental Biology 24, 337–45 (2010).

67

86. V. Pétrilli et al., Activation of the NALP3 inflammasome is triggered by low intracellular

potassium concentration., Cell death and differentiation 14, 1583–9 (2007).

87. F. Di Virgilio, Liaisons dangereuses: P2X(7) and the inflammasome., Trends in

pharmacological sciences 28, 465–72 (2007).

88. F. Di Virgilio, P. A. Borea, P. Illes, P2 receptors meet the immune system., Trends in

pharmacological sciences 22, 5–7 (2001).

89. L. Yip et al., Autocrine regulation of T-cell activation by ATP release and P2X7 receptors.,

FASEB journal : official publication of the Federation of American Societies for Experimental

Biology 23, 1685–93 (2009).

90. B. C. Suh, J. S. Kim, U. Namgung, H. Ha, K. T. Kim, P2X7 nucleotide receptor mediation of

membrane pore formation and superoxide generation in human promyelocytes and neutrophils.,

Journal of immunology (Baltimore, Md. : 1950) 166, 6754–63 (2001).

91. F. Aswad, H. Kawamura, G. Dennert, High sensitivity of CD4+CD25+ regulatory T cells to

extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors.,

Journal of immunology (Baltimore, Md. : 1950) 175, 3075–83 (2005).

92. S. R. J. Taylor, D. R. Alexander, J. C. Cooper, C. F. Higgins, J. I. Elliott, Regulatory T cells

are resistant to apoptosis via TCR but not P2X7., Journal of immunology (Baltimore, Md. : 1950)

178, 3474–82 (2007).

93. S. Hubert et al., Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment

through the ART2-P2X7 pathway., The Journal of experimental medicine 207, 2561–8 (2010).

94. K. Wilhelm et al., Graft-versus-host disease is enhanced by extracellular ATP activating

P2X7R., Nature medicine 16, 1434–8 (2010).

68

95. U. Schenk et al., Purinergic control of T cell activation by ATP released through pannexin-1

hemichannels., Science signaling 1, ra6 (2008).

96. T. Woehrle et al., Pannexin-1 hemichannel-mediated ATP release together with P2X1 and

P2X4 receptors regulate T-cell activation at the immune synapse., Blood 116, 3475–84 (2010).

97. A. la Sala et al., Extracellular ATP induces a distorted maturation of dendritic cells and

inhibits their capacity to initiate Th1 responses., Journal of immunology (Baltimore, Md. : 1950)

166, 1611–7 (2001).

98. A. la Sala et al., Dendritic cells exposed to extracellular adenosine triphosphate acquire the

migratory properties of mature cells and show a reduced capacity to attract type 1 T

lymphocytes., Blood 99, 1715–22 (2002).

99. M. Horckmans et al., Extracellular adenine nucleotides inhibit the release of major monocyte

recruiters by human monocyte-derived dendritic cells., FEBS letters 580, 747–54 (2006).

100. R. H. Straub et al., Neurotransmitters of the sympathetic nerve terminal are powerful

chemoattractants for monocytes., Journal of leukocyte biology 67, 553–8 (2000).

101. A. Kaufmann et al., “Host tissue damage” signal ATP promotes non-directional migration

and negatively regulates toll-like receptor signaling in human monocytes., The Journal of

biological chemistry 280, 32459–67 (2005).

102. F. B. Chekeni et al., Pannexin 1 channels mediate “find-me” signal release and membrane

permeability during apoptosis., Nature 467, 863–7 (2010).

103. C. Goepfert et al., Disordered cellular migration and angiogenesis in cd39-null mice.,

Circulation 104, 3109–15 (2001).

104. M. A. McCloskey, Y. Fan, S. Luther, Chemotaxis of rat mast cells toward adenine

nucleotides., Journal of immunology (Baltimore, Md. : 1950) 163, 970–7 (1999).

69

105. M. Idzko et al., Functional characterization of P2Y and P2X receptors in human

eosinophils., Journal of cellular physiology 188, 329–36 (2001).

106. M. Schnurr et al., Role of adenosine receptors in regulating chemotaxis and cytokine

production of plasmacytoid dendritic cells., Blood 103, 1391–7 (2004).

107. E. Panther et al., Expression and function of adenosine receptors in human dendritic cells.,

FASEB journal : official publication of the Federation of American Societies for Experimental

Biology 15, 1963–70 (2001).

108. M. Idzko et al., Nucleotides induce chemotaxis and actin polymerization in immature but

not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors., Blood

100, 925–32 (2002).

109. S. Honda et al., Extracellular ATP or ADP induce chemotaxis of cultured microglia through

Gi/o-coupled P2Y receptors., The Journal of neuroscience : the official journal of the Society for

Neuroscience 21, 1975–82 (2001).

110. S. E. Haynes et al., The P2Y12 receptor regulates microglial activation by extracellular

nucleotides., Nature neuroscience 9, 1512–9 (2006).

111. D. Davalos et al., ATP mediates rapid microglial response to local brain injury in vivo.,

Nature neuroscience 8, 752–8 (2005).

112. R. M. Lemoli et al., Extracellular nucleotides are potent stimulators of human

hematopoietic stem cells in vitro and in vivo., Blood 104, 1662–70 (2004).

113. L. Rossi et al., The extracellular nucleotide UTP is a potent inducer of hematopoietic stem

cell migration., Blood 109, 533–42 (2007).

114. B. McDonald et al., Intravascular danger signals guide neutrophils to sites of sterile

inflammation., Science (New York, N.Y.) 330, 362–6 (2010).

70

115. W. G. Junger, Immune cell regulation by autocrine purinergic signalling., Nature reviews.

Immunology 11, 201–12 (2011).

116. Y. Chen et al., ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.,

Science (New York, N.Y.) 314, 1792–5 (2006).

117. C. H. Y. Wong, B. Heit, P. Kubes, Molecular regulators of leucocyte chemotaxis during

inflammation., Cardiovascular research 86, 183–91 (2010).

118. B. S. Khakh et al., International union of pharmacology. XXIV. Current status of the

nomenclature and properties of P2X receptors and their subunits., Pharmacological reviews 53,

107–18 (2001).

119. M. F. Jarvis, B. S. Khakh, ATP-gated P2X cation-channels., Neuropharmacology 56, 208–

15 (2009).

120. S. R. Collins, T. Meyer, Calcium flickers lighting the way in chemotaxis?, Developmental

cell 16, 160–1 (2009).

121. A. A. Maghazachi, Intracellular signaling events at the leading edge of migrating cells., The

international journal of biochemistry & cell biology 32, 931–43 (2000).

122. E. J. Pettit, F. S. Fay, Cytosolic free calcium and the cytoskeleton in the control of

leukocyte chemotaxis., Physiological reviews 78, 949–67 (1998).

123. F. S. Fay, S. H. Gilbert, R. A. Brundage, Calcium signalling during chemotaxis., Ciba

Foundation symposium 188, 121–35; discussion 136–40 (1995).

124. B. A. MacVicar, R. J. Thompson, Non-junction functions of pannexin-1 channels., Trends

in neurosciences 33, 93–102 (2010).

125. C. D’hondt et al., Pannexin channels in ATP release and beyond: an unexpected rendezvous

at the endoplasmic reticulum., Cellular signalling 23, 305–16 (2011).

71

126. E. Scemes, S. O. Suadicani, G. Dahl, D. C. Spray, Connexin and pannexin mediated cell-

cell communication., Neuron glia biology 3, 199–208 (2007).

127. R. J. Thompson et al., Activation of pannexin-1 hemichannels augments aberrant bursting

in the hippocampus., Science (New York, N.Y.) 322, 1555–9 (2008).

128. T. E. Finger et al., ATP signaling is crucial for communication from taste buds to gustatory

nerves., Science (New York, N.Y.) 310, 1495–9 (2005).

129. R. A. Romanov et al., Afferent neurotransmission mediated by hemichannels in mammalian

taste cells., The EMBO journal 26, 657–67 (2007).

130. S. Locovei, L. Bao, G. Dahl, Pannexin 1 in erythrocytes: function without a gap.,

Proceedings of the National Academy of Sciences of the United States of America 103, 7655–9

(2006).

131. G. A. Ransford et al., Pannexin 1 contributes to ATP release in airway epithelia., American

journal of respiratory cell and molecular biology 41, 525–34 (2009).

132. T. H. Steinberg, S. C. Silverstein, Extracellular ATP4- promotes cation fluxes in the J774

mouse macrophage cell line., The Journal of biological chemistry 262, 3118–22 (1987).

133. R. Iglesias et al., P2X7 receptor-Pannexin1 complex: pharmacology and signaling.,

American journal of physiology. Cell physiology 295, C752–60 (2008).

134. P. Pelegrin, A. Surprenant, Pannexin-1 mediates large pore formation and interleukin-1beta

release by the ATP-gated P2X7 receptor., The EMBO journal 25, 5071–82 (2006).

135. P. Pelegrin, C. Barroso-Gutierrez, A. Surprenant, P2X7 receptor differentially couples to

distinct release pathways for IL-1beta in mouse macrophage., Journal of immunology

(Baltimore, Md. : 1950) 180, 7147–57 (2008).

72

136. W. R. Silverman et al., The pannexin 1 channel activates the inflammasome in neurons and

astrocytes., The Journal of biological chemistry 284, 18143–51 (2009).

137. T.-D. Kanneganti et al., Pannexin-1-mediated recognition of bacterial molecules activates

the cryopyrin inflammasome independent of Toll-like receptor signaling., Immunity 26, 433–43

(2007).

138. D. Brough, P. Pelegrin, N. J. Rothwell, Pannexin-1-dependent caspase-1 activation and

secretion of IL-1beta is regulated by zinc., European journal of immunology 39, 352–8 (2009).

139. S. P. Sorokin, R. F. Hoyt, Macrophage development: I. Rationale for using Griffonia

simplicifolia isolectin B4 as a marker for the line., The Anatomical record 232, 520–6 (1992).

140. M. E. Dailey, M. Waite, Confocal imaging of microglial cell dynamics in hippocampal slice

cultures., Methods (San Diego, Calif.) 18, 222–30, 177 (1999).

141. W. J. Streit, An improved staining method for rat microglial cells using the lectin from

Griffonia simplicifolia (GSA I-B4)., The journal of histochemistry and cytochemistry : official

journal of the Histochemistry Society 38, 1683–6 (1990).

142. R. J. Thompson, N. Zhou, B. A. MacVicar, Ischemia opens neuronal gap junction

hemichannels., Science (New York, N.Y.) 312, 924–7 (2006).

143. P. Thévenaz, U. E. Ruttimann, M. Unser, A pyramid approach to subpixel registration based

on intensity., IEEE transactions on image processing : a publication of the IEEE Signal

Processing Society 7, 27–41 (1998).

144. F. Cordelières, Manual Tracking (2005) (available at

http://rsbweb.nih.gov/ij/plugins/track/track.html).

145. L. Laitinen, Griffonia simplicifolia lectins bind specifically to endothelial cells and some

epithelial cells in mouse tissues., The Histochemical journal 19, 225–34 (1987).

73

146. K. Ohsawa, Y. Imai, H. Kanazawa, Y. Sasaki, S. Kohsaka, Involvement of Iba1 in

membrane ruffling and phagocytosis of macrophages/microglia., Journal of cell science 113 ( Pt

1, 3073–84 (2000).

147. Y. Imai, I. Ibata, D. Ito, K. Ohsawa, S. Kohsaka, A novel gene iba1 in the major

histocompatibility complex class III region encoding an EF hand protein expressed in a

monocytic lineage., Biochemical and biophysical research communications 224, 855–62 (1996).

148. D. Yanagisawa et al., Possible involvement of P2X7 receptor activation in microglial

neuroprotection against focal cerebral ischemia in rats., Biological & pharmaceutical bulletin 31,

1121–30 (2008).

149. L. Bernardino et al., Inflammatory events in hippocampal slice cultures prime neuronal

susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release.,

Journal of neurochemistry 106, 271–80 (2008).

150. M. Monif, G. Burnstock, D. A. Williams, Microglia: proliferation and activation driven by

the P2X7 receptor., The international journal of biochemistry & cell biology 42, 1753–6 (2010).

151. L. H. Jiang, a B. Mackenzie, R. a North, a Surprenant, Brilliant blue G selectively blocks

ATP-gated rat P2X(7) receptors., Molecular pharmacology 58, 82–8 (2000).

152. T. Woehrle et al., Pannexin-1 hemichannel-mediated ATP release together with P2X1 and

P2X4 receptors regulate T-cell activation at the immune synapse., Blood 116, 3475–84 (2010).

153. B. S. Khakh, W. R. Proctor, T. V Dunwiddie, C. Labarca, H. A. Lester, Allosteric control of

gating and kinetics at P2X(4) receptor channels., The Journal of neuroscience : the official

journal of the Society for Neuroscience 19, 7289–99 (1999).

154. A. Priel, S. D. Silberberg, Mechanism of ivermectin facilitation of human P2X4 receptor

channels., The Journal of general physiology 123, 281–93 (2004).

74

155. F. Marteau et al., Pharmacological characterization of the human P2Y13 receptor.,

Molecular pharmacology 64, 104–12 (2003).

156. G. Lambrecht, Agonists and antagonists acting at P2X receptors: selectivity profiles and

functional implications., Naunyn-Schmiedeberg’s archives of pharmacology 362, 340–50 (2000).

157. I. von Kügelgen, A. Wetter, Molecular pharmacology of P2Y-receptors., Naunyn-

Schmiedeberg’s archives of pharmacology 362, 310–23 (2000).

158. M. Kaulich, F. Streicher, R. Mayer, I. M�ller, C. E. M�ller, Flavonoids - novel lead

compounds for the development of P2Y2 receptor antagonists, Drug Development Research 59,

72–81 (2003).

159. G. Burnstock, Purinergic signalling: Its unpopular beginning, its acceptance and its exciting

future., BioEssays : news and reviews in molecular, cellular and developmental biology 34, 218–

25 (2012).

160. S. Locovei, J. Wang, G. Dahl, Activation of pannexin 1 channels by ATP through P2Y

receptors and by cytoplasmic calcium., FEBS letters 580, 239–44 (2006).

161. L. Bao, S. Locovei, G. Dahl, Pannexin membrane channels are mechanosensitive conduits

for ATP., FEBS letters 572, 65–8 (2004).

162. H. C. Castro-Faria-Neto, C. M. Penido, A. P. Larangeira, A. R. Silva, P. T. Bozza, A role

for lymphocytes and cytokines on the eosinophil migration induced by LPS., Memórias do

Instituto Oswaldo Cruz 92 Suppl 2, 197–200 (1997).

163. F. Kukulski et al., Endothelial P2Y2 receptor regulates LPS-induced neutrophil

transendothelial migration in vitro., Molecular immunology 47, 991–9 (2010).

75

164. F. Kukulski et al., The P2 receptor antagonist PPADS abrogates LPS-induced neutrophil

migration in the murine air pouch via inhibition of MIP-2 and KC production., Molecular

immunology 47, 833–9 (2010).

165. P. F. Bradfield, C. A. Johnson-Léger, C. Zimmerli, B. A. Imhof, LPS differentially

regulates adhesion and transendothelial migration of human monocytes under static and flow

conditions., International immunology 20, 247–57 (2008).

166. F. Kukulski et al., Extracellular nucleotides mediate LPS-induced neutrophil migration in

vitro and in vivo., Journal of leukocyte biology 81, 1269–75 (2007).

167. B. M. Tavares-Murta, J. S. Machado, S. H. Ferreira, F. Q. Cunha, Nitric oxide mediates the

inhibition of neutrophil migration induced by systemic administration of LPS., Inflammation 25,

247–53 (2001).

168. S. Aizawa, I. Amaki, J. Fujita, M. Tsurusawa, K. J. Mori, LPS induces migration of bone

marrow cells in LPS-nonresponsive C3H/HeJ mice., Journal of radiation research 25, 91–8

(1984).

169. M. W. Verghese, R. Snyderman, Endotoxin (LPS) stimulates in vitro migration of

macrophages from LPS-resistant mice but not from LPS-sensitive mice., Journal of immunology

(Baltimore, Md. : 1950) 128, 608–13 (1982).

170. M.-E. Fortier et al., The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats

via an interleukin-1-dependent mechanism., American journal of physiology. Regulatory,

integrative and comparative physiology 287, R759–66 (2004).

171. N. Thieblemont, R. Thieringer, S. D. Wright, Innate immune recognition of bacterial

lipopolysaccharide: dependence on interactions with membrane lipids and endocytic movement.,

Immunity 8, 771–7 (1998).

76

172. M. Rossol et al., LPS-induced cytokine production in human monocytes and macrophages.,

Critical reviews in immunology 31, 379–446 (2011).

173. I. Zanoni, F. Granucci, Differences in lipopolysaccharide-induced signaling between

conventional dendritic cells and macrophages., Immunobiology 215, 709–12.

174. H. Wolburg, W. Paulus, Choroid plexus: biology and pathology., Acta neuropathologica

119, 75–88 (2010).

175. M. E. Tulapurkar et al., Endocytosis mechanism of P2Y2 nucleotide receptor tagged with

green fluorescent protein: clathrin and actin cytoskeleton dependence., Cellular and molecular

life sciences : CMLS 62, 1388–99 (2005).

176. M. E. Tulapurkar, W. Laubinger, V. Nahum, B. Fischer, G. Reiser, Subtype specific

internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5’-O-(1-

boranotriphosphate) derivatives., British journal of pharmacology 142, 869–78 (2004).

177. V. Kanamarlapudi, S. E. Owens, K. Saha, R. J. Pope, S. J. Mundell, ARF6-dependent

regulation of P2Y receptor traffic and function in human platelets., PloS one 7, e43532 (2012).

178. M. E. Tulapurkar, G. Zündorf, G. Reiser, Internalization and desensitization of a green

fluorescent protein-tagged P2Y nucleotide receptor are differently controlled by inhibition of

calmodulin-dependent protein kinase II., Journal of neurochemistry 96, 624–34 (2006).

179. D. Giulian et al., Cell surface morphology identifies microglia as a distinct class of

mononuclear phagocyte., The Journal of neuroscience : the official journal of the Society for

Neuroscience 15, 7712–26 (1995).

180. A. Y. Hoi, M. N. Iskander, E. F. Morand, Macrophage migration inhibitory factor: a

therapeutic target across inflammatory diseases., Inflammation & allergy drug targets 6, 183–90

(2007).

77

181. J. S. Wiley, R. Sluyter, B. J. Gu, L. Stokes, S. J. Fuller, The human P2X7 receptor and its

role in innate immunity., Tissue antigens 78, 321–32 (2011).