Download EFFECTS OF INTERLEUKM 1p ON JSOLATED RAT

EFFECTS OF INTERLEUKM 1p ON JSOLATED RAT
SUBFORNICAL ORGAN NEURONS
SHEANA ELIZABETH DESSON
A thesis submitted to the Department of Physiology
in confomiity with the requirementsfor
the degree of Master of Science
Queen's University
Kingston, Ontario, Canada
Auaust, 2001
copyright Q Sheana Eliznbeth Desson, 2001
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ABSTRACT
Induction of fever is a physiological response to the presence of an
exogenous substance, which is benefiual to the organisrn.
This immune
challenge in the periphery initiates complex changes in metabolism and in
immune, endocrine and central nervous system (CNS) functions.
The
circumventricular organs (CVOs) are ideat centen for circutating pyrogens to
wrnmunicate with the CNS to initiate these changes, as their fenestrated
capillaries allow direct access of these circulating substances with CNS tissue
without irnpediment of the blood-brain barrier (BBB).
The subfomical organ
(SFO), a CVO involved in the febrile response, is a site of activity for the
circulating endogenous pyrogen niterleukin 1B (IL4 P).
The airn of this study was to detemine the response of rat SFO neurons
to IL-18 (100fM
- InM)
using whole cell current clamp and voltage clamp
techniques. We found that at subseptic concentrations of IL-lp (IpM, 500fM) a
transient depolarkation was seen in the neurons (5.4*1.2mV, n=7; 6.4M.9mV.
n=8 respectively). accompanied by a 5 W %
increase in spike frequency
(pe0.05), whereas at pathophysiological septic concentrations of ILIp (InM) a
sustained hyperpolaritation was observed (5.1 11 .OmV, n=8).
No depolarization was seen followïngconcurrent application of IL-1p with
IL-1rat an endogenous antagonist that prevents 11-18 response by binding to the
11-18 receptors (n=7).
However, hyperpolamations were still seen in 2 cells
suggesting that this response is not 11-18 receptor mediateci.
-
V
-
eletnp- anelpig FWA&
t ~ d'ffféFMe
s~bpopulatio~~
O# SFO
neurons defined by the presence of a dominant IA(DM or IK(DIK). The majonty
(85%) of cells responding with depolarkations (29 of 34) were DIKcells.
In ordet to detemine the ionic conductances involved in the IL4 P receptor
mediated depolarkation. a further series of experiments using voltage clamp
techniques were perfonned. The affects of IL43 on the non-selective cation
current (IN& were studied wing a slow (IZmVkec), depolarking ramp protocol.
A transient linear dîflerence current was produced having a dope of
0.60~.12pA/mV and a reversal potential of -38.811.8rnV
(n=9).
Further
investigation of other conductances revealed that Il-lp also pr~duced a
significant decrease in IKto û4.4*3.3% of control ( n 4 ) (p<0.005), which did not
show recovery.
Our data show that IL-lp exerts direct actionson SFO neurons. These
observations identify a mechanism through which circulating IL-1P influences the
activity of SFO neurons. They support the conclusion that during an immune
challenge, IL4 p increases the acüvity of SFO neurons leading to initiation of the
febrile response as a resutt of activation of additional autonornic centers
receiving afferent inputs fiom these cells.
ACKNOWLEDGEMENTS
First of all, I woukl like to thank Al for giving me a fresh and energked
perspective on science and for aiways keeping the door open, both literally and
figuratively.
To the Ferguson lab crew, thanks for ail the encouragement, guidance,
and pints. Thanks especially to Jim for al1 of your time, patience and support.
A big thank you to Shady for joining me on this very eventful two year
roller coaster ride on the 4' fioor, Botterell Hall.
Most importantiy, to my parents and brother whose constant support saw
me through the days when things just weren't going well and whose invaluable
advice has made those difficult decisions quite a bit easier to make. Siantel
TABLE OF CONTENTS
ABSTRACT...,........................................................................*.........ii
ACKNOWLEDGEMENTS............................................................
iv
TABLE OF CONTENTS................................................................ v
LIST OF FIGURES........................................................................
vii
LIST OF TABLES.......................................................................... viii.LlST OF ABBREVIATIONS.......................................................... ix
CHAPTER 1: 1NTRODUCTION..................................................i
Possible Pathways of Communication.................................... ..3
The Subfomical Organ.............................................................. s
Morphology.................................................................. ...5
Efferent Projections...................................................... ..6
ARerent Projections.. ..................................................... -8
Physiological Funcüons................................................. .9
Lntrinsic Conductances-
ii
~~~~~~~~~~~~~~~~~~~~
Fever.......................................................................................
-14
Inierieukin 1.................................................................. -15
lnterieukin 1 Receptor Antagonist.. .............................. 17
lnterleukin 1 Receptors.................................................. i 7
lnterfeukin 1 Signal Transduction.--......
........................-19
Biological Activ'i of Interieuh 1.................................. 21
.
.
Central Adions of lnterleukin 1t3 in Fever Generation...22
Rationale for This Study........................................................ 25
CHAPTER 2: MATERIALS AND METHODS.........................27
ElectrophysiologicalTechniques............................................. 29
Solution Concentrations.......................................................... -30
Statistical Analysis.................................................................. -30
CHAPTER 3: RESULTS............................................................
-31
Response to 11-18..................................................................-32
Receptor Specificity................................................................. 35
Effects on Spike Frequency..................................................... 37
Characteristics of Responsive Cells........................................ 38
Effectson Input Resistance..................................................... 41
Non-Selective Cation Channels... ............................................ 41
Potassium Currents................................................................. 43
CHAPTER 4: DISCUSSION......................................................46
CHAPTER 5: REFERENCES....................................................ss
VITA ..,.............................................................................................
-70
LIST OF FIGURES
FIGURE 1: The anatornical location of the SFO within the rat brain.
FIGURE 2:
Action potential and ionic conductances of the SFO neuron.
FIGURE 3:
IL4 receptor binding and signal transduction.
FIGURE 4:
Current clamp recordings during application of IL-1P.
FIGURE 5:
Dose dependent changes in membrane potential following
application of IL4 P.
FIGURE 6:
Current clamp recordings during application of IL-Ira and IL-1P.
FIGURE 7:
Voitage clamp recordings indicate the presence of two populations
of SFO neurons.
FIGURE 8:
Peak-to-steady state ratios of IL-1P responsive and non-responsive
neurons.
FIGURE 9:
Voltage clamp recordings display activation
1 application.
of NSCC following IL-
FIGURE 10: Voltage clamp recordings display a decrease in IKfollowing 11-18
application.
FIGURE t 1: Potential projection sites for 11-18 responsive SFO neurons.
LIST OF TABLES
TABLE 1:
Currents contributhg to action potential generation in SFO neurons
LIST OF ABBREVIATIONS
Accessory protein
Adrenocorticotrophin hormone
Angiotensin II
Anterovenhal region of the third ventricle
Area postrema
Arginine vasopressin
Atnal natnuretic peptide
Blood-brain barrier
Calcium receptor
Central nervous system
Circumventricular organ
c4un N-terminal kinase
Corticotrophin releasing hormone
Cyclooxygenase
Dominant IA
Dominant IK
Endogenous pyrogen
Fullide stirnulaüng hormone
Hypothalamic-pituitary-adrenal
lnterleukin 1 receptor antagonist
lnterleukin 1 receptor associated kinase
lnterleukin converting enzyme
Interieukin-1
Interieukin-1P
Interleukin-la
lntracerebroventricular
lntravenous
tipopoiysaccharide
Luteinking hormone
Medial preoptic nucleus
Media1septum
Median eminence
Median preoptic nucleus
Mitogenactivated protein kinase
Non-selective cation channel
Non-selective cation current
Nuclear -or
kappa B
Nucleus tractus solitafius
Organum vasculosum of the lamina terminalis
oxytocin
AcP
ACTH
ANG
AV3V
AP
AVP
ANP
BBB
CaR
CNS
CVO
JNK
CRH
COX
DIA
DIK
EP
FSH
HPA
IL-1ra
IRAK
ICE
IL4
lL-1p
IL-?a
icv
iv
tPS
LH
MePO
MS
ME
MnPO
MAPK
NSCC
iNSC
NFkB
NTS
OVLT
OXY
P a f a V e f ? ~ l a ~ - F t ~ & t h e h y p o t ~PVN
~~
POA
Praoptic area
PG
Prostaglandin
SFO
Subfomical organ
SON
Supraoptic nucleus
TTX
Tetrodotoxin
TLR4
Toll-like receptor 4
TRAF
Tumor necrosis factor receptor-associated factor
TNF
Tumor necrosis factor
IL-1RI
Type I interieukin-1 receptor
IL-?RiI
Type Il interieukin-? receptor
CHAPTER 1: GENERAL INTRODUCTION
Activation of the immune response can be initiatecl when certain
foreign substances are introduced into the body. These substances are
known as exogenous pymgens and they can trigger the synthesis of
proinflammatory mediators, known as endogenous pyrogens (EPs), by cells
of the immune system, especially monocytes. macrophages and neutrophils.
These mediators are released into the circulation and are transported
throughout the body to promote immune responses. They are able to signal
the œntral nervous system (CNS) to release central mediaton, such as
prostaglandins (PGs), which can direct and regulate further physiological
changes. The induction of the febrile response is one of the most weli-known
examples of these œntral changes. To effecüvely promote this response, the
immune system must interact with both the nervous and endocrine systems in
an efficient and cooperative manner. They must bring about the appropriate
rnetabolic, behavioral and endocrine changes in an atternpt to defeat the
challenge of the exogenous pyrogen and retum the body to homeostasis.
However, the rnechanism by which these peripheral EPs influence the
CNS to initiate the febrile response is not well understood. The brain is in a
unique situation, as it is surrounded by a blood-brain barrier (BBB), cornposed
of tight gap junctions between the endothelia1 cells that can exclude
peripherally circulating substances. While this bamer is absolutely essential
to the maintenanœ of normal CNS funcüon, it presents a problem for the
cimulating cytokines, as they may be too large or lipophobic to permeate
thmugh and thus, cannot di-
contact the brain.
POSSIBLE PATHWAYS OF COMMUNICATION
There
is much controversy sumunding the mechanism of
communication these cytokines adopt to converse with the CNS in order to
initiate the immediate immune response. There is some evidence suggesting
that cytokines can be actively transported through the BBB (Plotkin et al.,
I996), although the tirne course and quantity of this passage are probably too
slow and minimal to account for the rapid onset of fever. An alternative
explanation is direct neural communication with the CNS through cytokine
activation of the vagus nerve. This potential mechanism has lead to several
controvenial studies
with
conflictnig
results.
showing that
either
subdiaphragmatic vagotomy had no effect on (Luheshi et al., 2000). or
blocked (Gaykerna et al., 2000) the febrile response to systemic IL-Zp.
A further possibility that has received much support is that these
cytokines access the brain parenchyma at small, discrete areas at the brain
surface that are devoid of an intact BBB (Coœani et ai.. 1988).
Here,
circulating substances can make direct contact with CNS tissue via bloodbrain communication. This is possible because. instead of the usual tight
jundions
between endothelia1 cells, these capiilaries have a fenestrated
endothelium. These distinct
areas are called the circumventncular organs
(CVOs) and are believed to play essential roles in monitoring the constituents
of the systemic circulation. The CVOs are rkhly vascularized and contain
specific receptors for a wide variety of peptides and thus are ideally suited to
this sensoiy integrative mle at the BB i n t e d e . These unique properües of
CVOs make them a likely site at which cytokines may act to communicate
with the brain regarding the presenœ of immune challenges, resuiting in
initiation of the immune and febrile responses.
Lesioning çtudies have examined whether CVOs are responsible for
helping to trigger the febrile response (Takahashi et al., 1997). Discrete
lesions were placed in 3 CVOs: the subfomical organ (SFO), the organum
vasculosum of the lamina terminalis (OVLT) and the area postrema (AP) of
rats. Fever was induced by intravenous (N) administration of gram-negative
bacteria, lipopolysaccaflde (LPS), and while there was no affect on the fever
produced in OVLT and AP lesioned anirnals, the febrile response was
significantly attenuated in SFO lesioned anirnals.
Centrally induced
prostaglandin E l (PGEI) fevers were not reduced in any of the gmups of
lesioned anirnals, indicating downstream fever pathways within the brain had
not been interrupted by the SFO lesion (Takahashi et al., 1997). The
observation that SFO lesion prevented EP from generating the febrile
response supports the conclusion that this structure plays a crucial role in
communicaüng information from the periphery into the CNS during the
initiation of fever.
THE SUBFORNlCAL ORGAN
Morphology
The SFO is in a unique position, as it is a neural structure located at
the interface between both the circulatory and ventricular systems and the
CNS, where it can interact wiai circulating EPs. It is a srnall. semisphencal
protnision situated at the rostro-dorsal quadrant of the third œrebral ventricle,
attached to the hippocampal commissure (Dellman. 1985).
The cellular
makeup of SFO includes neuronal penkarya, glial cells and ependyrnal cells.
The neurons are parücularîy conœntrated within the central region, whereas
the caudal and rostral regions contain rnostly nerve fibres (Dellman and
Simpson, 1976).
The capillary network is composed of many distinct
subependymal loops. which fom a rich network that innervate SFO, along
with large adjacent perivascular spaœs. This network is seen especially in
the central neuronal region. This allows a large surface to be available for
interaction between these receptors and blood-borne ligands that are capable
of passing through the endotheliurn. Cerebrospinal-borne substances may
also enter SFO, as apart from the caudal end of the ventral surFace where the
choroid plexus is attached, the ventral surface is exposed to CSF.
The
morphology of the SFO neuron includes large dendriüc protrusions into the
ventricular lumen (Dellrnan and Simpson, 1979) and the perivascular spaces
of the fenestrated capillaries (Dellmann and Simpson, 1976), which allow
direct neuronal contact to both the CSF and blood.
--
--
-
Eflerent Projections
The location of SFO at the blood-brain interface allows it to receive
information fiom the periphery and then communicate this to neural structures
located deeper within the brain. A number of different techniques, including
anatomical tracing and electrophysiology, have identified projections of SFO
neurons to different brain regions; the rnost prolific being the anteroventral
third ventricle region (AV3V) and the hypothalamic neurosecretory area. The
AV3V region is richly innenrated by SFO projections, specifically the median
preoptic nucleus (MnPO), the OVLT (Miselis et al.. 1979), the preoptic
periventricular nucleus, adjacent medial parts of the medial preoptic area
(MePO) and the medial septum (MS) (Lind et al., 1982). Efferents were also
found to project to the tateral hypothalamus, perifomical area and the iateral
preoptic area (Miselis, 1981). SFO projections to the hypothalamus terminate
in the major neuroendocrine cell groups responsible for secretion of oxytocin
(OXY) and arginine vasopressin (AW), the paraventricular (PVN) and the
supraoptic (SON) nuclei (Miselis, 1981).
SFO celis also project to
parvocellular neurons wiaiin the PVN. which in tum, project to a number of
different areas including the intemiediolateral column of the spinal cord
(Hosoya and Matsushita, 1979), nudeus tractus solitarius (NTS) (Kannan and
Yamashita, 1983) and median eminenœ (ME) (Conrad and ffiff, 1976). The
ME projecting neurons are particularty important in mediatîng the production
of cortÎcotmphin releasing hormone (CRH) and later rdease at the median
eminenœ leading to the subsequent production of adrenocorticotrophin
FIGURE ?: The anabmical location of the SFO wifhin the
rat brain
Shown hem is a rnidsagittal view of the rat brain identifying
the SFO and its major projeCaion sites: the AV3V region and
the PVN
hormone (ACTH) (Csemus et al., 1975). This neuroendocrine systern is
activated by "stress", "immune activation' and stimuli resulting in the
generation of fever.
SFO neurons can also relay information concurrently to several
different efferent areas within the brain. Collateral branching of axons of SFO
neurons means that a single cell can innervate both the PVN and SON
(Weiss and Hatton, 1990). Polysynaptic pathways have also been identified
with efferent projections teminating at the medial septum, which in tum
acüvate secondary neurons projecüng to the PVN. SON and POA (Lind et al..
1982).
Aithough the hypothalamic and AV3V areas are the best understood
areas of SFO innervation, there are further projections to the infraiimbic
cortex, the rostral and ventral parts of the bed nucleus of the stria terminalis.
the zona incerta. and the dorsal and median raphe nuclei (Swanson and Lind,
1986).
Afférent Projections
Aithough much of the afhrent information received by SFO is derived
ftom both blood-borne and cerebrospinaCbome molecules, this structure also
receives affkrent neuronal input fiom a number of CNS structures. These
include the NTS (ZardettoSmith and Gray, 1987). MnPO, nucleus reuniens of
the thalamus, lateral hypothalamus (Lind et al., 1984). and midbrain raphe
(Lind. 1986).
Physiological Functions
The unique location of SFO at the blood-brain and CSF-brain interface,
when combined with the large number of receptors found on SFO neurons,
suggests involvernent of this structure in diverse functions. The following is a
brief summary of the primary physiological funcüons attributed to SFO:
Cardovascular Regulaüon
Traditionally, much of the focus has been plaœd on the role of SFO in
drinking and cardiovascular regulation. In the I ~ ~ O ' Sit,was established that
SFO is the primary CNS location where angiotensin II (ANG) acts to wuse
drinking (Simpson et al., 19?8), a conclusion confirmed by later microinjection
studies showing that direct administration of ANG into SFO also increased
blood pressure (Mangiapane and Simpson, 1980).
Other peptides also exert physiological effects as a consequence of
action in SFO. Atnal natriuretic peptide (ANP) antagonizes the excitatory
effects of ANG in drinking behaviour, as shown by studies in which ANP
injected into SFO after ANG administration, reduced the drinking response to
the latter peptide (Ehrlich and F i , 1990). AVP is another peptide involved in
modulating the expression of SFO. This peptide is closely linked with ANG,
as ANG appears to activate SFO neurons, promoting increases in blood
pressure, as welf as stimulating AVP release into the circulation from efferent
magnoceilular neurons in the PVN (Ferguson and Renaud, 1986). AVP then
acts in a negative feedback loop, as administration of AVP into SFO
decreases blood pressure (Smith and Ferguson, 1997).
SFO neurons also appear to possess intrinsic osmosensitivity as
illustrated in a recent study showing that changing the osmolarity of the fiuid
SFO neurons are bathed in can have significant effects on their membrane
potential and firing pattern. As compared with isotonic solution, hypoosmotic
solutions produced hyperpolarizations with deaeased neuronal firing and
hyperosmotic solutions produced depolarkations with increased neuronal
firing (Anderson et al*,2000). This suggests that SFO may play a key role in
responding to osrnotic stimuli within the nomal physiological range.
Reproductive Regulation
SFO lesions have also been shown to disrupt the estrous cycle in rats
(Limonta et al.. 1981), suggesting a role for this structure in reproductive
function. In accordance with such a hypothesis, activation of SFO neurons
also results in increased concentrations of luteinizing hormone (LH) in the rat
(Donevan et al., 1989). A further causal link with reproductive function is the
link that the SFO has with the putative OXYlAVP neurons in SON and PVN,
thus exerting control over the production of OXY. Activation of SFO neurons
can stimulate OXY secretion (Ferguson and Kasting, 1987) and increase
plasma OXY concentration (Ferguson and Kasüng, 1988).
FeverRegulaüon
The lesioning study performed by Takahashi et al. in 1997 (described
earfier) gave clear evidenœ that SFO is also implicated in the regulation of
the febrile response. SFO, which lacks an intact BBB, acts as a site where
EPs in the periphery can communicate with the CNS.
This information
--
--
transduction promotes the initiation of fever. However, the mechanism of
action as well as the cytokine responsibfe for the effect is stilf uncfear.
Therefore. the particular focus of this thesis is to detemine whether there are
direct effects by an EP on SFO neurons and the mechanism that mediates
these effects.
Intrinsic Currents
The fundons attrîbutedto SFO rnentioned in the previous section are
caused by the modulation of SFO neuronal firing and the subsequent
activation of other neurons through the synapses of efferent SFO projections.
A number of currents have been idenüfied that regulate the firing of action
potentials by these neurons. When combined, they create a coordinated and
dynamic neuronal state.
Table 1 identifies the currents that significantly
contribute to axon potential firing. Figure 1 depicts an SFO action potential
and the relative contributions of these currents to its generation. These are
the primary conductances found in neurons, although other currents such as
a persistent sodium current (INAP),a hyperpolarized-acüvated current (Ih)and
a non-selective cation current (Iwc) have been identified in the SFO which
help maintain resting membrane potential.
Changing the balanœ of intnnsic cunents results in profound effects
on neuronalfunction. In this way, stimuli evoke raçponses in SFO neurons by
modulating currents via opening. closing and inadkathg channels.
The
question of interest regarding Biis thesis ïs whether fever inducing substances
-
large inward sodium current
responsiblefor the depolarizing upstroke of the
action potenüal
(see Hille, 1992 for review)
-
large outward potassium current
repolarizes the neuron following the Na+influx
(Washbum et ai.. 1999)
-
high voltage activatecl inward calcium current
influences the length of the action potential
(Washburn and Ferguson, submitted)
-
transient outward potassium current
contributes to regulation of the interspike interval
by opposing the depolarizing stimulus
(Washbum et al., 1999)
TABLE 1: Curmnts contributhg tu action potentiat generation in SFO
neumns.
FIGURE 2: Acüon potential and ionic cunents of the SFO neuron
Shown here is a representative SFO action potential and the approm'mate
relative contributions of cunents to its generation.
can produce a response in the isolated SFO neuron and what ionic currents
are responsible for this response.
Fever is a universal response to immune activation. as it is seen
throughout the animal kingdom (Mackowiak, 1994).
It is an adaptive
mechanism that plays an important role in the survival of the host durîng
infedîonwith foreign substances (Mackowiak, 1994). Fever is manifested as
an increase in the hypothalamic temperature "set point" above that of normal
citcadian fluctuations.
To achieve this increase in core temperature,
mechanisms must be activated to increase heat production and conservation
(Elmquist et al., 1997).
This is achieved by initiating such responses as
shivering, increased metabolism and redistribution of blood flow, for example,
by cutaneous vasoconstriction. At this elevated set point, infection fighting
processes are amplifiecl, as demonstrated by the increased rate of enzymedependent inflammatory activities and amplifiad phagocytosis.
These
processes contribute to the host's ability to resist and fight infection (Marik,
2000)(Netea et al., 2000).
Fever occurs when exogenous pymgens are introduced to the body via
a break in its natural barriers.
These substanœs in turn, interact with
macrophages, which initiate the immediate immune response by producing
EPs (Netea et al.. 2000). These are camed via the circulation throughout the
body and trigger different physiological systems to commence the acute
phase response in order to fight the exogenous substance. This includes the
initiation of the febrile response by the triggering of changes in
thennosensitive areas of the hypothalamus, including the POA (Boulant,
2000), and the maintenance of the febrile response via activation of the
hypothalamic-pituitary-adrenal (HPA) axis in PVN (Saper, 4998).
Interfeukin 1
The search for the biological, molecular and chernical nature of EP has
received much attention during the past few decades. It was understood that
this substance. when injected into the peripheral circulation, acted on the
thermoreguiatory center of the hypothalamus to eiicit fever. Furthemore,
leukocytes that were stimulated by bacterial products, synthesize a mediator
that has EP-like properties (Bodel, 1974). This resulted in the labeling of the
first idenbified fever-inducing, leukocytederived EP, lnterieukin 1 (1 1-1)
(Lachman et al., 1980).
Iniüally, it was unclear as to whether IL4 was a single substance or
several different molecules. because four i s ~ e l e ~
points
c
and large and
small molecular weight foms had been described. In 1985, the human I L 1
DNA was identified. This revealed that there were in fact two distinct IL4
molecules: an acidic form that was labeled Interleukin l a (IL-la) and a
n e m l fom that was labeled Interleukin 18 (11-1 8) (March et al.. 1985). 80th
are found on the long a m of human chromosome 2, distrib~Zedover 430
kilobases. They elicit many of the same biological funcüons including the
induction of fever, hepatic protein synthesis. PGE2 synthesis, cartilage
breakdown, bone resorption, elevation of ACTH and augmentation of the Tlymphocyte response to antigens and mitogens (see rev. Dinarello, 1994).
This functional homogeneity is made possible because much of the structural
topology is sirnilar (Nanduri et al., 1991). It is thought that both peptides
share the same sequenœ at two specifk reœptor binding sites on the ligand,
which enables interaction with the receptor and subsequent signal induction
(Evans et al., 1995).
These two molecules have precurson with a molecular mass of 31kDa
(March et al., 1985). IL-la is active both in the pro-form and in the mature
17kDa form. However, proIl-1 requires cleavage to the 17kDa peptide for
release from the cytosol and transport out of the cell. It is only in this f o m
that it is biologically active. This structural change is accomplished via the IL1p converting enzyme (ICE), which is an intracellular cysteine protease that
cleaves the prolL-1p at the aspartic acid-alanine position (Thombeny et al.,
1992).
However, before ICE can become active -haIf, it must be
aut~catal~cally
acüvated and fomi an adive tetramer of two heterodimers.
Administration of fever inducing substances. for example. LPS, induces
transcription of ICE, which allows more to be available for the cfeavage of
proIl-1p (Thornberry et al.. 1992).
11-18 is also regulated via *ts
transcriptional and translational interactions. During normal conditions, large
----
-
amounts of IL-1P mRNA are transcribed, but a translational "block" is applied
that limits the production of the protein (Thomberry et al., 1992). This leads
to IL-lp mRNA degradation. However, upon LPS administration, this block is
removed and the proIl-IP is produced in abundance.
Interleukin 1 Receptor Antagonist
Another member of the IL4 family is the IL-1 receptor antagonist (ILIra), which is a naturally occumng, 17kDa, endogenous ligand (Eisenberg et
al., 1990; Hannum et al., 1990) that binds to, but does not produce any
response at IL-1 reœptors.
It too is found on human chromosome 2 and is
produced in the prolL-Ira form, which must be cleaved to produce its active
form. Although this molecule shares similar tertiary structure to IL-la and IL-
Ip, it lacks one of the two binding sites of the agonists (Evans et al., 1995).
This makes it unable to evoke a response (Dripps et al., 1991). By binding to
the IL4 receptors, there are fewer receptors available for the subsequent
binding of IL-1, resulting in less signal transduction. The induction of IL-Ira
usually occun following the sarne stimuli as IL4 induction,
although this
generally happens at a longer latency (Dinarello, 1994). Therefore, IL-Ira
expression offers another regulatory mechanism following the initiation of the
immune response, as, by binding to the receptor, it blocks potential IL-1
reœptor signal transduction.
lnterieukin 1 Receptors
The ILI reœptor group is composed of two different molecules: type 1
il-1 receptor (IL-1RI) and type II IL4 receptor (IL-IRII). Both are derïved
from a common receptor gene (Sims et al., 1995) found again on
chromosome 2 in humans. However. IL-IR1 is the receptor responsible for
signal transduction. It has a cybsolic domain of 213 amino acids (Sims et ai.,
1988), whereas IL-IR11 only has a short segment of 29 amino acids
(McMahon et al., 1991) and lacks the intemal mechanisms necessary to
transduce a signal. Instead, it sequesters the IL1 away from the active IL1RI (Sims et al., 1993). Therefore, R can still exert regulatory effects. as
increased binding of IL-1 to IL-1RI1 could lead to decreased availability for
binding to IL-1RI. There is both a soluble and receptor bound forrn of the IIIRII, and the soluble fom can bind circulating IL-I. therefore not allowing the
peptide to interact with IL-1RI. This is especially evident with 11-1B. which it
preferentially binds (Symons et ai.. 1995). Both reœptors are monomeric and
bind only one 11-1 molecule. IL-la has a higher affinity for IL4 RI and IL-1p
has a higher affinity for IL4 RI1 (Scapigliati et al., 1989). 11-1ra acts as a
cornpetitive antagonist and binds to both IL-1RI and IL-1RI1 (Dripps et al.,
1991; Granowih ef ai.. 1991). however it does preferentially bind to IL4 RI
(Svenson et al.. 1993). Therefore. it rnay block the biological effects elicited
by both IL-la and 11-18 by occupying the binding site. The afinities for the
binding of IL4 to recepton have been studied in different cell types
throughout the body by a number of investigators and are found to be within
-
P
-
the picomolar range (Matsushima et al., 1986; Takao et al., 1990). However,
biological responses can be observed at concentrations of 10 to 100-fold less
than that of the dissociation constant due to post-receptor binding
amplification such as multiple phosphoiylaüons of nuclear factors (Gallis et
al., 1989) and the IL4 RI itself (Takii et al., 1992; see rev. Dinarello, 1994).
Interleukin 1 Signal Transduction
IL-1RI activation by IL-1 results in association with an accessory
protein (AcP) (Gteenfeder et al.. 1995) and the adaptor ptotein MyD88 within
the intercellular domain. The L 1 R associated kinase (IRAK1 and II) is then
activated and complexes with AcP (Huang et al., 1997).
This leads to
activation of other proteins, including tumor necrosis factor (TNF) receptorassociated factor 6 (TRAFô),and the nuclear factor kappa B (NFkB) inducing
kinase. NFkB is released because the NFkB inhibitor, IkB is phosphorylated
and degraded. NFkB is then translocated into the nucleus where it promotes
transcription and upregulaüon of expression on the consensus sequence of a
target gene (DiDonato et al., 1997). 11-1 also activates kinases (Guy et al.,
1991) including p42/p44 mitogen-adivated protein kinase (MAPK), p38 MAPK
and cJun N-terminal kinase 1 (JNKI).
The significance and mechanism
linking this kinase expression to the rest of the signal transduction is still not
known.
Much of this signal transduction work has been canied out on
peripheral cells. However, the effects of IL4 on neurons, such as aiteration
of ion cunenis, occur very quickiy (Li et al., 1992; Akasu and Tsurusaki,
ACTIVATION OF GENES
FlGURE 3: 11-1 receptor binding and signal transduction
Show here is the preferential binding of the d i r e n t IL-1 molecules (prefemed
binding is displayed by a thicker line) at the IL4 receptors, and the subsequent
signal transducüon occuning at IL1RI, leading to NFk6 and kinase induced
activation of genes.
-
*
-,
--
l999), within seconds of administration and may be too rapid to rely on this
cornplex signaling cascade. Therefore, it is thougM that within the brain,
addiüonal signaling mechanisms are involved that have yet to be identifieci
(Rothwell and Luheshi, 2000).
It appears that such mechanisms could
involve direct influences on ion channels.
Biological Activity of lnterieukin 1
The concentration of 11-18 is low in the healthy body, usually in the
high femtomolar range in the periphery. Upon introduction of an immune
challenge, for example LPS, peripheral IL4P sources are activated leading to
a rapid 10 to 100 fold increase in concentration (Ma et al., 2000). However,
concentrations rarely increase beyond this initial upregulation.
High
picornolar IL-1P levels would be analogous to those seen during extrerne
sepsis.
Picomolar levels of IL-Ip introduced into the periphery led to
hypotension (Dinarello et al.. 1989) and general systern breakdown in rabbits.
analogous to the septic shock response.
W ~ i the
n brain, IL4 is widely expressecl along with its receptors and
lL
I Microglia are the primary initial source, however, neurons, astrocytes
and oligodendrocytes also produœ lL-1 (Rothwell and Luheshi, 2000).
Centrally, IL1 appears to exert its effects by enhancing the hast defense
response to infection, injury, and inflammaüon.
It mediates these effects
through a variety of coordinated actions in the immune, nervous and
endocrine systems.
These effects incfude increased rnetabolic rate
(Busbridge et al., 1990). sympathetic activation of brown fat (Busbridge et al.,
1990). sleep (Opp et al., 1989). increased 11-6 release (De Simoni et al.,
1990), suppressed appetite (Luheshi et al., 1999) and involvement in
neurodegeneraüveactivÎty (Touzani et al., 1999).
However, IL4 is probably best recognized for its role in the induction of
fever (Long et al., 1990; Rothwell, 1989). Although many of the effects of ILl a and IL4 p are similar, there appears to be a different mechanism of acto
in
of the two molecules on the generation of fever. IL-lp induced fever was
inhibited by central injections of corticotrophin releasing hormone (CRH)
receptor antagonists. whereas IL-la induced fever was not effected by the
antagonist (Busbridge et ai., 1989). This indicates that CRH mediates
therrnogenesis and fever induced by 11-18 but not by IL-la. In addition, there
are diffarances in the amplitude of 11-1B and IL-1a induced fever. Conscious
rats displayed significantly greater febrile and themogenic responses after
œntral injection of 11-18 as compared to IL-la (Busbridge et al., 1989).
Therefore, as IL-lp is the more potent pyrogen, it will be the focus of this
Central Actions of Interleukin 1P in Fever Generation
11-1$ exerts its central febrile inducing actions by modulating the
activii of thermoregulatory sites withh the brain. The preoptic area (POA) of
the anterior hypothalamus is one such area, as many of the thermosensitive
neurons are located here (Boulant, 2000). Peripheral information is refayed
to this œnter, where it alters the activity of these neurons so as to evoke
appropriate thermoregulatory responses.
During fever, the warrn-sensitive
neurons are inhibited by specific agonists, including IL-lp and PGs (Boulant.
2000). Several studies have shown that local application of IL-1P within the
MePO resulted in a decrease of firing of the wamsensiave neurons (Hori et
al., 1988; Nakashima et al.. 1989; Shibata and Blatteis, 1991). IL4 p can also
alter the levels of PG within POA by significantiy increasing cyclooxygenase-2
(COX-2) transcription (Rivest et al., 2000) and release of PGE2 (Kornaki et
al., 1992). The source of IL-lp within POA could be derived from neurons
projecting to this area, indicating that IL-lp may act as a neurotransmîtter
mediatirtg post synaptic responses.
The resufting inhibition of the w a n
sensitive neurons by IL-1P or PG tnggers the activation of efferent POA
projections to autonomie and endocrine centers where heat retention and
generation mechanisrns are applied (Scammell et al., 1996).
These
mechanisms include the redistribution of blood flow, shivering and increased
metabolism.
PVN is another area where 11-16 plays a role in fever, as shown by a
study in which microinjection of 11-18 directiy into PVN produced a
significantly
greater
fever
administration (Avitsur et al..
than
intracerebroventricular
1997).
tin
administration, idenwing the acvtiao
(icv)
11-1$
Fos studies following iv LPS
of POA precedirtg the parvocellular
neurons within PVN (Elmquist et al., 1996), indicate that PVN is likely a œnter
that receives information from POA, signaling fever initiation (Saper, 1998). It
is within these neurons of PVN where the putative CRH neurons are located
and where the HPA axis can be acüvated. CRH plays an essential role in IL1p induced fever, as icv administered IL-lp produced fever that was
significantly attenuated following an antagonist to CRH (Rothwell. 1989). The
endocrine response is not the only febrile response associated with PVN.
Efferent projections ftom PVN to autonomic areas within the spinal cord and
medulla can coordinate sympathetic activity and the redistribution of blood
flow (Porter and Brody. 1985) to further promote heat retention and
generation responses during fever.
Therefore, IL-1P can act directly on central themoregulatory sites,
including POA and PVN to promote fever initiation and the subsequent
activation of autonomic and endocrine areas during the febrile response.
RATIONALE FOR THIS STUDY
The reasoning for this study is based on evidenœ implicating a
coordinated role for IL4 P and SFO in the initiation of the febrile response. II18 is a potent EP and central injection of 11-18 produced fevers of greater
amplitude and duration as compared with central injecüon of IL-la in rats
(Busbridge et al., 19û9). Several studies have suggested a role for SFO in
transducing signals from circulating IL-18 into CNS generation of fever.
Takahashi et al. demonstrated that lesioning of SFO significantly attenuated
the febrile response following iv administration of LPS (1997). Furthemore.
microinjection of II4ra into SFO produced a significant reduction in the febrile
response in rats administered with LPS, yet had no efiect when injected into
OVLT (Cartmell et al.. 1999). There is also an upregulaüon of IL4 p mRNA in
SFO upon peripheral LPS administration (Eriksson et al.. 2000), suggesting
that this IL-lp originating within the structure could act as a neurotransmitter
and actviate
post synaptic neurons during the febrile response.
The location of SFO at the interface between the perighery and the
brain represents an ideal potential locus for circulating 11-18 interactions with
the CNS in view of its many fanestrated capillaries. which allow EP to directly
contact brain tissue. SFO has efferent projectÏons to POA, where many of the
thermosensiüve neurons are located and where fever can be initiated, as well
as projections to the putative CRH neurons within PVN, where the endocrine
response to the immune challenge can be modulated.
-
My studies were designed to test the following hypotheses:
1)
IL4 f3 exerts direct control over the excitabiiii of SFO neurons.
2)
Physiological concentrations of Il-1p exert these affects on SFO
neurons through interactions with IL4 6 reœptors.
3)
Rapid effects of IL-Ip are the result of IL-lp receptor mediated
interactions with specific ion channels.
CHAPTER 2: MATERIALS AND METHODS
SFO neurons were dissociated by the protowl previously described by
Ferguson et al. (Ferguson et al.. 1997). Male Sprague-Dawley rats (1251509) were decapitated. and the brains quickly removed and immersed in ice
cold Hank's buffer ( ~ a "and ~ g "free 0.03M sucrose, pH adjusted to 7.4
with NaOH). A tissue block containing the hippocampal commissure and
SFO was dissected out and placed in ca2+and ~
g free
~ Hank's
+
Balanced
Salt Solution. All surrounding tissue was removed from the SFO with the aid
~ ' Hank's
of a dissection microscope, and plaœd in ca2+ and ~ g frse
Balanced Salt Solution containing Img/mL Trypsin (Sigma), and incubated in
5% Co2/95%02,at 37'C for 30 minutes, with trituration through a tuberculin
syringe fitted with a 20 gauge needle. Cells were then resuspended in Hank's
solution containing
ca2+(1.3mM),
~ g ' +(0.9mM) and 0.1% bovine serurn
albumin (BSA) (Sigma type A-6003. essentially fatty acid-free) using
centrifugation. Following centrifugation, the pellet was resuspended in the
same solution. and recentnfuged. The resultant pellet was again resuspended
in BSA wntaining solution. and 0.2mL of this cell suspension was plated on
plastic culture dishes (Coming) to which isolated cells adhered rapidly.
Dishes were plaœd in the CO2 incubator and later. 2mL of Neurobasal-A
Medium (Gibco) containing IOOUlml penicillin/streptomycin and 0.5mM Lglutamine was added to culture dishes.
Experiments were camed out with
neurons prepared in this way 14 days following the dissociation.
Al
procedures conformed to the standards outiined by the Canadian Council on
Animal Care.
-
Electrophysiological Techniques
Whole cell patch clamp recordings were obtained using micropipettes
pulled using a P-87 FlaminglBrown pipette pulter (Sutter) and fire polished.
Tip resistances were 2-4 Mf2 when filled with a solution containing (in mM):
130 K gluconate, 10 HEPES, 10 EGTA, 1 MgCI2, 4 Na-ATP, 0.1 GTP, with
the pH adjusted to 7.2 with KOH. Following the establishment of a gigaohm
seal, the whole cell configuration was obtained with a brief pulse of suction.
The control bath solution consisted of aCSF of the following composition (in
mM):
140 NaCI, 5 KCI, 1 MgCI2, 10 HEPES. I O Glucose, 2 CaC12; pH
adjusted to 7.4 with NaOH. Tetrodotoxin F()(Alamone) was stored at a
concentration of 1OOpM in IOOpL aliquots of distilled water at -70C.
The IL-
I (Peprotech Inc) was stored at a concentration of 1OOnM in 100pL aliquots
of distilled water at -70°C.
IL4 ra (Serotec) was stored at a conœntration of
100pM also in 100pL aliquots in distilled water at -iû0C.
Cells were defined as neurons by the presence of voltage gated Na+
currents in voltage clamp and at least 60mV action potentials in response to a
depolarking pulse during cunent-clamp recordings.
Patch clamp recordings were proœssed using a List EPG? amplifier,
fiitered with an 8 pole Bessei filter at 1 kHz, digitaed using the CED 1401
Plus interface at 5 kHz, and stored on cornputer for offiine analysis. Data
were coliected using the EPCJSignal (Episode based capture) or Spike2
(continuous recording) packages (CED, Cambridge, UK). Junction potentials
were accounted for by subtmcüng the offset membrane potential obtained
----
-
following entry of the electrode into the aCSF bath from the membrane
potentials recorded.
Statisücal analysis
All data are presented as means
(SEM).
+ the standard error of the mean
Comparisons between two groups were performed using the
student's paired t-test, with a significance level of ~40.05. In al1 cases,
definitive changes in membrane potential during current clamp recording of
>2mV were used as an arbitrary cut off for effects of IL4 P.
CHAPTER 3: RESULTS
A total of 113 isolated rat SFO neurons were recorded ftom in either
current clamp or voltage clamp mode. These cells had resting membrane
itn
potentials between 4 5 m V and -48mV, eiicited aco
potentials with
amplitudes of 75mV to 115mV. and had input resistances betwaen 0.85GQ
and 2.2GQ.
Response to IL-1g
Continuous current clamp recordings were obtained from 69 SFO
neurons to determine whether IL-lfi affected the cell's membrane potential or
firing ftequency (n=69). After maintaining a control period of stable baseline
recording for at least 120 seconds. IL-1p was administered by bath perfusion
for 120 seconds at concentrations ranging between I O û f M and 1nM followed
by a retum to aCSF. Based on the established criteria, 45 of 69 cells (65%)
were classified as being responsive to 11-1p in this effective dose range.
In accordance with reports of picomolar concentrations of 11-1fi in the
periphery during initiation of the immune response (Ma et al.. 2000). we
began our experiments with application of lOpM
11-18. This concentration
produœd a transient depolarkation in 7 of 10 cells tested, this group of
ig
responsive cells shown
a mean depolarnation of 57.19.mV
which occurred
within a range of 6 to 28sec following 11-1$ administration. This was followed
by a sustained hyperpolarization of 3.W-7mV in 6 cells. Similar transient
Figure 4: Current clamp recordings durlng application of IL4 6
Shown here are current clamp traces depicüng responses to 120 sec
application of 11-1B. 4A, B and C show transient depolarkations with reaivery
at low dose 5OOfM. IpM and 10pM 1143 respectiveiy. 4D shows a transient
depolarnationfollowed by a sustained hyperpolarization at 1OOpM 11-1 B. 4E
shows only the sustained hyperpolamation at high dose InM 11-1B. Dotted
lines represent resting membrane potentials.
Figure 5: Dose dependent changes in membrane potentia! following
application of IL-IB
Shown here are peak changes in membrane potential for neurons responding
with either a transient depolarization at low dose (100 5 0 o 11-1B. a
transient depolarkation followed by a sustained hyperpolarizationat mid dose
(1-100pM) IL4 B, and a sustained hyperpolarization at high dose (1nM) 1143.
-
depolarizations were also seen in response to IpM IL-lp in 7 of 9 cells tested
(mean depolarization 5.4f1.2mV; latency 43 to 100sec) and again. this was
followed by a sustained hyperpolarization in 5 cells (-4.211.lrnV). A further
reduction in the dose of 11-18 to 500fM also produced depolaritaüons in 8 of
12 cells tested with a mean depolarization of 6.4k9mV and a latency of 55 to
208sec. 2 of 4 cells responded with a depolarization of 31lmV following
applicaüon of 1OOfM IL-1P.
lncreasing the dose of IL-1s to high picomolar and nanomolar levels
results in concentrations that are seen in the periphery only during extreme
sepsis (Ebong et a/., 1999). lncreasing the dose of 11-18 to 100pM resulted in
a sustained hyperpolarization in 13 of 21 cells rather than the depolarization
obsetved at lower doses. The mean change in membrane potential was
-
8.8Il.OmV (n=13) with a latency within the range of 110 to 300sec and no
retum to baseline membrane potential. However, 10 of these 13 responding
neurons displayed a biphasic response as the hyperpolarizations were
preceded by transient depolarizations (mean 4.4I0.6mV; latency 6 to I6sec).
Further increasing the dose of IL-la ten-fold to InM resulted in a similar
sustained hyperpolarization without recovery in 8 of 13 cells tested (mean
5.lf 1BmV; latency 55 to 100sec). Application of vehicle did not produce a
response (n=3).
Bii
Figure 6: Current clamp recordings durhg application of IL-Ira and IL-1B
6A shows a current clamp trace of 120 sec application of IL-1 ra (open bars) and
mid dose (100pM) IL1B (shaded bars) responding with only a hyperpolarization
with no depolarkation preceding it 25pA hyperpolarizing pulses were given at
10 sec intervals to elicit the acüon potentials. 6Bi shows no response to 120
sec application of low dose @ O M M ) IL4 B following 60 sec pretreatment of ILIra. 6Bii shows a depolarnation after a second application of 50ûfM 1143 in
the same ceII as 6Bi. followÎngwash of Il-lra. Dotteâ lines represent r a n g
membrane potentials.
--
P
A
-
-
Receptor Specificity
IL-1ra is an endogenous competiüve antagonist of lL-1@as it binds to
the IL4 receptor and does not promote signaling (Dripps et al.. 1991). No
response to 1OOnM IL4 ra application was seen in 3 cells tested. We next
examined whether the IL-Ira would block the responses of SFO neurons to
IL-?P. Application of 1OnM or IOOnM IL-Ira 60 seconds before application of
1OOpM 11-18 blocked the depolarkation nomally observed in response to IL-
Ip, as
O of 7 cells responded following such pretreatrnent.
However.
hyperpolariration was still seen in 2 cells, with a membrane potential change
of 7.3I0.3mV occum'ng at 114 and l7Osec following IL1P administration (see
Figure 6). The maintenance of the hyperpolaritation in the presence of the
IL-1ra implies that acüvation of the IL4 p receptor is not responsible for this
response. These data do however suggest that depolarizations are 11-18
receptor mediated. a conclusion supported by the additional observation that
depolarking responses to 500fM 11-1f3 were not observed following
pretreatrnent with iOOpM IL4ra (n=4).
Effects on Spike Frequency
The rnajority of cells, which depolarked in response to IL-la, also
showed an increase in action potential fraquency. There was an overall
increase in spike frequency in the responsive celis that displayed
spontaneous, non-bursting spike profiles during lOpM to lOûfM 11-38
-
-
application (n=8) (pc0.05). The spike frequency dunng the 60-second control
period before application of the drug was 0.80I0.25Hz and this increased to
1.20*0.37Hz during the initial 3 minutes after 11-1 application. The peak
spike frequency within any single 20 second bin for each cell during the
control period was 0.88a.23Hz. and increased to 1.98I0.52Hz during 11-1p
application (n=8) (p<0.005).
Characteristics of Responsive Celb
Voltage clamp recordings obtained ftom al1 cells prier to testing with IL-
I p penitted a broad classification of recorded neurons based on the
expression of voltage gated potassium channels elicited in response to a step
protocol starting at -70mV and increasing by increments of lOmV for a
duration of 0.25sec.
One population of neurons exhibited a prominent
transient outward potassium current (h) which was either not observed or
was considerably smaller in the remaining cells which displayed primarily
more sustained potassium current (IK). The cells could be readily quantifieci
into these two populations by measunng the peak current and the steady
state current elicited by the step to -1 0mV (see Figure 7). Cells displaying a
peak-to-steady state ratio greater than 2 were temed dominant k (DIA) cells
and cells w i h a peak-to-steady state ratio less than 2 were classified as
dominant IK(DIK) celk. The DIK cells had a peak-to-steady state ratio of
1-39rt0.06(n=37), whereas the D k cells had a peak-to-steady state ratio of
Pk current = 1OOOpA
Pk current = 200pA
Steady-state current = 25ûpA
Steady-state current = 20ûpA
Pk/ss ratio = 4
>2 +DIA
Pkks ratio = 1
4 +DIIK
Figure 7: Voltage clamp recordinga indicate the presence of two populatîons
of SFO neurons
Cells were held at -70mV and as illusbrâted Ri Figure ?A. currents were elicitecl by
250msec voltage steps h m -70 to +30mV in 10mV increments. The trace on the
left depicts a typical dominant 1, cell (DM and the trace on the right depids a typical
dominant IKceIl (DiK). In 7B. the pie chart displays the ratio of DIAto DIKcells
(60%). The single traces show the 40mV Hep. where the peak C)€0steady
state C) ratio was determined. Cells with a peak-to-steady state ratio greater than 2
at this-10mV step were classified as Dl,celIs; those with a ratio less than 2 were
classifieci as DIKcells.
RespoOnve Ceiis
Non-Responsive Cells
Figure 8: Peak-to-steady state ratios of IL46 responsive and non-responsive
neurons
Shown here are the peak-to-steady states of cells within the effective IL4 LS
depolarizing dose range (100pM 100fM). 34 of 56 cells depolarized, 29 of which
were ctassified as DIKas they had a peak-to-steady state ratio less than 2. The
mean values ISEM for the responsive and non-responsive ceIl groups were
1-73I0.17and 2.78I0.35 respectively. The niset depids aie perœntage of
depolarking cells in the DlK(78%. 29 of 37 cells) and DIA(26%. 5 of 19 cells) œll
populations.
-
---
- - -- - -- -
-
3.57I0.34 (n=19).
Analysis of cells within the depolarizing dose range
(100pM-10ûfM 11-18, n=56) revealed that 29 of 34 cells (85%) responding to
IL-IP with depolarizations were DIK cells, suggesting that it is a particular
subpopulation of SFO neurons that respond to IL-1P.
Efkcts of Input Resistance
IL4p also influenced input resistance, as deterrnined by the application
of hyperpolarizing pulses during control and response periods. There was a
significant decrease in input resistanœ to 84.3k3.01 of the original control
value during the depolarizing response (n-8) (p<0.001). In some cases full
currenthdtage (VV) relationships were obtained by applying successive
hyperpolarizing pulses of decreasing magnitude (-25 to -5 PA) before and
after IL-le application, and measuring peak changes in membrane potential
(n=3).
The reversal potential estirnated from such IN relationships was
- 5 M .5mV suggesting potential effects on a non-seleciive cationic
conductance (INSC).
Non-Sefective Cation Channels
Voltage clamp experiments were undertaken to determine the idenüty
of the current underiying the efbcts of
11-48 observed in the DIKcells. Our
input resistance data and previous studies suggesting that depolarizing
effects of a variety of peptides are mediated by opening of non-selme
Figure 9: Voltage clamp recordings display activation of NSCC following
IL48 application
-
9A depictç the cunents pmduced by a 12mVlsec depolarizing ramp from
80mV to +20mV. Show hem is the control current (i),the cunent elicited by
500fM IL-1 B (ii)and the current bllowing IL4 i3 wash (iii). The differenœ
cunent between 9Ai and 9Aii Ïs displayed in the inset, revealing a linear current
within the range of -80 to -20mV. 9B shows the average dbrence current
obtained fiom üte 9 of 15 œk that responded to 500fM 11-1B duhg this slow
ramp protocol. The slope of this cumnt was O.GOrH).llpAlrnV and had a
reversal poîential of 38.8fj.8mV.
cation channels (NSCCs) (Washbum et ai., 1999; Oliet and Bourque, 1993;
Takano et al., 1994), led us to examine the effects of 11-18 on this current
assessed by the application of slow (i2mVlsec) depolarizing voltage ramps (80
- +20mV) (see Figure 9).
Bath application of 500fM I L 4 P produced an
increase in conductance over the voltage range in 9 of 15 DIK cells tested
(60%). The subtracted current was Iinear through the range of -80 - - 15mV,
indicaüng a non-rectifying current The slope was O.6OH.lZpAlmV and had a
mean reversal potential of -38.811.8mV.
Recovery was seen in al1 cells
following return to aCSF and the response was repeatable following
subsequent similar IL-lp applications (n-3).
This current was not seen in
response to 50UfM IL-1p in the presence of 1OOpM IL-1ra (mean slope
0.013I0.008pAlmV. n=4).
Potassium Currents
The above slow ramps also revealed th& the difference current
between control and 11-18 application was non-linear in the upper depolarized
voltage range (-20 - +20mV). suggesüng the influence of a rectifying current.
The input resistanœ data estimated a reversal potential lower than the
reversal potential elicited by the Iwo This introduced the possibility that a
potassium conductance was also involved. as this ion has a reversal potential
around -70rnV.
A previous study showing the efkct of activin 1 on FSH
tumor cells supported this hypothesis as these cells also responded with a
-
control
a
Figure 10: Voltage clamp recordinas display a decrease in 1, followinq IL1B application
A voltage clamp step protocol was used to isolate 1, by applying a 500msec
prepulse at 3 0 m V to inactivate .1, followed by 250msec steps from -70mV to
+30mV in the presenœ of TiX. 10A shows these voltage steps before (left)
and after (right) 5OOfM application of tL-18, revealing a decrease in 1,
Reaivery was not seen. 10B shows the current-voltage relationship of the 40f
7 cells that responded to 500 fM IL43. resulang in a significant decrease in
current at the + I O to +30mV range (*) (pc0.05). The inset shows single traces
befoe and alter 11-1[3 application at the +30mV Hep. At this step. there was a
significant decrease in current to 84.4&3.3% of control in the responsive cells
(pc0.005).
depolarkation and an increase in firing frequency while showing an increase
in lNsCand a decrease in IK(Takano et al., 1994). Therefore, to determine
whether a potassium conductance was involved. a voltage step protocol was
used in the presence of a sodium channel blocker. m(, to isolate both the IA
and IK currents and detemine the effects of IL-lp on these currents.
A
500msec prepulse at -30mV inadivated IA and was followed by 250msec
steps from -70mV to +3OmV in increments of 10mV. This isolated IK and 1
was quantifiad by measunng the mean steady state cunent elicited at the end
of the voltage step (see Figure 10). Following 500fM IL-IP, there was a
significant decrease in the IKto 84.4&3.3% of control in 4 of 7 (pc0.005) at the
+30mV step.
Conductance and slow capacitance rneasurements did not
change before and after drug application (190I50nS and 6.7k0.4pF
respecüvely, n=4). Although no recovery was seen from this decrease in
current, this effect was unlikely the result of cunent rundown as subtradion
of the IAshowed no significant effect on this conductance in response to
application of 11-18 (n=7). As well, there was no change in conductance and
access resistanœ values. No significant change was seen in DIK cells after
500fM 11-18 and 100pM IL-Ira administration (n=3) and conductance and
slow capacitanœ measurements again remained the same before and alter
dmg application (23W7OnS and 8.M.5pS respectiveiy. n=3).
CHAPTER 4: DISCUSSION
The resuits of this study support the argument that SFO plays a
primary role in fever induction, by showing that SFO neurons responded to IL1p in concentrations (1nM
- 100fM) that are similar to those seen in the
periphery doring an immune challenge (Ma et al., 2000).
Endogenous
concentrations of IL-IP in the circulation are very low, in the femtomolar
range. However, upon an immune challenge, concentrations rapidly increase
in the circulation, generated primarily by localized monocytes and
macrophages. If SFO is involved in sensing this initial peripheral response, a
rnodest increase in IL-1B should be suffcient to trigger activation of this
structure.
lncreasing the dose of Il-1p to InM did not produce the
depolarkations seen previously at the low doses, but rather, resulted in a
sustained hyperpolarization. Concentrations above 100pM I L 1p are rarely
seen within the circulation, although they could occur in the latter stages of
severe sepsis (Ebong et al., 1999), a condition associated with general
system shutdown and a significant decrease in blood pressure (Dinarello et
al., 1989). It can be speculated that since SFO is also intimately involved in
carûiovascular regulation (Mangiapane and Simpson, 1980; Smith and
Ferguson. 1997). this hyperpolarizing response could inffuenœ the
hypotension. Previous studies have shown that electrÏcalstimulaüon of SFO
is accompanied by an increase in blood pressure (Ferguson and Renaud,
1984). Correspondingly, the decrease in activity following high dose IL-If3
could correlate with a decrease in blood pressure. In this way. IL4P could
potentially mediate the pressor response
Observed
during sepsis by
decreasing the activity of SFO neurons and influencing their subsequent
effects on cardiovascular regulatory centres.
Having established the response of SFO neurons to dwerent doses of
IL-1P. we were interested in determining whether these effects were due to
activation of IL4p receptors. There are two receptors that bind 11-18: IL4RI
and IL-1RN. IL4RI is capable of signal transduction by activation of MAP
kinases and NFkB, which leads to subsequent gene activation (Sims et al.,
1993). Conversely, IL4RII. is unable to activate signal transduction
mechanisms because it lacks the extensive intracellular domain of IL-1RI
(McMahon et al., 1991). IL-Ira was applied along with IL-lp to determine
whether the responses would be blocked in the presence of the antagonist. A
previous study showed that simultaneous application of IL-ira with IL-Ip in
the POA significantly reduœd IL-Ip initiated firing of cold-sensiüve neurons
(Xin and Blatteis, 1992). IL-lra binds to IL4 RI and I L 1RI1 (Dripps et al.,
1991). although it lacks one of the two binding sites found on 11-18 required
for promoting receptor activity (Evans et aL, 1995). The inability of IL4p to
evoke a depolarkation in the presence of IL-Ira suggests that IL-Ira was
bound to IL-1P reœptors, blocking any possible initiation of response.
Therefore, we can hypotheske mat the depolarkation was mediated by 11-18
receptors.
However, it remains unclear as to the receptor mechanism
responsible for the hyperpolarization. The maintenance of this response in
the presence of IL-ira suggests that 11-18 recepton do not mediate the
hyperpolarization.
We speculate that tolClike receptor 4 (TLR4) maybe
involved, as ais receptor has similar homology to the IL-1p reœpton and is
also involved in the immune response. Recently, TLR4 was identified in SFO
(Laflamme et al.. 2001). Much is still unknown about this receptor and its
agonists, although it is activated by a variety of stimuli including both gram
negative and gram positive bacteria (Means et ai.. 2000). High dose IL-IP
(1nM) could potentially activate this receptor and promote signal transduction
resulting in hyperpolarization.
Unlike the depolarization. this hyperpolarization did not show recovery.
Aithough the physiological significanœ and the receptor through which this
response is mediateci are unclear. it appears as though the SFO neuron does
not require the continuous presenœ of IL48 to maintain the hyperpolarizing
response. This could be an efficient mechanism for sustaining neuronal
effects to IL-Ip during aie initiation and maintenanœ of fever. as IL-lp has
been shown to produce long lasting efkcts on neuronal activity (Jeanjean et
al.. 1995). Another possible explanation is that the ce11 requires a stimulus to
tum off the initial hyperpolarizing signal, which is not found in this isolated cell
preparation.
Perfiaps endothelia! derived IL-Ira (Wong et al.. 1995) is
required to bind to 11-18 receptors and stop the response. An aftemative
possibility is that within the in vitro environment, there is an imbalanœ in the
intemal concentrations of the cell, resulting in an inability to terminate the
signal.
Having established that the IL-lp mediated depolarkation was due to
interactions with I L l p receptors, we were interested in determining the
intrinsic conductances responsiblefor this response. Based on voltage clamp
studies, we believe that the depolarization is due to both an increase in
current produced by the opening of NSCCs and by a decrease in IK.
Peptide
induced changes in these conductances have previously been seen in activin
A activation of human FSH-secreting tumor cells, which also respond with a
transient increase in membrane potential and increase in spike frequency
(Takano et al.. 1994). Previous wotk in Our laboratory has identified NSCCs
in SFO neurons that are activated by the calcium receptor (CaR) (Washburn
et al., 1999). This CaR induced current was linear and had a depolarized
reversal potential as compared wioi the SFO neuron's normal resting
membrane potential. The INSCevoked by IL-IP in the present study was
again Iinear between -80 and -20mV (see Figure 9).demonstrating the nonrecüiing propeitîes.
This current had a reversa1 potential of -39mV,
indicating that at the resting membrane potential of these SFO neurons
(between -55 and 48mV). opening of these channels will pmduce an inward
current Afthough the
appears to be relativeiy small compared with the
Na' and K* conductances, previous studies have shown that it plays an
extremely important role in the regulation of membrane potential. suggesting
that any subtle modulation of lNsc
can mediate cellular responses (Bae et al.,
1999; Oliet and Bourque, 1993). In the present study, the theoretical change
in membrane potential following IL-1p application can be detemined by
taking into account the difference in resting membrane and reversal
potentials, the slope of the lNsc
elicited by IL-lp (see Figure 96) and the input
resistance. These mdimentary calculations resuk in depolarkations that are
similar to those actually seen during IL-1P application in current clamp mode
(approximately 10mV). This is of course making several general assumptions
including that the opening of the NSCCs was instantaneous at the resting
membrane potential and that the inward lNsc
was maximal throughout the
depolarization despite the change in the inherent driving force. It is apparent
can
Rom the results of this study that activation of the seemingly srnall lNsc
have significant effects on the membrane potential and firing frequency of the
neuron and thus potenüally on the physiological function of the cell.
Beyond the -20mV range, the difference current was no longer linear,
indicating the influence of a voltage dependent current The input resistance
data and the relatively high voltage activation of this current suggested the
involvement of potassium.
Subtraction protocols isolating both IA and IK
revealed that 11-18 evoked a significant decrease in lK- This current is
responsible for the outwerd rectification of the neuron following the initial Na*
influx. A decrease in this outward current hinders the repolarization of the
neuron during action potential recovery. This results in a slower retum to
baseline and a smaller after hyperpolarization, leading to an increase in
membrane potential.
This brings the neuron closer to its spike firing
threshold, resulting in increased firing ftequency.
The decrease in IKdid not rewver following IL4 P wash, indicating that
different, long lasting, cellular mechanisms are activated as compared with
those mediating the transient INsc.This lengthy effect is most likely produced
by second messenger systems, involving activation of NFkB or MAP kinases
(DiDonato et al., 1997), which are associated with IL4 RI. During current
clamp recording, qualitative observations revealed that the neuron was more
application and displayed an increase in spike
likely to burst following ILI@
broadening and a decrease in after spike hyperpolarization. The decrease in
IKresulting in a delayed repolarization could account for these observations.
In concert, the modulation of these two currents has profound effects
on the activity of the SFO neuron. The inward lNsc
and the decrease in IK
help bring the membrane potential closer to spike threshold, thus increasing
the action potentialfrequency. However, at potentials more positive than -39,
as seen in the differenœ current obtained frorn
there is a reversal of the lNsc
the voltage rarnp protocol (see figure QA). This outward current impedes the
depolarization, comtering the efkct that the decrease in IKhas on membrane
potential. As well, the resulüng decrease in K+ effîux reduces the inward
cationic dnving force of the INsC at potentials below -39mV.
Therefore, the
interactions of both these currents modulate the activii of the neuron.
In this study, we have shown that there is a direct effect of IL-lp on
neurons by modulation of intrinsic conductances. This is significant because
IL-IP effects have been primarily seen in endothelial rather than neuronal
cells. The binding of IL-IP at IL4 receptors on endothelial cells triggers
subsequent release of other messenger molecules. such as PGs, which
further promote the febrile response. Systemic iv iniection of recombinant rat
IL-1S resuited in a significant increase in COX-2 activation within endothelial
celîs (Lacroix and Rivest, 1998). However, the present study indicates that
IL-la also has direct effects on neurons. This activity allows rapid synaptic
communication with other areas within the brain, which would be appropriate
during the initiation of fever. Activation of the SFO neuron by IL-18 triggers
responses in the post-synaptic neuron by either direct modulation of ion
channels or by the activation of second messenger systems.
The rapid
increase in IL4 p mRNA within SFO following iv LPS administration (Eriksson
et al., 2000) suggests that IL-la could itseif act as a potential neurotransmitter
when released from the SFO neuron during an immune challenge. In this
way, IL4 p coufd interact with receptors on the post-synaptic cell and rnediate
actions important to further trigger the immediate febrile response.
The present study indicates that a particular population of SFO
neurons responds to IL-18 administration. This subset of neurons has a
dominant k which is essential for repolarizing the neuron after the initial Na*
influx. In contra* the k is very smaii in these cells. The relative magnitudes
of these potassium cunents act as electrophysiological markers in
detemining the IL4 P sensitive population of neurons.
However, having established that ütere are direct effects of IL4 p on a
parücular subset of SFO neurons. the question of where this IK dominant
population projects to remains unclear. SFO sends eferent projections to a
number of sites, although the most prominent are the AV3V and
neurosecretory areas of the hypothalamus. the PVN and SON.
These
regions are intimately involved in the febrile response (Elmquist et al., 1997;
Boulant. 2000; Matsunaga et al., 2001).
The specific AV3V projection sites of SFO are to MnPO (Miselis et al.,
1979). MePO (Lind et al., 7982). OVLT (Miselis et al., 1979) and MS (Lind et
al., 1982). POA plays an important role in the initiation of fever, as many of
the thennosensitive neurons are locateci here (Boulant, 2000).
These
neurons help maintain the hypothalamic temperature set point; peripheral
information is relayed to this œnter and influences neuronal activity,
producing an
integrated level of output to
prornote appropriate
therrnoregulatory responses. It is here where the set point can be modulated
during the febrile response by the cornpanson of excitatory and inhibitory
inputs from both the warm-sensitive and coki-sensitive neurons (Boulant,
2000). Specific messengers, including IL4 P and PGs, are required to change
these neurons' activity and initiate adjustment of the set point level. Several
studies have shown that local application of IL4 8 within MePO resulted in
increased firing of cold-sensitive neurons and decreased firing of mm-
sensitive neurons (Hon et al., 1989; Nakashima et al., 1989; Shibata and
Blatteis, 1991). It can be hypothesized that the SFO neuron releases IL-1P at
POA and triggers changes in the activity of the themosensiüve neurons. This
alteration in firing pattern results in suppression of heat loss and potentiation
of heat producing responses by downstream autonornic and endocrine
mechanisms (Boulant, 2000). IL-lp has also been shown to affect PG levels
within POA by significantly increasing COX-2 transcription (Rivest et al.,
2000) and release of PGE2 (Komaki et al., 1992). This suggests that IL-lp
released from the SFO neuron not only acts directly on the thennosensitive
neurons within the POA, but also promotes PO synthesis. Thus, both SFOderived
il-le,
as well as PGs. could influence the neuronal activity within
POA, leading to subsequent actions at efferent projection sites, where both
endocrine and autonomic acüvity could further promote the febrile response.
The OVLT is another possible projection site for IL-lp sensitive
neurons. A number of studies have suggested its involvement in the febrile
response, as it is an alternative proposed entry site to the CNS for circulating
11-18 (Blatteis and Sehic, 1997; Stitt, 1990). This CVO also sends projections
to the POA and produces PGE mediaton that are thought to diffuse to this
thermosensitnre area (SM, 1990). However, the study by Takahashi et al.
showed that lesioning of the OVLT did not e
M the febrile response induced
by iv LPS (1997), suggesting that this area is probably not innervated by the
IL-l$ sensiove SFO neurons.
The MS, which contains projections to both POA and PVN (Lind et al.,
1982). i
s another possible projection site for this subpopulation of neurons.
The polysynaptic pathways modulate the information at both the initial febrile
enter within POA as well as at the subsequent endocrine and autonornic
sites within PVN.
The SFO neurons influenceâ by IL-1P could also project directly to the
parvocellular neurons of the PVN. where they could influence subsequent
endocrine and autonomic rnechanisms to further promote the febrile
response. The activation of the HPA axis by enhancing the expression of
CRH within this nucleus has been shown to be important in the regulation of
the immune and febrile responses (Elmquist et al., 1997; Plagemann et al..
1998; Rivest et al*, 2000). IL-iP influences this HPA activation, as it is a
potent stimulator of CRH neurons (Berkenbosch et al.. 1987).
11-48
adrninistered into the PVN caused a fever of greater magnitude and duration
than when injected icv (Avitsur et al., 1997). However, icv injection of an
antagonist to CRH prevented IL-18 induœd fever (Rothwell, 1989).
emphasizing the importance of CRH to the febrile response.
hypothesized that the SFO
It can be
11-18 sensitive neurons project directly to
parvocelIular neurons m i n PVN, where neumtransmitters, including possibly
IL-18 itseff. bind to receptors on the putative CRH parvocellular neurons and
trigger the activation of aie HPA axis. Direct SFO projections to PVN could
also activate the autonomic response required for the production of fever.
These stimulated parvocelluhr neurons send efFerent projections to areas
Media!
Septum a
rn
D
-
4
EI
*HPA
autonomie responses
+ heat sensitive neuronal activity
4 cold sensitive neuronal activity
4
4
set-point temperature
-
FEVER
FlGURE 11: Potential projection sites for Il4B responsive SFO
neurons
Activation of the SFO by circulating 11-1I5 tnggen subsequent efkrent
projection sites tu promote the febde response.
within the spinal cord and medulla (Porter and Brody, 1985), which could
subsequently mediate sympathetic activity and the redistribution of blood fiow
to promote both heat retention and generation mechanisms.
Further investigations using cell labeling techniques are required to
detemine the projection site of this IL4P sensitive population of neurons.
Interestingly, preliminary work by our laboratory using retrograde fluorescent
beads injected into the PVN revealed that none of the fluorescently labeled
SFO cells projecting to this nucleus responded to IL4 P.
In summary, this study indicates that a subpopulation of isolated SFO
neurons, which has a dominant IK responds to IL-IP by activation of IL-lp
receptors, resulting in a depolarization and an increase in firing frequency.
This occurs at I L I P concentrations that correspond with those produced at
the onset of the immune response. immediately afier N LPS administration
(Ma et al., 2000). These results are consistent with the idea that circulating
IL-If3 activates the SFO eariy during an immune challenge. This activation
then induces further central mechanisms to cause an increase in central IL-1p
and PG concentrations, which subsequently promote the febrile response.
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