Download The Role of Interleukin-6 in the Febrile Response Namik Hamzic

Linköping University Medical Dissertation
No. 1349
The Role of Interleukin-6 in the Febrile Response
Namik Hamzic
Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
Linköping University, SE-581 85, Linköping, Sweden
Linköping 2012
Published articles and figures have been reprinted with the permission of the respective
copyright holder.
Printed in Sweden by Liu-Tryck, Linköping 2012.
ISBN: 978-91-7519-722-7
ISSN: 0345-0082
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To my family
If at first you don`t succeed, you are running about average
M.H. Alderson
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LIST OF ARTICLES
This thesis is based on the following papers, referred to in the text by their Roman numerals:
I.
Nilsberth C, Hamzic N, Norell M, Blomqvist A. (2009) Peripheral
lipopolysaccharide administration induces cytokine mRNA expression in the
viscera and brain of fever-refractory mice lacking microsomal prostaglandin E
synthase-1. J Neuroendocrinol 21(8): 715-721
II.
Nilsberth C, Elander L, Hamzic N, Norell M, Lönn J, Engström L, Blomqvist A.
(2009) The role of interleukin-6 in lipopolysaccharide-induced fever by
mechanisms independent of prostaglandin E2. Endocrinology 150(4): 1850-1860
III.
Hamzic N, Blomqvist A, Nilsberth C. (2012) Immune-induced expression of
lipocalin-2 in brain endothelial cells: relationship to interleukin-6,
cyclooxygenase-2 and the febrile response. J Neuroendocrinol doi: 10.1111/jne.
12000
IV.
Hamzic N, Tang Y, Eskilsson A, Kugelberg U, Ruud J, Jönsson JI, Blomqvist A,
Nilsberth C. (2012) Interleukin-6 produced by non-hematopoietic cells mediates
the lipopolysaccharide-induced febrile response. Manuscript.
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TABLE OF CONTENTS
ABBREVIATIONS
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ABSTRACT
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INTRODUCTION
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The acute phase response
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Fever
The historical view
The febrile phases elicited by LPS
The intracellular signaling of LPS
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14
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Proinflammatory cytokines
Interleukin-1
Tumor necrosis factor α
Interleukin-6
The structure of interleukin-6
The signaling of interleukin-6
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16
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18
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Lipocalin-2
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The communication between the immune system and the brain
The role of the circumventricular organs
Cytokine transport over the blood-brain barrier
Vagal signaling
The role of the blood-brain barrier
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Prostaglandin E2
The synthesis of PGE2
The role of PGE2 in fever
Prostaglandin E2 receptors and their implication in fever
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Fever generating pathways
The preoptic area in hypothalamus
The role of raphé pallidus nucleus and dorsomedial hypothalamus
The cooling-evoked thermogenesis – similarities to PGE2
The implication of proinflammatory cytokines in BAT thermogenesis
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AIMS
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METHODOLOGY
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Animals
Genetically modified animals
Generation of Interleukin-6 chimeric mice
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Administration of drugs
Parecoxib – selective Cox-2 inhibitor
Etanercept – the TNF-α suppressor
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Inflammatory model and monitoring of the body temperature
Lipopolysaccharide
Telemetry
Administration of recombinant interleukin-6
Intracerebroventricular administration of PGE2
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Protein analysis
Immunohistochemistry
Western Blot
Quantitative immunoassays
Bio-Plex Pro Cytokine Assay
Enzyme immunoassay
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Quantitative real-time PCR
TaqMan®-based detection
SYBR green-based detection
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Microarray technology
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RESULTS AND CONCLUSIONS
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The lipopolysaccharide-induced peripheral and central mRNA expression of
IL-1β, TNF-α and IL-6 occurs independently of prostaglandin E2 (paper I)
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The critical role of interleukin-6 in lipopolysaccharide-induced fever is not
exerted through prostaglandin E2 dependent mechanism (paper II)
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The identification of lipocalin-2 as a novel factor in the pathway of
inflammatory IL-6 signaling and its relationship to the
lipopolysaccharide-induced febrile response (paper III)
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Interleukin-6 synthesized by non-hematopoietic cells is important for the
development of the lipopolysaccharide-induced fever (paper IV)
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GENERAL DISCUSSION
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The implication of lipocalin-2 in fever and its connection with IL-6 and Cox-2
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The importance of hematopoietically-derived IL-6 in fever
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ACKNOWLEDGEMENTS
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REFERENCES
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ABBREVIATIONS
ABC
ACTH
AP
AP-1
BAT
BNST
cAMP
CD
CeA
CNTF
Cox
CRP
CT-1
CVO
DAMP
DAB
DMH
DMARD
ECSIT
EP
ES cells
G-CSF
GABA
gp130
Gi
Gs
HPA
HRP
IκB
IL
IL-6R
IL-1R
IL-1 Ra
IRAK
JAK
LIF
LBP
LC
lcn2
LPS
MAP
ME
MIP-1
MK2
MnPO
mPGES
MPO
mIL-6R
avidin-biotin-complex
adrenocorticotropic hormone
area postrema
activator protein-1
brown fat thermogenesis
bed nucleus of stria terminalis
cyclic adenosine monophosphate
cluster of differentiation
central nucleus of the amygdala
ciliary neurotrophic factor
cyclooxygenases
C-reactive protein
cardiotrophin-1
circumventricular organs
damage-associated molecular pattern
3.3´-diaminobenzidine tetrahydrochloride
dorsomedial hypothalamus
disease-modifying ant-rheumatic drugs
evolutionary conserved signaling intermediate in Toll pathways
prostaglandin E2 receptor
embryonic stem cells
granulocyte-colony stimulating factor
γ-amino butyric acid
glycoprotein 130
inhibitory G protein
stimulatory G protein
hypothalamic-pituitary-adrenal
horseradish peroxidase
inhibitory protein-κB
interleukin
interleukin-6 receptor
interleukin-1 type 1 receptor
interleukin-1 receptor antagonist
interleukin-1 receptor associated kinase
Janus kinase
leukemia inhibitory factor
LPS-binding protein
locus coeruleus
lipocalin-2
lipopolysaccharide
mitogen-activated protein
median eminence
macrophage inflammatory protein-1
MAPK-activated protein kinase-2
median preoptic nucleus
microsomal prostaglandin E synthase
medial preoptic area
membrane-anchored interleukin-6 receptor
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MyD88
NF-κB
NSAID
NTS
OSM
OVLT
PAMP
PB
PGE2
PI3K
PIAS3
PLA
POA
PRR
PVH
RA
RP
SAPE
sgp130
sIL-6R
SH2
SFO
SOCS
STAT
UCP-1
TAK1
TGF-β
TIR
TLR
TNF-α
TRAF6
TRP
VEGF
VGLUT3
VLM
VMH
myeloid differentiation factor 88
nuclear factor-kappa B
non-steroidal anti-inflammatory drug
nucleus of the solitary tract
oncostatin M
organum vasculosum of the laminae terminalis
pathogen-associated molecular pattern
parabrachial nucleus
prostaglandin E2
phosphoinositide-3 kinase
protein inhibitor of activated STAT3
phospholipase A
preoptic area
pattern recognition receptor
paraventricular hypothalamus
rheumatoid arthritis
raphé pallidus
streptavidin-phycoerythrin
soluble form of the gp130
soluble interleukin-6 receptor
src homology 2
subfornical organ
suppressors of cytokine signaling
signal transducers and activators of transcription
uncoupling protein-1
transforming growth factor β-activated kinase 1
transforming growth factor-β
Toll/IL-1R
Toll-like receptor
tumor necrosis factor-α
TNF-receptor-associated factor 6
transient receptor potentials
vascular endothelial growth factor
vesicular glutamate receptor 3
ventrolateral medulla
ventromedial hypothalamus
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ABSTRACT
Everyone who has been exposed to influenza or a bacterial infection knows how it feels to be
sick. Apart from not being willing to participate in social activities, losing your appetite and
experiencing pain, you have also most likely suffered from increased body temperature,
which defines fever, one of the most prominent signs of an acute ongoing infection. Invading
the body, the infectious microorganisms are combated by the activated innate and adaptive
immune systems, and the impaired balance is thus restored. While fever is an event that is
controlled by the central nervous system, it has long been debated how the inflammatory
signals generated in the periphery communicate with the brain that is protected by the bloodbrain barrier that prevents large molecules such as cytokines from entering into the brain
parenchyma.
Previous studies from our group have provided evidence in support of the existence of a
pathway across the blood-brain barrier by demonstrating that proinflammatory cytokine
interleukin-1 transfers the inflammatory message to the brain through binding to its receptors
situated in the brain vessels. This will subsequently trigger the production of the prostaglandin
E2 (PGE2) that enters the brain and exerts its effect by binding to the receptors located on the
thermoregulatory neurons. Interleukin-6 (IL-6) is another cytokine essential for fever
signaling; however, the mechanism has not yet been identified. The research on which this
thesis is based aimed at elucidating the role of IL-6 in inflammatory induced fever.
In paper I, we demonstrated that mice incapable of producing inflammatory PGE2 still
responded with an intact cytokine production in the brain upon peripheral LPS-stimulation.
Thus, although the mice had induced expression of inflammatory cytokines in the brain, this
was not sufficient for a fever response without simultaneous production of PGE2. The
relationship between IL-6 and PGE2, both essential for fever, was further investigated in
paper II, focusing on clarifying the mechanism by which IL-6 controls fever. We
demonstrated that mice deficient in IL-6 did not respond with fever upon peripheral LPSadministration despite an intact expression of PGE2 in the brain. In contrast, upon
intracerebroventricular administration of PGE2 into the brain, a dose-dependent fever
response was monitored in IL-6 deficient mice. Thus, we suggest that IL-6 exerts its effect
neither up- nor downstream from PGE2, and propose instead that IL-6 may act alongside the
PGE2 and regulate the process that deals with the transport of and binding of PGE2 onto its
receptors. To further investigate the elusive role of IL-6 in fever, we performed a microarray
analysis to identify the genes that were differentially expressed in the brain of LPS-challenged
IL-6 deficient mice compared to wild-type mice (paper III). We demonstrated that mice
lacking IL-6 displayed two-times lower expression of lipocalin-2 in the hypothalamus. IL-6
and lipocalin-2 were directly related to each other since peripherally administrated IL-6
induced the expression of lipocalin-2 in cells associated with the brain vessels. Lipocalin-2
induced by LPS was expressed by brain endothelial cells and partly co-localized with
cyclooxygenase-2, one of the enzymes essential for inflammatory PGE2 production in the
endothelial cells. We also demonstrated that lipocalin-2 in a sex-dependent and ambient
temperature-specific manner may be implicated in thermogenesis. We have thus identified a
new factor in the IL-6 regulated fever pathway, but the pathway is still not understood. One
important question that remained to be answered was in which compartment IL-6 was needed
for the signaling. This question was studied further in paper IV, where we investigated the
role of hematopoietically produced IL-6 in fever by constructing chimeric mice. We
concluded that IL-6 produced by cells of non-hematopoietic origin is critical for the LPS-
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induced fever while hematopoietically produced IL-6 plays only a minor role in contributing
to fever.
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INTRODUCTION
We are unavoidably and constantly being exposed to potentially harmful pathogens such as
viruses, bacteria, fungi or protozoa yet we are often completely unaware of this ongoing
threat. When left unchecked, these potentially infectious organisms may invade the body and
compromise the health and survival of the host. In order to protect us from these harmful
microorganisms, nature has endowed us with an immune system consisting of two branches:
the innate and the adaptive immune systems. As indicated by their names, the innate immune
system represents the inborn and evolutionarily old defense whereas the adaptive immune
system develops during our whole life-time. Although these two branches of the immune
system act in different ways, they are still related to each other and are highly cooperative in
acting to neutralize the invading pathogens (Medzhitov and Janeway, 1997). The innate
immune system, representing the first line of defense, acts in a non-specific manner and
provides an immediate response against the foreign intruders, but it gives no information
about the earlier presence of the pathogen in the body meaning that it does not confer any
protective immunity on the host. The cells of the innate immune system that become activated
during an inflammatory response include mast cells, eosinophils, macrophages, dendritic cells
and natural killer cells. However, in certain cases the pathogen manages to escape the
defensive actions of the innate immune system, and this event will trigger the adaptive
immune system. In this case, the innate immune system will inform the adaptive immune
system about the nature of the infiltrating pathogens through a process known as antigen
presentation by specialized antigen-presenting cells, the most important of which are the
dendritic cells. Following the activation of the adaptive immune system, immune cells such as
T-and B lymphocytes, which are specialized to recognize and eliminate foreign invaders, are
activated. The adaptive immune system remembers that it has seen the pathogen before and
can subsequently mount a stronger response if the same pathogen is detected again, hence
providing us with acquired immunity against the specific pathogen.
The recognition of the pathogens by the innate immune system relies on the cells equipped
with pattern recognition receptors (PRRs) that can be expressed on the cell surface,
intracellularly or released into the blood (Medzhitov and Janeway, 1997). There are several
families of PRRs all able to identify the structurally conserved products among the microbial
organisms, collectively known as pathogen-associated molecular patterns (PAMPs).
(Medzhitov and Janeway, 1997). The PRRs have also been shown to recognize the
endogenous cell components released by stressed, damaged or dead tissues and as a result of
this recognition activate the immune system. These components are known as damageassociated molecular patterns (DAMPs) and include heat-shock proteins, nucleotides,
extracellular-matrix breakdown products, neuromediators and cytokines (Gallucci and
Matzinger, 2001, Kono and Rock, 2008).
The acute phase response
The maintenance of the stable internal environment termed “homeostasis”, controlled by the
autonomic nervous system, is a necessary property for the survival of the host and was
already recognized by Claude Bernard in the middle of the 19th century. The innate immune
system, which is always awake through the PRRs sensing of the PAMPs works silently to
maintain homeostasis. However, upon entrance of the harmful agents, the PRRs are highly
activated and an inflammatory response, which mostly remains local, is developed resulting in
a subsequent release of the proinflammatory cytokines and the synthesis of cyclooxygenases
(Cox) (Janeway and Medzhitov, 2002). In cases of more severe infection, a rapid immune
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response, often referred as the acute phase response, results (Hart, 1988, Konsman et al.,
2002). This response comprises an organized constellation of disease signs, including fever,
activation of the hypothalamic-pituitary-adrenal (HPA) axis, increased pain sensitivity,
sleepiness, social avoidance, decreased food intake and increased release of sympathetic
hormones. These inflammatory responses are necessary for the protection and survival of the
host but may become destructive during sustained inflammatory conditions or when they no
longer can be regulated. The febrile response is the most manifest sign of infection but is also
a well-known element of many immunological and inflammatory responses, which makes this
non-isolated event of interest in studying the brain-elicited acute phase response.
Fever
The historical view
Fever has been identified as a hallmark of disease since ancient times and can be defined as
the rise in body temperature due to elevated thermoregulatory set-point. As such, it is to be
separated from hyperthermia that occurs when the body is overwhelmed by excessive external
heating without being the consequence of an elevated set-point. Fever is a highly conserved
response and is nowadays among the most common reasons for people to ask for medical
care. Whether fever is harmful or beneficial has been debated for decades and there still are
arguments in favor of both views. Feared by most of the people among the oldest
civilizations, fever was apprehended as a punishment from God, caused by the evil spirits
(Hart, 1988). Hence, one of the earliest treatments of fever included exorcism. Whereas the
Greeks interpreted the appearance of disease as an imbalance among the four body fluids
(blood, phlegm, yellow bile and black bile well corresponding to the air, water, earth and fire)
with fever being particularly associated with overproduction of the yellow bile, the European
population assigned fever as a “death marker” during an epidemic that harvested
approximately 25 million lives (Atkins, 1982). Along with the discovery of blood circulation
in the 17th century, medical thinking about fever among physicians divided them into two
opposing camps. While fever was considered by one group to be a consequence of blood
friction through the body and was proposed to be implicated in maintaining temperature level,
others believed that fever was generated by a fermentation process in the blood. During the
course of the 18th century, different kinds of fever based on the external symptoms were
described. Moreover, the new physiology of disease was constructed on the basis of the
discovery by the French physician, Broussais that the various changes in the diseased tissues
possibly corresponded with the different manifestations of the febrile response (Atkins, 1982).
In the 19th century, fever was still considered both as a symptom (as it is viewed today) and,
as a disease itself. However, the involvement of the central nervous system in the basal
thermoregulation and in the pathogenesis of fever was not revealed until the end of 19th
century. Von Liebermeister was the first to declare that fever arises from an illness that “sets”
body temperature to a higher level. Shortly after this discovery, the French physiologist,
Claude Bernard proposed that maintenance of the normal temperature was achieved by the
balanced actions of heat production and its dissipation and showed that a temperature rise of
5-6°C had detrimental effect on the survival of the exposed animals, thus suggesting that
fever could be harmful. Conversely, several years later William Welch showed that fever
itself either could destroy the invading pathogens or assist the host in its defense against the
infection, thus suggesting the febrile response to be beneficial. However, the biological role of
fever in disease was recognized a long time ago by many ancient physicians, including
Hippocrates, who believed that fever was beneficial to the infected host. Because of this
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common belief at that time, various diseases, including epilepsy, syphilis and gonorrhea were
treated by inoculation with fever-inducing malaria parasites (Atkins, 1982, Hart, 1988). The
beneficial role of fever has been associated in several studies with the improved survival in
many species, both endothermic (warm-blooded) and ectothermic (cold-blooded). The
survival of the lizards, Dipsosaurus dorsalis, (ectothermic) was demonstrated to be
significantly improved with the development of the febrile response as a result of bacterial
infection (Kluger et al., 1975). In good accordance with these findings, the mortality of
infected lizards increased when they were physically prevented from seeking the preferred
temperature or when treated with antipyretic drugs (Bernheim and Kluger, 1976). The
increased mortality has also been observed in various endothermic vertebrates. In a study by
Vaughn et al. employing rabbits, an infection-induced fever was observed, that when
attenuated with the administration of antipyretic drugs, resulted in a lethal outcome (Vaughn
et al., 1980). However, the mechanisms behind the advantageous effect of infection-induced
fever have not yet been clarified. It has been suggested that fever causes the rise in body
temperature when the pathogens no longer can function properly and enhances the efficacy of
the innate immune system by optimizing the conditions for the defensive cells and enzymes
(Kluger, 1991). Hence, when cultivated at temperatures above 37°C, the Paracolobactrum
ballerup, a Gram-negative bacterium was shown to become markedly sensitive to serum
(Osawa and Muschel, 1964) and the replication of many viruses was reported to be decreased
at temperatures above 40°C (Lwoff, 1959). Furthermore, the bactericidal activity of
antibiotics was shown to be significantly increased in vitro as the temperature was elevated
(Mackowiak et al., 1982). The temperatures within the febrile range have also been believed
to enhance the activity of leukocytes (Grieger and Kluger, 1978). More recently, several
studies have provided substantial evidence that febrile temperatures recruit the migration of
circulating lymphocytes across high endothelial venules in the peripheral lymphoid tissues,
where the invading pathogens are being combated more efficiently (Evans et al., 2000, Chen
et al., 2004, Appenheimer et al., 2005) and importantly, the trans-signaling of interleukin-6
(IL-6) during fever has been demonstrated to enhance this trafficking process (Chen et al.,
2004, Chen et al., 2006). However, although the beneficial role of fever is supported by many
studies, the presence of too high fever can be dangerous to the infected host (Kluger et al.,
1998). In a study employing rabbits infected with Pasteurella multocida, the moderately
enhanced body temperature was shown to have a favorable effect on survival whereas the
high-range febrile response was associated with decreased survival (Kluger and Vaughn,
1978). In addition, an elevated body temperature has been associated with harmful outcomes
and an increased mortality rate in the case of experimentally-induced brain trauma (Diringer
et al., 2004). However, given the aid now provided by various antibiotic compounds, the
biological value of fever reflected by its antibacterial and immunostimulant effects is no
longer considered as a crucial part of present day healthcare.
Experimentally, the febrile response is often evoked by injection of lipopolysaccharide (LPS)
a toxic and heat-stable protein which comprises the main surface component of the endotoxins
derived from the Gram-negative bacteria such as Escherichia coli, Salmonella and Neisserie
(Brooks et al., 2004). Upon destruction of the bacterium, LPS (a term used here
interchangeably with the term endotoxin) is released and a large part of the LPS-elicited
febrile response has been ascribed to the increased production of proinflammatory cytokines.
Because its effects imitate the consequences of infection, the administration of LPS has been
widely used as a well-established model for studying the febrile response and for this reason a
brief overview of its fever-inducing effect will be given below.
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The febrile phases elicited by LPS
The patterns of the LPS-associated thermoregulatory response have been characterized and
are now recognized to be influenced by several different factors, some of them including the
ambient temperature in which the study has been performed and the dose and administration
route of LPS employed to induce the elevation of the body temperature (Romanovsky et al.,
2005). Whereas low dose of systemically administrated LPS (10 µg/kg) at the thermoneutral
conditions (30±1°C) causes monophasic fever, the moderate dose of LPS (50-100 µg/kg), thus
being within the range of the dose used in the study for paper I-IV, was shown to produce the
febrile response consisting of at least three sequential elevations in body temperature, the so
called febrile phases in rats (Romanovsky et al., 1998, Steiner et al., 2005a) and mice (Rudaya
et al., 2005). Whereas the first febrile phase normally occurs within 30-50 min after the
systemic administration of LPS (Rudaya et al., 2005), the second phase has been observed to
peak approximately 2 h after LPS challenge, being followed by the third phase 4 – 5 h after
LPS as shown in papers I, III, and IV but also by others (Chai et al., 1996, Rudaya et al.,
2005). Within this context, it should also be mentioned that an appearance of the first phase
has been shown in many studies including the studies for this thesis to be masked by the
stress-induced hyperthermia associated with the intraperitoneal injection of LPS. A typical
polyphasic fever response in mice at the thermoneutral conditions elicited by intraperitoneal
injection of LPS is shown in figure 1.
Fig. 1. Body temperature of wild-type mice (WT) injected intraperitoneally with LPS or
saline. Mice that have been implanted with the transmitters recording their body
temperature were injected with LPS or saline at time point 0. For more details, see text.
Since LPS has been used as stimulus to elicit the febrile response in our research a brief
overview of its signaling will be given below.
The intracellular signaling of LPS
Once pathogens have circumvented the protective barriers and invaded the host, the body
turns all efforts to combating and neutralizing them. The mechanisms for microbial
recognition long remained unknown, and it was not until 1996 that a receptor for Toll was
shown to be involved in the host defense of adult Drosophila, as their survival after a fungal
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infection was reduced following mutations in the Toll signaling pathway (Lemaitre et al.,
1996). A family of human Toll-like receptors 1-5 (TLR1-5) structurally related to Drosophila
Toll was discovered in 1998 and was recognized as representing human counterparts to the fly
molecule, thus revealing an important component of innate immunity in humans (Rock et al.,
1998). Among these receptors, TLR4 was shown to induce the expression of inflammatory
cytokines controlled by nuclear factor-kappa B (NF-κB) (Medzhitov et al., 1997). However,
the agonist was not known and a major step was taken in understanding how bacteria could
trigger the immune system and cause illness with the discovery that TLR4 was responsible for
the lack of response to LPS in mice (Poltorak et al., 1998a, Poltorak et al., 1998b).The
importance of TLR4 as critical for in vivo response to LPS was later confirmed by the
unresponsiveness to LPS in TLR4-deficient mice and the finding that this defect could be
restored in these mice by reintroducing the wild-type copy of the disrupted gene (Hoshino et
al., 1999). Thus, the TLRs, which appear to be well conserved throughout evolution from flies
to humans, were suggested to collectively act as recognizers of the invading pathogens and
provide the critical link between the immune stimulus and cellular initiation of the innate host
defense. So far, 11 TLRs have been identified in human and mouse. The different TLRs
diverge from each other in the specificity for a particular microbial product, the cell-type
specific expression patterns but probably also in induction of the targeted genes. Thus, TLR2
has been implicated, in most cases by acting in concert with TLR6 or TLR1, in the
recognition of bacterial lipoproteins (Aliprantis et al., 1999, Brightbill et al., 1999) and a
gram-positive bacteria component called peptidoglycan (Takeuchi et al., 1999, Ozinsky et al.,
2000). In addition, TLR3 has been implicated in the recognition of double-stranded viral RNA
(Alexopoulou et al., 2001), TLR5 in the recognition of bacterial flagellin (Hayashi et al.,
2001), TLR7 and 8 in recognizing single-stranded viral RNA (Heil et al., 2004), TLR9 in
response to CpG DNA (Hemmi et al., 2000) and TLR11 in the recognition of profilin
(Yarovinsky et al., 2005).
In 1990 it was shown that LPS binds to a liver-produced acute phase protein, (LPS-binding
protein; LBP) that interacts with a receptor of cell surface protein CD14 (Wright et al., 1990)
and may induce the immune response. Later on, it was proposed that the role of CD14 was to
load LPS onto the glycoprotein MD-2, which was identified as a link between LPS signaling
and the TLR4 (Shimazu et al., 1999).
The intracellular signaling cascade of TLR4 involves the intracellular domain, named Toll/IL1R (TIR) domain, which upon receptor activation associates with the myeloid differentiation
factor 88 (MyD88) that serves as an adaptor protein and recruits the interleukin-1 receptor
associated kinase 1 (IRAK-1) and IRAK-2 to the receptor complex (Muzio et al., 1997,
Wesche et al., 1997, Burns et al., 1998, Medzhitov et al., 1998). Following phosphorylation,
IRAK-1 becomes dissociated from the receptor complex and interacts with TNF-receptorassociated factor 6 (TRAF6) (Cao et al., 1996b), which in turn associates with and activates
the mitogen-activated protein (MAP) kinases (Ninomiya-Tsuji et al., 1999). This leads
subsequently to phosphorylation of the inhibitory protein-kappa B (IκB) and activation of the
NF-κB or the activation of p38 and c-jun N-terminal kinase (JNK) pathway (Shirakabe et al.,
1997) that in turn results in the formation of the activator protein-1 (AP-1) complex
(Krappmann et al., 2004). Furthermore, it has been proposed that an adapter protein ECSIT
(evolutionary conserved signaling intermediate in Toll pathways) interacts with TRAF6 and is
implicated in the signaling to NF-κB via MEKK1 (Kopp et al., 1999). As a result of the LPSinduced signaling, the activated NF-κB and AP-1 can translocate into the nucleus and induce
the transcription of cytokines, such as IL-6, interleukin-1β (IL-1β) and tumor necrosis factorα (TNF-α) as well as prostaglandin E2 (PGE2) synthesizing enzymes.
15
Proinflammatory cytokines
Cytokines are large proteins that primarily regulate and coordinate the immune responses in
the nervous system and the immune system. They are involved in regulating the growth and
differentiation of the cells, mainly exerting their effect in an autocrine or paracrine manner.
They are also synthesized, secreted and active in the inflammatory processes, including fever,
anorexia and malaise and are highly inducible in response to pathogens and stress stimuli.
Thus, not only do they regulate the local processes but they also act more systemically in an
endocrine manner.
Three of the most investigated proinflammatory cytokines are IL-1β, TNF-α and IL-6. It is
generally accepted that these cytokines are produced locally at the site of inflammation and
leak into the bloodstream to be transported to target the distant structures and trigger the
febrile response. However, although the contribution of these cytokines to fever has been
addressed by numerous studies, the results obtained have often been of conflicting nature,
thus making it hard to interpret their importance. There are several reasons that could explain
the inconsistency between the different studies. The available studies diverge both in terms of
the examined species (human, rat, mouse or rabbit) and in the models of inflammation
employed (localized or systemic, and in the administration of recombinant cytokines or
bacterial fragments). In addition, the degree of purity and biological activity of the cytokines
employed as well as the characteristics of different batches and serotypes of LPS can diverge
even though the same inflammatory model and species are being used. Furthermore, the lack
of a particular cytokine and its importance in fever may be difficult to interpret because of the
compensatory mechanisms that may be exerted by other cytokines that take over the function
of the deleted cytokine. For this reason and because the expression levels of these cytokines
were examined in mice that were unable to produce fever to LPS in paper I, I briefly
summarize their individual role in fever below, particularly focusing on IL-6, which is the
main topic in this thesis work.
Interleukin-1
The IL-1 family consists of 11 members, some of which have been described as proinflammatory inducing local and systemic inflammation such as IL-1β, whereas others
(interleukin-1 receptor antagonist; IL-1 Ra) have been shown to protect the infected host
against inflammation, thus exerting anti-inflammatory effects. In 1985, IL-1α and IL-1β were
cloned and sequenced as two separate cDNA encoding proteins, sharing 30 % homology
(March et al., 1985) and binding to the same receptors, which later were identified as
interleukin-1 type 1 receptor (IL-1R1) and interleukin-1 type 2 receptor (IL-1R2) (Sims et al.,
1988). Whereas the former receptor (IL-1R1) has been shown to be the receptor responsible
for mediating all of the actions of IL-1β (Sims et al., 1993), the latter (IL-1R2) has been
identified as a decoy receptor incapable of transducing the biological signal upon ligand
binding (Colotta et al., 1993). In contrast to IL-1α that primarily has been attributed to the
intracellular signaling (Auron et al., 1987) and considered to play a minor role as an
endogenous pyrogen in the immune-to-brain signaling (Kluger, 1991), IL-1β is an important
mediator of the inflammatory response. IL-1Ra has been demonstrated to competitively bind
to the IL-1R1 and block the activity of both IL-1α and IL-1β (Carter et al., 1990, Eisenberg et
al., 1990, Eisenberg et al., 1991, Granowitz et al., 1991). IL-1β is synthesized as an inactive
precursor and converted into a mature IL-1β protein through proteolytical cleavage by
caspase-1 (Cerretti et al., 1992, Thornberry et al., 1992) but has also been reported to be
generated by proteinase-3 mediated cleavage (Coeshott et al., 1999, Greten et al., 2007). After
16
binding to the IL-1R1, the IL-1β induces an intracellular cascade similar to that occurring
after activation of the TLR4 since both receptors share the same intracellular TIR domain
(Poltorak et al., 1998b, Dinarello, 2009), thus serving as the unifying linkage that is of
considerable interest for the pathogenesis of fever. However, the cellular responses produced
by these two receptors may differ.
The historical background in support of the view of IL-1β as a fever-inducing pyrogen comes
from the studies in which the administration of recombinant IL-1β directly into the circulation
or peritoneal cavity was shown to induce fever in various species (Dinarello et al., 1986,
Alheim et al., 1997, Saha et al., 2005), and the reported attenuation (although not elimination)
of the LPS-induced febrile response following peripheral or central treatment with IL-1Ra
(Luheshi et al., 1996, Dinarello, 2004). Although the systemic fever-inducing effects of IL-1β
have mostly been studied in animals there are a considerable number of studies on IL-1β
effects in humans. The febrile response has, for instance, been reported to occur in human
cancer patients injected systemically with very low doses of IL-1β as a part of the
antineoplastic treatment (Tewari et al., 1990) and IL-1β was later reported to induce fever in a
dose-dependent manner in patients with bone marrow failure (Nemunaitis et al., 1994). IL-1β
has also been associated with several highly febrile diseases such as Still`s disease since the
injection of IL-1Ra resulted in the complete abolishment of fever and symptoms characteristic
for this condition (Rudinskaya and Trock, 2003, Fitzgerald et al., 2005). Furthermore, the
dysregulated secretion of IL-1β caused by mutations in the genes governing the activation and
production of IL-1β and resulting in the elevated plasma levels of IL-1β has been implicated
in various genetically inherited febrile diseases, such as familial Mediterranean fever, which
is characterized by intermittently irregular bouts of fever that are completely abolished by the
drugs antagonizing the action of IL-1β (Dinarello, 2004). It can thus be concluded that IL-1β
by itself can induce fever.
On the other hand, the contribution of IL-1β to the endotoxin-induced pyresis has been
questioned and accumulating evidence has indicated that endotoxin-elicited pyresis is not
dependent on endogenously produced IL-1β. Accordingly, IL-1β could not be detected in
plasma of the human patients injected with LPS and even in cases when IL-1β was detected, it
was found in concentrations that are below those required for the induction of fever when
being injected artificially into the circulation (Kluger, 1991). Furthermore, the febrile
response elicited by LPS in humans remained intact following pretreatment with IL-1Ra,
although a number of various subjective symptoms associated with the LPS was decreased
(Granowitz et al., 1993, Van Zee et al., 1995, Preas et al., 1996) and the abatement of fever
was not observed in a patients suffering from severe sepsis or septic shock following
treatment with IL-1Ra (Fisher et al., 1994, Opal et al., 1997). This illustrates the complexity
of the fever response suggesting that different pathways may lead to the same response.
With the aid of genetically modified mice, the role of IL-1β in fever has been suggested to
depend on the type and route of inflammatory stimuli used to provoke the febrile response.
Thus, whereas the febrile response in mice deficient in IL-1β or IL-1R was unaltered or even
augmented following challenge with low dose of peripheral LPS (Alheim et al., 1997, Labow
et al., 1997) these mice were resistant to the fever-inducing effect of Listeria Monocytogenes
and turpentine (Zheng et al., 1995, Leon et al., 1996, Horai et al., 1998, Kozak et al., 1998), a
compound frequently used as a model of local inflammation. However, it should be
mentioned that an intact LPS-induced fever response observed in mice with deletion of IL1R1 might likely be due to the functional redundancy exerted by TNF-α since the blockade of
17
TNF-α could attenuate fever in mice deficient in IL-1β but not in wild-type mice (Bluthe et
al., 2000).
Taken together, the data dealing with the role of IL-1β in mediating the febrile response in
experimental animals suggest that endogenously produced IL-1β is important as a local
mediator of inflammation whereas its role in the systemic inflammatory response is not so
well-established and requires further investigation.
Tumor necrosis factor
The history of TNF begins with the discovery of a factor called lymphotoxin produced by
lymphocytes and recognized to exert cytotoxic effect on malignant mesenchymal tumor cells
(Kolb and Granger, 1968). Several years later, another factor possessing the similar cytotoxic
activity as lymphotoxin was found to be released from macrophages upon treatment with
endotoxin and was because of its observed tumor necrotic activity named TNF (Carswell et
al., 1975). However, when the cDNA encoding these two factors was cloned, a 30 %
homology was observed explaining some of their overlapping biological activities (Pennica et
al., 1984). Later, based on the fact that the genes for lymphotoxin and TNF were found next to
each other, it was suggested that they probably have evolved by gene duplication (Ware et al.,
1992). Furthermore, the activities of TNF-α and cachectin, a protein described to be involved
in mediating cachexia, was found to be attributable to the same protein (Beutler et al., 1985).
TNF-α is mainly synthesized as a homotrimer in a transmembrane-integrated form
(memTNF) at the cell surface of activated monocytes (Kriegler et al., 1988) but also by other
cells such as NK-cells, mast cells, T and B cells and granulocytes (Conti et al., 1991,
Baumgartner et al., 1996, Echtenacher et al., 1996). The soluble form of TNF (sTNF) can be
proteolytically cleaved from the extracellular domain of the memTNF by metalloproteinase
TNF-α-converting enzyme (Black et al., 1997), and has been recognized to neutralize the
circulating TNF, thus acting as an inhibitor of the TNF activity (Himmler et al., 1990). TNF-α
can interact with two receptors, TNF receptor type-1 (TNF-R1) or TNF receptor type 2 (TNFR2) and induces a variety of effects. In contrast to TNF-R1 which has been reported to be
expressed by almost all tissues, the distribution of TNF-R2 is tightly controlled and restricted
to the lymphoid tissue (Grell et al., 1998).
Studies examining the role of TNF-α in fever, have yielded conflicting results. The biphasic
dose-dependent febrile response accompanied by increased levels of PGE2 in the
cerebrospinal fluid was observed following intravenous injection with TNF (Nakamura et al.,
1988, Kawasaki et al., 1989, Morimoto et al., 1989). In line with this, the fever-inducing
effect of LPS and turpentine was suppressed by pretreatment with a TNF-α antiserum (Cooper
et al., 1994, Luheshi et al., 1997). In contrast, an attenuation of fever to LPS was seen when
TNF-α was injected intraperitoneally while the administration of sTNF or TNF-α antiserum
resulted in the enhanced fever response (Klir et al., 1995, Kozak et al., 1995), suggesting that
TNF-α exerts an anti-pyrogenic effect. Moreover, the febrile response following sepsis was
unaffected in studies utilizing mice deficient for TNF-R (Leon et al., 1998).
Interleukin-6
The molecule that we now call IL-6 was originally given various designations by different
groups, designations including interferon-β2 (IFN-β2), B cell stimulatory factor 2 (BSF-2),
hybridoma growth factor (HGF) and hepatocyte-stimulating factor (HSF), each reflecting the
18
different characteristics of the protein. These factors seemed unrelated at first, but the cloning
of their cDNA revealed without a doubt that they were identical (Hirano et al., 1985,
Haegeman et al., 1986, Hirano et al., 1986, Andus et al., 1987, Brakenhoff et al., 1987,
Gauldie et al., 1987, Van Damme et al., 1987).
IL-6 is a pleiotropic cytokine with a wide range of effects in inflammation but also in
regulating the processes associated with the immune system as well as in processes that are
not coupled to the immune system. Thus, IL-6 is implicated in the differentiation of activated
B cells into antibody-secreting cells (Hirano et al., 1986), activation and proliferation of T
cells (Ceuppens et al., 1988, Uyttenhove et al., 1988), induction of primitive hematopoietic
stem cells into a cell cycle (Ikebuchi et al., 1987), macrophage differentiation (Miyaura et al.,
1988, Chiu and Lee, 1989), release of adrenocorticotropic hormone (ACTH) from the anterior
pituitary (Naitoh et al., 1988) and ACTH-stimulated release of corticosterone from the adrenal
gland (Salas et al., 1990), proliferation of fibroblasts and vascular smooth muscle cells via
induction of plateled-derived growth factor (Nabata et al., 1990, Ikeda et al., 1991),
permeability of endothelial cells (Maruo et al., 1992), and regulation of vascular endothelial
growth factor (VEGF) (Nakahara et al., 2003). Additionally, IL-6 has been shown, in contrast
to IL-1 and TNF which only showed moderate effect on the induction of the acute phase
response (Castell et al., 1989), to act as a major regulator of the acute phase response in
hepatocytes via induction of specific acute phase proteins, such as α2-macroglobulin, α1-acid
glycoprotein, cysteine proteinase inhibitor, β-fibrinogen in rat (Gauldie et al., 1987, Andus et
al., 1988a, Andus et al., 1988b, Geiger et al., 1988) and serum amyloid A and C-reactive
protein (CRP) in human (Castell et al., 1988, Moshage et al., 1988, Castell et al., 1989)
suggesting that it may also be of importance in the initiation of the peripheral inflammatory
response.
The early observations employing IL-6 deficient mice revealed that although these mice were
viable, they were less susceptible to the induction of various autoimmune disorders, such as
antigen-induced arthritis (Ohshima et al., 1998), collagen-induced arthritis (Alonzi et al.,
1998), experimental-induced encephalomyelitis (Eugster et al., 1998) and Castleman`s disease
(Screpanti et al., 1996). In line with these observations, the elevated concentrations of IL-6 in
complex with its soluble receptor (sIL-6R) have been found in the synovial fluid in patients
with rheumatoid arthritis (RA) and have been suggested to induce the formation of osteoclastlike cells that are associated with the degree of joint damage (Kotake et al., 1996). IL-6 has
also been shown to mediate the enhanced development of osteoclasts as a result of
postmenopausal estrogen loss (Jilka et al., 1992) and furthermore it has been shown that IL-6
stimulate the production of VEGF in the synovial fibroblasts of patients suffering from RA
(Nakahara et al., 2003) in which some of the pathological manifestations, such as enhanced
angiogenesis and the increased vascular permeability of synovial tissue are attributable to the
exaggerate production of VEGF. Collectively, these studies indicated that IL-6 is a pivotal
cytokine in the development of RA and fueled the interest in IL-6 as a therapeutic target for
treatment of autoimmune defects, interest that resulted in the development of tocilizumab, a
humanized anti-IL-6 receptor antibody.
Since its introduction on the pharmaceutical market, the therapy with tocilizumab has been
appreciated for its inhibitory effect on joint destruction (Nishimoto et al., 2007, Smolen et al.,
2008, Kremer et al., 2011) and is now approved for the treatment of patients suffering from
the moderate to severe forms of RA either as monotherapy or in combination with other
arthritis-modulating pharmaceuticals in over 90 countries. In addition, the treatment with
tocilizumab has been demonstrated to decrease the severity of RA when anti-TNF-α blockers
19
and disease-modifying anti-rheumatic drugs (DMARD) have proven to be of inadequate
efficacy or to not be tolerated by the patients (Emery et al., 2008, Genovese et al., 2008). The
treatment with tocilizumab has also been shown to improve the disease outcomes, including
normalization of CRP levels and rapidly diminished febrile response in patients suffering
from juvenile idiopathic arthritis, which is the most common form of chronic arthritis in
children, manifested by joint damage and associated with systemic inflammation (Yokota et
al., 2005, Yokota et al., 2008). In fact, tocilizumab was found to be so effective in the general
improvement of arthritis and clinical characteristics that it was approved as a drug of choice in
Japan.
In addition, it has been found that for Castleman`s disease, a rare lymphoproliferative disorder
characterized by benign growths affecting the lymph nodes and associated with several
systemic manifestations, such as low-grade fever, anorexia and fatigue, long-term therapy
with tocilizumab markedly suppresses the disease activity and results in overall improvement
of the symptoms, including the disappearance of fever (Nishimoto et al., 2000, Nishimoto et
al., 2005, Song et al., 2010), thus suggesting that IL-6 produced by the lymph nodes is the key
component in regulation of the different symptoms.
One of the common adverse effects reported in patients treated with tocilizumab was the gain
in weight, indicating that IL-6 is involved in the regulation of metabolic processes. In support
of this, IL-6 has been shown to be expressed and released from the adipose tissue, and
circulating levels of IL-6 have been correlated with several metabolic parameters, such as
body mass index and insulin sensitivity (Mohamed-Ali et al., 1997, Fried et al., 1998, Bastard
et al., 2000). Moreover, although high levels of peripherally produced IL-6 are required, it has
been shown that IL-6 increases the lipolysis (Mattacks and Pond, 1999, Path et al., 2001) and
the oxidation of free fatty acids (Lyngso et al., 2002, van Hall et al., 2003, Petersen et al.,
2005), hence contributing to the loss of body fat. In accordance with this, the IL-6 deficient
mice were found to develop mature-onset obesity (Wallenius et al., 2002b). However, the
obesity in the IL-6 deficient mice could only partly be restored by an intraperitoneal injection
of IL-6 and the fat suppressing effect of IL-6 was instead ascribed to the centrally produced
IL-6 as it was demonstrated that IL-6 directly delivered into the brain was able to both induce
the loss of weight and elevate the energy expenditure (Wallenius et al., 2002a, Wallenius et
al., 2002b). Furthermore, it has been demonstrated that the enhanced energy expenditure
induced by the central expression of IL-6 may be due to the induced expression of uncoupling
protein-1 (UCP-1), thus activating the thermogenesis in brown adipose tissue (BAT) (Li et al.,
2002).
Although a large amount of evidence has identified IL-6 as a necessary factor for the
generation of fever (Chai et al., 1996, Kozak et al., 1998, Lenczowski et al., 1999, Cartmell et
al., 2000, Cao et al., 2001, Nilsberth et al., 2009), the underlying mechanism of its action in
the fever-generating pathway has been elusive, and interest in this mechanism therefore
became the focus of the research for this thesis. The importance of IL-6 in fever began with
findings in which the levels of IL-6 were readily found to be elevated during the course of the
endotoxin-elicited pyresis (Nijsten et al., 1987, LeMay et al., 1990a, LeMay et al., 1990c) and
was later further strengthened by findings that neither mice deficient in IL-6 nor animals
treated with IL-6 antiserum developed fever in response to systemic challenge with moderate
doses of LPS, IL-1β, TNF-α (Chai et al., 1996, Cartmell et al., 2000, Rummel et al., 2006) or
subcutaneous injection of turpentine (Kozak et al., 1997). Collectively, these studies provided
undoubted support for IL-6 being a crucial component of the febrile response. However, in
contrast with this, an intact febrile response of about the same magnitude as that seen in the
20
wild-type mice has been observed in mice lacking IL-6 when they were injected with high
sepsis-like dose of LPS or being subjected to infection evoked by the influenza virus (Kozak
et al., 1997, Kozak et al., 1998). This apparent discrepancy may, however, have found its
explanation in the finding that sepsis-like dose of LPS may trigger the redundant pathways of
fever mediated by the remaining members of the IL-6 family, such as ciliary neurotrophic
factor (CNTF), which is pyrogenic (Shapiro et al., 1993) and which in line with IL-6 can
transduce the signal through the glycoprotein 130 (gp130)-dependent pathway (further
described in the section about the IL-6 signaling). In addition, a high dose of LPS may trigger
the secretion of other non-gp130 related pyrogenic cytokines, such as IL-2, IL-8, interferon-α,
β and γ, macrophage inflammatory protein-1 (MIP-1) which in turn may induce fever in an
IL-6 independent manner.
Interestingly, IL-6 itself has been shown, in contrast to IL-1β, to not be pyrogenic or possibly
only weakly pyrogenic when large doses of recombinant IL-6 were used in mice and rats
(LeMay et al., 1990c, Chai et al., 1996, Wang et al., 1997, Rummel et al., 2006), thus
suggesting that circulating IL-6, at least in these species, is insufficient in eliciting the febrile
response. However, the inability of systemically administrated IL-6 to induce the febrile
response could be overcome by co-administration of a low, non-pyrogenic dose of IL-1β with
a moderate dose of IL-6 (Cartmell et al., 2000), suggesting that IL-6 in order to be able to
induce pyresis needs to be primed by IL-1β. Moreover, IL-1β has been shown to potentially
induce the production of IL-6 and it has been suggested that the pyrogenic action of IL-1β
elicited by LPS are mediated by IL-6 (LeMay et al., 1990b, Klir et al., 1994). This has
however been challenged by findings in which it was demonstrated that LPS-induced plasma
levels of IL-6 were elevated even in the absence of IL-1β since mice deficient in IL-1β
displayed induction of IL-6 comparable with that seen in wild-type mice (Zheng et al., 1995,
Kozak et al., 1998), thus suggesting that IL-1β is not required for the induction of IL-6
following an immune challenge with LPS.
A febrile response has been readily observed in several studies evaluating the fever-inducing
effect of centrally administrated IL-6 (Dinarello et al., 1991, Rothwell et al., 1991, Chai et al.,
1996, Lenczowski et al., 1999, Harden et al., 2008) and it has been suggested that this is due
to the elevated expression of cyclooxygenase 2 (Cox-2) in the cells associated with the bloodbrain barrier (Cao et al., 2001) and the concomitant increase of PGE2 in the cerebrospinal
fluid (Dinarello et al., 1991). However, we were not able to reproduce these results in our
laboratory (C. Nilsberth personal communication). Additional support for this idea was
provided by showing that mice devoid of Cox-2 as well as rats that had been pretreated with a
Cox-2 inhibitor were resistant to fever induced by central IL-6 (LeMay et al., 1990c, Rothwell
et al., 1991, Cao et al., 2001, Li et al., 2003). These findings collectively indicate that
centrally delivered IL-6 induces the production of PGE2 in brain endothelial cells in a Cox-2
dependent manner and that PGE2 produced in this way could be secreted into the brain
parenchyma and by acting on the thermoregulatory neurons in hypothalamus evoke fever. The
research for this thesis aimed at addressing these questions, and new data are presented
suggesting that IL-6 does not regulate the production of PGE2.
Overall, the above mentioned findings provide support for IL-6 being an essential cytokine
for the febrile response; however the mechanism(s) still remains to be clarified.
The structure of interleukin-6
21
The molecular weight for human and murine IL-6 varies between 20-30 kDa depending on
the cellular source and preparation and is probably the result of extensive posttranslational
modifications undergone by this molecule, such as N-and O-linked glycosylations and
phosphorylations (May et al., 1988a, May et al., 1988b). However, these variations do not
seem to account for any significant role in the biological functionality of the molecule. The
protein sequence of mouse (Van Snick et al., 1988) and rat (Northemann et al., 1989) IL-6
consists of 211 amino acids whereas human (Hirano et al., 1986) IL-6 consists of 212 amino
acids that except for 28 amino acids at the N terminus are all necessary for the biological
activity (Brakenhoff et al., 1989, Kruttgen et al., 1990, Snouwaert et al., 1991).The human
IL-6 and mouse IL-6 display 65 % homology at the DNA level and 42 % at the protein level
whereas the murine and rat amino acid sequences are 93 % identical. Both human IL-6 and
mouse IL-6 consist of five exons and four introns (Yasukawa et al., 1987, Tanabe et al., 1988)
and their gene organization is similar to that of granulocyte-colony stimulating factor (GCSF) suggesting an evolutionary relation between these molecules. The genes for human and
mouse IL-6 have been located at chromosome 7 and 5, respectively (Sehgal et al., 1986, Mock
et al., 1989).
The signaling of interleukin-6
The transduction of the IL-6 signal starts with the ligand-binding to a complex that consists of
interleukin-6 receptor (IL-6R) and gp130. It was reported in 1990 that an association between
IL-6R and a non-ligand binding gp130 takes place only in the presence of IL-6 (Hibi et al.,
1990) suggesting gp130 to be the possible component for the transduction of the signal. Upon
ligand-binding, the IL-6R can transfer the signals either via the classical way of signaling
where IL-6 binds to its membrane-anchored receptor (mIL-6R) (Yamasaki et al., 1988) which
in turn associates with gp130 (Taga, 1996) or via trans-signaling which involves the binding
of IL-6 to the soluble form of the interleukin-6 receptor (sIL-6R) (Novick et al., 1989)
subsequently forming a complex with gp130. The levels of sIL-6R are in addition to being
found in normal human urine and serum (Novick et al., 1989, Narazaki et al., 1993) also
found to be increased in connection with several diseases including multiple myeloma,
juvenile chronic arthritis and multiple sclerosis (Gaillard et al., 1993, De Benedetti et al.,
1994, Keul et al., 1998, Padberg et al., 1999). The formation of the sIL-6R has been
demonstrated to occur through the proteolytical cleavage of the mIL-6R (Mullberg et al.,
1993a, Mullberg et al., 1993b) but also to a minor extent through alternative splicing of the
IL-6R transcript (Lust et al., 1992, Muller-Newen et al., 1996). Many cells have been
observed in vitro to be highly responsive to IL-6/sIL-6R but not when only exposed to IL-6
(Rose-John et al., 2006). The affinity of IL-6 for sIL-6R is similar to that of the mIL-6R and
the formation of IL-6/sIL-6R complex has been suggested to extend the half-life of circulating
IL-6 and also to enable the propagation of IL-6 signaling in cells that lack the mIL-6R and
that accordingly are unresponsive to this cytokine (Peters et al., 1996). The importance of
IL-6 trans-signaling has been underscored by the findings in which the soluble form of the
gp130 (sgp130) has been reported to act as an exclusive inhibitor of the responses mediated
by the IL-6/sIL-6R complex resulting in a prevented association of the complex to the gp130
expressed on cell membranes without interfering with IL-6 responses mediated via the
mIL-6R (Rose-John et al., 2006). In contrast to the gp130 that is ubiquitously expressed
among all cell types, the distribution of the mIL-6R is limited to a few cell/tissue types such
as hepatocytes and hematopoietic cells (Rose-John et al., 2006) and is found to be induced in
the brain (Vallieres and Rivest, 1997) following immune challenge. The IL-6R provides
ligand specificity whereas gp130 is shared with all the other members of the IL-6 family of
cytokines, including interleukin-11 (IL-11), leukemia inhibitory factor (LIF), oncostatin M
22
(OSM), CNTF and cardiotrophin-1 (CT-1). As a consequence of shared usage of gp130,
similar and overlapping responses can be elicited within this group of cytokines and may also
explain the possible functional compensation mediated by the remaining members of the IL-6
family of cytokines in cases where a certain cytokine is lacking. Indeed, the IL-6 transsignaling has been suggested to imitate or supplement the autocrine and paracrine actions of
other gp130-related cytokines (Peters et al., 1997). The formation of IL-6/IL-6R complex and
following recruitment of gp130 induces the homodimerization of gp130 and subsequent
activation of gp130-associated tyrosine kinases of the Janus kinase (JAK) family, JAK1,
JAK2 and TYK2 (Murakami et al., 1993, Darnell et al., 1994, Lutticken et al., 1994, Narazaki
et al., 1994, Stahl et al., 1994) which phosphorylate gp130, thereby creating docking sites for
the activated transcription factors called signal transducers and activators of transcription
(STAT), mainly STAT3 and to a minor extent STAT1 (Zhong et al., 1994, Stahl et al., 1995,
Gerhartz et al., 1996, Ihle, 1996) (Fig. 2). The favoured view of STAT-association involves
the src homology 2 (SH2) domain mediated recruitment of these signal transducers to the
activated receptors (Heim et al., 1995, Hemmann et al., 1996) but there are also reports that
STATs can associate prior to receptor stimulation (Stancato et al., 1996, Ndubuisi et al., 1999,
Haan et al., 2000). Following cytoplasmic phosphorylation by tyrosine these STATs are
translocated into the nucleus where they become further phosphorylated by serine in order to
be fully activated (Boulton et al., 1995, Wen et al., 1995, Zhang et al., 1995) and subsequently
regulate transcription of the target genes containing STAT response elements.
B
A
IL-6
IL-6
sIL6-R
gp130
gp130
gp130
mIL-6R
gp130
P SH2
Ras
JAK
P
STAT3
SOCS
PI3K
STAT3
PIAS3
P
MAPK
STAT3
NF-κB
cytoplasm
STAT3
nucleus
NF-κB
P
STAT3
NF-IL-6
Fig. 2. IL-6 signaling pathway. The transduction of IL-6 signal starts via binding of IL-6 to
the membrane-anchored IL-6 receptor (mIL-6R) (A) or to the soluble IL-6 receptor (sIL-6R)
(B) present in the circulation and the concomitant association of IL-6/IL-6R complex with
the signal transducer molecule gp130. A cascade of different cellular events is initiated
including: the activation of JAK/STAT pathway (depicted by black arrows), the activation of
Ras/MAPK mediated pathway (depicted by pink arrows) and the activation of PI3K/NF-κB
pathway (depicted by blue arrows). The negative regulation is mediated by PIAS3 and SOCS
(depicted by dashed red arrows). For more detailed
23 description, see text.
IL-6 has also been shown to induce the activation of Ras/MAPK mediated pathway with
subsequent activation of the transcription factors NF-IL-6 and Elk1 (Poli et al., 1990, Akira
and Kishimoto, 1992) which in turn can act on their response elements in the genome.
Additionally, IL-6 has been shown to activate the signaling cascade involving
phosphoinositide-3 kinase (PI3K) that has been described to be implicated in the inhibition of
transforming growth factor-β (TGF-β) induced apoptosis (Chen et al., 1999) as well as in the
expansion of multiple myeloma cells (Hideshima et al., 2001, Shi et al., 2002).
Since the activation of STAT is transient, several mechanisms involved in its deactivation
have been proposed. The dephosphorylation of STAT1 by tyrosine phosphatases has been
demonstrated to be responsible for the inactivation of IL-6 signaling (Haspel et al., 1996)
whereas a protein inhibitor of activated STAT3 (PIAS3) has been shown to specifically block
the DNA binding of activated STAT3 and inhibit the gene activation mediated by this protein
(Chung et al., 1997). PIAS1 has been shown, in a similar manner, to inhibit STAT1-mediated
gene activation (Liu et al., 1998). Furthermore, the discovery of classical feedback inhibitors
induced via JAK/STAT pathway, named suppressors of cytokine signaling (SOCS), provided
a new mechanism for the negative regulation of STAT activation. SOCS 1-3 were shown to
inhibit tyrosine phosphorylation of gp130, STAT1 and STAT3 (Endo et al., 1997, Naka et al.,
1997, Starr et al., 1997). The regulation of IL-6 signaling has also been shown to be
modulated by reduction of its mRNA. The IL-6 mRNA half-life has been efficiently reduced
by inactivating the MAPK-activated protein kinase-2 (MK2), (Neininger et al., 2002) a
protein that has been observed to be essential for the stabilization of IL-6 at mRNA level
(Winzen et al., 1999). The protein availability of IL-6 has also been described to be regulated
by proteolysis at sites of inflammation (Bank et al., 1999).
Lipocalin-2
Lipocalin-2 (lcn2) is a member of the lipocalin family, a large group of small diverse
extracellular proteins that share a tertiary structure but only limited amino acid sequence
similarity. This unique structure allows them to bind and transport a range of small
hydrophobic molecules such as retinol and to form complexes with macromolecules,
including fatty acids, prostaglandins, and steroids (Flower et al., 1996). Recent reports have
shown that lcn2 is directly implicated in neuroprotection against bacteria due to its ability to
limit the bacterial growth by iron sequestration (Flo et al., 2004).
The communication between the immune system and the brain
Upon their biosynthesis, the proinflammatory cytokines summarized above are released into
the bloodstream upon pathogen-induced fever and have been described as the triggers of the
immune system. However, in order to activate the central component of the febrile response,
the blood-borne cytokines carrying the inflammatory message from the periphery need to
communicate with the brain which is protected by the blood-brain barrier. This barrier is
constituted of tight junctions between the endothelial cells of the cerebral vasculature and
normally restricts the passage of large and hydrophilic molecules such as cytokines while
allowing the diffusion of small lipophilic molecules. Since peripherally derived cytokines due
to their size and physical-chemical properties cannot freely enter the brain parenchyma, they
must in some other way signal across the blood-brain barrier to affect the neuronal activation.
24
The mechanisms that underlie this immune-to-brain communication have been debated for a
long time and four major routes have been suggested including a) entry of circulating
cytokines to the brain via the circumventricular organs (CVO), b) an active transport of
cytokines over the blood-brain barrier through a carrier-mediated pathway, c) the recognition
of cytokines by afferent nerve fibers, in particular the vagal nerve, d) the binding of cytokines
to their receptors located on cells associated with the blood-brain barrier (Fig. 3).
Circumventricular
organs
A
D
B
Blood Brain Barrier
Prostaglandins
Cytokine
Cytokine
transporter
Cytokine
receptor
C
Prostaglandin
synthesis
Vagus nerve
Fig. 3. Pathways for cytokine-to-brain signaling. Four main hypotheses have been proposed
describing the possible ways of entry for circulating cytokines into the brain. A: Cytokines
enter the brain via the circumventricular organs. B: Cytokines are transported to the brain via
an active carrier-mediated pathway. C: The peripheral inflammatory signals are transferred to
the brain via the vagus nerve. D: The circulating cytokines bind to their receptors on brain
endothelial cells and induce the synthesis of Cox-2 and mPGES-1, which produce PGE2.
Modified from Engblom et al. (2002).
The role of the circumventricular organs
The cytokines may enter the brain parenchyma and pass into the deeper brain structures
through CVO, the specific brain regions devoid of a blood-brain barrier which can be reached
by the bloodstream. These brain structures include the organum vasculosum of the laminae
terminalis (OVLT), the area postrema (AP), the subfornical organ (SFO) and the median
eminence (ME). As the name indicates they are positioned around the brain ventricles
surrounding the third and the fourth ventricle. In particular OVLT is situated adjacent to the
preoptic area of the hypothalamus that is known as the thermoregulatory control station.
Thanks to their fenestrated blood vessels the passage of large molecules into the extracellular
space of the CVOs is enabled and the concentration of various chemical compounds in the
25
blood is sensed by the neurons of the CVOs without requirement of a specialized transport
system. The expression of the genes encoding CD14 that is highly inducible upon immune
challenge, and TLR4 has been reported within these structures (Lacroix et al., 1998,
Laflamme and Rivest, 2001). Furthermore, the expression of the genes encoding IL-1β (Quan
et al., 1998b, Konsman et al., 1999), IL-1R (Ericsson et al., 1995) and IL-6R (Vallieres and
Rivest, 1997) as well as the transcriptional activation of the gene encoding Cox-2 (Lacroix
and Rivest, 1998) has been observed in the CVOs following systemic LPS challenge. The
neurons of the CVOs have been reported to be activated during the immune challenge
(Ericsson et al., 1994, Elmquist et al., 1996) and tracing studies have revealed the existence of
bidirectional nerve connections from the CVOs to hypothalamus (Silverman et al., 1981,
Larsen and Mikkelsen, 1995), hippocampus and amygdala (Silverman et al., 1981, Ciriello
and Gutman, 1991) thus making it possible to convey the peripheral immune signals to the
brain. However, even if these structures, based on the findings briefly described above, seem
important in the communication of an activated immune system with the brain, their role as
the gateway for cytokine entry into the brain is still obscure. This controversy originates from
the inconsistent results produced by different CVO lesion studies. Thus, whereas lesioning the
OVLT unexpectedly resulted in an enhancement of the LPS-induced febrile response (Stitt,
1985), lesioning a different part of the CVO, the SFO and not the OVLT was reported to
reduce the temperature response following LPS challenge (Takahashi et al., 1997).
Furthermore, many “side effects” such as hyperthermia that probably developed as a result of
increased thermogenesis were observed following lesion of the OVLT (Romanovsky et al.,
2003) making the interpretation of the results difficult. To make the situation even more
complex, a barrier formed by the specialized ependymal cells, the tanycytes, between the ME
and brain parenchyma have been identified, hindering the entrance of molecules in the CVOs
to adjacent brain regions (Peruzzo et al., 2000, Rodriguez et al., 2010).
Cytokine transport over the blood-brain barrier
Another way by which circulating cytokines may reach the brain is to be actively transported
across the blood-brain barrier through an action of cytokine-specific carriers (Banks et al.,
1995). This transendothelial transport mechanism has been demonstrated for IL-1β (Banks et
al., 1991), TNF-α (Gutierrez et al., 1993) and IL-6 (Banks et al., 1994) but it is slow, becomes
rapidly saturated and has relatively low capacity. These obvious limitations, together with the
rapid onset of the CNS response to inflammation, suggest that this pathway plays either a
minor or no role at all in transmitting the blood-borne signals to the brain.
Vagal signaling
An alternative mechanism for conveying the pyrogenic signals to the brain, acting
independently of the blood-brain barrier, was proposed to occur via neural signaling. The
vagus nerve in particular, innervating various visceral organs, was suggested to convey the
sensory information about the state of the immune system to the brain. The functional
importance of neuronal transmission resulted from the observation that vagotomy attenuated
the polyphasic febrile response induced by systemically administrated IL-1β (Watkins et al.,
1995) or LPS (Sehic and Blatteis, 1996). The significance of this signaling was later further
strengthened by demonstrating the expression of TLR4 mRNA and receptors for IL-1β and
PGE2 on the vagal afferent fibers (Goehler et al., 1997, Ek et al., 1998, Hosoi et al., 2005) and
also by demonstrating an attenuation of the febrile response to LPS following desensitization
of the abdominal afferent fibers with capsaicin (Szekely et al., 2000). However, other studies
showed that vagotomy only affects the early fever responses to low doses of LPS (Van Dam
26
et al., 2000) or IL-1β (Hansen et al., 2001), when local action could occur, and that this was
not the case when the larger doses of LPS or cytokines were administrated either
intraperitoneally or intravenously (Romanovsky et al., 1997b, Van Dam et al., 2000, Hansen
et al., 2001). The discrepancy between these studies and their apparently different outcomes
was suggested to, at least in part, have been due to the many severe side effects caused by
vagotomy, one such being malnutrition which itself could impair the thermoregulatory
responses and lead to the fever attenuation observed in the initial studies (Romanovsky et al.,
1997a). Furthermore, it has been shown that the decrease in LPS-induced fever achieved by
capsaicin desensitization had nothing to do with the vagal signaling as proposed initially
(Dogan et al., 2004). Hence, vagal signaling does not appear to play any significant role in
polyphasic febrile response to inflammatory stimuli.
The role of the blood-brain barrier
The role of the blood-brain barrier as an active relay station in transferring the pyrogenic
signals from the periphery to the brain has been suggested by several groups. The brain
endothelial cells have been demonstrated to express the receptors for IL-1β (Van Dam et al.,
1996, Ek et al., 2001, Konsman et al., 2004), IL-6 (Vallieres and Rivest, 1999) and TNF-α
(Bebo and Linthicum, 1995) in response to immune challenge. Furthermore, the mRNA
expression of the IκB known to serve as an indicator of NF-κB activity was rapidly enhanced
in brain endothelial cells following various immune stimuli (Laflamme and Rivest, 1999).
More recently, the role of brain endothelial cells in the immune-to-brain signaling was
directly supported by using the transgenic mice with specific deletion of IL-1R in these cells.
It was shown that mice devoid of IL-1R on their endothelial cells were afebrile to the
pyrogenic effect of IL-1β (Ching et al., 2007), thus strongly suggesting that the endothelial
cells of the blood-brain barrier are responsible for the febrile response (Engstrom et al., 2012).
Additional support comes from a study in which the expression of transforming growth factor
β-activated kinase 1 (TAK1), a key mediator of proinflammatory signals and activator of NFκB, was specifically ablated in brain endothelial cells. As a result of this ablation, the febrile
response to intravenously administrated IL-1β was blunted (Ridder et al., 2011). The several
lines of evidence presented above support the hypothesis that circulatory cytokines can bind
to their receptors expressed on the endothelial cells and affect the central mechanisms of
fever. These mechanisms are now generally accepted to be mediated by PGE2 that is
synthesized within the blood-brain interface and that has been recognized through extensive
research as a principal mediator of the febrile response.
Prostaglandin E2
The history of prostaglandins started with the discovery of a substance thought to be derived
from the prostate that was found in seminal fluid and seminal vesicles. Since it was originally
identified in the prostate, this novel substance was given the name prostaglandin (Goldblatt,
1935, von Euler, 1936). The first prostaglandins, shown to be produced by unsaturated fatty
acids, were purified during the 1950s by Bergström and his colleagues and their chemical
structures were determined. However, although identified as a substance that could cause both
the constriction of smooth muscles and reduction of blood pressure, the role of prostaglandin
E in the fever generation was not revealed until 1971 when the central administration of this
compound was shown to elevate the body temperature of cats and rabbits (Milton and
Wendlandt, 1971) and with the discovery that its synthesis was inhibited following
administration of non-steroidal anti-inflammatory drugs (NSAID), such as acetyl salicylic
27
acid (more commonly known as aspirin) (Vane, 1971). However, the advantageous effects of
salicylic acid have been known for a long time and the legend is that Hippocrates himself had
advised his patients to use bark and leaves from the willow tree in order to relieve pain and
fever. The substance responsible for these effects was much later identified as salicylic acid.
The synthesis of PGE2
The first step in the synthesis of prostanoids involves the conversion of membrane
phospholipids into arachidonic acid, the major prostanoid precursor, by cleavage with
different phospholipase A2 (PLA2) enzymes which are present in most cells (Clark et al.,
1991). At least 19 mammalian enzymes, divided into four subfamilies, exhibiting PLA2
activity have been identified so far (Kudo and Murakami, 2002), of which cytosolic PLA2-α
(cPLA2-α) and secretory PLA2-IIA (sPLA2-IIA) have attracted a considerable interest in the
inflammatory response since cPLA2-α in the hypothalamus and sPLA2-IIA in hypothalamus as
well as in the liver and lungs have been found to be up-regulated following LPS challenge
(Ivanov et al., 2002). The arachidonic acid is thereafter, by the action of Cox, converted to
prostaglandin endoperoxide H2 (PGH2) which is then, depending on the cell type and the
presence of terminal isomerases, converted to different prostanoids which include PGI2
(prostacyclin), TXA2 (thromboxane), PGD2, PGF2 and PGE2 (Fig. 4).
A
B
Constitutive
Inflammation
membrane
phospholipids
membrane
phospholipids
PLA2
PLA2
Arachidonic acid
Arachidonic acid
Cox-1
Cox-2
PGH2
PGH2
cPGES
mPGES-2
PGD2
PGE2
mPGES-1
PGF2
PGI2
TXA2
PGD2
PGE2
PGF2
PGI2
TXA2
Fig. 4. Schematic illustration of the prostanoid synthesis pathways during constitutive (A)
and inflammatory (B) conditions. For more details, see text. Modified from Engblom, et al.
(2002).
Two different isoforms of Cox have been identified, cyclooxygenase 1 (Cox-1) and Cox-2
which due to their catalyzing activity have also been called prostaglandin endoperoxide H
synthases-1 and 2, respectively (Vane et al., 1998, Smith et al., 2000). Although these two
isoforms display some common features, such as 63 % identity on the amino acid level within
the same species, 600 amino acid length and similar kinetic properties, they differ with
respect to their physiological functions (Vane et al., 1998). Cox-1, encoded by the Ptgs1 gene
and with a primary structure characterized in 1988 (DeWitt and Smith, 1988, Merlie et al.,
28
1988, Yokoyama et al., 1988), is constitutively expressed in nearly all cells, and prostanoids
produced by Cox-1 have been described to be involved in the protection of the stomach lining
against acids as well as in the maintenance of normal kidney function. Based on the studies
employing genetically modified animals, an essential role of Cox-1 for survival of the fetus
has been established with a strongly compromised survival rate of the offspring born to
homozygous Cox-1 deficient mice (Langenbach et al., 1995). Thus, Cox-1 is generally
considered to produce prostanoids that mediate “housekeeping” functions, although
conflicting data exist, suggesting that Cox-1 could participate in chronic (Gilroy et al., 1998)
as well as in acute inflammation (Schwab et al., 2000).
Cox-2 on the other hand, encoded by the Ptgs2 gene (Kujubu et al., 1991, O'Banion et al.,
1991, Xie et al., 1991, Hla and Neilson, 1992, Simmons et al., 1992) has been given the most
attention with respect to cytokine-to-brain communication. During basal conditions, i.e.
constitutively, Cox-2 has in particular been found to be expressed in neurons of the cerebral
cortex, the hippocampus and the paraventricular hypothalamus (PVH) and the OVLT
(Yamagata et al., 1993, Breder et al., 1995). Its neuronal expression was, however, rapidly
and transiently induced by synaptic stimuli (Yamagata et al., 1993) and has also been
suggested to be implicated in pain hypersensitivity and mechanical pain (Samad et al., 2001,
Vardeh et al., 2009).
During inflammatory conditions, Cox-2 is rapidly and heavily induced by stimuli such as LPS
and cytokines and has generally been recognized as an isoform of Cox responsible for the
synthesis of inflammatory PGE2 (Dubois et al., 1998). An essential role of Cox-2 in fever
(more fully described in the section about the role of PGE2 in fever below) was recognized by
findings in which it was demonstrated that Cox-2 was induced in cerebral vasculature upon
peripheral LPS-challenge (Cao et al., 1995) and that its induction in blood vessels, but not in
neurons, was closely associated with the febrile response (Cao et al., 1997). Moreover, in a
model of adjuvant-induced arthritis, Cox-2 has been shown to be induced in rat brain
endothelium (Engblom et al., 2002). In line with these findings, agents inhibiting the
production of Cox-2 have been efficiently used in the abolishment of the many central
nervous responses associated with the acute inflammation and arthritis (Hori et al., 2000). It
deserves to be mentioned that Cox-2 is not only involved in the inflammatory signaling but
has also been described to participate in the regulation of physiological functions such as
reproductive processes (Lim et al., 1997), and ablation of Cox-2 has been shown to result in
severe renal failure (Morham et al., 1995). Moreover, some studies have suggested that Cox2, at least during late stages of inflammatory responses, might also possess anti-inflammatory
properties (Gilroy et al., 1999, Gilroy and Colville-Nash, 2000, Rossi et al., 2000).
However, which terminal enzyme was responsible for the metabolism of PGH2 to PGE2 has
remained unclear until quite recently when Jakobsson with co-workers recognized that a
member of protein family consisting of membrane associated proteins involved in eicosanoid
and glutathione metabolism had the ability to specifically and efficiently catalyze the
formation of PGE2 (Jakobsson et al., 1999). In addition, since this protein was found to be
distributed in organs known to produce PGE2 and was found to be induced by cytokines in a
way similar to that for Cox-2, it was thus referred to as PGE synthase. Since the membraneassociated fraction of this protein was found to possess the PGE synthase activity it was given
the name microsomal prostaglandin E synthase-1 (mPGES-1). Apart from the inducible
mPGES-1, two additional PGE2 synthesizing enzymes have been identified, microsomal PGE
synthase-2 (mPGES-2) and cytosolic PGES (cPGES) (Ogorochi et al., 1987, Watanabe et al.,
1997, Tanioka et al., 2000). These two enzymes are constitutively expressed, weakly affected
29
following inflammatory response and functionally linked to Cox-1 (Tanioka et al., 2000,
Guay et al., 2004), whereas the inflammation-induced mPGES-1 has been shown to be
associated with Cox-2 both in vitro (Murakami et al., 2000, Thoren and Jakobsson, 2000) and
in vivo (Ek et al., 2001, Yamagata et al., 2001), thus suggesting that they are functionally
coupled.
The role of PGE2 in fever
The essential role of prostanoid-like activity in fever generation was recognized with the
observations that PGE1 and PGE2 administrated directly in the preoptic area (POA) of the
anterior hypothalamus caused an almost immediate but short-lasting rise in body temperature
in several different species (Milton and Wendlandt, 1970, Feldberg and Saxena, 1971, Stitt,
1973, Williams et al., 1977, Morimoto et al., 1988, Oka and Hori, 1994, Scammell et al.,
1996) and by Vane`s discovery that the synthesis of prostaglandin was inhibited following
administration of NSAIDs, such as acetyl salicylic acid (Vane, 1971). An indication that
PGE2 is synthesized within the blood-brain interface was recognized with the findings that
peripheral immune challenge rapidly induced the expression of PGE2-synthesizing enzymes,
Cox-2 (Cao et al., 1995, 1996a, 1997, Lacroix and Rivest, 1998, Matsumura et al., 1998,
Quan et al., 1998a) and mPGES-1 (Ek et al., 2001, Yamagata et al., 2001) in the cells
associated with the blood brain vessels. In addition, the expression of Cox-2 in the cerebral
endothelium as well as the elevated levels of PGE2 in the cerebrospinal fluid following
peripheral immune challenge were suggested to correlate with the rise of body temperature in
various species (Bernheim et al., 1980, Coceani et al., 1988, Cao et al., 1995, 1996a). In
support of these findings, an attenuation of the LPS-induced synthesis of PGE2 in the anterior
hypothalamus and the concomitant fever response was observed in rats (Cao et al., 1997,
Dogan et al., 2002, Zhang et al., 2003) and rabbits (Cranston and Rawlins, 1972, Morimoto et
al., 1988, Scammell et al., 1998) upon peripheral and central administration of Cox-2
inhibitors, respectively. Furthermore, the LPS-induced fever was observed to be abolished in
animals devoid of Cox-2 (Li et al., 1999, Steiner et al., 2005b) and mPGES-1 (Engblom et al.,
2003).
In good accordance with the induction of PGE2 synthesizing enzymes along the cerebral
microvasculature, no elevation of body temperature was seen in mice expressing mPGES-1
only in hematopoietic cells whereas an intact febrile response was observed in mice
expressing mPGES-1 only in cells of non-hematopoietic origin, identified as endothelial cells
(Engstrom et al., 2012). However, although all these findings point out the brain’s endothelial
cells as the critical cell type in the transduction of the peripheral immune signals to the brain,
there are also some conflicting results from other studies suggesting that the perivascular
macrophages throughout the brain are the contributing source of PGE2 synthesizing enzymes
during the systemic immune challenge (Elmquist et al., 1997, Schiltz and Sawchenko, 2002).
Following its synthesis at the blood-brain interface, the PGE2 would exit the blood-brain
barrier either by passive diffusion and/or by a specific carrier-mediated transport mechanism
shown to be driven by multidrug resistance protein 4 (Reid et al., 2003), and then enter the
brain parenchyma and activate its neuronal receptors in the thermoregulatory center of the
hypothalamus.
30
Prostaglandin E2 receptors and their implication in fever
The prostanoids mentioned above are released outside the cells following their synthesis and
are ready via binding to the specific membrane receptors to exert their effects in multiple
tissues. Cloning studies have revealed the existence of multiple prostaglandin E2 receptors,
called EP receptors, encoded by specific genes. EP receptors belong to the family of Gprotein coupled receptors with seven transmembrane domains and so far, four different
subtypes (denoted EP1-EP4) have been identified. They differ in their ligand-binding
characteristics, tissue distribution and cellular localization, regulation of expression and
different intracellular messenger signaling, thus accounting for the diverse effects of PGE2.
Furthermore, despite the presence of conserved sequences, the EP receptors display higher
similarity on amino acid level to the corresponding receptor homologues from other species
than to each other, the overall homology among prostanoid receptors ranging between
20 – 30 % (Breyer et al., 2001).
The EP1 receptor was upon its cloning found to be most abundantly expressed in kidneys
(Watabe et al., 1993) and its activation has been associated with increasing concentration of
intracellular Ca2+ (Funk et al., 1993, Watabe et al., 1993). While the distribution of the EP1
receptor has been mapped to the PVH and the central nucleus of the amygdala (CeA) in the
mouse brain (Matsuoka et al., 2003), its mRNA expression in the POA of the rat brain was
reported to be weak and diffuse (Oka et al., 2000). The EP1 receptor has been implicated in
the control of behavior since mice deficient in this receptor exhibited impaired ability to
control their impulsive behavior during stress (Matsuoka et al., 2005). The role of EP1
receptor in inflammatory signaling has mostly been studied with the aid of pharmacological
substances, serving as agonists or antagonists of its action. However, the role of the EP1
receptor in the thermoregulatory response has yielded contradictory results. While the central
administration of an EP1 receptor agonist has been reported to elevate the body temperature in
rats in a manner similar to that of PGE2 (Oka and Hori, 1994) and while in line with this, the
hyperthermic response elicited by PGE2 or IL-1β was blocked by EP1 receptor antagonist
(Oka and Hori, 1994, Oka et al., 1998), only a small elevation of body temperature, not
different from that seen in the vehicle-treated animals, was observed upon central
administration of the EP1 receptor agonist in pigs (Parrott and Vellucci, 1996), suggesting the
differential outcomes to be due to species variation. Furthermore, whereas only an early phase
of the febrile response induced by low dose of the intraperitoneal LPS was observed to be
partially attenuated in the absence of this receptor (Oka et al., 2003), a completely normal
fever response was seen in mice lacking the EP1 receptor when LPS or IL-1β were given
intravenously or when PGE2 was administrated centrally (Ushikubi et al., 1998).
The EP2 receptor, cloned in 1994 mediates the increase in concentration of cyclic adenosine
monophosphate (cAMP) (Regan et al., 1994) and had originally been identified as the
“relaxant” receptor. Mice deficient in EP2 receptors were shown to be resistant to
hyperalgesia induced by spinally administrated PGE2 (Reinold et al., 2005) and its mRNA
distribution under basal conditions, detected by in situ hybridization, has been found in the
bed nucleus of stria terminalis (BNST), the SFO, the ventromedial hypothalamus (VMH), the
locus coeruleus (LC) and the AP (Zhang and Rivest, 1999). The immune-induced expression
of the EP2 receptor was by the same authors reported in the majority of the above mentioned
nuclei (Zhang and Rivest, 1999). However, while the centrally given agonist for the EP2
receptor (butaprost) was shown to elevate body temperature in pigs (Parrott and Vellucci,
1996), the EP2 deficient mice that were challenged with PGE2 or IL-1β given centrally
31
displayed a febrile response that was indistinguishable from that seen in wild-type mice
(Ushikubi et al., 1998).
The EP3 receptor (Sugimoto et al., 1992) is unique among the PGE2 receptors, a feature that is
reflected by the existence of multiple splicing variants which all bind PGE2 but display
different sequences at their cytoplasmatic carboxy-terminals and may both activate or block
adenylate cyclase through an employment of stimulatory G proteins (Gs) or inhibitory G
proteins (Gi). However, most of the isoforms are inhibitory. As a result of the alternative
splicing, the functional differences in the signal transduction and the intracellular signaling
have been suggested (Breyer et al., 2001). The EP3 receptor is broadly expressed in the brain
and has been found in the structures known to play an important role in immune-to-brain
communication. Thus, the EP3 receptor positive signal has been found in the structures that
are important in the transmission of the incoming signal to the endocrine hypothalamus [the
nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM)], structures that have
been associated with the control of sleep and wakefulness [the median raphé nuclei, the LC
and the tuberomammilary nucleus], and structures that are involved in the nociceptive and the
analgesic pathways [the thalamic nuclei, the parabrachial nucleus (PB), the LC, the
periaqueductal gray matter, the raphé nucleus and the nucleus raphé magnus] (Ek et al., 2000,
Engblom et al., 2000). In addition, the EP3 receptor-like immunoreactivity was detected in
most of the serotonergic neurons and in some catecholaminergic neurons (Nakamura et al.,
2001). However, the most prominent expression of the EP3 receptor has been found within the
preoptic nucleus of the hypothalamus (Ek et al., 2000, Engblom et al., 2000), the structure that
is known to play a central role in the thermoregulation. The crucial and undisputed role of the
EP3 receptor in the febrile response has been established by numerous studies. Thus, the
complete absence of fever, irrespectively of the inflammatory stimuli used and their route of
administration, was observed in mice lacking the EP3 receptor (Ushikubi et al., 1998, Oka et
al., 2003, Lazarus et al., 2007). In line with this, the rise in body temperature has been shown
upon central administration of an EP3 receptor agonist (Parrott and Vellucci, 1996).
In line with the EP2 receptor, the activation of the EP4 receptor couples with increased cAMP
concentrations (Bastien et al., 1994, Regan et al., 1994), and functions in a redundant manner
with the EP2 receptor in some processes. In contrast to the EP3 receptor, the distribution of the
EP4 receptor transcript is more restricted. The expression has been found mostly in the
structures implicated in neuro-endocrine control such as the PVH, the LC, the PB, the NTS
and the VLM under basal conditions. The induced level of the gene encoding the EP4 receptor
elicited by intravenous LPS or IL-1β has been detected in most of the above mentioned nuclei
(Zhang and Rivest, 1999). However, the role of EP4 receptor in the immune-to-brain
communication has been difficult to study since mice deficient in EP4 receptor do not survive
due to failure of closing ductus arteriosus (Nguyen et al., 1997) and the available EP4 receptor
agonists have displayed a lack of specificity.
Fever generating pathways
The preoptic area in hypothalamus
As mentioned above, the EP3 receptor is most abundantly expressed in the neurons of the
POA and especially in its subregions, the median preoptic nucleus (MnPO) and medial
preoptic area (MPO) (Nakamura et al., 1999, Nakamura et al., 2000). The majority of the
POA neurons expressing the EP3 receptor have been found to contain γ-amino butyric acid
32
(GABA), an inhibitory neurotransmitter. These EP3 receptor-expressing neurons have been
found by use of an anatomical tracing technique to project to the ventral medullary regions
(Murphy et al., 1999), including not only to the rostral part of raphé pallidus (rRPa) in the
medulla (Nakamura et al., 2002) but also to other brain regions such as the dorsomedial
hypothalamus (DMH). Under basal, non-infectious conditions, the EP3 receptor-expressing
neurons in POA have been suggested to tonically inhibit the neurons in the rRPa and DMH
whereas under inflammatory fever-producing conditions, the pyrogenic action of PGE2 on its
cognate receptors has been suggested to suppress the activity of POA neurons (Ranels and
Griffin, 2003) by their negative coupling to adenylate cyclase via inhibitory GTP-binding
proteins (Narumiya et al., 1999). Subsequently this leads to removal of the inhibitory
signaling from EP3 receptor-expressing neurons to rRPa and DMH neurons. The disinhibited
neurons, innervated by the descending projections from POA, target the preganglionic
neurons in the spinal cord and consequently activate the peripheral sympathetic
thermoeffectors, the BAT (recruited to increase the body temperature) and the cutaneous
vasoconstriction particularly in the tail (to decrease blood flow and prevent heat loss from the
skin to the environment), thus contributing to the generation of fever. The febrile response
evoked by pyrogenic stimulation with PGE2 also activates shivering (Nakamura and
Morrison, 2011), which is an involuntary somatic thermogenic response that produces heat
due to the rapid and repeated skeletal muscle contractions.
The role of raphé pallidus nucleus and dorsomedial hypothalamus
Based on extensive experimental evidence, the rRPa in particular has been considered as
essential in generating the febrile responses. For instance, the implication of PGE2-activated
sympathetic premotor neurons in rRPa in the increased thermogenesis and decrease of heat
loss has been demonstrated through their innervations of the BAT and the preganglionic
neurons of the spinal cord (Nakamura et al., 2004, Nakamura et al., 2005a). In line with this,
the neuronal inhibition of the rRPa through local microinjection of the GABA receptor
agonist muscimol resulted in the block of PGE2-evoked thermogenesis in BAT (Nakamura et
al., 2002, Morrison, 2003) and markedly decreased the constriction of cutaneous blood
vessels in the rat tail induced by PGE2 (Rathner et al., 2008). Conversely, the disinhibition of
rRPa neurons evoked by local microinjection of GABA-antagonist, bicuculline was shown to
elicit the sustained increase of BAT thermogenesis and generate shivering (Nason and Mason,
2004).
The role of DMH in driving the thermogenesis has been difficult to dissect due to
interconnections with other adjacent areas. However, its role in the thermogenesis is
supported by several lines of evidence including: 1) the DMH receives projections from many
EP3 receptor-expressing neurons in POA (Nakamura et al., 2005b, Nakamura et al., 2009),
2) the neuronal disinhibition of DMH neurons achieved by blockade with GABA antagonist
bicuculline leads to increased thermogenesis (Zaretskaia et al., 2002, Cao et al., 2004) and
conversely the inhibition of DMH neurons by GABA agonist attenuates the PGE2-evoked
thermogenesis in BAT (Madden and Morrison, 2004, Nakamura et al., 2005b, Rathner et al.,
2008). Furthermore, the PGE-evoked increase in BAT thermogenesis has been observed to be
attenuated following electrolytic and chemically-generated lesions of the VMH (Monda et al.,
1997, Monda et al., 2001). Although these studies undoubtedly suggest that activated DMH is
implicated in the thermogenesis, the activation of its neurons is probably not critically
involved in the PGE2-induced cutaneous vasoconstriction (Rathner et al., 2008) and the
33
increase of the body temperature, thus suggesting that differential regulation of peripheral
thermoeffectors is mediated by distinct descending pathways.
However, very few EP3 receptor-expressing POA neurons have been observed to project to
both the DMH and rRPa and it has been suggested that the transmission of pyrogenic signals
to these two regions is mediated by different EP3 receptor-expressing neuronal populations
within the POA (Nakamura et al., 2009). Whereas the EP3 receptor-expressing neurons
projecting to the DMH were found to be distributed to almost the same extent between the
MPO and MnPO, the neurons projecting to the rRPa appeared to be more densely localized in
the MPO than in the MnPO (Nakamura et al., 2009). Based on these and previous findings it
has thus been proposed (which also is the prevailing hypothesis) that while the PGE2-evoked
signaling through suppression of the activity of EP3 receptor-expressing neurons in the POA
leads to the removal of the tonic inhibition in the DMH that subsequently activates the
sympathetic premotor neurons in the rRPa resulting in the drive of thermogenesis in BAT, the
simultaneous increase in cutaneous vasoconstriction is thought to involve the disinhibition of
neurons in the rRPa without implication of neurons in the DMH (Nakamura et al., 2004,
Morrison et al., 2008, Rathner et al., 2008, Nakamura et al., 2009).
Beyond the disinhibition of rRPa and DMH either evoked by the action of PGE2 in the MnPO
or by blocking the tonic inhibitory signaling of GABA these regions need to be excited in
order to elevate the thermogenesis and several studies have suggested the glutamatergic and
serotonergic receptors to be of importance in exerting these excitatory actions. However, the
source of these excitatory inputs to the rRPa is yet to be determined. The PGE2-evoked
thermogenesis has been reported to be blocked following administration of glutamate receptor
antagonists to rRPa or DMH (Madden and Morrison, 2003, 2004) and markers indicative of
glutamatergic and serotonergic neurons, such as vesicular glutamate receptor 3 (VGLUT3)
and tryptophan hydroxylase have been found in rRPa (Nakamura et al., 2004, Stornetta et al.,
2005). The functional role of these glutamate-expressing and serotonin-containing neurons in
elevating the thermogenesis comes from the anatomical studies which have revealed a
substantial co-expression of VGLUT3-containing neurons in the rRPa with c-fos (marker of
neuronal activation) following intracerebroventricular administration of PGE2 (Nakamura et
al., 2004) and findings demonstrating an increased firing activity among the serotonergic
neurons in the rRPa upon administration of GABA antagonist into the ventromedial medulla
(Martin-Cora et al., 2000). Consistently, the immunoreactive terminals for VGLUT3 and
serotonin were found to synapse on the preganglionic neurons in the intermediolateral (IML)
nucleus in the spinal cord (Vera et al., 1990, Stornetta et al., 2005) and a rapid increase in
BAT thermogenesis was clearly demonstrated following administration of glutamate
(Nakamura et al., 2004). In contrast, serotonin injected into the thoracic segment of the IML
only evokes a delayed elevation in the BAT thermogenesis and itself seems to be unable to
elicit depolarization of BAT-controlling preganglionic neurons (Madden and Morrison, 2006).
However, when serotonin is co-administrated with the glutamate agonist in the IML the
potentiated elevation in BAT thermogenesis has been observed (Nakamura et al., 2004,
Madden and Morrison, 2006).
The cooling-evoked thermogenesis – similarities to PGE2
The role of POA in conveying the pyrogenic signaling evoked by PGE2 and thus controlling
the activation of peripheral thermal effectors (increased thermogenesis in BAT and the
decreased cutaneous vasoconstriction) during the febrile response has also been suggested to
34
play an essential role during the cold-defense responses when animals are exposed to the
environment of reduced temperature (Nakamura and Morrison, 2008). The environmental
changes in temperature are sensed by the transient receptor potentials (TRP) in the cutaneous
terminals and conveyed to the dorsal horn of the spinal cord (Lumpkin and Caterina, 2007)
which then project to the lateral parabrachial nucleus (LPB) (Cechetto et al., 1985, Bebo and
Linthicum, 1995). Recently, the cool-responsible LBP have been shown to target the MnPO
and display an increased firing activity at the onset of skin-cooling that was in concordance
with the enhanced temperature in BAT but without evoking any changes in the rectal or brain
temperature (Nakamura and Morrison, 2008). In addition, the complete reversal of the skin
cooling-evoked thermogenesis was seen upon administration of the muscimol to the LPB
(Nakamura and Morrison, 2008) thus being in line with the neuronal inhibition of rRPa and
DMH and the following attenuation of the PGE2-evoked thermogenesis (Nakamura et al.,
2002, Morrison, 2003, Madden and Morrison, 2004, Nakamura et al., 2005b, Rathner et al.,
2008). The ascending thermoregulatory pathway mediating the cutaneous thermosensation
described here differs from the spinothalamocortical pathway which mediates the perception
and discrimination of coolness. In the latter, the cutaneous thermosensitive signals are
transmitted from the dorsal horn to the thalamus and are further relayed to the primary
somatosensory cortex but are not involved in mediating the thermoregulatory responses
(Osaka, 2004, Nakamura and Morrison, 2008).
The implication of proinflammatory cytokines in BAT thermogenesis
The ability of BAT to produce heat is due to the induced expression of UCP-1, controlled by
the sympathetic nerves, that has been shown to uncouple the oxidative phosphorylation in
mitochondria, thus enabling fast oxidation of substances and generation of heat (Klingenberg
and Huang, 1999). The essential role of UCP-1 in heat generation was demonstrated by
showing that cold-exposed mice with inactivated UCP-1 were not able to defend their body
temperature (Enerback et al., 1997). However, as this is considered to be a necessary
molecule for BAT thermogenesis, the involvement of this protein in the febrile response
evoked by LPS or IL-1β has been a matter of debate. Presumably, the mechanism by which
the proinflammatory cytokines influence the thermogenesis has been thought to be similar to
that of centrally acting PGE2. In vitro studies have provided evidence supporting the role for
LPS- and IL-1β-induced thermogenesis during fever as measured by the increased marker of
UCP-1 activity (Jepson et al., 1988, Dascombe et al., 1989, Rothwell, 1989), the increased
blood flow in BAT (Dascombe et al., 1989) and the LPS-induced expression of mRNA for
UCP-1 (Cannon et al., 1998). Furthermore, IL-1α and IL-1R have both been reported to be
expressed by the brown adipocytes in mice under basal and stimulated conditions (Burysek et
al., 1993, Burysek and Houstek, 1996, 1997). Contrary to this view, in vivo studies have
yielded opposite results. It was thus shown that the febrile response evoked by peripheral
IL-1 was completely intact in mice lacking UCP-1. Moreover no induction in the expression
of UCP-1 mRNA was seen after the treatment of wild-type mice with IL-1β (OkamatsuOgura et al., 2007) suggesting that UCP-1 and thermogenesis in BAT are independent of IL1β.
The importance of IL-6, previously shown to be induced in mouse brown adipocytes (Burysek
and Houstek, 1997) in the activation of BAT, has not been examined in detail and the
available evidence comes from the recent study in which the chronic expression of IL-6 in rat
hypothalamus was found to elevate the mRNA and protein levels of UCP-1, indicating
35
activated thermogenesis in BAT. In addition, the IL-6 induced increase in UCP-1 levels was
entirely erased when BAT was surgically denervated (Li et al., 2002).
36
AIMS
The overall aims of the research on which this thesis is based was to study the mechanism by
which IL-6 enables the inflammatory induced fever response and the relationship between
IL-6 and PGE2 in the elevation of the body temperature.
The specific aims were:
Paper I:
To examine the relationship between PGE2 and the LPS-induced cytokine
response in the brain.
Paper II:
To investigate if the critical role of IL-6 in the LPS-induced febrile response is
connected to the mPGES-1 regulated synthesis of PGE2.
Paper III:
To identify genes regulated by IL-6 in the hypothalamus of LPS-treated mice
that can explain the role of IL-6 in fever regulation.
Paper IV:
To clarify the role of IL-6 produced by cells of hematopoietic origin in the
regulation of the febrile response evoked by LPS.
37
38
METHODOLOGY
Animals
Genetically modified animals
In the late 1980s, the ability to obtain genetically modified mice lacking a particular gene of
interest was demonstrated (Thomas and Capecchi, 1987, Mansour et al., 1988, Koller et al.,
1989, Thompson et al., 1989). This so called “knockout” technique based on the homologous
recombination of embryonic stem (ES) cells containing the disrupted gene provided an
opportunity to generate mice of any desired genotype. Briefly, the DNA sequence for the
neomycin resistance gene is inserted in the cloned fragment of the gene which then is
transfected to the ES cells. Subsequently, these cells are injected into the blastocytes obtained
from the wild-type mouse and then implanted into the pseudopregnant female mice, thus
generating a mouse with the mutated germ line cells. Mice lacking the endogenous production
of IL-6, mPGES-1 and lcn2 in the entire body were used in papers Ι – IV and the
construction of these mice strains have previously been described by their originators (Kopf et
al., 1994, Trebino et al., 2003, Flo et al., 2004).
Some of the benefits from the use of genetically modified mice are that they offer a valuable
tool in studying the importance of the “knocked” gene and that especially in situations when
pharmaceutical agents acting as agonists or antagonists are not available. In addition, the
antagonizing drugs do not provide a complete inhibition of the targeted gene and the
difficulties including use of the appropriate drug, dose and the route of administration are thus
avoided. However, an employment of these modified mice is not entirely without caveats.
One of them is that compensatory mechanisms may emerge thus making the outcome of the
targeted ablation difficult to interpret (Luquet et al., 2005). Secondly, the introduction of the
selection marker together with the neomycin cassette can influence the phenotype of the
mutation and make it difficult to distinguish the effects of the targeted gene ablation from
those obtained by insertion of the selection marker (Fiering et al., 1995). Thirdly, the targeted
gene ablation can cause severe abnormalities both before birth and in adult life as previously
reported when mice lacking c-fos were generated (Johnson et al., 1992, Wang et al., 1992).
Fourthly, the loss of the particular gene can be directly lethal as has been demonstrated for the
EP4 receptor which, on being mutated by homologous recombination, resulted in failure to
close ductus arteriosus and consequently in a markedly decreased survival among the
neonates (Nguyen et al., 1997, Segi et al., 1998). Despite these obstacles caused by specific
ablation of the individual genes, the knock-out technology has clearly contributed to better
understanding of mammalian physiology and its utilization has in fact literally exploded since
its introduction.
Generation of Interleukin-6 chimeric mice
The widely used procedure in creating chimeric mice involves the utilization of whole-body
irradiation accompanied by bone marrow transplantation. When exposed to lethal irradiation,
cells situated in the bone marrow are destroyed and shortly after replaced by the bone marrow
cells derived from another mouse, thus generating a mouse that contains two separate cell
sources. In paper IV, mice with the deletion of IL-6 and wild-type mice were exposed to
whole body γ-irradiation which killed the bone marrow stem cells. Following irradiation,
hematopoietic donor cells from mice of the other genotype (non-irradiated donor animals) that
express the green fluorescent protein (GFP) were given through an intravenous tail injection
39
and in that way repopulated the recipients. The bone marrow cells were transplanted from
wild-type mice to IL-6 knock-out mice, and vice versa, as well as to animals of the same
genotype and were allowed to repopulate the recipients for five months. Thus, IL-6 knock-out
mice transplanted with wild-type bone marrow lacked IL-6 expression in all cells but those of
hematopoietic lineage, such as immune cells, whereas wild-type mice transplanted with bone
marrow derived from the IL-6 knock-out mice had an intact expression of IL-6 in all cells but
those of hematopoietic origin. Based on the fact that IL-6 knock-out mice are unresponsive to
the LPS-induced fever we reasoned that if the febrile response was seen in knock-out mice
that are transplanted with wild-type hematopoietic cells, and absent in wild-type mice
transplanted with knock-out hematopoietic cells, this would imply that cells of the
hematopoietic lineage are important. Conversely, if IL-6 knock-out mice transplanted with
wild-type hematopoietic cells remained afebrile, whereas wild-type mice transplanted with
knock-out hematopoietic cells displayed fever upon intraperitoneal LPS-challenge, this would
then suggest that the critical mechanism is not dependent on IL-6 expressed in hematopoietic
cells (Fig. 5). Since donor mice whose bone marrow-derived cells were used to replace the
destroyed bone marrow in the recipients also were equipped with GFP this allowed us to track
and distinguish these cells from the resident cells and thus evaluate the success of irradiation
and reconstitution by flow cytometry.
WT
IL-6 KO
Radiation
Transplantation
KO with KO
bone marrow
No Fever
2
WT with WT
bone marrow
KO with WT
bone marrow
LPS
1
No Fever
Fever
1
WT with KO
bone marrow
LPS
2
Fever
Fig. 5. Flow chart illustrating generation of the chimeric mice. IL-6 knock-out (IL-6 KO)
and wild-type (WT) were exposed to whole-body irradiation which destroyed their bone
marrow. Following irradiation, the IL-KO mice and WT mice were transplanted with the
bone marrow from the same or opposite genotype. Five months later, mice were injected
intraperitoneally with LPS and their body temperature was monitored. For more details, see
text.
40
Briefly, the bone marrow cells to be transplanted were extracted from femur and tibia
dissected from 8-10 weeks old IL-6 knock-out and wild-type mice by gently crushing them in
the phosphate buffered saline containing 5 % fetal calf serum. After dissociating the
remaining bone fragments, the bone marrow was filtered, centrifuged and suspended in the
same medium as above. The obtained bone marrow cells were labeled with CD45 antibody
conjugated with magnetic microbeads and then enriched by passing them through a magnetic
field. The cells were counted in the Bürken chamber and diluted to a final concentration of
2x106 CD45 positive cells which then were injected in the tail vein of irradiated mice. Five
months later, the degree of reconstitution in the peripheral blood was assessed by tail-vein
bleeding and analyzed using FACSCanto flow cytometer.
However, although the irradiation procedure provides a possibility to examine the importance
of a particular gene in the hematopoetic system, especially when no floxed mice strain is
available, it is not completely free of drawbacks. It has been observed, for instance, that
whole-body irradiation may transiently increase the permeability of the blood-brain barrier in
rats (Diserbo et al., 2002), induce hippocampal dysfunction (Monje et al., 2002) and lead to a
substantial infiltration of bone marrow-derived microglia into the brain (Simard et al., 2006).
Furthermore, the lethal irradiation may correlate with the influx of cytokines, and the
transplanted bone marrow from donor mice may result in a non-physiological persistence of
the circulating hematopoietic cells (Ransohoff, 2007). Although these observations do
indicate the possibility of compromised immune response in the brains of the recipients and
may complicate the interpretation of the results, there are reasons to believe that the
irradiation technique itself does not alter the LPS-induced response in the immune system
(Turrin et al., 2007) and this validates the use of this technique in examining the LPS-evoked
effects in chimeric mice.
Administration of drugs
The doses of commercially available pharmaceutical agents used in the research for the thesis
were based on their reported biological activity in the literature. The main objection against
utilization of the pharmacological agents can be ascribed to possible weaknesses in existing
knowledge about their targeting (specific/non-specific) and use of appropriate doses in order
to evoke a relevant biological effect. In fact, the differing results obtained by different studies
may be directly related to the differences in drug doses and the varying routes of their
administration. The use of a particular drug can further be complicated if it only is soluble in
organic solvents that are inappropriate for use in vivo.
Parecoxib – selective Cox-2 inhibitor
Parecoxib (Dynastat) is a commercially available prodrug of valdecoxib. This selective Cox-2
inhibitor was used in the study for paper II and it does not have any inhibitory effect on
Cox-1 when used at therapeutic doses, at least not in humans. It was dissolved in an isotonic
solvent (NaCl 0.9 %) and injected intraperitoneally 30 min before LPS using the same route
of administration. The dose of Parecoxib used was based on reports in the literature.
Etanercept – the TNF-α suppressor
Etanercept is a soluble TNF receptor fusion protein, commercially better known as Enbrel.
This TNF-α neutralizing drug was used in the study for paper II and it was administered both
intraperitoneally and intracerebroventricularly. When the former route of administration was
41
applied, the etanercept was injected 24 h prior LPS, due to its pharmacokinetic properties with
maximum plasma concentration reached 1-2 days following peripheral administration. In
contrast, when etanercept was administered intracerebroventricularly, it was given 30 min
before LPS, to take into account the rapid turnover of the cerebrospinal fluid. The doses used
were comparable to those previously shown to neutralize the action of TNF-α (Hermann et al.,
2002, Abdulla et al., 2005, Hu et al., 2007).
Inflammatory model and monitoring of the body temperature
Lipopolysaccharide
In the research for papers I – IV, the LPS from Escherichia coli (serotype 055:B5) was
diluted in sterile 0.9 % saline solution and administered intraperitoneally. Except for some
experiments in paper II where the dose of LPS was 2 µg/mouse (~ 100 µg/kg body weight),
the dose of 120 µg/kg body weight, being considered as a medium dose was used. This dose
has previously been recognized as suitable in generating a robust immunological response
with a regular biphasic febrile response (Engblom et al., 2003) without evoking hypothermia
that usually is associated with higher doses of LPS (Blanque et al., 1996).The standardized
way of handling and administration, generation of highly reproducible results and no need to
use live bacteria are only some of the benefits associated with the use of LPS as an immune
stimulus. The battery of cytokines induced by LPS offers a much more complete
physiological picture and contributes to the better understanding of processes involved in the
regulation of fever than would result if only a single pyrogenic cytokine, such as IL-1β were
to be used. In addition, the longer duration of LPS makes it more relevant as immunological
stimulus than some short-lived cytokines that are rapidly metabolized (Reimers et al., 1991,
Givalois et al., 1994). The major drawback coupled to the use of LPS is that this highly
inflammatory stimulus, despite its broad effects which have been described to be of similar
nature in mice and humans (Michie et al., 1988, Burrell, 1994) does not reflect the complex
pathophysiological picture that occurs during human bacteria-evoked diseases.
Telemetry
The telemetric recordings were employed in the studies for papers I – IV to efficiently and
accurately monitor the body temperature of freely moving (non-restrained) mice, thus offering
a clear advantage in comparison with measurement of rectal temperature which requires the
mice to be restrained. The animals were implanted intra-abdominally at least one week before
administration of an appropriate stimulus with the calibrated radio transmitters recording their
core body temperature. The surgical insertion of these transmitters was carried out under
continuous anesthesia with isoflurane and was generally well tolerated by the operated mice.
Those animals that post-surgically displayed any kind of suffering or increased burden were
immediately excluded.
Administration of recombinant interleukin-6
In the study for paper II, two different doses of IL-6 were injected intraperitoneally (500 and
900 ng/mouse) and for paper III, only the higher dose of these two was employed. Although,
IL-6 was purchased from different suppliers and diluted in sterile saline (paper II) or sterile
water (paper III) both were expressed in E.coli and were reported to have comparable
biological activity. In contrast to the intravenously administrated IL-6 which rapidly is
degraded in plasma with a half-life of only ~ 7 min in serum, the significantly prolonged
42
duration of IL-6 has been observed following an intraperitoneal route of administration (Mule
et al., 1990). In this case, the half-life of circulating IL-6 was determined to be ~ 3 h and the
biologically active levels of IL-6 were detected for 6 h (Mule et al., 1990). The benefit
achieved by use of a single cytokine is that the evoked effects specifically are attributed to
that particular cytokine but, on the other hand, the effects achieved are of restricted nature.
Intracerebroventricular administration of PGE2
In the study for paper II, isoflurane-anesthetized mice were mounted in a stereotactic device
and a drill hole was made in the skull. PGE2 was diluted in artificial cerebrospinal fluid and
graded concentrations of PGE2 were administrated during 1 min in a total volume of 2 µl to
the lateral ventricle. Two minutes later, the injection needle was removed, the skin was
sutured and the anesthesia was turned off. During the whole procedure, the mice were kept at
37°C through a feedback controlled heating pad. All mice were awake 5 min after the
injection and were immediately returned to their home cages for monitoring of body
temperature using telemetry.
Protein analysis
Immunohistochemistry
Immunohistochemistry is a technique widely used to detect, localize and visualize different
biomarkers at the protein level. It is based on the interaction between the applied antibody and
a tissue epitope which is recognized by the employed antibody and the following detection of
this interaction either by enzymes, such as peroxidase that can catalyze a color-producing
reaction or by conjugating the antibody to fluorescent dyes, such as Alexa Fluor.
Mice were flushed transcardially with ice-cold saline (0.9 %) to rinse out the blood followed
by perfusion with ice-cold paraformaldehyde (4 %). The brain and peripheral organs of
interest were dissected and post-fixated for 3 h using the same fixative as during the perfusion
and subsequently cryopreserved with sucrose until they were saturated, usually over night. 30
µm thick coronal tissues sections were obtained by cutting the tissue with a freezing
microtome and were kept in a cold-protectant at -20°C until used for immunohistochemistry.
In papers II, III and IV, the avidin-biotin-complex (ABC) system with a peroxidase as an
enzyme label was used to detect Cox-2, lcn2 and GFP, respectively. To evaluate if lcn2 colocalize with Cox-2 and vWF in paper III and to examine the re-constitution in brain in
paper IV, double staining immunohistochemistry was used.
In brief, the freely-moving tissue sections were incubated with the primary antibodies which
consequently were recognized by the secondary biotin-labeled antibodies raised against the
species in which the primary antibodies were produced. The complex system constituting of
avidin-biotin and peroxidase was added, thus binding to the secondary antibodies and the
brown color was developed using 3.3´-diaminobenzidine tetrahydrochloride (commonly
abbreviated as DAB) as a chromogen. The staining intensity of DAB, which polymerizes
upon oxidation, was further enhanced by adding ammonium nickel sulfate. The precipitated
brown end product produced by DAB is highly insoluble in alcohol and other organic solvents
which results in the stable conservation of immunohistochemically stained sections for a long
time.
43
One of the most important aspects in immunohistochemistry is the specificity of the primary
antibodies used. In order to ascertain that an antibody employed really recognizes the protein
of interest and nothing else, it helps to have some controls validating the specificity of the
antisera (Saper and Sawchenko, 2003). The preferable scenario results if the antibody does
not stain the tissue from which the targeted antigen has been eliminated and this can easily be
accomplished by employing the conventional knock-out animals lacking the antigen of
interest. However, this approach, which so far is only applicable in mice, requires that such
genetically modified animals are available, which unfortunately is not always the case. In
addition, keeping in mind that antibodies are biological affinity agents and not to be
considered as standard chemical reagents, it may happen that the antibody being employed
cross-reacts with and stains a variety of structurally related antigens other than the one that it
was raised against. It is thus, possible that the antibody still stains something in the tissue
from which the targeted molecule has been deleted. The rationale for this, however rarely
occurring situation, may depend on the deletion strategy that may render a truncated,
biologically non-functional part of the protein to be expressed in the knock-out animal that is
recognized and stained by an antibody (Lorincz and Nusser, 2008). An obvious but premature
conclusion that may be drawn is that the obtained labeling is not to be viewed as specific but
it does not mean that an antibody itself is non-specific. In cases when mice lacking the
targeted protein are not available, the specificity of the antibody used can be examined by
subjecting the tissue of interest to Western Blot where the ideal outcome should reveal that
the antibody used only recognizes one antigen corresponding to the single band of the
expected molecular weight. However, there are situations when the antibody, due to the
various posttranslational modifications, stains several bands but this does not mean that the
antibody is not specific. If the targeted antigen or the antibody used is well-characterized, i.e.
when it is known how the staining pattern should appear, one could conclude that the
antibody is specific even if it stains several bands on the blot. Another control experiment
employs the use of the pre-adsorption test. In this test, the primary antibody is pre-adsorbed
with the purified immunizing antigen and if the staining is absent after following this
procedure it probably reflects the condition that the antibody recognizes the antigen against
which it was raised. Thus, the pre-adsorption test is a valuable tool for use in obtaining a valid
control for distinguishing specific from unspecific staining. Unfortunately, it is not cost
effective, not always available and does not provide the user with the information if an
employed antibody cross-reacts with other structurally related antigen sequences. This can,
however, be solved by re-performing the pre-adsorption test in which the immunizing antigen
is excluded but including these related antigens. In this case, the labeling should persist.
Furthermore, this test is not applicable on the monoclonal antibodies, which always will be
adsorbed to their antigen. The alternative approaches in the verification of an appropriate
staining specificity include: 1) the combination of immunohistochemistry with in situ
hybridization and the comparison of the staining pattern obtained by these two techniques, 2)
the close overlap in the staining pattern obtained with two different antibodies raised against
the distinct epitopes on the same antigen, 3) the comparison of the well known staining
pattern with that previously reported by others, 4) the concordance in staining specificity with
that detected by reporter molecules (such as Cre-recombinase or GFP) or 5) to examine if the
expression of antibody used co-localize with that obtained by an antiserum that is better
characterized in a double-staining study. However, one should keep in mind that not only the
specificity of the primary antibody may affect the experimental outcome. Thus, an attention
must also be addressed to other parts of the procedure such as type and duration of fixative,
the thickness of tissue sections, pH and salt concentration of buffers, ambient temperature,
blocking reagents and detergents. In the present thesis, these recommendations have been kept
in mind to secure and optimize the reliable stainings of the used antibodies.
44
Western Blot
Western blotting is a routine technique in protein analysis and has been used in the study for
paper III. The term “blotting” refers to the transfer of the gel-separated proteins to a
membrane that subsequently can be visualized by antibodies. In brief, the denaturated proteins
were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and individually
separated based on their size. The separated proteins were then electrophoretically transferred
to the polyvinylidene difluoride membrane and the non-specific binding was blocked. The
membrane was incubated with the primary antibody directed against the antigen of interest
followed by addition of a secondary horseradish peroxidase (HRP) enzyme-conjugated
antibody that was directed against the primary antibody and used as a probe for protein
detection. A developing solution that constituted of a chemiluminescent substrate which
produces light in the reaction with the HRP enzyme was applied to the membrane and the
light output could be captured and protein bands visualized by using a light-sensitive camera.
The light intensity was directly correlated to the abundance of the protein on the membrane.
Quantitative immunoassays
Immunohistochemistry is a valuable technique for use in the detection and localization of the
antibody-targeted molecules at the cellular level in tissue. However, in order to quantify the
concentration of certain molecule in a given sample, this technique is not number one in
choice. Quantitative immunoassays are based on the same principle as immunohistochemistry, i.e. the interaction between an antibody and an antigen. In contrast to
immunohistochemistry, the analysis is done in biological fluids such as urine, plasma and
serum or lysed tissue samples containing a complex mixture of substances.
In the studies for papers II and IV, two different techniques were employed. The enzyme
immunoassay was used to measure the concentration of PGE2 in cerebrospinal fluid in paper
II and the Bio-plex pro cytokine assay was used to measure the concentration of IL-6 in
plasma and cerebrospinal fluid in paper IV.
Briefly, the mice were asphyxiated by CO2 and blood was drawn from the heart, transferred to
EDTA-coated tubes and centrifuged at 4°C to obtain plasma which consequently was
aliquoted and directly frozen on dry ice. Immediately after, the mouse was fixed in a
stereotactic device with the head flexed anteriorly. Neck muscles were divided in the midline
to expose the atlanto-occipital membrane and the cerebrospinal fluid was collected from the
cisterna magna using the Hamilton syringe. The cerebrospinal fluid was frozen on dry ice
right away and blood-contaminated samples were discarded.
Bio-Plex Pro Cytokine Assay
The Bio-plex system offers simultaneous measurement of multiple biological substances in
relatively small sample volume thus being both cost-effective and less time-consuming. The
bio-plex cytokine assay is formatted on 6.5 µm fluorescently dyed magnetic beads meaning
that each of them is internally assigned with a specific spectral code obtained by loading the
beads with various fractions of red and infrared fluorophores. This arrangement allows
distinguishing the specific antibody-antigen interactions within a multiplex suspension and at
least theoretically, the detection and analysis of 100 different analytes is possible. The system
usually employs two antibodies. The capture antibody raised against the molecule that is to be
measured is covalently linked to the certain magnetic bead immobilizing the analyte and the
45
biotinylated detection antibody binding the same antigen is coupled to the complex
constituting of streptavidin-phycoerythrin (SAPE) where the latter serves as a fluorescent
indicator.
The detection of multiplexed reactions was acquired using a Luminex 200 system. The
discrete measurement of individual molecules was based using the principles of flow
cytometry allowing a stream of suspended magnetic beads to line up in a single line before
passing the detection chamber. While the red laser excites the internal fluorescent dyes within
each bead and provides the proper classification and identification of the bead, the green laser
excites the fluorescence that is attributable to the binding of an analyte thus generating a
reporter signal. The concentration of bead-bound analyte that is directly proportional to the
median fluorescence intensity of the reported signal was calculated against the fluorescence
intensity of the standard curve run in parallel on the same plate. The major drawback
associated with the use of this technique is that antibodies directed against the antigens
present in an analyzed sample can cross-react thus leading to erroneous interpretation of
results. An alternative but more expensive approach is to use the competent immunoassay.
Enzyme immunoassay
In contrast to the multiplex assay described above, the use of an enzyme immunoassay only
employs the detection and quantification of a single analyte. In the study for paper II, the
concentration of antigen (in this case PGE2) in the cerebrospinal fluid was measured using a
competitive immunoassay. The sample containing unknown levels of PGE2 was applied to
wells that were pre-coated with the primary antibody against the PGE2 molecules. This was
followed by addition of the enzyme to which the PGE2 was bound covalently, thus competing
with PGE2 in the sample for binding sites. The excess of reagents was washed away and
chromogenic substrate was added to develop a yellow product. The absorbance was read in a
spectrometer with the intensity of yellow color being inversely proportional to the
concentration of PGE2 in the samples, meaning that the greater the absorbance, the fewer
molecules of PGE2 were present in the samples to compete with the enzyme-conjugated PGE2
for binding sites. The concentration of PGE2 in the individual samples was calculated by
relating their absorbance against the standard curve.
Quantitative real-time PCR
The quantitative real-time PCR or Real-time RT-PCR is based on the same principle as
conventional PCR technique and has become a method of choice in quantifying the mRNA.
The first step involves the reverse transcriptase-catalyzed conversion of RNA to
complementary DNA (cDNA) and in the second step the utilization of fluorescent reporter
dyes allows the simultaneous detection, amplification and quantification of targeted DNA
sequence during the PCR in real time. The qPCR reaction can be carried out either by using
so called non-specific SYBR green dye or probe-based TaqMan® chemistry.
TaqMan®-based detection
In the studies for papers I, II and III we used TaqMan® pre-designed primers and probes.
The principle of TaqMan rely on the 5`- 3`exonucleae activity of the Taq polymerase and the
use of specific oligonucleotide probes between the two primer pairs (Holland et al., 1991).
These probes are designed to hybridize within the target sequence and consist of fluorescently
labeled reporter attached at the 5`-end and a quencher dye at the opposite end of the probe,
46
which prevents the detection of fluorescence from intact probes (Livak et al., 1995). Upon
annealing of the probe to its target sequence, the Taq polymerase degrades the probe at the 5`end, thus allowing the emission of previously quencher-suppressed fluorescence which then is
detected and monitored. This process is repeated after each cycle causing an accumulation of
fluorescence which is directly proportional to the amount of amplified product. However, this
technique is not without caveats which may originate from various sources and markedly
influence the reliability of the results (Nolan et al., 2006). In order to standardize the
measurements of the gene expression and thus maximize its reproducibility we constantly
used the same kit in extracting the total RNA from the desired tissues followed by quality
check (purity and integrity) of the isolated RNA. Subsequently, the same amount of RNA was
used as a template in the synthesis of cDNA and the priming strategy consistently employed
random hexamer primers, which, as the name implies, randomly target the RNA and as a
consequence of this may cause variation in the targeting efficiency between the individual
samples. On the other hand, the use of random hexamer priming is associated with increased
yields and is suitable for samples with poor integrity and low concentrations (Nolan et al.,
2006). The TaqMan probes used contained minor groove binders at the 3` end which stabilize
the probe-DNA hybridization, thus increasing the target sensitivity. Furthermore, these
stabilizing molecules are not cleaved by 5`exonuclease activity of Taq polymerase and make
it possible to use the shorter probes, thus increasing the design flexibility. The samples are
compared during the exponential phase of the reaction, i.e. when the target quantity doubles
with each cycle and at a certain user-defined threshold of the fluorescence (Ct) which reflects
the point when the amount of target reaches the point of detection. Expression of the gene in
individual samples was calculated relative to the expression of endogenous control gene (∆Ct)
that should be constant and independent of the genotype or treatment, and the relative
expression (2-∆Ct) was normalized to the average expression in wild-type mice treated with
saline (2-∆∆Ct).
SYBR green-based detection
In the study for paper IV, the SYBR green chemistry was used to detect the expression of
IL-6 in the chimeric mice. This detection technique is based on the amplification of all
double-stranded DNA without inhibiting or compromising the efficiency of the PCR.
Furthermore, the need for probes is circumvented by using the SYBR green dye, thereby
reducing the running costs. The reaction starts with the addition of the SYBR green dye to the
sample which immediately binds to all double-stranded DNA present and generates
fluorescence. Upon denaturation of the DNA, the SYBR green dye is released from the
sample causing the decrease in the fluorescence. During PCR, the AmpliTaq® DNA
polymerase amplifies the target sequence thus generating the PCR products or “amplicons”.
The SYBR green dye binds then to each new product of the double-stranded DNA with a net
increase in the fluorescence being directly proportional to the amount of the PCR products
produced. One of the major disadvantages associated with SYBR green detection chemistry is
that it binds both to specific and non-specific products, and hence may increase the
probability of generating false positive results. The second drawback is that it may result in
the formation of primer-dimers which, as the name implies, are composed of primer
molecules that have hybridized to each other. Consequently, the DNA polymerase may begin
to amplify the primer-dimers formed which in this way compete for reaction components and
in turn may reduce the amplification efficiency of the targeted sequence, and the reliability of
the quantification. However, the risk of these potential pitfalls is according to the
manufacturer (Applied Biosystems) reduced with the design of SYBR green master mix
(paper IV) which has been created to optimize the efficiency and accuracy of the reaction
47
without compromising its sensitivity. In addition, the presence of formed primer dimers can
be detected by melting curve analysis, which provides a powerful assessment in
characterizing the dissociated DNA double strands. The primer-dimers usually consist of
shorter sequences with lower melt temperature than the targeted sequence and become clearly
discriminated from the amplified products when the temperature gradually is increased in
melting curve analysis (Nolan et al., 2006).
Microarray technology
The use of DNA microarray platforms offers simultaneous monitoring of the expression of
multiple genes and was first reported in the mid-1990s (Pease et al., 1994, Schena et al., 1995,
Lockhart et al., 1996). Expression analysis is performed by following two different principles.
The first is manufactured by robotic spotting of the complementary DNA (cDNA) or small
PCR fragments, known as probes at the selected locations on the array surface (Schena et al.,
1995). Because of the way they are factorized, these platforms are called the spotted
microarrays with each probe having its own specific hybridization characteristics (one single
probe for each target transcript) and each array is measuring two biological samples at the
same time. These arrays are sometimes called two-color microarrays since the two samples to
be compared are labeled with two different fluorophores (Shalon et al., 1996), with Cy3 and
Cy5 being the two most commonly used dyes in labeling of cDNA. Post hybridization, these
two colors are scanned individually and the excited fluorescence from the two fluorophores is
visualized, thus enabling the relative differences in gene expression between two samples to
be reported (Tang et al., 2007). The second technology (oligonucleotide microarrays)
measures one sample at the time and reports the quantitation of the absolute expression levels.
This technique is based on the in situ synthesis of thousands of specific short oligonucleotide
probes at specific locations representing a particular gene or family of gene-splicing variants
on silicon surface by using the photolithographic chemistry (Pease et al., 1994, Lockhart et
al., 1996). The use of oligonucleotide probes circumvents the need of cloning, spotting or
PCR-performance which are some of the advantages with this technology. Every probe
consists of 25 bases and is designed to be complementary to the sequence of the targeted
gene. Also each one of these probes is constructed to possess similar hybridization properties
and binding affinities. The probes completely matching the transcript are referred as perfect
match (PM) probes whereas an equal number of these measure any cross-hybridization that
may have occurred and are referred to as mismatch (MM) probes, only differing in a single
base that is altered to measure the background. Before analyzing the obtained microarray data,
the signal intensities from the numerous probes are collected during a process called
summarization combining the data for a probe-set into a single expression value level.
However, the availability of MM probes is not always utilized to obtain the summarized
expression and depends on the type of the microarray chip used. Also the type of algorithm
used in analysis of the data determines whether the information provided by MM probes is
used or not.
In paper III, we utilized an oligonucleotide array (GeneChip 1.0 ST array) covering the
entire mouse genome. Each of the mouse genes was represented by approximately 27 probes
distributed along the entire length of the transcript, thus offering an accurate and reliable
picture of gene expression. The Gene 1.0 ST system where the “ST” stands for sense target
covers well-interpreted genes and only uses the PM-design with background being evaluated
by a collection of generic background probes, which are not present in the mouse genome.
48
In brief, the assay process began with extraction of the total hypothalamic-RNA that
concomitantly was subjected to quality control. The concentrations and purity of isolated
RNA were measured and 200 ng of total RNA was converted to double stranded cDNA
according to the Affymetrix GeneChip® whole transcript sense target labeling assay. The
cDNA produced was amplified and used as a template to synthesize cRNA which
concomitantly was reversely transcribed to single stranded DNA (ssDNA) by using random
hexamers (Fig. 6). The dUTPs introduced in the DNA in the previous step was subsequently
recognized by a combination of uracil DNA glycosylase and apurinic/apyrimidine
endonuclease 1 resulting in an efficient fragmentation of the ssDNA. In the next step, the
ssDNA was labeled with the terminal deoxynucleotidyl transferase covalently linked to the
DNA labeling reagent (biotin allonamide triphosphate). Thereafter, 2 µg of ssDNA was
hybridized to the microarray chip according to manufacturer’s instructions (17 ± 1 h in 45°C).
Prior to hybridization, a number of various controls were added to the hybridization cocktail.
bioB, bioC and bioD representing the genes involved in the biotin synthesis in E.coli and cre
representing the recombinase from P1 bacteriophage were used to control how well the
hybridization, washing and staining procedures have succeeded. The chip was washed and
stained using the SAPE, a fluorescent staining agent that binds to the biotin-labeled ssDNA.
The washing and staining steps were carried out on the GeneChip® fluidics station 450
operated using the GeneChip® operating software (GCOS). The chip was then checked for
bubbles and scanned on the GeneChip® Scanner 3000 7G. By using the laser light, the SAPE
was excited and the emitted signal recorded, creating an image of the whole chip. From this,
raw probe intensity values were calculated and written into the CEL files. Subsequently, these
files were imported into the GeneSpring software for data analysis.
cRNA
cDNA
RNA
extraction
Total RNA
Reverse
transcription
IVT
ssDNA
Transcription
Hypothalamus
Fragmentation
CEL files
Analyze
GeneSpring
software
Scanning
Absolute gene
expression levels
Staining
GeneChip Mouse 1.0 ST array
Hybridization
Labeling
Biotin-labeled
Fragmented ssDNA
fragmented ssDNA
Fig. 6. Microarray analysis. RNA was extracted from hypothalamus and subsequently
transcribed to complementary DNA (cDNA). In the following steps, the single-stranded
(ssDNA) was fragmented, labeled and hybridized to a microarray chip covering the whole
mouse genome. After being stained, the chip was scanned and a digital image was created.
The probe intensity values were calculated and converted to CEL files which then were
subjected to data analysis.
The experiments employing the microarray platforms are readily exposed to many sources of
variation including the disproportional amounts of starting RNA, the differences in
hybridization quality between the arrays, the variability between the factorized arrays,
49
scanner-setting differences etc. These unwanted and obscuring variations may be of technical
or non-biological nature and must regardless of their origin be minimized in order to reveal
the true biological picture. This is done in a process called pre-processing or normalization. A
variety of different mathematic algorithms can be used for this purpose with the common
denominator that they all include the background correction and summarization of the raw
probe intensities to convert them into useful gene expression measures. However, these steps
are, dependent on the algorithm used, carried out differently. In our application (paper III),
the robust multi-array analysis (RMA) algorithm was used to pre-process/normalize the probe
intensities (Bolstad et al., 2003, Irizarry et al., 2003). This algorithm only uses the PM
information completely ignoring the MM in all steps and is usually suitable for use when the
background is believed to be minimally distributed across the experiment. Also, the MM
probes do not ideally represent the background and subtracting them when adjusting the
background is thought to add more noise. In addition, it is preferable to perform the
normalization at the probe level, thus only reproducing the probe-specific dependent bias. The
RMA uses so called quantile normalization, a non-linear symmetry that corrects for array
biases. It assumes that the general gene expression is evenly distributed across the samples
and normalizes the input data so that the distribution of the probe intensities is the same for
each array.
50
RESULTS AND CONCLUSIONS
Below, I will briefly reproduce the main findings with short comments on each of the papers.
For more details, I refer the reader to the individual papers.
The lipopolysaccharide-induced peripheral and central mRNA expression of IL-1β,
TNF-α and IL-6 occurs independently of prostaglandin E2 (paper I).
The febrile response evoked by LPS is accompanied by the expression of several cytokines,
such as IL-1β. In general, these cytokines are thought to exert their pyrogenic effects via
synthesis of PGE2 in cells associated with the brain blood vessels. However, these cytokines
may also exert their actions by pathways that do not necessarily involve the formation of
PGE2 since they have been found to be induced in the brain following an immune challenge.
Here, we examined the induced mRNA expression of IL-1β, TNF-α and IL-6 and their
receptors evoked by peripheral LPS challenge in several brain regions (hypothalamus, cortex
and striatum) but also in peripheral tissues (lung and liver) in mice lacking the gene encoding
mPGES-1, the terminal enzyme in the formation of PGE2.The mPGES-1 deficient mice were
shown to be incapable of mounting the febrile response upon peripheral LPS challenge
compared to wild-type mice which displayed a prominent fever response. The mRNA levels
of IL-1β, IL-6 and TNF-α induced by peripheral LPS challenge were not different in
mPGES-1 deficient mice from those quantified in wild-type mice. Rather, there was a
tendency toward higher expression of these cytokines in the absence of mPGES-1. More
specifically, although the expression of IL-1β, both in wild-type mice and mPGES-1 mice,
was induced to various degrees in all brain regions examined with the highest expression
quantified in hypothalamus, there was no difference in expression of IL-1β between these
genotypes. Further, the expression of the IL-1R was, independently of the genotype, similarly
up-regulated in all brain regions. Also in the periphery, the expression of IL-1β did not differ
between the genotypes. In line with this, the LPS-treated mPGES-1 knockout mice displayed
neither different central nor peripheral expression of IL-6 and TNF-α nor different expression
of their respective receptors in comparison to wild-type mice.
The main conclusions from this study are that the LPS-induced expression and signaling of
the three examined cytokines remain intact in mPGES-1 deficient mice, thus being
independent of the induced PGE2 synthesis and that these cytokines regardless if being
expressed centrally or peripherally are by themselves unable to elicit a febrile response.
The critical role of interleukin-6 in lipopolysaccharide-induced fever is not exerted
through prostaglandin E2 dependent mechanism (paper II).
IL-6 and PGE2 are both necessary for the establishment of the febrile response as
demonstrated by the complete absence of fever in animals lacking the endogenous production
of IL-6 or PGE2. However, the mechanism of IL-6 in the regulation of fever has been a matter
of debate and the results have pointed in different directions. Hence, some studies have
suggested IL-6 to act upstream of PGE2 whereas others claim that it rather acts downstream of
PGE2. Here, using mice devoid of IL-6, the direct relationship between IL-6 and PGE2 was
examined. The body temperature of mice lacking IL-6 was unaffected by peripheral injection
with LPS, despite the fact that they upon the same treatment showed similar expression of
Cox-2 in endothelial-like cells in brain as wild-type mice. Furthermore, the LPS-induced
mRNA expression of Cox-2 and mPGES-1 in hypothalamus was elevated to the same degree
51
in both genotypes. In good accordance with this, the LPS-induced levels of PGE2 in
cerebrospinal fluid of IL-6 deficient mice were not different from those quantified in wildtype mice. However, the quantification of IL-6 mRNA levels in the hypothalamus of
mPGES-1 deficient mice revealed that IL-6, following an intraperitoneal LPS injection, was
not only significantly increased compared to the saline-injected mice but also displayed a
tendency towards higher expression than in wild-type mice (paper I). Surprisingly, when
PGE2 was injected intracerebroventricularly, mice deficient in IL-6 developed the febrile
response that was indistinguishable from that observed in wild-type mice. Although the
inducible expression of TNF-α mRNA in comparison to wild-type mice was somewhat higher
in IL-6 deficient mice, neither central or peripheral inhibition of TNF-α, a cytokine that due to
its hypothermic action was thought to prevent the development of fever in these mice, helped
in restoring the febrile response in mice lacking IL-6. We also examined whether the absence
of fever in IL-6 deficient mice was due to presence of some Cox-2 dependent factor, but the
peripheral treatment with Cox-2 inhibitor did not change the body temperature in these mice.
The main conclusions from this study can be summarized as follows: The LPS-induced
synthesis of PGE2 occurs independently of IL-6 and endogenously produced PGE2 cannot
evoke fever in the absence of IL-6. Importantly, although the induced expression of IL-6 is
intact in the absence of PGE2, endogenous IL-6 is unable to provoke an elevation of body
temperature without PGE2. However, since fever is generated in IL-6 deficient mice when
exogenous PGE2 is administrated centrally, IL-6 cannot be considered as a component in the
thermogenic pathway downstream of PGE2. Instead, an alternative possibility that may
resolve this discrepancy is that IL-6 may be involved in the transport of PGE2 so that it in the
absence of IL-6 cannot reach the EP3 bearing receptors in the thermogenic preoptic region and
thus exert its pyrogenic effect.
The identification of lipocalin-2 as a novel factor in the pathway of inflammatory IL-6
signaling and its relationship to the lipopolysaccharide-induced febrile response (paper
III).
In order to further clarify the mechanism by which IL-6 regulates the febrile response, we
investigated the role of IL-6 in the inflammatory cascade provoked by peripheral LPS
challenge by using a genome wide microarray analysis which allowed us to identify the
differentially expressed genes in hypothalamus of LPS-treated IL-6 knockout mice compared
to wild-type mice. Despite the fact that IL-6 knockout mice in contrast to the wild-type mice
are unable to mount a febrile response elicited by peripheral LPS only a few genes were
differentially expressed between these two genotypes. The largest difference in gene
expression induced by LPS between the two genotypes was displayed by lipocalin-2 and mice
lacking IL-6 displayed two times lower expression of this gene in the hypothalamus. The
results from the microarray analysis were confirmed by qPCR and western blotting. A direct
relationship between IL-6 and lipocalin-2 was shown by injecting wild-type mice with
peripheral IL-6 which, as detected by an immunohistochemical analysis, induced the
expression of lipocalin-2 in brain vascular cells. The expression of LPS-induced lipocalin-2
was shown to be localized in cells associated with the brain blood vessels and these cells were
positively identified as endothelial cells. Furthermore, we also showed that the induced
expression of lipocalin-2 in brain endothelial cells was partly co-localized with Cox-2, the
rate limiting enzyme for the production of PGE2. The direct role of lipocalin-2 in fever was
examined by employing mice lacking the production of lipocalin-2 and monitoring their body
temperature following treatment with LPS. While the telemetric recordings revealed that there
was no difference in fever between lipocalin-2 deficient mice and wild-type mice at
52
conditions near thermoneutrality, the body temperature of female mice lacking lipocalin-2
was significantly lower during the third phase of fever compared to the wild-type mice at
room temperature. Subsequently, the attenuated fever response in lipocalin-2 deficient female
mice was reflected in lower expression of Cox-2 mRNA in the hypothalamus of these mice
following treatment with LPS.
In summary, we have in this paper identified lipocalin-2 as a new factor in the IL-6 dependent
fever generating pathway and shown that lipocalin-2 is partly controlled by IL-6.
Furthermore, we have shown that lipocalin-2 co-localizes with Cox-2 in brain endothelial
cells and that it in a sex-dependent and ambient-specific manner is implicated in
thermogenesis, possibly by influencing the expression of Cox-2. However, lipocalin-2 only
has a minor and restricted effect in LPS-induced thermogenesis and its lower expression in
mice devoid of IL-6 cannot explain the complete absence of fever in these mice.
Interleukin-6 synthesized by non-hematopoietic cells is important for the development of
the lipopolysaccharide-induced fever (paper IV).
IL-6 can be synthesized by a variety of different cells in response to different pathological
stimuli but the source of IL-6 in immune-induced fever has not been completely identified. In
this paper, we aimed to examine the role of hematopoietically and non-hematopoietically
derived IL-6 in mediating the febrile response elicited by peripheral LPS. This approach was
enabled by using chimeric mice which either had an intact expression of IL-6 in the cells of
hematopoietic or non-hematopoietic origin. Mice lacking the expression of IL-6 in
hematopoietic cells displayed an intact fever response that was indistinguishable from that
seen in wild-type mice whereas mice only expressing IL-6 in the cells of hematopoietic
origin, except for the slightly increased body temperature during the second phase of fever,
were resistant to LPS-induced fever. In good accordance with this, mice with an intact
expression of IL-6 in cells of non-hematopoietic origin were quantified to have comparable
levels of IL-6 in plasma and cerebrospinal fluid as wild-type mice. Furthermore, the
expression of IL-6 mRNA in brain of mice that were only expressing IL-6 in nonhematopoietic cells was not different from that in wild-type mice. In contrast, the IL-6 levels
in plasma and cerebrospinal fluid were only moderately elevated in mice with intact
production of IL-6 in the hematopoietic cells, thus being consistent with the minor elevation
of the body temperature in these mice following challenge with LPS.
In summary, it can be concluded that IL-6 primarily synthesized in the cells of nonhematopoietic origin is critical for the LPS-induced fever whereas IL-6 produced by
hematopoietic cells only contributes to the second phase of fever. However, although this
study has identified the non-hematopoietic cells as critical for the systemic IL-6 production
and associated them with the generation of fever, the phenotype of these cells is still to be
revealed.
53
54
GENERAL DISCUSSION
IL-1β, TNF-α and IL-6 belong to the three most extensively studied cytokines mediating the
febrile response. The implication of these highly inducible proteins in fever has been based on
several lines of experimental findings demonstrating that: i) fever coincides with the
appearance of cytokines in plasma and cerebrospinal fluid (Cannon et al., 1990, LeMay et al.,
1990a, LeMay et al., 1990c, Coceani et al., 1993, Givalois et al., 1994, Jansky et al., 1995,
Kakizaki et al., 1999) ; ii) cytokines are induced in the brain following an immune challenge
and their receptors are presented on various brain cells (Ban et al., 1991, Vallieres and Rivest,
1997, French et al., 1999); iii) fever is evoked by exogenous administration of cytokines and
likewise abolished by application of agents that antagonize or neutralize their action (Opp and
Krueger, 1991, Luheshi et al., 1996, Lundkvist et al., 1999, Cartmell et al., 2000, Rummel et
al., 2006); iv) generation of fever is compromised in mice lacking the endogenous production
of the proinflammatory cytokines, such as IL-6 (Chai et al., 1996, Horai et al., 1998, Kozak et
al., 1998).
Although the involvement of secreted proinflammatory cytokines in mediating the febrile
response has been well established, it has been debated for a long time how these cytokines
transfer the pyrogenic signals to the brain. Several hypotheses have been suggested over the
years, some of them including the direct effect of the induced cytokines on the brain and the
implication of the neural pathway through the vagal afferents in conveying the inflammatory
message to the brain (se also “the communication between the immune system and the
brain”). However, our findings in paper I show that it is unlikely that this is the case, at least
not when the expression of IL-1β, TNF-α and IL-6 is induced endogenously. Hence, the
peripheral and central expression of these cytokines was, following the peripheral challenge
with LPS, induced at the same or even at higher levels in the mice lacking mPGES-1 than in
the wild-type mice, thus implying that the induced cytokine expression occurs independently
of the PGE2 synthesis. Furthermore, it was shown that the cytokines despite being markedly
induced were unable to alter the unresponsiveness of the mPGES-1 knockout mice to LPS
(paper I). The situation may however be different when the exogenous cytokines, as
demonstrated for the IL-1β (Ching et al., 2007), are administered into the peritoneal cavity. In
that case, mice lacking the endothelial-specific expression of IL-1R displayed a fever
response when IL-1β was injected intraperitoneally but not when the same stimulus was
administered intracerebroventricularly or intravenously. However, the onset of the febrile
response in mice devoid of IL-1R in brain endothelial cells was delayed, appearing 3 ½ h after
the intraperitoneal injection of IL-1β. These results implied that the activation of the central
nervous system and the concomitant fever response may not depend on the intact expression
of the IL-1R in the endothelial cells and suggested that the neural pathway may partly be
involved in the transduction of IL-1β elicited signals in the periphery to the brain by-passing
the cerebral endothelium. However, the implication of the neural transmission has been
controversial for several reasons (see “the communication between the immune system and
the brain”). Rather, the predominating view supported by convincing evidence is that the
synthesis of PGE2 in endothelial cells of the blood-brain barrier plays a critical role in the
generation of fever following an immune challenge (Ek et al., 2001, Yamagata et al., 2001,
Engblom et al., 2003, Ching et al., 2007, Vardeh et al., 2009). The additional support favoring
this pathway of immune-to-brain communication has quite recently revealed that mPGES-1
produced by the cells of non-hematopoietic origin, identified as endothelial cells, is crucial for
the development of fever upon peripheral immune challenge (Engstrom et al., 2012).
However, the synthesis of PGE2 in brain endothelial cells needs to be induced and it has been
suggested that IL-1β can be the “triggering signal” behind its induction. Although this idea is
55
supported by the findings that the IL-1R1 is expressed on the vascular endothelium (Ericsson
et al., 1995, Konsman et al., 2004) and that it co-localizes with Cox-2 and mPGES-1 in brain
endothelial cells (Ek et al., 2001, Engblom et al., 2002, Konsman et al., 2004), along with the
evidence demonstrating that the IL-1β inducible expression of Cox-2 in the brain is dependent
on the intact expression of the IL-1R1 on the endothelial cells (Ching et al., 2007), the
presence of the detectable levels of IL-1β in plasma of immune challenged humans and rats
have yielded conflicting results, thus making the role of IL-1β in immune-to-brain
communication ambiguous (Michie et al., 1988, Cannon et al., 1990, Givalois et al., 1994,
Van Zee et al., 1995). Moreover, while mice deficient in IL-1β or IL-1R1 are resistant to fever
elicited by Listeria Monocytogenes or turpentine (Zheng et al., 1995, Leon et al., 1996, Horai
et al., 1998, Kozak et al., 1998), they display an augmented fever response upon
administration of LPS or IL-1β (Alheim et al., 1997, Labow et al., 1997). On the contrary,
mice deficient in IL-6 remain afebrile when challenged with LPS or IL-1β (Chai et al., 1996,
Kozak et al., 1998) suggesting the critical role of IL-6 in eliciting the febrile response to
peripheral immune challenge. However, peripherally administrated IL-6 itself does not induce
or only weakly induces fever and the expression of Cox-2 in the brain (Lacroix and Rivest,
1998, Cartmell et al., 2000, Rummel et al., 2006) (paper II) or the production of PGE2 in the
cerebrospinal fluid (paper II), thus being in contrast to IL-1β, which has been reported to
imitate the LPS-inducing effects on the febrile response (Dinarello and Savage, 1989, Saper
and Breder, 1992, Chai et al., 1996). It has therefore been suggested that IL-6 exerts its effect
downstream of PGE2, this being supported by the findings that the expression of Cox-2 in
brain induced by peripherally injected IL-1 could occur in the IL-6 deficient mice without
eliciting the febrile response (Kagiwada et al., 2004). In line with this, the inhibition of Cox-2
activity was shown to be associated with the suppressed induction of IL-6 with concomitantly
abolished fever response (Kagiwada et al., 2004) and the hyperthermia evoked by centrally
administrated PGE2 was significantly attenuated by injecting the neutralizing antibody against
IL-6 into the preoptic area of rats (Fernandez-Alonso et al., 1996). However, our findings in
paper I do not lend support for this idea since the absence of mPGES-1 did not affect the
expression of either peripheral or central IL-6. Furthermore, we showed that centrally
administrated PGE2 was able to evoke the febrile response in IL-6 deficient mice thus being in
agreement with what was observed by others (Chida and Iwakura, 2007) and importantly
excluding the possibility that IL-6 acts downstream of PGE2 (paper II). On the other hand, a
considerable bulk of evidence has suggested that IL-6 signaling upstream of the PGE2 instead
is important in eliciting the febrile response to peripheral immune challenge. It was suggested
that IL-6 by binding to its receptors in the blood or in the brain (Vallieres and Rivest, 1997),
similarly to what was suggested for IL-1β, could trigger the synthesis of PGE2 in the vascular
endothelium. Subsequently, PGE2 could then enter the brain parenchyma and induce fever.
The increased levels of circulating IL-6 have been measured during the course of the LPSinduced febrile response with the abolishment of fever observed when IL-6 was neutralized in
the bloodstream (Cartmell et al., 2000, Rummel et al., 2006). Further evidence in support for
this idea was provided by findings in which the intracerebroventricular administration of IL-6,
in contrast to the peripherally injected IL-6, was shown to induce the expression of Cox-2 in
the brain endothelial cells (Cao et al., 2001) thus elevating the levels of PGE2 in the
cerebrospinal fluid which coincided with the rise in body temperature (Dinarello et al., 1991).
Moreover, centrally administrated IL-6 was not only able to elevate body temperature in wildtype mice but also to rescue the febrile response in the IL-6 deficient mice (Chai et al., 1996)
in a Cox-2 dependent manner since centrally administrated IL-6 did not alter body
temperature in mice devoid of Cox-2 (Li et al., 2003) and the pretreatment of the rats with
intraperitoneally injected Cox-2 inhibitor was shown to completely block the fever response
evoked by intracerebroventricular IL-6 (LeMay et al., 1990c, Rothwell et al., 1991, Cao et al.,
56
2001). However, our findings in paper II are not compatible with IL-6 being upstream of
PGE2 because mice lacking the endogenous production of IL-6, i.e IL-6 deficient mice,
despite being afebrile to peripheral immune challenge with LPS, had an intact expression of
Cox-2 and mPGES-1 both peripherally and centrally and displayed comparable levels of
PGE2 in the cerebrospinal fluid as those seen in the wild-type mice.
Our findings in paper II show that whereas the endogenously produced PGE2 is not sufficient
in eliciting the febrile response in LPS-challenged mice that lack IL-6, the exogenously
administrated PGE2 is shown to, in a dose-dependent manner, evoke an elevation of body
temperature in the IL-6 deficient mice. While the immune-induced production of PGE2
represents the condition which encompasses the fully activated inflammatory cascade, the
exogenously injected PGE2 represents an artificial stimulus with limited response that
circumvents the need of being transported across the blood-brain barrier. Furthermore, the
PGE2 concentration is much higher when this pyrogenic substance is injected directly into the
brain compared to the concentration achieved during normally occurring inflammatory
conditions. However, despite the obvious inconsistency between these two conditions, our
data still demonstrate that the fever-generating pathway downstream of PGE2 is clearly
functional in IL-6 deficient mice and this implies that some immune-induced hypothermiapromoting factor that normally is suppressed by IL-6 is enhanced in the absence of IL-6, thus
keeping IL-6 knockout mice resistant to the pyrogenic action of PGE2. One such factor could
be TNF-α which in some studies has been suggested to induce hypothermia (Klir et al., 1995,
Kozak et al., 1995, Kozak et al., 1998, Leon et al., 1998). Furthermore, IL-6 deficient mice
have in response to LPS been shown to display increased local and plasma levels of TNF-α
(Kozak et al., 1998, Xing et al., 1998). However, our findings in paper II show that this is
unlikely the case since pretreatment of the IL-6 knockout mice with a TNF-α neutralizing
agent failed in generating the fever in these mice. We also showed that such a factor, if
present, is not Cox-2 dependent as the body temperature in LPS-treated IL-6 deficient mice
did not rise following pretreatment with selective Cox-2 inhibitor (paper II).
The other possibility is that endogenously produced PGE2 require the presence of IL-6 in
order to reach its receptors in the preoptic region. This idea has been supported by the
findings in which it has been demonstrated that the IL-6 signaling governs the entrance of the
circulating lymphocytes across the blood vessels (Vardam et al., 2007) and furthermore, it has
been shown that the vascular permeability of the blood-brain barrier was significantly reduced
in the IL-6 knockout mice or in rats treated with IL-6 neutralizing antibodies when being
exposed to the pneumococcal infection (Paul et al., 2003). Hence, but still only speculatively,
it can be hypothesized that PGE2 in the absence of IL-6 is not transported to its cognate
receptors in sufficiently high concentrations to exert its pyrogenic effect in the IL-6 knockout
mice.
The implication of lipocalin-2 in fever and its connection with IL-6 and Cox-2
In paper III, lipocalin-2 was identified as a new factor in the fever-generating pathway that
was controlled by IL-6. Lipocalin-2 was found to be differentially expressed in the brain of
the IL-6 knockout mice following the peripheral immune challenge with LPS, and displayed
half the expression in these mice compared to wild-type mice. However, our findings
demonstrated that IL-6 was not the only factor influencing the expression of lipocalin-2 in the
brain since its induction was present also in the mice devoid of IL-6. Similar to our results
(paper III), the expression of lipocalin-2 was recently found to be regulated by IL-6 in the
57
liver (Sultan et al., 2012).The gene expression of lipocalin-2 was shown to be markedly
suppressed in the liver of mice lacking IL-6 following intramuscular treatment with
turpentine-oil. Furthermore, the expression of lipocalin-2 in hepatocytes was shown to be
directly induced by IL-6 (Sultan et al., 2012), thus being comparable to our findings in paper
III which showed that the peripherally injected IL-6 was able to induce the production of
lipocalin-2 in the brain and provided evidence for the direct relationship between IL-6 and
lipocalin-2. The implication of lipocalin-2 in the inflammatory signaling began to clarify with
studies showing that this protein not only is a major acute phase protein in mice (Flo et al.,
2004, Sultan et al., 2012) but also is a key participant in the anti-microbial host-defense
response that limits the growth of the invading bacteria (Flo et al., 2004) and that can be used
as a biochemical marker of disease, such as kidney injury (Shemin and Dworkin, 2011). In the
brain, systemic administration of LPS has been shown to induce the expression of lipocalin-2
in choroid plexus epithelial cells and in the endothelial cells (Marques et al., 2008, Ip et al.,
2011). These findings were confirmed and further extended in paper III, demonstrating that
lipocalin-2 expressed by brain endothelial cells co-localizes with Cox-2. The functional
interrelationship between the LPS-induced expression of lipocalin-2 and Cox-2 by brain
endothelial cells can however, only be speculated about. It is possible that lipocalin-2, via its
receptors which have previously been found on the endothelial cells (Ip et al., 2011, Lee et al.,
2011), influence the expression of Cox-2 in an autocrine manner since the available data do
not lend support for the explanation that lipocalin-2 control the LPS-induced mRNA
expression of proinflammatory cytokines that are able of inducing Cox-2 (Ip et al., 2011).
The available data not only provide evidence in support for lipocalin-2 and IL-6 being
mutually related during the inflammation-induced responses but also suggest that they may be
linked in the control of thermogenesis. Thus, it has been observed that lipocalin-2 deficient
mice, in a similar manner to that observed for mice lacking IL-6 (Wernstedt et al., 2006),
were unable to defend their body temperature when they were acutely placed in a cold
environment (4°C), an exposure that when lasting for more than 10 h subsequently resulted in
a lethal outcome for these mice (Guo et al., 2010). However, during the thermoneutral
conditions, our findings showed that mice lacking lipocalin-2, in contrast to the complete
absence of LPS-induced fever in the IL-6 knockout mice, were fully capable of mounting the
typical febrile response when challenged with LPS, displaying an elevation of body
temperature that was no different from that seen in the wild-type mice. The situation became
different when the LPS-induced fever was examined at the ambient temperature where female
(but not male) mice lacking lipocalin-2 displayed a significant attenuation of the fever
response. It is worth noting that only the late phase of the LPS-induced fever was affected by
the absence of lipocalin-2 in the female mice with the two earlier phases remaining intact. The
two earlier phases of fever have been suggested to employ the mechanisms that are
differentially regulated from the mechanism eliciting the late phase. However, while our
findings suggest that lipocalin-2 in sex-dependent and ambient-specific manner may regulate
the late phase of the LPS-induced fever, other studies have suggested this phase to be reduced
by vagotomy (Szekely et al., 2000) and to be associated with the induced mRNA expression
of cPLA2-α in the hypothalamus (Ivanov et al., 2002). In addition, our findings suggested that
the attenuated fever response in female mice lacking lipocalin-2 was associated with the lower
expression of prostaglandin-synthesizing enzyme Cox-2 in the hypothalamus, hence giving
support to the previous observation in which the expression of Cox-2 in cultured astrocytes
was induced by lipocalin-2 (Lee et al., 2011).
Another possibility that may help in explaining the observed difference in fever response
between the sexes in paper III evolved from the observation by Guo et. al. (Guo et al., 2012)
58
demonstrating that female mice lacking lipocalin-2 displayed reduced levels of 17β-estradiol
in serum and had significantly decreased adipose tissue levels of aromatase, a key enzyme in
the biosynthesis of estradiol. Further, the expression of the estrogen receptor α was found to
be down-regulated in female lipocalin-2 deficient mice (Guo et al., 2012), thus suggesting the
link between lipocalin-2 and estrogen. The idea has however been challenged by a study in
which it was demonstrated that the fever response evoked by intraperitoneal LPS was
attenuated in the ovariectomized rats following treatment with the ovarian hormones
(estrogen/progesterone) (Mouihate and Pittman, 2003). Furthermore, it was shown that
reduction in the LPS-induced fever was accompanied by the decreased levels of Cox-2 in the
hypothalamus in rats receiving the treatment with estrogen/progesterone (Mouihate and
Pittman, 2003). The association of gender specific hormones with Cox-2 was thus
demonstrated in a way that is in a disagreement with our findings in paper III since we
demonstrated that female mice lacking lipocalin-2, which possibly also had reduced levels of
the circulating ovarian hormones, displayed an attenuated fever response elicited by
peripheral LPS.
The importance of hematopoietically-derived IL-6 in fever
IL-6 has been shown to be produced by many different cells following an immune challenge.
However, the role of hematopoietically derived IL-6 in LPS-induced fever has not been
elucidated. Our findings in paper IV show that IL-6 synthesized by cells of the hematopoietic
origin play minor role in fever and identify IL-6 synthesized by non-hematopoietical cells as
the major contributor in the LPS-elicited febrile response. In agreement with this, it was
previously shown that the elevated levels of circulating IL-6 were associated with the
development of fever in children with completely depleted hematopoeisis, thus suggesting
that cells of non-hematopoietic origin may account for the production of IL-6 and the
concomitant generation of fever (Pechumer et al., 1995). Our findings in paper IV
demonstrated that mice deficient of IL-6 in hematopoietic cells but with an intact expression
of IL-6 in the cells of non-hematopoietic lineage displayed a typical polyphasic fever response
to peripheral LPS. The body temperature in mice with impaired production of IL-6 in nonhematopoietic cells was slightly elevated 2 – 3½ h following peripheral LPS challenge and
coincided with increased levels of IL-6 in plasma and cerebrospinal fluid. Thus the finding
that mice with IL-6 production only in hematopoietic cells were able to mount a short increase
in body temperature during the second fever phase at a magnitude of approximately one third
of the fever response in wild-type mice implies that peripherally produced IL-6 of the
hematopoietic origin may have a role in development of the second febrile phase elicited by
LPS. However, our data in paper IV do not provide support for involvement of
hematopoietically derived IL-6 in the third febrile phase, thus only being partly in agreement
with the previous observation in which the generation of LPS-elicited second and third fever
phase was ascribed predominantly to the peripheral production of pyrogenic cytokines by the
hematopoietical cells (Chakravarty and Herkenham, 2005). It is also worth noting that the
initial phase of fever, which normally appears within the first 30-50 min after the nonstressful injection of LPS, was masked in the study for this paper due to the stress-induced
hyperthermia associated with the handling of mice during the injection procedure. However,
the establishment of the first phase of fever has not been attributed to the presence of IL-6 in
the circulation since the onset of this phase was shown to occur before IL-6 appears in the
blood (Givalois et al., 1994, Cartmell et al., 2000). Although we in paper IV have identified
the non-hematopoietic cells as critical for LPS-induced production of IL-6 and concomitant
fever response, the phenotype of these cells remain to be clarified which in turn may involve a
variety of cell types, as suggested by findings in both in vitro and in vivo studies. Thus, the
59
induced production of IL-6 has been demonstrated in brain endothelial cells (Reyes et al.,
1999, Verma et al., 2006), astrocytes, microglia and neurons (Benveniste et al., 1990, Sawada
et al., 1992, Ringheim et al., 1995, Vallieres and Rivest, 1997, Beurel and Jope, 2009),
fibroblasts (Helfgott et al., 1987, May et al., 1988a), hepatocytes (Saad et al., 1995, Panesar et
al., 1999) and mesangial cells (Rugo et al., 1992). Although speculative it can thus be
hypothesized that IL-6 signals by binding to either its soluble receptors in the bloodstream
(Novick et al., 1989) or to the receptors that are anchored to brain blood vessel cells. In
support to this idea are the previous findings demonstrating that both the membrane bound
form of the IL-6 receptor and the adapter molecule gp130 which is required for the
transduction of the signal (Taga et al., 1989, Hibi et al., 1990) were found on the cells
associated with the brain blood vessels (Vallieres and Rivest, 1997). Furthermore, it has been
demonstrated that STAT3, which upon activation translocate to the nucleus and acts as a
transcription factor, was induced by peripherally administrated IL-6 and its immunoreactivity
was shown to be associated with brain blood vessels (Rummel et al., 2005). Perhaps, an
endothelial-specific ablation of gene encoding IL-6 (or IL-6R) could help in elucidating the
functional significance of this mechanism in the LPS-evoked fever response.
Another issue that could not be answered by the experimental approach used in paper IV,
thus requiring further examination, is whether the induced increase of IL-6 in cerebrospinal
fluid in mice only expressing IL-6 in the non-hematopoietical cells, which also responded
with fever, is derived from the periphery or from the brain since the levels of IL-6 were
elevated in both compartments. Previously, it has been suggested that circulating IL-6 can
alter the brain function by travelling across the blood-brain barrier (Banks et al., 1994), which
favors the idea that IL-6 in cerebrospinal fluid, at least to some extent, may originate from the
periphery. In addition to this, the vascular permeability of blood vessels in the blood-brain
barrier was suggested to be enhanced by IL-6 that is known to activate STAT1 and STAT3
(Rummel et al., 2006) which in turn have been associated with the impaired integrity of the
blood-brain barrier (Chaudhuri et al., 2008).
In summary, the findings presented in this thesis suggest that the immune-induced expression
of proinflammatory cytokines is insufficient in eliciting the febrile response in the absence of
PGE2. Examining the role of IL-6 and its relation to PGE2 in the febrile response revealed that
these two molecules probably mediate fever through closely related but different pathways. In
addition, investigating the gene expression profile of mice deficient in IL-6 resulted in the
identification of lipocalin-2 as a new factor in the IL-6 mediated fever signaling. Furthermore,
with the aid of chimeric mice IL-6 produced by non-hematopoietic cells was found to mediate
the immune-induced pyresis.
60
ACKNOWLEDGEMENTS
This journey has taught me many things, some of them having a big portion of patience in
research and appreciating even the smallest progress. Many people have helped me through
this period of life and if I forget to mention someone I apologize in advance. I would like to
acknowledge:
Camilla Nilsberth, my supervisor, for letting me be part of the never-ending interleukin-6
story and for allowing me to develop into an independent PhD student. Further, I thank you
for your support, patience, time and for believing in me through the years.
Anders Blomqvist, my assistant supervisor, for giving me the chance to work in your
laboratory. Your knowledge about the neuroscience alongside with your ability to improve a
certain proof has been a source of inspiration for me.
Johan Ruud, my college and friend for your help, all fruitful discussions, an inexhaustible
optimism and reflections not just when it comes to the interpretation of different laboratory
outcomes but also regarding more general things in life. The time spent at floor 11 would not
have been the same without you.
David Engblom, for always having time and always having an explanation to whatever the
problem is. You are one of the few people that have made a strong influence on me with your
great enthusiasm, knowledge and great personality.
Ludmilla Mackerlova, for being such a wonderful person, nice conversations and for having
patience with me during the many telemetry sessions. I cannot thank you enough for your
technical skills.
Yanjuan Tang for your help with the bone marrow transplantations and for analyzing
reconstitution.
Louise Elander for your help when I was new in the lab and for making a good team during
the ICV sessions. Linda Engström Ruud for showing me the art of mounting sections and for
always having a positive attitude. Anna Eskilsson for your help, for being a good friend and
nice discussions. I admire your integrity and loyalty. Daniel B. Wilhelms for always having
cheering-up anecdotes to tell and for your help and advices when I so badly needed them. Unn
Kugelberg for being helpful and pleasant conversations about parenthood (now you don`t
have to apologize any longer when entering the office). Anna Nilsson, Anna-Maria Vasilache
and Sofie Sundberg for nice conversations around the coffee-table. Robert Ihnatko for nice
conversations and for sharing the office with you. Milen Kirilow for your skills in making the
transgenetic mice and nice sense of humor. Simin Mohseni for good team-work with students.
Other people currently or previously working at floor eleven: Christine, Kiseko, Takashi,
Jakob, Nina, Sarah, Johan, Ulrika, Sara, Anders, Björn, Elin, Elahe, Maryam, Amir, Nesar,
Sivert, Fredrik, Sivert, Michael, Anna K and Maarit for making a nice atmosphere to work in.
The staff at the animal facility, especially Anders Delleskog, for always being nice and trying
to help.
61
Åsa Schippert at floor nine for helping me with troubling qPCR machine, I will never forget
the Saturday when you manage to fix the machine so I could run that big 384 well plate.
Larry Lundgren for reviewing this thesis.
Kalle Wallin for input on statistics.
My friends and relatives outside the lab for your friendship and support although you most
likely will find this thesis boring.
My parents in law, Sead and Sabina for your support and for always making me feel welcome
into your home.
My dear parents, Agan and Menura for endless support, love and never stop believing in me
even in the most difficult situations through the life. You showed me the right way and have a
great part of where I stand today. You have told me that as a parent you never stop caring
about your child but it is first now that I truly understand the meaning of it.
Sunita, my darling wife for your love, unconditional support and for understanding what I am
going through. Thank you for believing and having confidence in me and also for blessing us
with Nejla and in such way providing me the different aspect of life. I am sorry for sometimes
being “absent” when I arrive home but all this struggling and effort was hopefully for the
better tomorrow.
62
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