Download Alcohol and neuroinflammation: Involvement of astroglial cells and

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Revisión
Inmunología
Vol. 25 / Núm 3/ Julio-Septiembre 2006: 188-200
Alcohol and neuroinflammation:
Involvement of astroglial cells and TLR4/IL-1RI receptors
A.M. Blanco, C, Guerri
Unidad de Patología Celular, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain.
ALCOHOL Y NEUROINFLAMACIÓN: PAPEL DE LAS
CÉLULAS ASTROGLIALES Y DE LOS RECEPTORES TLR4/IL-1RI.
Recibido: 200 Junio 2006
Aceptado: 26 Septiembre 2006
RESUMEN
La expresión de mediadores inflamatorios y citocinas están implicadas en la patogénesis de diversas enfermedades neurodegenerativas.
Una característica importante de la neuroinflamación es la activación de
las células gliales, especialmente microglia y astroglia, que producen citocinas, compuestos pro-inflamatorios y tóxicos, desencadenando una respuesta inflamatoria y daño cerebral. Estudios recientes sugieren que los
receptores TLRs (Toll-like), junto a las células gliales desempeñan un papel
relevante en la respuesta inmune del sistema nervioso central (SNC), y
una alteración en la regulación de dicha respuesta puede causar neurodegeneración.
El abuso de alcohol y el alcoholismo inducen daño cerebral y, en algunos casos causan neurodegeneración. Los procesos neuropatológicos implicados en estos efectos no están totalmente esclarecidos. Evidencias recientes sugieren que el etanol es capaz de activar a las células gliales y de inducir procesos inflamatorios en el cerebro que pueden conducir a muerte
neural. Estas evidencias demuestran que el etanol favorece la activación
de vías de señalización intracelular (IKK, MAPKs) y factores de transcripción (NF-κB, AP-1) que conllevan a la producción de citocinas y de
mediadores inflamatorios (iNOS, NO, COX-2) en cerebro y en células
astrogliales. La respuesta inflamatoria inducida por el etanol parece estar
mediada por una activación de los receptores TLR4/IL-1RI, ya que cuando se bloquea su función, se eliminan los efectos del etanol sobre la inducción de mediadores inflamatorios y muerte celular. Aunque los mecanismos que subyacen a la activación de estos receptores se desconoce, proponemos que el etanol a través de su interacción con los lípidos de membrana, podría facilitar el reclutamiento de los receptores TLR4 e IL-1RI en
los microdominios de membrana (lipid rafts), conllevando a un aumento en su respuesta y señalización. En conclusión, aunque se requieren más
trabajos para evaluar el mecanismo de la activación de los TLR4/IL-1RI
por el etanol, en esta revisión se presentan estudios que apoyan la idea
de que un aumento en la respuesta innata inmune a través de los receptores TLR4/IL-1RI, podría participar en el daño cerebral asociado al consumo de alcohol.
PALABRAS CLAVE: Etanol / Daño cerebral / Inflamación / Células gliales / Receptor Toll-Like.
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ABSTRACT
Inflammatory mediators and cytokine expression are implicated in
the pathogenesis of several neurodegenerative diseases. The hallmark of
brain inflammation is the activation of glial cells, especially microglia and
astroglia, which produce a variety of pro-inflammatory and toxic compounds that can induce brain damage. Recent developments in our understanding of neurodegeneration implicate glial cells and Toll-like receptors
(TLRs) as vital players in the immune response within the central nervous
system, and that deregulation of this response plays an important role in
brain injury and neurodegeneration.
Alcohol abuse and alcoholism induce brain damage, and in some
cases, neurodegeneration. The neuropathologic processes underlying these
effects remain poorly understood. Recent data demonstrate that ethanol
promotes inflammatory processes in the brain and in glial cells by upregulating cytokines and inflammatory mediators (iNOS, NO, COX-2),
and by activating signalling pathways (IKK, MAPKs) and transcriptional
factors (NF-κB, AP-1) implicated in inflammatory injury. TLR4 and IL1RI are involved in the signalling of ethanol-induced inflammatory response, since blocking these receptors abolishes the production of ethanolinduced inflammatory mediators and cell death in astrocytes. Although
the mechanisms involved in the ethanol-induced activation of TLR4/IL1RI receptors are unknown, we propose that ethanol can facilitate TLR4/IL1RI recruitment into lipid raft microdomains through its interaction with
membrane lipids, leading to the activation and signalling of these receptors. In summary, although further work is needed to evaluate this hypothesis, this review presents evidences supporting the notion that the activation of innate immune system and TLR4/IL-1RI by ethanol triggers
inflammatory mediators in the brain and causes brain damage.
KEY WORDS: Ethanol / Brain injuries / Inflammation / Glial cells / TollLike Receptor.
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INMUNOLOGÍA
ROLE OF INFLAMMATION IN BRAIN DAMAGE
Inflammation is an important host defence response to
injury, tissue ischemia, autoimmune responses or infections
and it often elicits a generalized sequence of events known
as the acute phase response, which can limit the proliferation
of invading pathogens. However, while local and generalized
inflammatory responses offer clear benefits in infectious
states, sustained or inappropriate inflammation can cause
numerous diseases including inflammatory bowel disease,
rheumatoid arthritis and psoriasis. Inflammation is also an
important component of the damage caused by autoimmune
diseases, and fundamentally contributes to diseases such
as cancer, diabetes and cardiovascular diseases(1, 2).
The central nervous system (CNS) was considered as
an immune privileged organ, which was not susceptible to
inflammation or immune activation, and was thought to
be largely unaffected by systematic inflammatory and
immune responses. This point of view has changed drastically
during the last decade. It is now accepted that the brain
coordinates and regulates many aspects of the host defence
response, which may explain the behavioural responses to
diseases such as fatigue and depression, and how the
psychological state influences susceptibility to disease and
recovery(3). Furthermore, emerging evidence indicates that
inflammation represents a potential pathogenic factor in
many CNS diseases, including chronic neurodegenerative
diseases, such as Parkinson´s disease(4), Alzheimer´s disease(5),
Creutzfeldt-Jacob disease(6), and more recently in some
psychiatric disorders such as depression, anxiety and
schizophrenia(3). In these diseases, inflammation is atypical
and occurs in the absence of leukocyte over infiltration(7).
There is no doubt that the brain differs significantly
from other tissues in its response to infection or central
inflammation. One evident example in this response is
leukocyte recruitment, which is rapid in systemic organs,
but modest and delayed in the brain. However, the brain
exhibits key features of inflammation and in response to
acute insults, and the resident cellular elements, including
glial cells, release inflammatory mediators within minutes
or hours in response to acute insults(3).
Glial cells, particularly microglia and astrocytes, are
responsible for the immune functions within the brain,
and they play roles in inflammatory response. Microglia,
the macrophages of brain parenchyma, are in a downregulated state as compared with other tissue macrophages
in the healthy brain. However, they are rapidly stimulated
in response to injury or infection, and their morphology
changes and acquires an array of functions, including
phagocytosis, up-regulation of cell-surface molecules and
the production and secretion of inflammatory mediators(8).
A.M. BLANCO, C. GUERRI
In addition to microglia, astrocytes are also important in
the immune response, contributing to the establishment
and maintenance of the blood-brain barrier (BBB)(9) and
modulating the migration of monocytes and lymphocytes
across the BBB(10). Astrocytes also respond vigorously to
brain injury and seem to play an important role in the fine
tuning of brain inflammation(11). Indeed, injury to CNS
damage is inevitably accompanied by astrocytic hypertrophy,
proliferation, and altered gene expression, a process
commonly referred to as reactive astrogliosis(12) which is
associated with inflammation(13). Depending on the disease
context, however, astrogliosis can be seen as either a positive
event that promotes neuronal and glial survival via the
production of neurotrophins and growth factors, or as a
negative influence on regeneration via the inhibition of
neuronal and glial growth and migration. The diffuse
nature of reactive astrogliosis in the CNS suggests a role
either for soluble mediators, such as cytokines, and/or the
presence of an integrated astrocyte-to-astrocyte syncytium
that enables the transfer of information across extended
distances.
The cytokines for which the evidence is most compelling
in the initiation and modulation of reactive gliosis include
IL-1-β, TNF-α, IFN-γ, and TGF-β. Glial cells express receptors
for all these cytokines, and each one appears to fulfil a
different functional role in the astrocyte response(14). Stimulation
of astrocytes in response to a neuropathologic process
triggers the activation of an innate immune response (see
below), leading to the production of inflammatory cytokines
and free radicals, as well as to the expression of major
histocompatibility complex II molecules(15). This functional
reprogramming may be essential for maintaining homeostasis
and the local regulation of inflammatory and immune
responses(16). Among the cytokines, IL-1-β has been considered
an important mediator of inflammatory responses in the
CNS. It has been implicated in a number of neurodegenerative
conditions, including Alzheimer´s disease(17), and it is vastly
produced under conditions of brain damage, disease, or
stress(18). Although the mechanisms of these effects are
unclear, IL-1 is initially released by glial cells, acting on
astrocytes and microglia to induce the production of additional
cytokines and growth factors, thereby promoting inflammatory
activity in the brain(19, 20). Indeed, IL-1 promotes glial scaring
or astrogliosis when directly injected into the CNS, thus
suggesting a potential role of IL-1 in mediating astrocytic
hypertrophy upon neuronal damage(21).
Finally, although extensive data suggest that inflammation
contributes to the development of many CNS diseases,
controversy regarding whether neuroinflammation and
glial (microglial and astroglial) activation are beneficial or
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ALCOHOL AND NEUROINFLAMMATION: INVOLVEMENT OF ASTROGLIAL CELLS AND IL-1RI/TLR4 RECEPTORS
detrimental remains. Indeed, many mediators produced as
a result of microglial and astroglial activation have a dual
role; they can be neuroprotective as well as neurotoxic, and
may participate in long-term repair and recovery. The
complex interplay and balance between diverse mediators
and environmental factors may ultimately determine the
outcome of acute CNS injury and the initiation and progression
of inflammatory brain damage. A deeper knowledge of the
mechanisms underlying the glial response may lead to the
identification of potential therapies that will selectively
encourage repair in the injured brain.
INNATE IMMUNITY IN THE CNS:
ROLE OF TRL4/ IL-1RI SIGNALLING
The immune system is subdivided into two interactive
branches, namely, the innate (cellular) and adaptive (humoral)
immune systems. Both systems have been shown to participate
in infectious and autoimmune responses in the CNS. In
general, innate immunity represents the first line of defence
against pathogens and does not require prior exposure to
foreign antigens to be triggered. Cell types that make up
the innate immune system include macrophages, neutrophils,
dendritic cells, natural killer cells, and microglia and
perivascular macrophages in the CNS. In contrast, the
induction of adaptive immunity requires signals provided
by the innate immune system to facilitate the expansion of
antigen-specific T and B lymphocytes, which are important
for antibody production and the formation of long-lived
memory cells. Unlike adaptive immunity, in which a vast
number of potential antigens can be recognized by T and
B cells because of random gene rearrangements of their
specific antigen receptors, innate immune system cells must
recognize their cognate antigens by virtue of a predetermined
subset of germ line-encoded receptors. As a result of this
limited receptor expression, cells of the innate immune
system may not be able to recognize every possible antigen;
but may instead focus on a few highly conserved structures
expressed by large groups of microorganisms. These conserved
structural motifs are referred to as pathogen-associated molecular
patterns (PAMPs), and the receptors of the innate immune
system that recognize these molecules are known as the
«Toll-like receptors» (TLRs). At present, eleven TLRs have
been identified in humans, while 13 can be found in the
mouse genome. TLRs 1-9 are conserved between both species
and they all share a common cytosolic TIR domain (see
below)(22). Among these receptors, TLR4 was the first human
TLR to be identified. It interacts with lipopolysaccharide
(LPS), a major component of the gram-negative bacterial
cell wall. This interaction and the subsequent signalling
response of TLR4 has been extensively studied(22).
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Interestingly, the stimulation of TLRs triggers proinflammatory signalling pathways similar to those activated
by IL-1-β. TLRs have been shown to be a member of a large
superfamily that includes the interleukin-1 receptors (IL1Rs)(23, 24). TLRs and IL-1Rs have a conserved region of ~ 200
amino acids in their cytoplasmic tails, which is known as
the Toll/IL-1R (TIR) domain(24-27). By contrast, the extracellular
regions of TLRs and IL-1Rs differ markedly(28). Activation
of IL-1R and TLRs shares downstream signalling molecules,
which culminate in the activation of nuclear factor-κ B (NFκB), a transcription factor that regulates the expression of
a wide array of genes involved in immune responses. IL-1R
and TLRs also signal through the mitogen-activated protein
kinases (MAPKs), such as extracellular signal-regulated
kinase (ERK), p38 and c-jun N-terminal kinase (JNK)(29). A
diagram depicting the major steps involved in TLR4 and
IL-1RI signalling pathways is presented in Figure 1.
The activation of TLR4 and IL-1RI triggers the association
of MyD88 (myeloid differentiation primary-response protein
88)(30), which in turn recruits IRAK-4 (IL-1 associated kinase4)(31) thereby allowing the association of IRAK-1(32). IRAK4 is activated during the formation of this complex (complex
I), leading to the hyperphosphorylation of IRAK-1, which
then induces the interaction of TRAF6 (tumour necrosis
factor receptor-associated factor 6) with complex I(33). The
association IRAK-4-IRAK-1-TRAF6 causes some
conformational change in one or more of these factors,
leading to their disengagement from the receptor complex.
Complex I interacts at the membrane level with another
preformed complex consisting of TAK-1 (transforming
growth factor-α-activated kinase-1), TAB-1 (TAK-binding
protein) and TAB-2(34) forming a second complex or complex
II. This interaction induces phosphorylation of TAB-2 and
TAK-1, which then translocate together with TRAF6 and
TAB-1 to the cytosol. TAK-1 is subsequently activated in
the cytoplasm, leading to the activation of the Iκ-B-α-kinase
complex (IKK). The phosphorylation of Iκ-B-α leads to its
degradation, the release of NF-κB and the activation of NFκB-dependent genes, such as IL-1-β, TNF-α, IL-6, COX-2,
iNOS(35).
Biochemical evidence also indicates that the activation
of complex II triggers the stimulation of p42/p44 ERK, p38
and JNK(23), which in turn regulate the nuclear activation of
the transcriptional factors NF-κB and AP-1 (activator protein1)(36). Notably, because NF-κB and AP-1 binding sites have
been found in the promoters of many genes that are induced
during inflammation, it has been claimed that the large
number of specific receptors of the innate immune system
exerts their manifold gene activations principally through
the MAP kinase and the IKK-NF-κB pathways(22, 24, 37).
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INMUNOLOGÍA
A.M. BLANCO, C. GUERRI
Figure 1. TLR4 and IL-1RI signalling pathways. Activation of TLR4 and IL-1RI by their ligands, LPS and IL-1-β share downstream signalling molecules (MyD88,
Traf-6, IRAKs) forming complex I, that activates complex II formation (Traf-6, IRAKs, TAB-1, -2 and TAK-1), which culminates in the activation of MAPKs and
transcriptional factors (NF-κB and AP-1), and regulates the expression of target genes in immune response and cell death.
The aforementioned signalling pathways suggest that
stimulation with either IL-1 or LPS could lead to the
transcriptional induction of common sets of genes that
encode pro-inflammatory proteins such as chemokines,
cytokines, proteases and metabolic enzymes, like inducible
nitric oxide synthase (iNOS) and ciclooxygenase-2 (COX2)(36). Indeed, iNOS and COX-2 are important components
of the post-lesion inflammatory cascade in various types of
brain damage(38-40).
Finally, significant advance in recent years has led us to
understand the role of the innate immune response in the
CNS and the involvement of TLRs as vital players in this
orchestrated response in the brain. In the brain and spinal
cord, the expression of TLRs in glial cells has been documented
and this expression is increased during neuroinflammation
events(41). Recent evidences also indicate that a chronic
activation of the innate immune response and signalling
may lead to neuronal cell loss and may be involved in
neurodegeneration(42). The innate immune response in the
CNS is necessary to resolve potential pathogenic conditions,
and although the transient upregulation of inflammatory
events in the brain may not lead to cell death(43), overactivation
of innate immunity may lead to neurodegeneration(42). Indeed,
cell damage and apoptosis occur concomitantly as a result
of the stimulation of signalling pathways and inflammatory
mediators associated with TLR4/IL-1RI, which in turn,
induce a highly inflammatory response in the CNS under
conditions of damage, disease, or stress(44).
ETHANOL AND INNATE IMMUNE SYSTEM
Clinical and experimental studies revealed that ethanol
intake affects both the immune and inflammatory systems(45),
resulting in specific defects in the cellular components of
the innate immune responses to bacterial and viral pathogens(46,
47). Ethanol is known to alter cytokine levels in a variety of
tissues including lung, liver and brain. However, the regulation
of the immune response by ethanol is complex and depends
on dose, duration of ethanol treatment (acute vs. chronic)
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ALCOHOL AND NEUROINFLAMMATION: INVOLVEMENT OF ASTROGLIAL CELLS AND IL-1RI/TLR4 RECEPTORS
and the type of cell and pathogen(48). Acute ethanol treatment
impairs immune functions, interfering with the inflammatory
response and increasing the host susceptibility to a variety
of infections, such as bacterial pneumonia(49). Accordingly,
ethanol suppresses the TLR3(50) and TLR4(51) response in
monocytes or macrophages in vitro, decreasing the synthesis
and secretion of numerous cytokines(52). Conversely, chronic
ethanol consumption is associated with elevated serum
levels of proinflammatory cytokines(53, 54), and the activation
of some of these cytokines, such as IL-1-β and TNF-α and
their signalling transduction response(55) is involved in
ethanol-induced liver damage(56).
Increasing evidence demonstrate that the activation of
the innate immune response plays a crucial role in the
development of alcoholic liver disease(57). The gastrointestinal
tract seems to be the initial target of ethanol, leading to an
activation of the innate immune response. Alcohol consumption
is associated with the impaired barrier function of the
intestinal mucosa in patients with various stages of alcoholic
liver injury(58), increasing the plasma levels of endotoxins
such as lipopolysaccharide (LPS) (a component of gramnegative bacterial cell wall). Increased exposure to LPS is
an important contributor to the activation of the innate
immune response in the liver. In addition, chronic ethanol
also exacerbates the response of Kupffer cells to LPS, resulting
in an increased production of inflammatory cytokines(57).
Recent studies have identified a number of intermediates
in the TLR-4 signalling cascade that are affected by chronic
ethanol, including an increased expression of CD14, as well
as enhanced activation of NF-κB and the MAPK family
members, p44/p42 ERK and p38(57). The importance of TLR4
in the development of alcohol liver diseases is exemplified
by the decrease in hepatic damage in mice that have a natural
mutation and lack a functional TLR4(59).
ETHANOL AND BRAIN DAMAGE:
ROLE OF THE INFLAMMATORY MEDIATORS
The brain is one of the major target organs for ethanol
actions, and heavy alcohol consumption results in significant
alterations of the brain structure, physiology and function.
Alcoholics have reduced brain weight compared with nondrinking controls(60), and the degree of brain atrophy correlates
with the rate and amount of alcohol consumed over a
lifetime. The reduction in brain weight and volume has
been attributed to a loss of white matter, which occurs
primarily in the frontal lobe and is specifically susceptible
to alcohol-related brain damage(61, 62). Furthermore, neuronal
loss has also been documented in specific regions of the
cerebral cortex, hippocampus and cerebellum from alcoholic
brains(62, 63).
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Current neuroimaging studies also indicate that chronic
alcohol use induces important changes in brain morphology,
such as cortical and subcortical atrophy, and confirms that
frontal lobe structures are specifically vulnerable to the effects
of ethanol with shrinkage in this area, largely owing to a loss
of white matter(64). The mechanisms involved in white matter
loss remain unclear, although glial impairments in conjunction
with astrocytic loss and death have been reported in the
prefrontal cortex and hippocampus from human alcoholic
brains(65-68). Changes in myelination might also occur during
chronic alcoholism, since both glial fibrillary acidic protein
(GFAP) gene expression (a marker of astrocytes) and myelinassociated genes were down-regulated in the brain of
alcoholics(69, 70). A recent report demonstrates that astrocytes
promote myelination in response to electrical impulses(71),
suggesting that alterations and cell death in astrocytes could
cause deficits and loss of myelin in alcoholics. Alcohol-related
neuronal loss has been also documented in specific regions
of the cerebral cortex, hippocampus and cerebellum(62).
Experimental evidence also demonstrates that alcohol
is toxic for neural cells in culture(72-75) and that acute intoxication
can cause brain damage and even neurodegeneration in
some cases(76, 77). Indeed, neural damage and neurodegeneration
have been demonstrated in short-term binge drinking animal
models, leading to neuronal loss in specific brain regions
including, olfactory and forebrain corticolimbic association
areas, entorhinal and pirirhinal cortex and hippocampus,
brain areas involved in many aspects of learning and spatial
memory(77). These results suggest that a drinking pattern,
specifically binge drinking in which high blood and brain
alcohol levels are achieved, is an important factor in the
ethanol-induced neuropathology, and that alcoholic
neurodegeneration could occur primarily as a result of binge
drinking episodes(76-78).
The neuropathological processes underlying the effects
of ethanol on neural damage are largely unknown, although
several mechanisms and concurring factors have been
proposed to contribute to neurodegeneration. Among the
mechanisms proposed include: the participation of excitotoxic
events and nitric oxide generation(79), the involvement of
glial swelling and brain edema(78), and the production of
free radicals causing oxidative stress(80). The latter mechanism
includes the ability of ethanol to enhance free radical species
by inducing the cytochrome P450 2E1 (CYP2E1)(81), which
leads to the generation of hydroxyethyl radicals(82), reactive
oxygen species (ROS)(83) and the activation of NF-κB(80).
Interestingly, the induction of both CYP2E1 and ROS was
also noted in astrocytes exposed to ethanol(81), suggesting
that glial activation might contribute to the induction of free
radical species by ethanol in the brain.
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INMUNOLOGÍA
Recent findings also suggest the involvement of
inflammation in alcohol-induced brain damage. These studies
demonstrate that chronic ethanol treatment not only increases
the levels of cytokines (IL-1-β, TNF-α) and inflammatory
mediators (iNOS and COX-2) in the rat's brain and in cultured
astrocytes, but also activates signalling pathways that are
classically associated with inflammation (MAPKs, NF-κB,
AP-1, see below). Notably these inflammatory events are
associated with an increase in cell death(75). This suggests that
activation of glial cells by ethanol might trigger the production
of toxic compounds, such as ROS or nitric oxide(73), inflammatory
cytokines and glutamate, which might contribute to ethanolinduced brain damage, similar to what occurs in several brain
disorders and neurodegenerative diseases(4, 5, 84).
The evidence indicating the presence of ethanol-induced
inflammatory mediators in the brain originates from several
studies mainly performed in neural cells in culture. These
studies demonstrate that ethanol influences the expression
of iNOS and COX-2 in both the brain and neural cells exposed
to ethanol(75). It is known that both enzymes are induced
only in response to different stimuli including stress,
inflammation or neural damage(38-40).
Ethanol is known to influence NO production and iNOS
expression in a number of cellular systems, and in the
presence or absence of additional stimuli, such as cytokines
or LPS. For example, ethanol enhances the cytokine-induced
iNOS gene and protein expression in C6 glioma cells, in
immortalized astrocytes(85, 86), in BBB cells(87) and in embryonic
cortical neurons(88). Stimulation of NO production and iNOS
expression also occurs as a direct effect of ethanol, as
demonstrated in brain homogenates from the guinea pig
model of prenatal ethanol exposure(89), in brain lysates from
chronic-alcohol fed rats(75) and in cerebral pial cultures(90).
In fact, the induction of iNOS can occur in astrocytes
upon 30 min treatment with ethanol(73).
Up-regulation of COX-2 expression has also been observed
in the rat’s brain after acute (91) and chronic ethanol
administration(75, 91, 92), as well as in ethanol-exposed astrocytes(73,
93). Interestingly, the study by Luo et al (2001)(93), demonstrated
that ethanol selectively increased COX-2 levels in astrocytes,
but not in neurons. However, a clearer demonstration that
ethanol induces COX-2 in astrocytes is seen in a recent study
showing a fast induction of COX-2 upon 30 min of ethanol
treatment(73). This study also demonstrates that the activation
of NF-κB is critical for the ethanol-induced up-regulation
of iNOS and COX-2 in astrocytes, since inhibition of NF-κB
activity either by pyrrolidine dithiocarbamate (PDTC) or
BAY 11-7082 suppresses the induction of iNOS and COX2, suggesting a transcriptional regulation of these inflammatory
mediators by NF-κB(73).
A.M. BLANCO, C. GUERRI
ETHANOL ACTIVATES SIGNALLING PATHWAYS
AND TRANSCRIPTION FACTORS INVOLVED IN
INFLAMMATORY BRAIN DAMAGE
Stimulation of the innate immune system triggers the
activation of NF-κB (see Figure 1) and the induction of
numerous immune and inflammatory response genes(94)
encoding cytokines, chemokines, enzymes (iNOS and COX2)(95-97) and adhesion molecules.
Several in vitro and in vivo studies clearly demonstrate
that both acute and chronic ethanol treatments cause the
activation of NF-κB. Short-term ethanol treatment (25-100
mM) has been shown to induce NF-κB DNA binding in
human astroglial cells(98), similarly to long-term ethanol–treated
rat astrocytes(75). Chronic ethanol treatment also induces
activation of NF-κB in liver(99), brain(100) and in the cerebral
cortex of ethanol-fed rats(75). In the last study the stimulation
of NF-κB was accompanied by a significant decrease in the
cytoplasmic levels of IκB-α, and by elevated levels of cytokines,
as well as COX-2 and iNOS(75). These findings suggest that
NF-κB is activated in response to challenge by both acute
and chronic ethanol, although the mechanism involved
remains unclear.
The activity of NF-κB is controlled at multiple levels,
most notably by the regulation of its subcellular localization.
NF-κB is retained in the cytoplasm in resting cells, and it
is transported to the nucleus in response to a diverse range
of stimuli(101, 102), where it binds specifically to κB enhancer
elements of DNA and alters the expression of a great number
of proinflammatory genes(35). Following stimulation, the
duration of NF-κB activation may be transient or persistent,
depending on the cellular stimulus and cell type. The temporal
profile is of considerable clinical relevance because whereas
rapid induction of NF-κB is beneficial for immune response
to infection or injury, long-term activation has been
demonstrated to be associated with chronic inflammatory
diseases, such as multiple sclerosis. Notably, chronic ethanol
treatment triggers sustained activation of NF-κB in the rat’s
brain and in astrocytes in culture, and this event is associated
with elevated levels of inflammatory mediators, IL-1-β and
TNF-α(75). Likewise, a recent study demonstrated that NFκB is sustained in astrocytes in response to stimulation with
IL-1-β(103), suggesting that elevated levels of IL-1 could
mediate the persistent activation of NF-κB observed in both
astrocytes and in the brain of alcohol-fed rats, and might
contribute to a prolonged induction of inflammatory mediators
and ethanol-induced damage in the brain.
Furthermore, despite the fact that the induction of NFκB is directly regulated by IKK, there is evidence demonstrating
that IKKs themselves are also activated through
phosphorylation by an upstream kinase(s). Candidates
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for this kinase include NF-κB-inducing kinase (NIK) and
mitogen-activated protein kinase (MAPK) pathways (p44/p42
ERK, JNK and p38)(104-106) (see Figure 1). Cumulative data
demonstrates the participation of the MAPKs pathway in
inflammation processes(107). Moreover, the stimulation of
this pathway, apart from activating NF-κB, can also trigger
the activator protein-1 (AP-1) transcriptional activity. The
AP-1 family is dependent on its phosphorylation by MAPK
or MAPK-activated kinases(108), resulting in the transcriptional
activation of genes of the jun and fos-family or in the activation
at the protein level. Ethanol withdrawal hyperactivity is
associated with the expression of the early genes c-fos and
c-jun(109), with transient selective increases in the DNAbinding activity of immediate early genes (IEGs)-encoded
AP-1 in the brain(110, 111). Notably, chronic ethanol treatment
increases the MAPKs and AP-1 expression in liver, brain
and astrocytes(57, 75), and these effects are associated with
inflammation and cell damage. Finally, although the activation
of MAPKs can mediate the inflammation and release of
neurotoxic molecules, it is obvious that the activation of
MAPK under certain circumstances may be beneficial for
the plasticity of the CNS. Nevertheless, based on the few in
vivo and in vitro studies available to date, the MAPKs, which
mediate the activation of glial cells by ethanol with neurotoxic
consequences, represent promising targets for the
pharmacotherapy for acute brain insults and
neuroinflammatory injury induced by ethanol.
Finally, neural injury and cell death have been associated
with many neurological and neuroinflammatory disorders(112),
and several reports demonstrate that one of the mechanisms
implicated in ethanol-induced neurotoxicity is the promotion
of apoptosis. Our recent studies suggest a role for inflammation
in ethanol-induced cell death in brain and in astrocytes: we
found that ethanol-induced activation of MAPKs, NF-κB,
AP-1 were associated with increased apoptosis in the brain
and in astrocytes(74, 75). Furthermore, we were able to prevent
ethanol-induced apoptosis in astrocytes by blocking the
receptors TLR4 and IL-1RI(74) (see below), suggesting the
involvement of the innate immune response in the ethanolinduced astrocytic death. Finally, although apoptosis occurs
in neurodegenerative disorders(113, 114), we cannot exclude
the possibility that ethanol also causes necrotic cell death
since it has been suggested that necrosis often triggers a
prominent inflammatory reaction, while apoptosis results
in the uneventful removal of dying cells, with little or no
inflammation 115).
Figure 2 illustrates the cascade of events by which ethanol
induces glial activation through the stimulation of intracellular
signalling pathways, and triggers the production of
inflammatory mediators and toxic compounds that could
194
VOL. 25 NUM. 3/ 2006
exacerbate an inflammatory response leading to astrocytic
and neural death.
INVOLVEMENT OF TLR4/IL-1RI IN ETHANOLINDUCED INFLAMMATORY RESPONSE IN
ASTROCYTES
As already discussed, stimulation of TLRs triggers the
initial innate immune response, which ultimately leads to
inflammatory gene expression and the clearance of infectious
agents(24), although an excessive production of inflammatory
molecules contributes to the pathogenesis of inflammatory
diseases(42). Ethanol is known to affect the innate immune
response in several organ systems(45, 57). Nevertheless, very
little is known about the potential action of ethanol on the
CNS immune system, as well as the participation of
inflammation in alcohol-induced brain damage.
The CNS exhibits well-organized innate immune reactions.
In response to injury(116, 117) and infections(43, 118), glial cells
are capable of mounting a quick and effective response to
control an infection until cells of the peripheral adaptive
immune system can be recruited (119, 120). Microglia and
astrocytes express TLR2, TLR4, TLR5 and TLR9(41, 121, 122),
respond functionally with cytokine and chemokine
production(121, 122), and are capable of contributing to an
inflammatory environment in the CNS(15, 123) following a
variety of infectious or inflammatory insults(124).
Our recent studies demonstrate that astrocytes respond
to ethanol, by secreting cytokines and other inflammatory
mediators(73, 75), and by contributing to an inflammatory
environment in the brain of alcohol-fed animals(75). These
effects seem to be mediated by the ethanol-induced activation
of TLR4/IL-1RI in astrocytes (74). In fact, we recently
demonstrated that ethanol, at physiological relevant
concentrations (10 mM or 50mM), is capable of inducing a
rapid activation of the TLR4/IL-1RI signal-transduction
pathways(74) in a similar manner to the situation when cells
are stimulated with LPS and IL-1-β(125-128). Thus, we observed
a rapid phosphorylation (within 10 min) of IRAK, p44/p42
ERK, SAPK/JNK and p38 MAPK, upon ethanol stimulation.
In addition, a subsequent downstream activation of the
transcription factors NF-κB and AP-1, as well as the upregulation of iNOS and COX-2 were noted after a 30-min
ethanol treatment. We also found a time lag between the
maximal COX-2 and iNOS expression and the increase in
cell death, observed at 3 h of ethanol treatment(74). Consistent
with the hypothesis that ethanol mediates inflammatory
events by activating TLR4/IL-1RI, we showed that both the
ethanol-induced inflammatory mediators and the cell death
in astrocytes were abolished by blocking the activation of
these receptors with neutralizing antibodies(74). These results
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INMUNOLOGÍA
A.M. BLANCO, C. GUERRI
Figure 2. Potential mechanisms of ethanol-induced brain damage. Ethanol triggers signalling inflammatory responses and the production of inflammatory mediators
and toxic compounds by activating TLR4 and IL-1RI in glial cells, and this could exacerbate the inflammatory response leading to brain damage by astrocytic
and neural death.
support the notion that ethanol-induced inflammatory
mediators in the brain are mediated via activation of glial
TLR4/IL-1RI signalling pathways.
At present, it is not clear how ethanol might interact
with TLR4/IL-1RI to either activate or inhibit their signalling
response, which depends on the cell type and ethanol
concentration. It is well established that ethanol interacts
with membrane lipids and influences the function of membrane
proteins(129). A plausible explanation is that ethanol can either
facilitate or disrupt the recruitment of these receptors,
depending on the ethanol concentration and the
physicochemical characteristics of the cell membrane. A
high ethanol concentration can perturb membrane lipid
microdomains, such as lipid rafts, resulting in disruption
of the receptor function. On the contrary, low ethanol
concentrations might facilitate the aggregation and interaction
of proteins within the membrane, thus allowing their activation
and signalling through the lipid rafts. We suggest a schematic
model of low and high concentrations of ethanol effects on
lipid rafts and the signal transduction of TLR4 and IL-1RI
(Figure 3).
Lipid rafts are cholesterol/sphingomyelin-enriched
membrane microdomains, which are involved in the
recruitment of molecules with signal transduction capabilities(130,
131), and are now recognized as important sites for initial
immune cell activation. The presence of TLR2 and TLR4
within lipid rafts and their subsequent clustering in response
to LPS(132, 133) support their role in the innate immune response(132135). Indeed, disruption of lipid rafts leads to an inhibition
of TLR internalization and signalling(132). High concentrations
of ethanol appear to interfere with lipid raft clustering,
leading to the suppression of TLR4 signalling in murine
macrophages(51, 136). However, low concentrations of ethanol
might promote receptor recruitment into lipid rafts, leading
to dimerization and signalling. Furthermore, as several
studies have demonstrated that ethanol suppresses both
cytokine-induced expression of iNOS(85, 86) and LPS-induced
NO production(137) in glial cells, ethanol may possibly interfere
with the recruitment of TLRs into the lipid rafts in the
presence of other stimuli (e.g. LPS, cytokines), leading to
an inhibitory, rather than an additive effect on the TLR
signalling transduction.
195
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ALCOHOL AND NEUROINFLAMMATION: INVOLVEMENT OF ASTROGLIAL CELLS AND IL-1RI/TLR4 RECEPTORS
VOL. 25 NUM. 3/ 2006
Figure 3. Suggested model by which ethanol mediates activation of TLR4/IL-1RI through its interactions with lipid rafts. We hypothesize that low concentrations
of ethanol (10-50 mM) might facilitate TLR4 and IL-1RI aggregation and their recruitment into lipid rafts, leading to the activation of their signal transduction.
On the contrary, high ethanol concentration can perturb membrane lipids, including lipid rafts, and result in a disruption of the receptors function and signalling.
AcP= Accessory protein.
Finally, since lipid rafts are considered important for
TLRs signalling, it should be expected that the levels of
ethanol intake would cause distinct alterations in the
membrane lipid composition and subsequently, affect the
response of TLRs in different ways. Indeed, while acute
ethanol treatment down-regulates the production of proinflammatory cytokines induced by LPS in human
monocytes(138, 139), chronic ethanol treatment (7 days in vitro),
results in augmentation of LPS-induced TNF-α(48).
To summarize, although ethanol effects on TLR4/IL1RI are complex, data suggest that TLR4/IL-1RI are targets
of ethanol-induced inflammatory damage in many organs,
including the brain.
CONCLUSIONS
Results of studies reviewed in this article indicate that
ethanol consumption activates a wide range of inflammatory
mediators and signalling pathways in the brain, which
are associated with inflammation and the immune response.
In particular, the ethanol-induced inflammatory response
seems to be related to the activation and signalling of TLR4
196
and IL-1RI, the LPS and IL-1β specific receptors. Further
studies are required to understand the mechanisms by which
ethanol activates TLR4/IL-1RI, although it is clear that these
receptors are targets of ethanol-induced inflammatory
damage in astrocytes and the brain. However, there is still
much to be understood about the nature of CNS inflammation
and the molecular effects of ethanol in the brain before the
development of a clinical treatment may be successfully
explored. These results contribute to our understanding
of the brain injury caused by ethanol, and they may as well
lead to the possibility of new treatments and/or intervening
strategies to restore the brain damage induced by ethanol.
ACKNOWLEDGMENTS
The work of the authors is supported by Grants from
Ministerio de Educación y Ciencia (SAF 2003-06217 and
SAF 2006-02178), Instituto Carlos III, FIS-Red RTA G03/005,
Dirección General. Drogodependendecias, GV, and Fundación
de Investigación Médica Mutua Madrileña. We wish to
thank Marisa March for her assistance in the preparation of
the manuscript.
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INMUNOLOGÍA
DISCLOSURES
The authors have no financial conflict of interest.
CORRESPONDENCE TO:
Dra. Consuelo Guerri Sirera
C/ E. P. Avda. Autopista del Saler, 16-3 (junto Oceanográfico)
46013 Valencia (Spain)
E-mail: [email protected]
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