TLR4, Ethanol, and Lipid Rafts: A New Mechanism of
Ethanol Action with Implications for other
Gyongyi Szabo,1,2* Angela Dolganiuc,* Qun Dai,†and Stephen B. Pruett1†
Ethanol (EtOH) is the most widely abused substance in
the United States, and it contributes to well-documented
harmful (at high dosages) and beneficial (at low dosages)
changes in inflammatory and immune responses. Lipid
rafts have been implicated in the regulation and activa-
tion of several important receptor complexes in the im-
mune system, including the TLR4 complex. Many ques-
tions remain about the precise mechanisms by which rafts
regulate the assembly of these receptor complexes. Results
summarized in this review indicate that EtOH acts by al-
tering the LPS-induced redistribution of components of
the TLR4 complex within the lipid raft and that this is
ceptor clustering, and subsequent signaling. EtOH pro-
at least in part by modifying lipid rafts, and it could rep-
resent a model to probe the relationships between rafts,
receptor complexes, and signaling. The Journal of Im-
munology, 2007, 178: 1243–1249.
conducted (1). When such studies were done, they demon-
strated a strong association between excessive EtOH consump-
tion and an increased risk of infections, such as pneumonia and
tuberculosis (1). Experimental studies with human subjects
given EtOH in a controlled setting and with animals have con-
sistently revealed suppression of innate immunity and inflam-
mation (2–5). However, the proximate molecular mechanism
by which EtOH exerts these effects is not known.
Because EtOH per se is relatively unreactive under physio-
logical conditions, attention initially focused on its physical
properties. Over 100 years ago, a correlation was reported be-
tween the lipid solubility of a series of compounds and their
uppression of innate immunity to infection by ethanol
(EtOH)3was reported by Benjamin Rush in 1785,
many years before scientific studies on the subject were
refined and alterations in membrane volume or curvature,
membrane disordering, and changes in lipid phase transitions
have all been suggested as mechanisms by which EtOH could
affect the function of membrane proteins (9). However, objec-
example, the effects of anesthetics, including EtOH, at relevant
concentrations on the temperature of phase transition are ex-
ceedingly small. Similarly the amount of increase in membrane
fluidity caused by a relevant concentration of EtOH is very
small and can be produced alternatively by an increase in tem-
perature of just a few tenths of a degree Celsius, a temperature
change that certainly does not produce the same changes in the
nervous or immune systems as EtOH.
It is not surprising that interest began to shift to direct action
of EtOH on proteins to explain its effects in the CNS. Direct,
consistent evidence has been difficult to obtain (10), but recent
results with ?-aminobutyric acid (GABA)Areceptor subunits
(inhibitory receptors whose function is enhanced by EtOH)
suggest that EtOH can bind to a water-filled pocket in some
subunits (11), thereby altering the subunit conformation and
increasing the sensitivity of the receptor to GABA. Similar re-
5-hydroxy-5-methyl-4-isoxazolepropion acid receptors (stimu-
latory receptors that are inhibited by EtOH). However, the
EtOH concentrations required to cause significant changes in re-
mediated by lower concentrations have sometimes not been repli-
cated. This suggests a situation in which simple, pharmacological
be fully applicable (10). Thus, it is interesting that results have re-
cently been reported indicating that GABAA, N-methyl-D-aspar-
ceptors are associated with lipid rafts (12–15).
Lipid rafts and alcohol
Lipid rafts, also called detergent-resistant domains, are plasma
membrane microdomains characterized by insolubility in non-
ionic detergents (at cold temperatures) and enrichment in
University Health Sciences Center, Shreveport, LA 71130
Received for publication October 16, 2006. Accepted for publication December 5, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
1G.S. and S.B.P. contributed equally.
2Address correspondence and reprint requests to Dr. Gyongyi Szabo, Director of Hepa-
tology and Liver Center, Department of Medicine, University of Massachusetts Medical
Center, 364 Plantation Street LRB125, Worcester, MA 01605. E-mail address:
3Abbreviations used in this paper: EtOH, ethanol; GABA, ?-aminobutyric acid; TIR,
cholesterol and sphingomyelin (16, 17). They range from a few
nanometers to a few hundred nanometers in diameter and rep-
resent ?50% of cellular membrane (16, 18). Certain proteins
reside preferentially in rafts, whereas others are recruited or ex-
cluded from the rafts upon cell activation (16–21). By protein
composition, the lipid rafts are heterogeneous: some are en-
riched in caveolin and others concentrate clathrin and GPI-an-
chored proteins or dynamin (16, 17). It is generally believed
plexes, their internalization and triggering of signaling path-
ways, thus facilitating cell activation (19, 21). Lipid rafts had
not yet been characterized when most of the experimental evi-
dence was obtained to evaluate the hypothesis that EtOH pro-
duces biological effects primarily by interacting with mem-
brane lipids. The identification of lipid rafts and their
important role in cellular signaling provides ample rationale
to revisit this hypothesis. Any direct interaction of EtOH
with lipids would probably not occur in the lipid core but
within the more hydrophilic inner and outer margins (9). It
is not clear whether this would alter protein conformation to
a sufficient degree to explain the actions of EtOH on the
innate immune system, but this should be a productive line
We propose that a comprehensive hypothesis regarding the
mechanisms of EtOH action on cellular signaling should in-
clude possible direct action on proteins, nonspecific interac-
tions with lipid components, and disruption of lipid protein
interactions that alter protein conformation. Any of these
mechanisms could potentially limit entry or exit of receptor
components to and from lipid rafts or among different raft
types. This could prevent assembly of an optimum receptor
complex. Conformational change in a single receptor compo-
nent (e.g., by direct binding of EtOH or alteration of protein-
lipid interactions) could yield similar effects.
brane proteins by disrupting protein-lipid interactions (22). If
this occurred relatively specifically in lipid rafts, the movement
of proteins into or out of rafts or among different raft subpopu-
lations, which is normally part of signaling through several re-
ceptor types, could be adversely affected. There is evidence that
neurological adaptation (tolerance) to chronic administration
of EtOH is associated with increased cholesterol in cell mem-
with the remainder of the membrane. Thus, additional raft mi-
crodomains permitted by the presence of additional cholesterol
in the membrane may represent additional “targets” for EtOH,
to disrupt. This is a quantitatively feasible proposal because rel-
ecule in the membrane for every 200 phospholipid molecules
ditional target sites to diminish the action of EtOH. Recent ev-
the actions of EtOH on macrophage activation in humans and
It is possible that some form of membrane adaptation to
chronic EtOH exposure (such as increased cholesterol incorpo-
ration) could explain the disparate effects reported for chronic
tious diseases and inflammatory responses. Chronic excessive
exposure to EtOH is typically associated with increased risk of
infection but paradoxically also to increased risk of inflamma-
tory processes such as alcoholic hepatitis in the liver. In part,
this may reflect a balance between anti-inflammatory effects of
EtOH and proinflammatory effects of bacteria that enter the
circulation due to decreased barrier function in the gastrointes-
tinal tract (29). However, there is also evidence that cells ob-
tained from animals that have been exposed to chronic EtOH
treatment are more sensitive to inflammatory stimuli such as
LPS than cells isolated from untreated animals (30). We ob-
served that macrophages from mice treated with EtOH for 1
mo were less affected by acute EtOH administration than un-
treated mice with regard to production of cytokines (31). This
EtOH exposure. It should also be noted that effects of chronic
EtOH exposure on the immune system can be relatively persis-
tent, and mechanisms such as increased production of reactive
oxygen species and cellular damage caused by the reactive
effects of EtOH on the immune system (32, 33). In addition,
the effects of acute EtOH exposure on lipid rafts discussed later
in this review are relatively short-lived. In contrast, some effects
of chronic EtOH exposure on cells of the immune system last
mediated by short-term alterations in lipid rafts. All of these
findings suggest that inhibition of signaling by EtOH is prob-
ably a more important mechanism in acute EtOH exposure
than in chronic exposure.
sive in humans is accumulating. For example, people with de-
or burn injury have increased morbidity (35), and ?25% of
convincing case can also be made that moderate EtOH con-
sumption exerts beneficial effects by inhibiting inflammation
that contributes to the development of cardiovascular disease
(37). Thus, inhibition of signaling by the mechanism discussed
ful and beneficial effects of acute EtOH exposure.
TLRs and lipid rafts
The innate immune system, macrophages, and TLRs are criti-
cally involved in the initial phase of microbial detection (38,
39). TLRs recognize pathogen-associated molecular patterns
that induce expression of proinflammatory cytokines and initi-
ate pathogen-specific immune responses (38, 39). Each TLR
has a unique extracellular domain that allows specific ligand
recognition. The intracellular Toll/IL-1 receptor (TIR) do-
main of TLRs associates with the TIR domain of their respec-
tive adaptor molecules to initiate intracellular signaling.
TLR3, which uses TIR-domain-containing adapter-inducing
IFN-? (41). Uniquely, TLR4 can associate with both of these
ceptor that has a GPI-linked transmembrane but no intracellu-
lar domain, transduces signals by interacting with other signal-
ing molecules, including TLRs (41). CD14, similar to other
1244 BRIEF REVIEWS: TLR4, EtOH, AND LIPID RAFTS
GPI-linked proteins, resides in lipid rafts (42). Upon stimula-
tion with specific ligand the recruitment of membrane-associ-
ated TLRs, such as TLR2 and TLR4, and other components of
TLR complex occurs into the lipid rafts (19, 25, 42–44). Lipid
rafts appear to provide a platform for interaction of endosomal
TLRs, such as TLR3, TLR7, and TLR9, with their ligands in
macrophages (45–47). Of interest, it remains to be determined
whether CD14 has a role in TLR association with lipid rafts.
The mechanisms by which CD14 amplifies TLR signaling re-
mains to be explored; however, multiple lines of evidence sug-
gest that CD14 association with lipid rafts may be a common
element where the CD14-lipid raft association provides a plat-
form for recruitment of not only TLRs but also key signaling
molecules for potent interaction and signal initiation.
In previous independent studies in our labs, we noted that
EtOH inhibits TLR-mediated signaling in macrophages and
that both early and late events are inhibited (25–28, 31, 48–
50). This led both of us to evaluate the possibility that EtOH
acted at the earliest points in signal transduction, possibly by
acute EtOH exposure alters LPS-induced redistribution of
TLR4 receptor components to lipid rafts, that LPS-induced
TLR4 signaling is dependent on normal raft structure, that
EtOH also alters the reorganization of the actin cytoskeleton
(which is required for full cellular activation by LPS), that
EtOH decreases TLR4 and CD14 clustering and colocaliza-
tion, and that these changes are associated with decreased pro-
duction of TNF-? a key proinflammatory cytokine (27, 28).
Results were similar for a mouse macrophage like cell line,
mouse peritoneal macrophages, and human monocytes.
In a recent report from one of our labs (S. B. Pruett), it was
demonstrated that acute EtOH treatment of cells or mice al-
most completely prevented the redistribution of CD14 (a com-
ponent of the TLR4 receptor complex) between different lipid
raft fractions that is normally induced by LPS (28). This is as-
sociated with decreased production of TNF-?, and agents that
inhibit lipid raft function and rearrangement of the actin cy-
toskeleton similarly inhibited TNF-? production, suggesting
that lipid raft-mediated events and rearrangement of the actin
cytoskeleton are required for optimum signaling and TNF-?
expression. In a subsequent study, this relationship was con-
and clustering were also implicated in activation of macro-
phages for TNF-? production (27). Confocal microscopy re-
vealed that TLR4 and CD14 colocalization and clustering oc-
curred rapidly (3 min) and persisted for at least 1 h. Inhibiting
this clustering with cholesterol sequestering agents also inhib-
ited TNF-? production. Interestingly, increased expression of
membrane-associated TNF-? (from which the secreted form is
derived) was observed almost exclusively in the same cells that
had TLR4 and CD14 colocalization and clustering (?5% of
total cells). EtOH altered actin reorganization in LPS-treated
cells, and alteration of actin reorganization by cytochalasin D
a mechanism of inhibition of this activation by EtOH consis-
tent with that depicted in Fig. 1. Three other groups have re-
ported rapid clustering of the TLR4 receptor complex induced
by LPS (19, 42–44), but our studies are the only ones to date
that provide evidence for the involvement of the actin cytoskel-
eton in this process or in signaling (27, 28).
demonstrated that both TLR2 and TLR4 are localized outside
of lipid rafts, whereas CD14, a coreceptor of TLR4, is distrib-
uted both in and outside of rafts in resting cells (25). TLR4
ligand activation with LPS resulted in concentration of both
TLR4 and CD14 in lipid rafts. Acute EtOH treatment inter-
tenuation of TLR4 and CD14 recruitment to lipid rafts by
EtOH was associated with reduction in LPS-induced TLR4
downstream signaling events and inflammatory cell function.
Our studies found reduced NF-?B activation and attenuation
ministration in vitro in monocytes (25, 26, 51).
It remains to be explored whether EtOH would affect simi-
demonstrated that TLR2 is also associated with lipid rafts after
NF-?B activation was not prevented by EtOH administration
It remains to be investigated whether these results reflect a se-
lective effect of EtOH on TLR4-induced signaling or, for ex-
ample, on specific lipid rafts involved in TLR4 signaling. Pre-
vious studies have demonstrated the presence of different lipid
rafts (16, 17), and at this time, it is unknown whether TLR2
systems, TLR2-mediated murine macrophage activation was
inhibited by acute alcohol and involved impaired p38 and
ERK1/2 MAPK activation (52, 53). Alcohol administration in
IL-6 and TNF-? (52). While TLR9 intracellularly is localized
to the endoplasmic reticulum and is translocated to the lyso-
somes upon ligand activation, association of TLR9 with lipid
rafts has been also demonstrated recently (47, 54). Interest-
ingly, TLR3, unlike TLR2, 4, and 9, was found in associa-
tion with lipid rafts by some investigators (45) but not by
others (47). Thus, it remains to be evaluated whether inhi-
bition of TLR3-induced proinflammatory cytokine produc-
tion and mRNA levels of the IFN-related amplification loop
after in vivo administration of acute alcohol in mice involves
lipid rafts (49). Based on these observations and additional
evidence that EtOH also modulates intracellular pathways
that are not lipid-raft associated, we can conclude that dis-
ruption of signaling events associated with lipid rafts may
represent just one of the multiple intracellular effects
It is unclear whether alteration of lipid rafts by EtOH is a
major mechanism by which EtOH alters TLR signaling in cell
types that do not express CD14. Some types of cells (e.g., epi-
on soluble CD14 for LPS-induced signaling (55). It is not yet
known whether lipid rafts are involved in TLR signaling under
these circumstances. However, if TLR4 mobilization to rafts is
important in these cells, as it is in monocytes (which express
attached CD14), it would not be surprising to find the EtOH
alters TLR4 signaling in cells that do not express CD14 on the
1245 The Journal of Immunology
other receptors (such as the BCR for Ag), a sequence of events
is proposed in Fig. 1. Although this figure is simplified (e.g.,
LPS-binding protein, MD-2, and other components of the re-
ceptor complex are not shown), it illustrates the following key
points: 1) LPS induces conformational changes causing TLR4
to enter lipid raft microdomains to interact with CD14 and
LPS; 2) this causes initial signaling leading to rearrangement of
the actin cytoskeleton; and 3) actin mediates massive clustering
of the CD14 and TLR4 complex and possibly the addition of
other components leading to optimal cellular signaling. As in-
dicated in Fig. 1, we hypothesize that EtOH alters this series of
the testable conclusion that some cellular signaling should be
detectable in cells in which step 1 is inhibited, for example, by
cholesterol-sequestering agents, but this signaling will be quan-
titatively and/or qualitatively different and less effective than
signaling induced by the same stimulus in normal cells.
It is not clear whether ligand-induced alterations in the dis-
tribution of receptor components are mediated by an initial
(perhaps weak) cellular signal induced by interaction of the li-
gand with its nominal receptor or by direct physical changes in
the receptor and its components mediated by ligand binding
and causing conformational changes that lead to migration
from, to, or among rafts. There are indications that in some
cases initial transmembrane signaling events are induced by
binding of ligand and that this induces rapid rearrangement of
the actin cytoskeleton, which is required for assembly of the
fully functional receptor complex. This sequence of events has
been best documented in the case of signaling through the
BCRs for Ag (56, 57). There are important commonalities be-
tween BCR and TLR signaling such as multicomponent recep-
of the actin cytoskeleton. Therefore, it seems reasonable to sug-
gest a sequence of events in TLR4 signaling similar to that re-
ported for BCR signaling.
Lipid rafts: implications for the effect of alcohol on microbial defense
to amplify efficient signaling in microbe recognition, microbes
can independently alter the dynamics of lipid rafts to modulate
these host defense mechanisms. Pathogens have evolved strate-
gies to ensure their own survival in some cases by “hijacking”
lipid rafts. Thus, in addition to modification of host receptors
involved in pathogen recognition and pathogen-induced im-
mune responses, alcohol may directly affect pathogen-lipid raft
interactions. A broad spectrum of bacteria (Legionella, Pseudo-
monas, Brucells, Salmonella, Shigella, Chlamydia, Streptococcus,
Listeria, etc.), viruses (HIV, Ebola, HSV, hepatitis C virus, in-
fluenza, EBV, etc.), and some protozoa (toxoplasma, plasmo-
intracellular survival, or replication (58). Many of these infec-
tions are more frequent in patients with excessive alcohol use
rafts that triggers downstream TLR signaling events (left panel). In the presence of EtOH, LPS-induced recruitment of TLR4 and CD14 into the lipid rafts is
decreased with a corresponding reduction in activation of downstream pathways.
EtOH disturbs TLR4 association with lipid rafts. Stimulation with the TLR4 ligand, LPS, results in recruitment of CD14 and TLR4 into the lipid
1246 BRIEF REVIEWS: TLR4, EtOH, AND LIPID RAFTS
Lipid rafts and immune receptors: relevance to alcohol-induced immune
The association and dissociation of particular proteins with
lipid rafts is necessary for cellular signal transduction mediated
through TLRs, the BCRs for Ag, and the TCRs for Ag (20, 21,
25, 28, 56). Thus, lipid rafts are involved in the induction of
has been discerned by several experimental approaches, includ-
ing: 1) disruption of signaling by agents known to disrupt lipid
rafts (e.g., ?-methylcyclodextrin and nystatin) (25, 28); 2) li-
gand-induced changes in the distribution of receptor compo-
nents among raft and non-raft fractions (25, 28); and 3) micro-
scopic analysis indicating coalescence (61) of rafts or clustering
(27) of membrane proteins that are known to occur in rafts.
However, only a few reports have described receptor clustering
in the case of TLRs (62, 63), and the relationships between ini-
ceptor clustering, movement of proteins into, out of, or among
raft fractions, and assembly of an optimally responsive receptor
complex are just beginning to be investigated. As an agent that
alters all of these events, EtOH has already been useful in sug-
gesting that they are connected. In addition to defective micro-
cohol use is also associated with defects in adaptive immune
responses. Ag-specific T cell activation, Th1/Th2 cytokine bal-
dysfunction, NK cell abnormalities, defects in macrophage
phagocytic activity, and Ag-presenting function of dendritic
use conditions (64–67). Importantly, all of these cellular func-
tions involve receptor complexes associated with lipid rafts, in-
cluding MHC class II, TCRs, BCRs, TNFRs, and FcRs (20,
21, 68–71). While a direct role for lipid raft modulation by
alcohol in these receptors and/or cellular defects is yet to be in-
vestigated, based on the previously identified role of lipid rafts
lipid raft-associated signaling events may be common targets of
EtOH to result in such diversity of immune cell defects. A re-
cent report from Joshi-Barve et al. (72) showed that EtOH
treatment interferes with efficient TCR assembly within lipid
T cells, and phospholipase C phosphorylation and leads to de-
creased IL-2 gene expression. MHC class II molecules involved
in the formation of the immunological synapse between APCs
(B cells or monocytes) and T lymphocytes are associated with
lipid rafts, and disruption of rafts results in decreased Ag pre-
deficient Ag presentation after alcohol administration or in
vitro treatment (75). It is intriguing that reduced Ag-specific T
cell activation by monocytes or dendritic cells was associated
in alcohol-treated cells consistent with the possibility that the
signaling function rather than the absolute levels of these pro-
teins are affected by EtOH. This is also suggested by inhibition
of anti-CD3-induced Ca2?mobilization, inositol-(1,4,5)-
trisphosphate production, and T cell proliferation in the pres-
ence of alcohol (76). In addition, alcohol-induced treatment
by the same investigators (76). It is interesting that CD45 ex-
clusion from raft fractions is required for formation of an im-
munological synapse and for optimal T cell signaling (77).
for lipid raft alterations in the effects of EtOH on cellular re-
ceptor signaling and await further investigation.
Conclusions and implications for effects of EtOH in other physiological
Recent discoveries on the effects of EtOH on lipid rafts and
TLR4 demonstrate that EtOH has significant modulating po-
tential on receptor recruitment to lipid rafts, resulting in func-
tional changes in inflammatory cell functions. Based on our
findings related to acute alcohol administration and TLR4 sig-
subsequent actin polymerization is attenuated by alcohol (Fig.
1). However, our results cannot rule out alternate possibilities
that may contribute to alcohol-induced alterations in lipid raft
associated receptor functions. First, EtOH may modulate not
only the recruitment of the raft-associated molecules but also
the interactions and/or the stoichiometry of proteins associat-
ed/recruited in the raft, which itself could result in conforma-
tional changes potentially modulating the efficiency of down-
stream signaling. Second, EtOH may differently affect
recruitment of receptors and their adaptor molecules, thereby
affecting downstream signaling. Third, EtOH may change the
lipid composition of the detergent-resistant domains. Finally,
based on the observation that EtOH can inhibit actin filament
dimerization, it remains to be determined whether EtOH
would affect trafficking of lipid rafts to intracellular compo-
nents. These possibilities for modulation of lipid rafts and as-
sociated cellular function are likely to occur beyond TLRs and
of different receptor components to rafts and actin polymeriza-
tion were inhibited in rat adipocytes and mouse neurons by ad-
ministration of EtOH, suggesting that this mechanism may
contribute to the effects of EtOH on signaling through a num-
ber of receptor types in which lipid rafts play a role. In conclu-
sion, we propose that the influence of EtOH on lipid raft-me-
diated dynamics and signaling of TLRs, and possibly other
receptors, constitutes a previously unrecognized mechanism
that may contribute, along with other mechanisms, to alcohol-
cific receptor-mediated events by acute or chronic alcohol
administration in other systems.
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