By
From Merck Research Laboratories, Rahway, New Jersey 07090
Innate recognition of bacterial products constitutes a
principal bulwark of our defenses against infection. In
vertebrates, innate mechanisms both instruct the adaptive
immune response and provide immediate protection from
infectious challenge. In all lower phyla, innate mechanisms
represent the totality of immune protection, attesting to
both the power and evolutionary precedence of these mechanisms.
Although the workings of the adaptive immune system
may be traced in a satisfying way from generation of diversity to ligation of surface immunoglobulin to clonal expansion, a similarly complete and satisfying view of innate recognition is not in hand. Even the best characterized
example of innate recognition, the inflammatory response
to bacterial LPS (endotoxin), has a yawning gap in the steps
of its progression. Results of long anticipated work published in a recent issue of Science (1) and in this issue of The
Journal of Experimental Medicine (2) have now identified a
molecule with a privileged position in innate recognition
and the potential to fill this major gap.
Work
on innate recognition of LPS has generated information
from two polar starting points. The first has followed LPS
and its binding partners during the initial interaction with
cells. This work has shown that LPS is first acted on by LPS
binding protein (LBP), a plasma lipid transfer protein that
moves LPS monomers from aggregates or bacterial membranes to a binding site on CD14 (3, 4). CD14, a protein
expressed both in plasma and as a glycosylphosphatidylinositol (GPI)-linked protein on the surface of leukocytes,
then transfers LPS monomers into the plasma membrane of
cells (references 3 and 5, and Vasselon, T., E. Hailman, R. Thieringer, and P.A. Detmers, manuscript submitted for
publication). LPS moves in turn to the Golgi apparatus via an as yet undefined vesicular pathway (reference 6 and Thieblemont, N., and S.D. Wright, manuscript submitted for
publication). The goal of this approach has been to identify
the binding partner or "receptor" that discriminates LPS
from host lipids and transduces signals across the membrane The second line of work began with gene expression in
the nucleus and has worked backward toward determining
the agents that initiate new transcription. LPS causes dramatic transcriptional regulation of a wide range of proinflammatory genes including TNF, IL-1, IL-8, IL-6, ICAM-1,
E-selectin, tissue factor, and many more. LPS-dependent activation of these genes has been shown to be controlled by
the transcription factors, nuclear factor (NF)- Both recent studies discussed here (1, 2) examine the molecular defect in
well-characterized mouse strains (C3H/HeJ and C57BL10/
ScCr) that exhibit impaired ability to respond to LPS.
Work on these mice over the past 20 yr has shown that hyporesponsiveness to LPS maps to a single autosomal locus
(lps), and impaired responses can be documented both in
whole animals and in cells taken from the animals (10). The
consequence of this hyporesponsiveness is a dramatically
enhanced susceptibility of lpsd animals to challenge with
Gram-negative pathogens. Importantly, the animals respond
normally to Gram-positive challenge and do not have other
health defects. This phenotype corresponds precisely with
that expected of a defect in innate recognition of LPS. It is
fair to say that studies of lpsd animals did not merely "confirm
expectations" about innate immunity. Rather, they showed
for the first time that a functional innate mechanism for detecting particular microorganisms actually exists in mammals,
that the range of microorganisms detected by this system is
roughly defined as "Gram-negative bacteria," and that this
mechanism plays an important role in resistance to infection. These studies have propelled research on LPS recognition as
a paradigm of innate immunity. The identification of the genetic defect in lpsd mice has long been a holy grail, with the
potential to reveal a protein at the heart of innate immunity,
and that protein is now in hand.
Both Poltorak et al. (1) and Qureshi et al. (2) performed
extensive genetic mapping to narrow the position of the lps locus to a region of chromosome 4, both assembled the target region on YACs and BACs, and both sought candidate
genes on the basis of sequence and expression. However,
for both groups, the most important clue came not from
mapping but from studies in Drosophila which identified the
Toll protein as a key player in the response to fungal infection (11). Several mammalian homologues of Toll have
been discovered (see below), and recent studies had shown that TLR4 (Toll-like receptor 4, also known as hToll) can
initiate signaling steps similar to those seen in response to
LPS (12). Both Poltorak et al. and Qureshi et al. found
TLR4 in the target region of chromosome 4, and both
groups identified a missense mutation in the cytoplasmic
domain of TLR4 in C3H/HeJ. Importantly, an independent mutation at the lps locus in the C57BL10/ScCr strain resulted in the absence of TLR4 message, offering a strong
confirmation of the correct identification of TLR4 as the
defective protein resulting in the lpsd phenotype. The formal proof that TLR4 accounts for impaired responses to
LPS still must come from reconstitution of the defect with authentic sequence, but the data in hand provide fair assurance that the identification is correct.
Toll is a transmembrane protein that was discovered as a necessary player
in the establishment of dorsal-ventral polarity in Drosophila
embryos (13). The cytoplasmic domain of Toll bears significant homology with the cytoplasmic domain of the IL-1
receptor, and Toll signals transcriptional changes in Drosophila through a cascade of proteins with remarkable similarity
to those used by the IL-1 receptor:
IL-1R Toll In addition to its role in embryogenesis, Toll also plays an
important role in antifungal defenses of flies, being necessary for the strong upregulation of drosomycin in response
to fungal infection (11).
Recent observations from Medzhitov et al. (12) indicate
that a human homologue of Toll (TLR4) can be an effective
signaling molecule in mammalian cells. A constitutively active TLR4 construct drives NF- Additional data suggest that, as expected with any important physiological function, redundancy exists with respect to LPS signaling. Although lpsd mice are hyporesponsive to LPS, they are not unresponsive, and LPS-dependent
gene transcription will occur if a very large dose of LPS is
administered (10). Moreover, the sensitivity of lpsd cells and
animals may be restored by activation with IFN- The best candidates for backing up TLR4 are other
members of the Toll family. In Drosophila, a homologue of
Toll known as "18 wheeler" also plays a role in host defense
but serves primarily in recognition of bacterial rather than
fungal pathogens (19). In humans, five homologues of Toll
have been described by three groups (12, 20, 21). Importantly, transfection of cell lines with TLR2 confers on them
the ability to respond to LPS with activation of NF- As outlined
above, TLR4 appears to be an early and necessary part of the
signal transduction machinery linking LPS with gene expression. Since TLR4 is a transmembrane protein, we may now ask the most important question: is TLR4 the critical "LPS
receptor" that discriminates LPS from host lipids and initiates
signaling? Is it the last piece in the puzzle, the "T cell receptor of endotoxin biology?" Use of Occam's razor makes this
the obvious suggestion. On the other hand, any casual inspection of a diagram of signal transduction will reveal that
nature has made scant use of Occam's razor in this area, and
the remainder of this commentary will address the known
and possible links between LPS and TLR4.
The extracellular domain of TLR4 contains 22 copies of
a leucine rich repeat (LRR) motif. The best characterized
binding partner of LPS, CD14, contains 10 copies of the
LRR motif. Despite this marvelous similarity, it is unlikely
that the LRRs of TLR4 represent an LPS binding site.
Mapping studies with CD14 have revealed that 7 out of the
10 LRRs can be deleted without affecting LPS binding (24), and additional mapping studies have defined residues
outside the LRR region of CD14 that are clearly necessary
for LPS binding (25). Yang et al. (22) have suggested that
LPS may bind directly to TLR2. However, the affinity and
stoichiometry observed were so low as to eliminate any biological significance of this phenomenon, and at present
there is no strong evidence for a direct, meaningful interaction of LPS with any member of the Toll family. An alternative to direct binding of LPS is an interaction of TLR4
with CD14. Although conceptually attractive, work with
CD14 has failed to uncover a binding partner in cells. Despite use of probes of extremely high sensitivity, it has not
been possible to measure binding of CD14 (26) or CD14-LPS complexes (Vasselon, T., and R. Thieringer, unpublished observations) to LPS-responsive cells.
A key to understanding how LPS might interact with
TLR4 (or any other adapter protein) comes from a consideration of the form and location of the LPS during recognition. LPS is an amphiphile that will incorporate into lipid
bilayers. Indeed, CD14 readily donates LPS to liposomes,
lipoprotein particles, and the membranes of responsive cells
(references 5, 27, and 28, and Vasselon, T., E. Hailman, R. Thieringer, and P.A. Detmers, manuscript submitted for
publication). Two lines of evidence suggest that LPS signals responses not as a monomer but as a component of the
lipid bilayer. Scientists at Eisai Pharmaceuticals have synthesized an LPS homologue, E5531, that acts as a powerful
antagonist of LPS action in cells and animals (29). Inversion
of all 13 chiral centers of E5531 yields a mirror image, and
this mirror image compound shows equal ability to antagonize LPS action (Christ, W.J. 1998. Advances in synthetic
LPS antagonists. Oral presentation at Fifth Conference of
the International Endotoxin Society, Santa Fe, NM). This
finding argues that LPS is not recognized in the stereospecific fashion expected of stoichiometric interactions with
proteins. An alternative is that the colligative properties,
which are identical in enantiomers, may be key to the biological action of E5531 and LPS. This possibility is suggested by the finding that another LPS analogue (from
Rhodobacter sphaeroides) may be converted from an antagonist to an agonist by simultaneous addition of the membrane-active agent, chlorpromazine (30). From these considerations, we are directed to seek recognition proteins
that either sense LPS in a bilayer or that sense the properties of a bilayer containing LPS.
There is only one well-characterized precedent in which
membrane composition is sensed and relayed to gene expression. The concentration of cholesterol in membranes of
the endoplasmic reticulum is gauged by a set of proteins
that control transcription of the genes for cholesterol synthesis and uptake and which thereby affect cholesterol homeostasis at the cellular level (for review see reference 31).
In brief, low levels of cholesterol in the endoplasmic reticulum are thought to be sensed by a multispan protein
known as SCAP. SCAP controls the action of a transmembrane protease (S1P). Interestingly, the proteolytic domain
of S1P is lumenally disposed (32), and its substrate is an intralumenal loop of SREBP (sterol response element binding protein), a transcription factor. Cleavage of the SREBP
by S1P in turn enables a second cleavage on the cytoplasmic face of SREBP, and this cut liberates a fragment of
SREBP that acts as a transcription factor. For the purposes
of this discussion, the message I wish to take from this diversion is that membrane composition can be sensed and
that, at least for cholesterol, the actuation device is a protease cascade that starts in the lumen of an intracellular vesicle. For completeness, it should be added that just as Toll
is involved in dorsal-ventral polarity in Drosophila, other
genes known as hedgehog and patched are involved in establishing anterior-posterior polarity (33). Hedgehog encodes a
protein covalently derivatized with cholesterol, and patched
encodes a homologue of SCAP.
Several observations suggest that LPS may activate a protease cascade and that a protease cascade may play a role in activation of receptors such as Toll. In Drosophila, the ligand for
Toll that drives dorsal-ventral polarity is a proteolytic fragment of the protein, spätzle. Spätzle is cleaved by the protease, easter, and easter in turn is activated by another protease
known as snake (13). It is also well known that LPS can initiate a protease cascade that leads to clotting in arthropods,
and the familiar Limulus amebocyte lysate (LAL) assay for LPS
exploits this cascade. Recent studies indicate a high similarity
between the LPS-induced clotting cascade and the Toll
cascade (34). Snake and easter are homologues of the Limulus
proteases factor B and proclotting enzyme, respectively. The
final step of the LAL reaction involves cleavage of coagulogen. As its name implies, coagulogen forms a fibrin-like clot
but recent crystal structure analysis (35) has revealed that, in
addition, coagulogen contains a cysteine knot fold also found
in spätzle, NGF, TGF- Could a protease cascade function in recognition of LPS
in mammalian cells? The only soluble factors needed for responses of mammalian cells to LPS in vitro are LBP and
CD14, arguing against a role for a plasma-borne protease
cascade. An alternative source of a cascade is the cell itself
with the proteases being membrane bound. Many transmembrane proteases have been characterized and are
known to function in sensing cholesterol concentration
(described above), processing of notch (36), and maturation
of signaling proteins such as insulin (37). Additionally, since
LPS is rapidly internalized and carried to the Golgi, soluble
components may exist in sufficient concentration in this
organelle. It is worth noting that like LPS, ceramide is also
transported rapidly to the Golgi (38) and lpsd cells fail to respond to ceramide (39). Membrane-bound proteases such
as furin are known to be concentrated in the Golgi (37), certain genes that act upstream of Toll (pipe and windbeutlel) are found in the Golgi (13), and the IL-1R has been reported to traffic to the Golgi after ligation (40).
In conclusion, the new work describing a role for TLR4
in responses to LPS clearly adds a piece to the puzzle of
LPS responsiveness and innate immunity. How important
is this piece? TLR4 clearly contributes a new part of the
signal transduction cascade but it is not clear that TLR4
contributes to the most vexing problem in innate immunity: how do cells discriminate LPS or other microbial molecules from similar structures in the environment?
Work in the next year should clarify this issue, but this author's view is that TLR4 may be well downstream of the
step or steps discriminating host from pathogen.
Article
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Article
References
but this goal has remained elusive: neither LBP nor
CD14 has the binding specificity to discriminate LPS from
host lipids (3, 7), and experiments to identify new binding
partners for LPS in cells have been uniformly frustrating.
B and AP-1.
These transcription factors are in turn controlled by kinases
that are rapidly activated by LPS (8, 9). For this approach as
well, the still unmet goal is to identify the receptor that discriminates LPS from host lipids at the membrane and initiates the kinase cascade. The new work reviewed here focuses on the potential interface between the molecules
defined by these two lines of investigation, and this article
will focus on the extent to which a connection can be made.
MyD88
IRAK
I
B
NF-
B
gene expression
Tube
Pelle
Cactus
Dorsal
gene expression.
B activation, AP-1 activation, and cytokine production in transfected cells (12). Additional data show that, like the IL-1R, TLR4 uses MyD88
and IRAK to activate NF-
B (14, 15). As described above,
NF-
B and AP-1 activation are key players in responses to
LPS, and these data suggest that activation of a receptor such
as TLR4 may be sufficient to explain the transcriptional responses to LPS. Together with the observation that the lpsd
phenotype derives from defective TLR4, we may conclude
that at least under some conditions TLR4 is necessary and
sufficient for responses to LPS. These findings now extend
our understanding of the steps in LPS signal transduction
from the nucleus all the way to the membrane.
(16, 17),
and cells from C3H/HeJ mice are nearly as sensitive as their
normal counterparts when stimulated with certain types of LPS (e.g., Porphyromonas gingivalis LPS) (18). These observations suggest that proteins other than TLR4 may replace
the function of TLR4 in signal transduction for responses
to LPS.
B (22,
23), thus directly suggesting that TLR2 may serve in place
of TLR4. It is interesting to note that Kirschning et al. (23)
examined not only TLR2 but also TLR4. Contrary to the
prediction from the lpsd mice, transfection of TLR4 led to
constitutive activation of NF-
B with no enhancement
upon addition of LPS. The reasons for this discrepancy are
not clear but could derive from the presence of the additional "Flag" sequence in the TLR4 construct or the requirement for a factor or subunit not present in the recipient cells. It is clear that a great deal of work needs to be
done to fully describe the cell distribution, regulation, and
contribution of individual members of the Toll family to
responses to LPS and other microbial products.
, and other signaling molecules. It is
thus a reasonable possibility that LPS may initiate a protease
cascade to generate ligands for receptors such as Toll.
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Footnotes |
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Address correspondence to Samuel D. Wright, Merck Research Laboratories, PO Box 2000, RY80W-250, Rahway, NJ 07090. Phone: 732-594-3086; Fax: 732-594-1169; E-mail: samuel_wright{at}merck.com
Received for publication 4 January 1999.
I wish to thank Drs. Nathalie Thieblemont, Thierry Vasselon, Rolf Thieringer, Katheryn Anderson, and Patricia Detmers for helpful discussions. ![]() |
References |
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