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B/Toll-Like Receptor Pathway in Induction of Inflammatory and Tissue-Repair Gene Expression by Necrotic Cells1
* Department of Biological Sciences, Columbia University, New York, NY 10027; and Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and induce expression of genes involved in
inflammatory and tissue-repair responses, including neutrophil-specific
chemokine genes KC and macrophage-inflammatory protein-2, in
viable fibroblasts and macrophages. Intriguingly, NF-B activation by
necrotic cells was dependent on Toll-like receptor 2, a signaling
pathway that induces inflammation in response to microbial agents.
These results have identified a novel mechanism by which cell necrosis,
but not apoptosis, can induce expression of genes involved in
inflammation and tissue-repair responses. Furthermore, these results
also demonstrate that the NF-B/Toll-like receptor 2 pathway can be
activated both by exogenous microbial agents and endogenous
inflammatory stimuli. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue damage can initiate not only an inflammatory response but also a tissue-repair and remodeling process mediated by induction of metalloproteinases (11) and factors involved in angiogenesis, such as vascular endothelial growth factor (VEGF; Ref. 12). Interestingly, enhanced VEGF expression was detected in areas adjacent to necrotic cells in various disease models (13, 14), although the inducing mechanisms are still largely uncharacterized. Two recent studies have proposed that necrotic cells, but not apoptotic cells, can potently induce dendritic cell (DC) maturation in vitro (15, 16). Sauter et al. (16) have further demonstrated that the immunostimulatory activity was present in necrotic tumor cells but not in primary necrotic cells. Thus, it is not known whether primary necrotic cells have any immunoregulatory effect on DCs.
The NF-B family of transcription factors are key regulators of genes
involved in immune and inflammatory reactions (17, 18, 19).
Studies with mice deficient in the RelA (p65) subunit of NF-B also
have shown that these proteins are important in the regulation of
apoptosis. The absence of RelA results in massive apoptotic death of
hepatocytes and embryonic lethality at E15 (20), which
appears related to the cytotoxic effect of TNF-
on
RelA-/- cells (21, 22). Among the
most potent NF-B inducers are microbial products such as bacterial
LPS (18). NF-B activation by LPS is dependent on
members of the mammalian Toll-like receptor (TLR) protein family
(homologous to Drosophila Toll; Refs. 23, 24, 25),
and downstream signaling molecules which include TNFR-associated factor
(TRAF) 6, myeloid differentiation primary response gene and IL-1
receptor-associated kinase (26, 27, 28). Our recent
studies have demonstrated a key role for RelA in activation of many
inflammatory genes in response to LPS stimulation, including those
encoding proinflammatory cytokines and chemokines
(29).
Here we have identified necrotic cells as novel and potent inducers of
NF-B. NF-B activation by necrotic cells resulted in dramatic
induction of expression of genes involved in the inflammatory response,
such as neutrophil-specific chemokines, and genes involved in the
tissue-repair and remodeling process. Interestingly, necrotic
cell-induced NF-B was found dependent on the TLR2 signaling pathway.
In contrast to necrotic cells, apoptotic cells did not induce
expression of chemokines because they did not significantly activate
NF-B. Thus, our results provide the first evidence for an
NF-B-dependent mechanism responsible for the strikingly different
pathophysiological affects of necrotic and apoptotic cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs were prepared from bone marrow suspensions obtained from
femurs and tibias of C57BL/6 mice (Taconic Farms, Germantown, NY).
Cells were grown in 6-well plates at 3 x
106 cells in 2 ml of RPMI 1640
supplemented with 5% FBS, 2-ME (50 µM), IL-4 (5 ng/ml; R&D Systems,
Minneapolis, MN), and GM-CSF (10 ng/ml; R&D Systems). Cells were
"fed" IL-4 and GM-CSF every 2 days. On day 5, nonattached cells
(DCs) were replated in fresh medium and treated with either LPS (5
µg/ml; Sigma, St. Louis, MO) or necrotic cells
(106). Mouse embryonic fibroblasts (MEFs) or
fetal liver macrophages were obtained and cultured as described
previously (21). TNF- and IL-1 were purchased from R&D
Systems and used at 10 and 4 ng/ml, respectively. pBIIX-luc is a
firefly luciferase report construct driven by a minimal murine
c-fos promoter with two Ig B motifs upstream. pRL-TK
is a renilla luciferase control reporter construct driven by the HSV
thymidine kinase promoter. The expression vector pLPC was a gift of S.
Lowe (Cold Spring Harbor Laboratory, NY). The pIB(S-A)
plasmid was constructed by cloning an IB mutant
(Ser32, Ser36 to Ala) into pLPC.
pIKK
DN was constructed by inserting a dominant-negative mutant of
IB kinase (IKK; a mouse IKK mutant consisting of a point
mutation of Lys44 to Ala) into pLPC. pTRAF2DN,
pTRAF6DN, pMyD88DN, and pTLR2DN were described previously (26, 30). pTLR2, pTLR4, and pTLR6 were constructed by cloning murine
TLR2, TLR4, and TLR6 cDNA into the pIRES vector (Clontech Laboratories,
Palo Alto, CA). Constructs were confirmed by sequencing.
Generation of necrotic and apoptotic cells
To obtain necrotic cells, MEFs or thymocytes were washed and
resuspended at 107 cells/ml of PBS. Dounce-lysis
was carried out by subjecting MEFs or thymocytes to 10 strokes with a
Teflon homogenizer, after which complete cell lysis was evident by
trypan blue inclusion. Freeze-thaw lysis was carried out by subjecting
cells to five cycles of freezing on dry ice followed by thawing at
37°C. Such treatment generally resulted in lysis of 90% of the cells
as judged by trypan blue inclusion. Apoptotic thymocytes were generated
by 
-irradiation. Briefly, thymocytes were subjected to 750 rad of
-irradiation. The cells then were cultured for 10 h, after
which 90% of the cells showed typical apoptotic morphology and were
annexin V positive. These cells were collected and resuspended in PBS.
Greater than 80% of these cells were typically trypan blue negative.
Necrotic or apoptotic cells were added to DC suspensions, MEFs, or
macrophages for indicated periods. All experiments were carried out
under sterile conditions. All cells were tested to be mycoplasma-free
with a mycoplasma detection kit from American Type Culture Collection
(Manassas, VA).
ELISA
Supernatants from cultured DCs, MEFs, or macrophages were used to determine the amount of IL-12 or KC. ELISA was carried out following recommendations of the manufacturer (R&D Systems).
EMSA and Western and Northern blots
EMSA was carried out as described previously (20).
RelA-specific antiserum was purchased from Rockland (Gilbertsville,
PA). Western blotting for IB was carried out with cytosolic
extracts from 
105 MEFs by using a rabbit
polyclonal IgG against the carboxyl terminus of IB (Santa Cruz
Biotechnology, Santa Cruz, CA). Northern blotting was carried out as
described previously (29) with probes made from cDNA
fragments amplified by RT-PCR with gene-specific primers.
Luciferase assays
Transfections in the mouse macrophage cell line, RAW 264.7, were carried out by using Fugene6 (Roche, Gipf-Oberfrick, Switzerland). 293 Cells were transfected by using calcium phosphate. 24 h after transfection, cells were either left untreated or treated with 106 dounce-lysed MEFs. After another 10 h, cellular extracts were made and tested for luciferase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We wished to investigate whether necrotic primary cells have any
immunoregulatory effect on DCs. DC activation induced by microbial
agents, such as LPS, results in enhanced production of IL-12, a key
cytokine that promotes differentiation of Th1 cells. Thus, we first
tested whether necrotic primary MEFs also could induce IL-12 production
by DCs. To this end, 106 dounce-lysed MEFs were
used to stimulate mouse bone marrow-derived DCs. After 12 h,
supernatants were collected and the amount of IL-12 produced by DCs
determined by ELISA. Only minimal levels of IL-12 were detected in
necrotic cell-treated supernatants (Fig. 1
A), whereas LPS-treated DCs
produced high levels of IL-12 (Fig. 1A). Necrosis in vivo
often is associated with recruitment of neutrophils (3).
Therefore, we tested whether necrotic cell treatment of DCs could
result in production of KC, a chemokine involved in neutrophil
recruitment during inflammation (6, 8, 9). Significantly,
necrotic MEF-treated DCs secreted high levels of KC (Fig. 1A). Interestingly, unlike IL-12, the amount of KC produced
by necrotic MEF-treated DCs was higher than that produced by
LPS-treated DCs (Fig. 1A), suggesting that necrotic cells
may preferentially induce genes involved in innate immunity. To
determine whether necrotic cells also could induce KC production in
macrophages and fibroblasts, two cell types that play a critical role
in regulation of innate immunity, we treated fetal liver-derived
macrophages and MEFs with dounce-lysed MEFs for 6 h. Significant
amounts of KC also were produced by these cells (Fig. 1B),
demonstrating that necrotic cells can induce KC production in multiple
cell types.
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-irradiation. These apoptotic cells were annexin V
positive and retained membrane integrity for several hours (data not
shown), making them suitable for these experiments. In contrast to
dounce-lysed thymocytes, no significant increase in KC was detected in
either macrophages or MEFs treated with apoptotic thymocytes (Fig. 1
C). Importantly, KC production took place if apoptotic
thymocytes were rendered necrotic by dounce-lysis (Fig. 1C),
although the levels of KC were lower than those induced by dounce-lysed
thymocytes. These results suggest that intracellular contents are
critical and sufficient for induction of KC expression. These results
also demonstrate that intracellular contents from different primary
cell types (fibroblasts or thymocytes) can induce KC production.
To determine whether the increased KC production was a consequence of
enhanced mRNA levels, Northern blot analysis was performed with RNA
from both macrophages and fibroblasts treated with necrotic thymocytes.
In both cell types, a significant increase in KC transcripts were
detected (Fig. 1D). Consistent with ELISA results, no
significant change in KC levels were detected in either macrophages or
fibroblasts treated with apoptotic thymocytes (Fig. 1D),
whereas dounce-lysis of apoptotic thymocytes resulted in enhanced KC
transcription (Fig. 1D). Consistent with enhanced production
of KC by DCs, increased KC transcripts were detected in necrotic
MEF-treated DCs (data not shown). Another neutrophil-specific
chemokine, MIP-2, also was induced by necrotic thymocytes in either
macrophages or fibroblasts (Fig. 1D). Increased KC and MIP-2
transcripts also were detected in necrotic MEF-treated fibroblasts and
macrophages (see below). These observations suggest that the
intracellular contents released from necrotic cells can induce
neutrophil-specific chemokine gene expression at the transcriptional
level in multiple cell types.
NF-B activation by necrotic cells but not apoptotic cells
Our recent studies have demonstrated a critical role for NF-B
in regulation of KC and MIP-2 (29). Thus, one intriguing
possibility was that induction of KC expression by necrotic cells was
mediated by activation of NF-B in viable cells, after interaction
with necrotic cells. To test this possibility, we determined whether
NF-B could be activated in viable cells exposed to necrotic cells.
NF-B normally is sequestered in the cytoplasm bound to the
inhibitory IB proteins (18). Degradation of IB
proteins by NF-B activators allows free NF-B to translocate to
the nucleus and activate target gene expression (18). EMSA
analysis showed that significant nuclear translocation of NF-B took
place in MEFs exposed to dounce-lysed necrotic thymocytes (Fig. 2A). In striking contrast, a
similar number of apoptotic thymocytes (Fig. 2A) were
significantly less capable of activating NF-B in MEFs. However,
lysis of apoptotic thymocytes resulted in NF-B activation to the
same extent as with necrotic cells (Fig. 2A). These results
indicate that cell lysis is required for activation of NF-B and
demonstrate a key difference between apoptotic and necrotic cells in
their ability to activate NF-B. Necrotic MEFs also could activate
NF-B in a dose-dependent manner (Fig. 2B). Nuclear
NF-B also was detected if necrotic MEFs were generated by
freeze-thaw lysis, although the activation was less strong than that
induced by dounse-lysed cells, suggesting the NF-B-inducing agents
present in necrotic cells may in fact be quite labile (data not shown).
These results indicate that NF-B activation is not dependent on the
cell type used or the manner in which necrotic cells are generated.
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treatment of MEFs results in activation of NF-B heterodimers
consisting of p50 and RelA (p65) (20). The NF-B complex
activated by necrotic cells was specifically supershifted by antisera
generated against RelA (Fig. 2C, lane 3). Furthermore,
necrotic cell-treated RelA-/- MEFs showed
significantly reduced induction of B site binding activity (Fig. 2C, compare lanes 2 and 5), as
observed previously after TNF- treatment (20). When
necrotic RelA-/- MEFs were added to
RelA+/+ MEFs, a significant increase in B
binding activity was detected (Fig. 2C, lane 7). The induced
B activity was supershifted by RelA-specific antisera (Fig. 2C, lane 8), demonstrating that NF-B was activated in
viable RelA+/+ MEFs, and not necrotic
RelA-/- cells. NF-B was activated within 30
min after addition of necrotic MEFs and persisted for at least 6 h
in nuclear fractions (Fig. 2D, lanes 1–4). Significant
NF-B activation by necrotic cells also took place in the presence of
cyclohexamide (Fig. 2D, lanes 5–8), suggesting that
activation was not dependent on protein synthesis. Inflammatory and tissue-repair gene induction in MEFs and macrophages by necrotic cells is dependent on RelA
To determine whether NF-B activation by necrotic cells may be
an important mechanism for induction of inflammatory genes such as KC
and MIP-2, we first investigated expression of IB, a key NF-B
target gene. Both RelA+/- and
RelA-/- MEFs were treated with necrotic MEFs
for different periods after which RNA was isolated to determine levels
of IB mRNA. IB was strongly induced in
RelA+/- but not in
RelA-/- MEFs after exposure to necrotic cells
(Fig. 3A). We then tested
whether KC and MIP-2 induction also was dependent on RelA.
RelA+/- MEFs showed dramatic induction of both
KC and MIP-2 mRNA after a 6-h treatment with necrotic cells (Fig. 3A). In contrast, induction of these transcripts was
significantly reduced in RelA-/- MEFs (Fig. 3A), indicating a key role for RelA in chemokine gene
induction by necrotic cell contents. Similar results also were obtained
when freeze-thaw MEFs were used (data not shown). These results
demonstrate that necrotic cells can induce chemokine gene expression
through activation of RelA-containing NF-B complexes.
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A). Thus,
necrotic cells also can induce expression of genes involved in the
tissue-repair process. These results also reveal a previously
uncharacterized function for RelA in regulation of genes involved in
the wound-healing response. Importantly, induction of KC, MIP-2, and
VEGF by necrotic cells was significantly higher than induction by
TNF-, whereas IB and MMP3 were similarly induced by both
stimuli. These results suggest that necrotic cells may preferentially
induce high expression of a subset of NF-B target genes.
We then tested whether RelA also plays an essential role in regulating
inflammatory genes in macrophages. To this end, fetal liver-derived
RelA+/+ and RelA-/-
macrophages were treated with necrotic cells for 6 h. Although
RelA-/- embryos do not survive past E15 because
of TNF--mediated liver degeneration (20, 21, 22), we have
readily obtained fetal liver macrophages from E13-E14
RelA-/- embryos (21). Northern
blot analysis of RNA from these macrophages demonstrated that induction
of IB, KC, and MIP-2 transcripts in RelA+/+
macrophages was significantly more than the induction in
RelA-/- macrophages (Fig. 3B). ELISA
analysis of supernatants showed that synthesis of both chemokines also
was significantly more in RelA+/- MEFs and
RelA+/+ macrophages than in
RelA-/- MEFs and macrophages (data not shown).
Thus, NF-B activation by necrotic cell contents results in dramatic
induction of chemokine production by fibroblasts and macrophages, two
cell types that play a key role in chemokine production.
IL-1/TLR signaling molecules are required for NF-B activation by
necrotic cells
Because NF-B activation by many agents, including the
proinflammatory cytokines TNF- and IL-1, is dependent on degradation
of IB proteins (18, 32), we tested whether levels of
the IB protein were affected by necrotic cells. A reduction in
cytosolic levels of IB was noticed, which was further decreased
in the presence of cyclohexamide (Fig. 4A). IB degradation
requires initial phosphorylation of two key serine residues by the
recently identified IKK proteins (19). NF-B activation
can thus be inhibited by expression of a phosphorylation-deficient
IB protein. As expected, treatment of the RAW macrophage
cell-line with necrotic cells significantly increased B luciferase
reporter activity (Fig. 4B; RAW macrophages also activated
NF-B and induced target gene expression after necrotic cell
stimulation; data not shown). The increase in luciferase activity was
dramatically suppressed by expression of a phosphorylation-deficient
IB protein (Fig. 4B), suggesting that IB
phosphorylation may be essential for NF-B activation by necrotic
cells. We then tested the effect of expression of a dominant-negative
mutant of IKK (DN-IKK), which appears most important for
phosphorylation of IB proteins (33, 34), on luciferase
activity induced by necrotic cells. Expression of DN-IKK
significantly reduced reporter activity (Fig. 4B),
suggesting a requirement for IKK in activation of NF-B by
necrotic cells. These results demonstrate that NF-B activation by
necrotic cells appears to be by the "classical" activation pathway,
which does not require new protein synthesis and depends on
phosphorylation and degradation of IB proteins.
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B activation pathways important for
inducing inflammatory gene expression have been identified: the TNF
pathway, which can be inhibited by a dominant-negative mutant of the
adapter protein TRAF2 (35) and the IL-1/TLR pathway, which
can be inhibited by dominant-negative mutants of MyD88, IRAK, and TRAF6
proteins (26, 27, 28). Expression of a dominant-negative TRAF2
did not significantly inhibit necrotic cell-mediated NF-B activation
in RAW cells (Fig. 4B), suggesting activation may not be
dependent on signaling components used by the TNF pathway. In contrast,
expression of MyD88 or TRAF6 dominant-negative proteins significantly
inhibited NF-B activation by necrotic cells (Fig. 4B).
These results suggest that NF-B activation by necrotic cells could
be mediated by IL-1/TLR signaling components.
TLR2 is sufficient for NF-B activation and IL-8 expression by
necrotic cells
To further study the involvement of the IL-1/TLR signaling pathway
in necrotic cell-mediated activation of NF-B, we used human 293
cells, which were found unable to activate NF-B after necrotic cell
stimulation (Fig. 5A).
However, IL-1 treatment of 293 cells resulted in strong NF-B
activation (Fig. 5A), suggesting that IL-1/TLR signaling
components were present in 293 cells. Thus, a possible reason for the
inability of 293 cells to activate NF-B by necrotic cells may be
that these cells lack expression of requisite TLRs (27).
We tested this by transiently expressing TLR2, TLR4, or TLR6 along with
a B reporter construct in 293 cells. As expected from EMSA results,
virtually no activation of the B reporter occurred when 293 cells
were transfected with a control GFP construct and treated with necrotic
MEFs (Fig. 5B). However, expression of TLR2, but not TLR4 or
TLR6, resulted in dramatic activation of NF-B activity in 293 cells
(Fig. 5B). TLR2 also was able to mediate necrotic
thymocyte-induced NF-B activation in 293 cells (data not shown).
These observations demonstrate that the intracellular contents from
different cell types can activate NF-B in a TLR2-dependent manner. A
recent study has revealed that TLR2 can functionally cooperate with
TLR6 to mediate responses to certain TLR2 activators (36).
To test whether TLR2-mediated NF-B activation of necrotic cells
could also be potentiated by TLR6, we cotransfected 293 cells with
constructs encoding TLR2, TLR6, or TLR2 and TLR6. Significant increase
of NF-B activity was detected when both TLR2 and TLR6 were expressed
(Fig. 5C), suggesting that TLR6 can facilitate TLR2
responses to necrotic cells.
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B
activation and gene induction. Nuclear extracts of TLR2-transfected and
necrotic cell-stimulated 293 cells showed significant NF-B
activation, which was abolished by coexpression of an IB (S to A)
mutant (Fig. 5D). Significantly, necrotic cell treatment of
TLR2-transfected 293 cells also induced expression of IL-8 (a human
neutrophil-specific chemokine), which also was inhibited by
coexpression of IB (S to A; Fig. 5E). These results
demonstrate that TLR2 expression is sufficient for NF-B activation
and expression of the IL-8 chemokine by necrotic cells. Importantly,
overexpression of a dominant-negative mutant of TLR2 significantly
reduced B reporter gene activity by necrotic MEFs in RAW cells (Fig. 5F), suggesting that TLR2 may play an essential role in
mediating necrotic cell-induced NF-B activation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a master regulator of genes involved in immune and
inflammatory responses. Induction of these genes typically occurs in
response to stimulation of cells with conserved microbial patterns such
as LPS or with proinflammatory cytokines. Here we have shown that
intracellular contents released from necrotic cells also function as
novel and potent inducers of NF-B. We believe these results may have
significant implications for our understanding of how cell death
affects inflammatory and tissue-repair responses. Foremost, we believe that necrotic cells may provide an endogenously generated "sentinel" signal that leads to production of inflammatory mediators and allows efficient removal of damaged tissue. Importantly, we have shown that exposure of viable cells to necrotic cell contents is sufficient for inducing expression of chemokines specific for neutrophils. Although the ability of neutrophils to infiltrate necrotic tissue is well known, it has not been clear whether necrosis itself causes neutrophil entry (5, 37). This is especially true where tissue damage is associated with microbial infections, because microbial products are themselves strong inducers of neutrophil recruitment (e.g., LPS). Thus, our results provide the first evidence for a direct role of necrotic cells in regulating expression of genes important for inducing inflammation. Importantly, we also have found that necrotic cells are strong stimulators of KC but weak stimulators of IL-12 production in DCs, suggesting that necrotic cells may preferentially induce genes important for innate immunity.
In addition to an inflammatory response, tissue damage can initiate a
tissue-repair and remodeling process. Here, we have provided evidence
that necrotic cells also may directly regulate the tissue-repair
response. Metalloproteinases can be rapidly induced after injury
(11) and play an essential role in tissue repair and
remodeling (31). Such induction has been proposed to be
mediated by proinflammatory cytokines and growth factors
(38). Our results indicate that necrotic cells also may
function as inducers of metalloproteinase expression and thus directly
initiate a wound-healing response after tissue damage. VEGF is an
important mediator of angiogenesis, another key component of the
tissue-repair pathway (12). Interestingly, VEGF expression
could be detected in areas adjacent to necrotic cells in various
disease models (13, 14) and in situ analysis of tumor
specimens also has demonstrated the localization of VEGF-producing
cells proximal to necrotic foci (39). Our results indicate
that necrotic cells may directly induce expression of VEGF and thus
also participate in stimulating angiogenesis. Similar to chemokine
genes, induction of both MMP3 and VEGF expression was dependent, at
least in part, on the RelA subunit of NF-B. These observations also
indicate that NF-B proteins may play an important role in regulating
expression of tissue-repair genes.
In addition, our results provide a possible explanation for well known
differences between apoptotic and necrotic cell death in induction of
inflammation. We have shown that KC and MIP-2 expression is induced by
necrotic but not by apoptotic cells. The inability of apoptotic cells
to induce chemokine expression appears linked to relatively weak
NF-B activation by apoptotic cells, which is typically at least
10-fold less than by an equivalent number of necrotic cells (Fig. 2A and data not shown). However, lysis of apoptotic cells
results in similar activation of NF-B as seen with necrotic cells,
demonstrating a key role for intracellular contents in NF-B
activation. Apoptotic cells are rarely detected in situ as a
consequence of rapid clearance by phagocytic cells such as macrophages
or by surrounding cells (2, 37). Our results indicate that
rapid removal of apoptotic cells before lysis may be important in
preventing release of intracellular contents that can activate NF-B
and induce expression of inflammatory genes. In addition, macrophages
that have phagocytosed apoptotic cells can produce antiinflammatory
cytokines such as TGF and IL-10 (40, 41, 42). Whether these
cytokines also can modulate NF-B-mediated induction of inflammatory
genes will be an interesting area for investigation.
Necrotic cell activation of NF-B is dependent on TLRs
TLRs are evolutionarily conserved receptors that regulate innate
and adaptive immune responses (43). This is mediated, in
large part, by activation of NF-B and subsequent induction of
expression of inflammatory genes by this transcription factor. We show
here that activation of NF-B by necrotic cells also requires
signaling molecules of the IL-1/TLR pathway. Recent studies have shown
that TLR2 can mediate signaling to multiple components of Gram-positive
bacteria, mycobacteria, and yeast (30, 44, 45, 46, 47, 48), whereas
TLR4 appears specific for LPS (25, 45). Our results
demonstrate that TLR2 also is sufficient to mediate responses to
necrotic cells. We believe these results are significant because they
show that the mechanism for inducing inflammatory gene expression by
exogenous agents, such as microbes, and endogenous signals, such as
necrotic cells, is remarkably similar. Interestingly, coexpression of
TLR6 and TLR2 could potentiate necrotic cell-induced NF-B
activation, although TLR6 alone had no significant effect (Fig. 5C). Thus, similar to microbial TLR2 activators
(36), necrotic cells might also engage a combinatorial
repertoire of TLRs to induce an inflammatory response. However,
expression of a dominant-negative mutant of TLR6 did not significantly
inhibit NF-B activation by necrotic cells in RAW macrophages (data
not shown). Because other TLRs, such as TLR1, can also augment TLR2
signaling (36), it is possible that TLRs besides TLR6 are
involved in necrotic cell-induced NF-B activation.
Nature of NF-B inducing agents present in necrotic cells?
Two recent studies have demonstrated that recombinant heat shock
proteins (HSPs) 60 and 70 can activate NF-B (49, 50),
raising the possibility that these endogenous proteins may be
responsible for induction of chemokine gene expression by necrotic
cells. A recent study has further demonstrated that TLR4 can mediate
responses to HSP60 (51). However, our results show that
TLR4 is not sufficient to mediate NF-B activation by necrotic cells.
In addition, chemokine induction by necrotic cells was unaffected in
macrophages derived from C3H/HeJ mice, in which TLR4 is nonfunctional,
whereas HSP60- and HSP70-induced chemokine gene expression was
compromised (unpublished observations). These results suggest that
HSP60 and HSP70 may not be the agents responsible for the induction of
chemokine expression by necrotic cells.
To identify the nature of inducing agents present in necrotic cells, we
have carried out fractionation studies and have found that both
mitochondrial and nuclear fractions can use TLR2 to activate NF-B.
Unfortunately, we were unable to further characterize these fractions
because of a virtually complete loss of NF-B-inducing activity when
subjected to standard fractionation and purification procedures. It is
possible that the labile nature of these activities represents a
requirement for maintenance of higher order structures and/or an
unusual susceptibility to inactivation following fractionation. Thus,
further characterization may first require identification of
appropriate conditions that preserve the structural integrity of
NF-B-inducing agents present in necrotic cells. Characterization of
these activities will eventually be required to determine whether
NF-B activation is mediated by unique molecule-specific interactions
or interactions with classes of molecules representing specific
structures (or patterns), as seen during recognition of microbial
components.
Concluding remarks
We have demonstrated here, for the first time, that necrotic cells
can directly enhance expression of genes important for inducing an
inflammatory and tissue-repair response, through activation of the
NF-B and TLR2 pathway. We believe that these results will help us
better understand different pathophysiological affects induced by
tissue damage. Because cell death forms an integral part of virtually
any disease, these findings also may have significant implications for
understanding disease mechanisms.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Amer A. Beg, 1110 Fairchild Center, Department of Biological Sciences, 1212 Amsterdam Avenue, Columbia University, New York, NY 10027. E-mail address: aab41{at}columbia.edu
3 Abbreviations used in this paper: MIP-2, macrophage-inflammatory protein 2; VEGF, vascular endothelial growth factor; DC, dendritic cell; TLR, Toll-like receptor; TRAF, TNFR-associated factor; MEF, mouse embryonic fibroblasts; IKK, IB kinase; HSP, heat shock protein.
Received for publication December 19, 2000. Accepted for publication April 5, 2001.
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  S. S. Iyer, W. P. Pulskens, J. J. Sadler, L. M. Butter, G. J. Teske, T. K. Ulland, S. C. Eisenbarth, S. Florquin, R. A. Flavell, J. C. Leemans, et al. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome PNAS, December 1, 2009; 106(48): 20388 - 20393. [Abstract] [Full Text] [PDF]
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 F. Angelot, E. Seilles, S. Biichle, Y. Berda, B. Gaugler, J. Plumas, L. Chaperot, F. Dignat-George, P. Tiberghien, P. Saas, et al. Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases Haematologica, November 1, 2009; 94(11): 1502 - 1512. [Abstract] [Full Text] [PDF]
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 K. Miles, D. J. Clarke, W. Lu, Z. Sibinska, P. E. Beaumont, D. J. Davidson, T. A. Barr, D. J. Campopiano, and M. Gray Dying and Necrotic Neutrophils Are Anti-Inflammatory Secondary to the Release of {alpha}-Defensins J. Immunol., August 1, 2009; 183(3): 2122 - 2132. [Abstract] [Full Text] [PDF]
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 M. Lalancette-Hebert, D. Phaneuf, G. Soucy, Y. C. Weng, and J. Kriz Live imaging of Toll-like receptor 2 response in cerebral ischaemia reveals a role of olfactory bulb microglia as modulators of inflammation Brain, April 1, 2009; 132(4): 940 - 954. [Abstract] [Full Text] [PDF]
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 J. Lichtnekert, V. Vielhauer, D. Zecher, O. P. Kulkarni, S. Clauss, S. Segerer, V. Hornung, T. N. Mayadas, B. Beutler, S. Akira, et al. Trif is not required for immune complex glomerulonephritis: dying cells activate mesangial cells via Tlr2/Myd88 rather than Tlr3/Trif Am J Physiol Renal Physiol, April 1, 2009; 296(4): F867 - F874. [Abstract] [Full Text] [PDF]
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 X. Urra, A. Cervera, V. Obach, N. Climent, A. M. Planas, and A. Chamorro Monocytes Are Major Players in the Prognosis and Risk of Infection After Acute Stroke Stroke, April 1, 2009; 40(4): 1262 - 1268. [Abstract] [Full Text] [PDF]
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 J. C. Gensel, S. Nakamura, Z. Guan, N. van Rooijen, D. P. Ankeny, and P. G. Popovich Macrophages Promote Axon Regeneration with Concurrent Neurotoxicity J. Neurosci., March 25, 2009; 29(12): 3956 - 3968. [Abstract] [Full Text] [PDF]
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 A. Laurenzana, L. A. Petruccelli, F. Pettersson, M. E. Figueroa, A. Melnick, A. S. Baldwin, F. Paoletti, and W. H. Miller Jr. Inhibition of DNA Methyltransferase Activates Tumor Necrosis Factor {alpha}-Induced Monocytic Differentiation in Acute Myeloid Leukemia Cells Cancer Res., January 1, 2009; 69(1): 55 - 64. [Abstract] [Full Text] [PDF]
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T. Eigenbrod, J.-H. Park, J. Harder, Y. Iwakura, and G. Nunez Cutting Edge: Critical Role for Mesothelial Cells in Necrosis-Induced Inflammation through the Recognition of IL-1{alpha} Released from Dying Cells J. Immunol., December 15, 2008; 181(12): 8194 - 8198. [Abstract] [Full Text] [PDF]
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K. Ckless, A. Lampert, J. Reiss, D. Kasahara, M. E. Poynter, C. G. Irvin, L. K. A. Lundblad, R. Norton, A. van der Vliet, and Y. M. W. Janssen-Heininger Inhibition of Arginase Activity Enhances Inflammation in Mice with Allergic Airway Disease, in Association with Increases in Protein S-Nitrosylation and Tyrosine Nitration J. Immunol., September 15, 2008; 181(6): 4255 - 4264. [Abstract] [Full Text] [PDF]
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M. Palazzo, S. Gariboldi, L. Zanobbio, S. Selleri, G. F. Dusio, V. Mauro, A. Rossini, A. Balsari, and C. Rumio Sodium-Dependent Glucose Transporter-1 as a Novel Immunological Player in the Intestinal Mucosa J. Immunol., September 1, 2008; 181(5): 3126 - 3136. [Abstract] [Full Text] [PDF]
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 A. O. Edwards, D. Chen, B. L. Fridley, K. M. James, Y. Wu, G. Abecasis, A. Swaroop, M. Othman, K. Branham, S. K. Iyengar, et al. Toll-like Receptor Polymorphisms and Age-Related Macular Degeneration Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1652 - 1659. [Abstract] [Full Text] [PDF]
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R. Torgler, S. E. Bongfen, J. C. Romero, A. Tardivel, M. Thome, and G. Corradin Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-{kappa}B Activation and Inducible NO Synthase Expression J. Immunol., March 15, 2008; 180(6): 3990 - 3999. [Abstract] [Full Text] [PDF]
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 A. Woywodt, T. Kirsch, and M. Haubitz Circulating endothelial cells in renal disease: markers and mediators of vascular damage Nephrol. Dial. Transplant., January 1, 2008; 23(1): 7 - 10. [Full Text] [PDF]
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M. Bevelander, J. Mayette, L. A. Whittaker, S. A. Paveglio, C. C. Jones, J. Robbins, D. Hemenway, S. Akira, S. Uematsu, and M. E. Poynter Nitrogen Dioxide Promotes Allergic Sensitization to Inhaled Antigen J. Immunol., September 15, 2007; 179(6): 3680 - 3688. [Abstract] [Full Text] [PDF]
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Y. Peng, G. Han, H. Shao, Y. Wang, H. J. Kaplan, and D. Sun Characterization of IL-17+ Interphotoreceptor Retinoid-Binding Protein-Specific T Cells in Experimental Autoimmune Uveitis Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4153 - 4161. [Abstract] [Full Text] [PDF]
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 D. Kim, M. A. Kim, I.-H. Cho, M. S. Kim, S. Lee, E.-K. Jo, S.-Y. Choi, K. Park, J. S. Kim, S. Akira, et al. A Critical Role of Toll-like Receptor 2 in Nerve Injury-induced Spinal Cord Glial Cell Activation and Pain Hypersensitivity J. Biol. Chem., May 18, 2007; 282(20): 14975 - 14983. [Abstract] [Full Text] [PDF]
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 J. Favre, P. Musette, V. Douin-Echinard, K. Laude, J.-P. Henry, J.-F. Arnal, C. Thuillez, and V. Richard Toll-Like Receptors 2-Deficient Mice Are Protected Against Postischemic Coronary Endothelial Dysfunction Arterioscler Thromb Vasc Biol, May 1, 2007; 27(5): 1064 - 1071. [Abstract] [Full Text] [PDF]
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A. A. Babcock, M. Wirenfeldt, T. Holm, H. H. Nielsen, L. Dissing-Olesen, H. Toft-Hansen, J. M. Millward, R. Landmann, S. Rivest, B. Finsen, et al. Toll-Like Receptor 2 Signaling in Response to Brain Injury: An Innate Bridge to Neuroinflammation J. Neurosci., December 6, 2006; 26(49): 12826 - 12837. [Abstract] [Full Text] [PDF]
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 R. M. Levy, J. M. Prince, R. Yang, K. P. Mollen, H. Liao, G. A. Watson, M. P. Fink, Y. Vodovotz, and T. R. Billiar Systemic inflammation and remote organ damage following bilateral femur fracture requires Toll-like receptor 4 Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2006; 291(4): R970 - R976. [Abstract] [Full Text] [PDF]
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 I. K. Demedts, K. R. Bracke, T. Maes, G. F. Joos, and G. G. Brusselle Different Roles for Human Lung Dendritic Cell Subsets in Pulmonary Immune Defense Mechanisms Am. J. Respir. Cell Mol. Biol., September 1, 2006; 35(3): 387 - 393. [Abstract] [Full Text] [PDF]
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 P. G. Tipping Toll-Like Receptors: The Interface between Innate and Adaptive Immunity J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1769 - 1771. [Full Text] [PDF]
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Y. Li, M.-C. Gerbod-Giannone, H. Seitz, D. Cui, E. Thorp, A. R. Tall, G. K. Matsushima, and I. Tabas Cholesterol-induced Apoptotic Macrophages Elicit an Inflammatory Response in Phagocytes, Which Is Partially Attenuated by the Mer Receptor J. Biol. Chem., March 10, 2006; 281(10): 6707 - 6717. [Abstract] [Full Text] [PDF]
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 M. E. Poynter, R. L. Persinger, C. G. Irvin, K. J. Butnor, H. van Hirtum, W. Blay, N. H. Heintz, J. Robbins, D. Hemenway, D. J. Taatjes, et al. Nitrogen dioxide enhances allergic airway inflammation and hyperresponsiveness in the mouse Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L144 - L152. [Abstract] [Full Text] [PDF]
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P Van der Meer, E Lipsic, R. Henning, K Boddeus, J van der Velden, A. Voors, D. van Veldhuisen, W. van Gilst, R. Schoemaker, J. Leemans, et al. Heart Failure after Myocardial Infarction--Benefit beyond Hemoglobin from Erythropoietin: Erythropoietin Induces Neovascularization and Improves Cardiac Function in Rats with Heart Failure after Myocardial Infarction J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3449 - 3454. [Full Text] [PDF]
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 K. Yaraei, L. A. Campbell, X. Zhu, W. C. Liles, C.-c. Kuo, and M. E. Rosenfeld Chlamydia pneumoniae Augments the Oxidized Low-Density Lipoprotein-Induced Death of Mouse Macrophages by a Caspase-Independent Pathway Infect. Immun., July 1, 2005; 73(7): 4315 - 4322. [Abstract] [Full Text] [PDF]
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 H. Yang, H. Wang, C. J. Czura, and K. J. Tracey The cytokine activity of HMGB1 J. Leukoc. Biol., July 1, 2005; 78(1): 1 - 8. [Abstract] [Full Text] [PDF]
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 C. Martens, S. Bilodeau, M. Maira, Y. Gauthier, and J. Drouin Protein-Protein Interactions and Transcriptional Antagonism between the Subfamily of NGFI-B/Nur77 Orphan Nuclear Receptors and Glucocorticoid Receptor Mol. Endocrinol., April 1, 2005; 19(4): 885 - 897. [Abstract] [Full Text] [PDF]
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 A.H. Schoneveld, M.M. Oude Nijhuis, B. van Middelaar, J.D. Laman, D.P.V. de Kleijn, and G. Pasterkamp Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development Cardiovasc Res, April 1, 2005; 66(1): 162 - 169. [Abstract] [Full Text] [PDF]
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 P. Tobias and L. K. Curtiss Thematic review series: The Immune System and Atherogenesis. Paying the price for pathogen protection: toll receptors in atherogenesis J. Lipid Res., March 1, 2005; 46(3): 404 - 411. [Abstract] [Full Text] [PDF]
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X. Meng, L. Ao, Y. Song, C. D. Raeburn, D. A. Fullerton, and A. H. Harken Signaling for myocardial depression in hemorrhagic shock: roles of Toll-like receptor 4 and p55 TNF-{alpha} receptor Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2005; 288(3): R600 - R606. [Abstract] [Full Text] [PDF]
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 A. Devitt, K. G. Parker, C. A. Ogden, C. Oldreive, M. F. Clay, L. A. Melville, C. O. Bellamy, A. Lacy-Hulbert, S. C. Gangloff, S. M. Goyert, et al. Persistence of apoptotic cells without autoimmune disease or inflammation in CD14-/- mice J. Cell Biol., December 20, 2004; 167(6): 1161 - 1170. [Abstract] [Full Text] [PDF]
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A. Grabiec, G. Meng, S. Fichte, W. Bessler, H. Wagner, and C. J. Kirschning Human but Not Murine Toll-like Receptor 2 Discriminates between Tri-palmitoylated and Tri-lauroylated Peptides J. Biol. Chem., November 12, 2004; 279(46): 48004 - 48012. [Abstract] [Full Text] [PDF]
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K. Kranzer, A. Eckhardt, M. Aigner, G. Knoll, L. Deml, C. Speth, N. Lehn, M. Rehli, and W. Schneider-Brachert Induction of Maturation and Cytokine Release of Human Dendritic Cells by Helicobacter pylori Infect. Immun., August 1, 2004; 72(8): 4416 - 4423. [Abstract] [Full Text] [PDF]
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M. Nikulina, C. Habich, S. B. Flohe, F. W. Scott, and H. Kolb Wheat Gluten Causes Dendritic Cell Maturation and Chemokine Secretion J. Immunol., August 1, 2004; 173(3): 1925 - 1933. [Abstract] [Full Text] [PDF]
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D. Messmer, H. Yang, G. Telusma, F. Knoll, J. Li, B. Messmer, K. J. Tracey, and N. Chiorazzi High Mobility Group Box Protein 1: An Endogenous Signal for Dendritic Cell Maturation and Th1 Polarization J. Immunol., July 1, 2004; 173(1): 307 - 313. [Abstract] [Full Text] [PDF]
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 C. Farina, D. Theil, B. Semlinger, R. Hohlfeld, and E. Meinl Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines Int. Immunol., June 1, 2004; 16(6): 799 - 809. [Abstract] [Full Text] [PDF]
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 N. Jiang and D. S. Pisetsky The Effect of Dexamethasone on the Generation of Plasma DNA from Dead and Dying Cells Am. J. Pathol., May 1, 2004; 164(5): 1751 - 1759. [Abstract] [Full Text] [PDF]
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H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [Abstract] [Full Text] [PDF]
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T. J. Murphy, H. M. Paterson, J. A. Mannick, and J. A. Lederer Injury, sepsis, and the regulation of Toll-like receptor responses J. Leukoc. Biol., March 1, 2004; 75(3): 400 - 407. [Abstract] [Full Text] [PDF]
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 J.-i. Oyama, C. Blais Jr, X. Liu, M. Pu, L. Kobzik, R. A. Kelly, and T. Bourcier Reduced Myocardial Ischemia-Reperfusion Injury in Toll-Like Receptor 4-Deficient Mice Circulation, February 17, 2004; 109(6): 784 - 789. [Abstract] [Full Text] [PDF]
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M. Cvetanovic and D. S. Ucker Innate Immune Discrimination of Apoptotic Cells: Repression of Proinflammatory Macrophage Transcription Is Coupled Directly to Specific Recognition J. Immunol., January 15, 2004; 172(2): 880 - 889. [Abstract] [Full Text] [PDF]
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M. Pierer, J. Rethage, R. Seibl, R. Lauener, F. Brentano, U. Wagner, H. Hantzschel, B. A. Michel, R. E. Gay, S. Gay, et al. Chemokine Secretion of Rheumatoid Arthritis Synovial Fibroblasts Stimulated by Toll-Like Receptor 2 Ligands J. Immunol., January 15, 2004; 172(2): 1256 - 1265. [Abstract] [Full Text] [PDF]
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T. Shishido, N. Nozaki, S. Yamaguchi, Y. Shibata, J. Nitobe, T. Miyamoto, H. Takahashi, T. Arimoto, K. Maeda, M. Yamakawa, et al. Toll-Like Receptor-2 Modulates Ventricular Remodeling After Myocardial Infarction Circulation, December 9, 2003; 108(23): 2905 - 2910. [Abstract] [Full Text] [PDF]
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R. Caricchio, L. McPhie, and P. L. Cohen Ultraviolet B Radiation-Induced Cell Death: Critical Role of Ultraviolet Dose in Inflammation and Lupus Autoantigen Redistribution J. Immunol., December 1, 2003; 171(11): 5778 - 5786. [Abstract] [Full Text] [PDF]
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B. McElhinney, M. E. Poynter, P. Shrivastava, S. L. Hazen, and Y. M. W. Janssen-Heininger Eosinophil peroxidase catalyzes JNK-mediated membrane blebbing in a Rho kinase-dependent manner J. Leukoc. Biol., November 1, 2003; 74(5): 897 - 907. [Abstract] [Full Text] [PDF]
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 S. Shi, C. Nathan, D. Schnappinger, J. Drenkow, M. Fuortes, E. Block, A. Ding, T. R. Gingeras, G. Schoolnik, S. Akira, et al. MyD88 Primes Macrophages for Full-Scale Activation by Interferon-{gamma} yet Mediates Few Responses to Mycobacterium tuberculosis J. Exp. Med., October 6, 2003; 198(7): 987 - 997. [Abstract] [Full Text] [PDF]
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 L. E. Nagy Recent Insights into the Role of the Innate Immune System in the Development of Alcoholic Liver Disease Experimental Biology and Medicine, September 1, 2003; 228(8): 882 - 890. [Abstract] [Full Text] [PDF]
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M. Lucas, L. M. Stuart, J. Savill, and A. Lacy-Hulbert Apoptotic Cells and Innate Immune Stimuli Combine to Regulate Macrophage Cytokine Secretion J. Immunol., September 1, 2003; 171(5): 2610 - 2615. [Abstract] [Full Text] [PDF]
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S. Chakrabarti, K. S. Kobayashi, R. A. Flavell, C. B. Marks, K. Miyake, D. R. Liston, K. T. Fowler, F. S. Gorelick, and N. W. Andrews Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice J. Cell Biol., August 18, 2003; 162(4): 543 - 549. [Abstract] [Full Text] [PDF]
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H. M. Paterson, T. J. Murphy, E. J. Purcell, O. Shelley, S. J. Kriynovich, E. Lien, J. A. Mannick, and J. A. Lederer Injury Primes the Innate Immune System for Enhanced Toll-Like Receptor Reactivity J. Immunol., August 1, 2003; 171(3): 1473 - 1483. [Abstract] [Full Text] [PDF]
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 J. A. Thomas, S. B. Haudek, T. Koroglu, M. F. Tsen, D. D. Bryant, D. J. White, D. F. Kusewitt, J. W. Horton, and B. P. Giroir IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction Am J Physiol Heart Circ Physiol, August 1, 2003; 285(2): H597 - H606. [Abstract] [Full Text] [PDF]
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 M. Witcher, D. T. Ross, C. Rousseau, L. Deluca, and W. H. Miller Jr Synergy between all-trans retinoic acid and tumor necrosis factor pathways in acute leukemia cells Blood, July 1, 2003; 102(1): 237 - 245. [Abstract] [Full Text] [PDF]
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M. P. Radsak, N. Hilf, H. Singh-Jasuja, S. Braedel, P. Brossart, H.-G. Rammensee, and H. Schild The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions Blood, April 1, 2003; 101(7): 2810 - 2815. [Abstract] [Full Text] [PDF]
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G. Melmed, L. S. Thomas, N. Lee, S. Y. Tesfay, K. Lukasek, K. S. Michelsen, Y. Zhou, B. Hu, M. Arditi, and M. T. Abreu Human Intestinal Epithelial Cells Are Broadly Unresponsive to Toll-Like Receptor 2-Dependent Bacterial Ligands: Implications for Host-Microbial Interactions in the Gut J. Immunol., February 1, 2003; 170(3): 1406 - 1415. [Abstract] [Full Text] [PDF]
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U. Andersson, H. Erlandsson-Harris, H. Yang, and K. J. Tracey HMGB1 as a DNA-binding cytokine J. Leukoc. Biol., December 1, 2002; 72(6): 1084 - 1091. [Abstract] [Full Text] [PDF]
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S. J. Kim, D. Gershov, X. Ma, N. Brot, and K. B. Elkon I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation J. Exp. Med., September 2, 2002; 196(5): 655 - 665. [Abstract] [Full Text] [PDF]
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 K. Bieback, E. Lien, I. M. Klagge, E. Avota, J. Schneider-Schaulies, W. P. Duprex, H. Wagner, C. J. Kirschning, V. ter Meulen, and S. Schneider-Schaulies Hemagglutinin Protein of Wild-Type Measles Virus Activates Toll-Like Receptor 2 Signaling J. Virol., July 29, 2002; 76(17): 8729 - 8736. [Abstract] [Full Text] [PDF]
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 J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
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G. Zhang and S. Ghosh Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip J. Biol. Chem., February 22, 2002; 277(9): 7059 - 7065. [Abstract] [Full Text] [PDF]
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