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* Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201;
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; and
Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation, p38
mitogen-activated protein kinase phosphorylation, and TNF-
gene
expression, documenting the induction of endotoxin tolerance. FACS and
Western blot analyses of LPS-tolerant monocytes showed increased TLR2
expression, whereas TLR4 expression levels were not affected.
Comparable levels of mRNA and protein for myeloid differentiation
factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and
TNFR-associated factor-6 were found in normal and LPS-tolerant
monocytes, while MD-2 mRNA expression was slightly increased in
LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and
increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant
monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1
was not activated in response to LPS stimulation. Moreover,
endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also
showed decreased IRAK-1 kinase activity in response to LPS despite the
failure of LPS to inhibit cell surface expression of transfected TLR4
and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with
subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant
phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and
mitogen-activated protein kinase (MAPK) induction (21),
and dendritic cell maturation (22). Stimulation of cells
via TLR4, but not TLR2, induces NO production (23), STAT-1
phosphorylation, and IFN-
gene expression (24),
suggesting the involvement of the MyD88-independent pathway. Unlike
TLR4, TLR2 interacts with TLR1 or TLR6 to elicit an optimal response to
certain TLR2 agonists (25). Gram-negative septic shock is characterized by tissue and organ damage resulting from hyperproduction of cytokines and low m.w. mediators by the immune system in response to large amounts of bacteria and LPS (26, 27). Septic shock survivors have an increased incidence of bacterial infections and suppressed monocyte responses to LPS (27, 28). This is reminiscent of endotoxin tolerance, a transient state of LPS refractoriness following an initial, non-lethal exposure to LPS. An understanding of the mechanisms that elicit endotoxin tolerance is critical for unraveling the molecular basis of the septic shock syndrome, yet despite numerous studies these mechanisms remain largely unknown. Whereas inhibition of cell surface expression of the TLR4/MD-2 complex has been suggested to underlie LPS tolerance in mouse macrophages (29), we have recently demonstrated that LPS induces tolerance in CHO cells without affecting cell surface expression of transfected TLR4 and MD-2 (30). In addition, LPS-tolerant THP-1 cells exhibit significantly suppressed LPS-induced IRAK activation and diminished IRAK-MyD88 association (31). Of note, tolerance induction to bacterial flagellin does not affect TLR5 protein levels, but is associated with an inhibition of IRAK release from TLR5 (32), and TLR4 and TLR2 agonists induce cross-tolerance in mouse macrophages (33) and in CHO cells that overexpress the corresponding TLRs (30). These data imply that tolerance induction may affect the expression and/or functions of intracellular intermediates downstream of TLRs.
In this study we have examined the expression of TLR4, TLR2, and numerous intracellular intermediates involved in the TLR pathway in normal and LPS-tolerant human monocytes and measured LPS-induced TLR4-MyD88 complex formation and IRAK-1 activation. Our data demonstrate that LPS tolerance may be induced under conditions where cells express comparable protein levels of TLR4, MD-2, MyD88, and TRAF-6. However, LPS-tolerant cells exhibited a markedly impaired capacity to recruit MyD88 to TLR4 in response to LPS, leading to significantly suppressed IRAK-1 phosphorylation and kinase activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocytes were prepared by counterflow elutriation and resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The CHO fibroblast cell line, CHO/TLR4/MD-2, stably transfected with expression constructs pCDNA3-huTLR4 and pEFBOS-Flag huMD-2, was described previously (30). CHO/TLR4/MD-2 cells were cultured in Ham’s F-12 medium (BioWhittaker) supplemented with 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 400 U/ml hygromycin B (Calbiochem, San Diego, CA). Protein-free, phenol/water-extracted Escherichia coli LPS K235 was prepared according to the method of McIntire et al. (34). Anti-Flag M2 mAb, mouse IgG1, mouse IgG2a, and anti-mouse IgG (Fab-specific) FITC conjugate were purchased from Sigma-Aldrich (St. Louis, MO). Phospho-p38 MAPK Ab and p38 MAPK Ab were obtained from New England Biolabs (Beverly, MA). Rabbit anti-human TLR4 antiserum was provided by Dr. R. Medzhitov (Yale University School of Medicine, New Haven, CT), mouse anti-TLR2 mAb, TL2.1, was a gift from Dr. T. Espevik (Norwegian University of Science and Technology, Trondheim, Norway), rabbit anti-human TLR4 Ab H80, goat anti-human TLR4 Ab C18, rabbit anti-human MyD88 Ab HFL 296, goat anti-human MyD88 Ab N19, and anti-human TRAF-6 Ab N20 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-IRAK Ab was purchased from Upstate Biotechnology (Waltham, MA), and mouse anti-Myc Ab was obtained from Invitrogen (Carlsbad, CA). SuperFect transfection reagent, endotoxin free plasmid Maxi Prep, and QIAprep Spin Miniprep kits were obtained from Qiagen (Valencia, CA). Immobilon P membranes were purchased from Millipore (Bedford, MA). Donkey anti-rabbit and anti-mouse HRP conjugates were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).
Flow cytometric analysis of TLR2 cell surface expression
Human monocytes (2 x 106/sample) were resuspended in medium and pretreated for 20 h with medium or 10 ng/ml LPS at 37°C in a 5% CO2 atmosphere. Cells were washed three times with PBS, then resuspended in medium, and TLR2 cell surface expression was measured by staining cells for 45 min with anti-TLR2 mAb TL2.1 or mouse IgG2a isotype control Ab (5 µg/ml each) on ice, washing twice with PBS/2% FCS, and incubating for 30 min with secondary FITC-labeled anti-mouse IgG (5 µg/ml). Thereafter, cells were subjected to flow cytometric analysis on a Coulter EPICS XL-MCL cell analyzer (Beckman Coulter, Fullerton, CA).
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared as described previously
(35). The protein concentration was determined using an
assay kit (Bio-Rad, Hercules, CA). The NF-B-specific oligonucleotide
probe 5'-AGTTGAGGGGACTTTCCCAGGC-3' from the murine Ig-B L chain gene
enhancer was synthesized by the BIC Synthesis and Sequencing Facility
(Uniformed Services University of the Health Sciences, Bethesda, MD)
and 32P end-labeled with T4 polynucleotide kinase
(Promega, Madison, WI) as recommended by the manufacturer. Nuclear
extracts (4 µg) were incubated with 0.2 ng radiolabeled DNA probe in
a binding buffer (final volume, 20 µl) containing 2 µg poly(dI-dC)
(Amersham Pharmacia Biotech), 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 4%
glycerol, 1 mM DTT, and 0.25 mg/ml BSA for 30 min at room temperature.
After incubation, a portion of each reaction (18 µl) was loaded onto
a 5% nondenaturing polyacrylamide gel, and the DNA-protein complexes
were resolved from free oligonucleotide by electrophoresis (0.25x Tris
borate/EDTA, 150 V, 2 h). The gels were dried (80°C, 2 h)
and exposed to x-ray films (X-OMAT AR, Eastman Kodak, Rochester,
NY).
Western blot analysis and immunoprecipitation
Cellular extracts and membrane fractions were prepared as previously described (36), boiled in Laemmli buffer for 5 min, resolved on 4–12% SDS-PAGE gradient gels (Invitrogen, San Diego, CA) in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, and 0.1% SDS), and transferred onto Immobilon P transfer membranes (100 V, 1.5 h, 4°C; Millipore). After blocking for 2 h in 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20 (TBS-T) containing 5% nonfat milk, membranes were washed three times in TBS-T and probed for 20 h at 4°C with the respective Abs diluted in TBS-T/5% nonfat milk. Following washing in TBS-T, membranes were incubated with secondary HRP-conjugated, donkey anti-rabbit IgG or goat anti-mouse IgG (1/10,000 dilution) and washed five times in TBS-T, and bands were detected using ECL Plus reagents (Amersham Pharmacia Biotech) according to the manufacturer’s directions. For immunoprecipitations (IP), 5 µg of the corresponding Ab was added to 800 µl each of the cellular extracts and incubated at 4°C for 18 h on a rotator. Fifty microliters of a 50% slurry of prewashed protein G-agarose beads (Amersham Pharmacia Biotech) was then added to each sample, followed by incubation for an additional 4 h at 4°C. The samples were washed four times in lysis buffer, solubilized in Laemmli buffer, and subjected to Western blot analyses as described above.
In vitro IRAK-1 kinase assay
The IRAK-1 kinase assay was conducted essentially as previously described (31). Briefly, the immunoprecipitated IRAK-1 complexes were washed four times with lysis buffer and twice with kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM glycerophosphate, 20 mM para-nitrophenylphosphate, 1 mM EDTA, 1 mM sodium orthovanadate, and 1 mM benzamidine). Fifty microliters of kinase buffer was then added to each sample, supplemented with 5 µM ATP, 1 µg myelin basic protein (MBP; Sigma), and 1 µl [32P]ATP, and incubated at 37°C for 30 min. Ten microliters of Laemmli sample buffer was added, and the samples were incubated at 50°C for 10 min and subjected to SDS-PAGE analysis. The gel was dried and exposed to x-ray film. The intensity of the radioactive signal was quantified using a PhosphorImager plate (Molecular Dynamics, Sunnyvale, CA).
Isolation of RNA and RT-PCR analysis of gene expression
Total RNA was isolated by using RNA Stat60 isolation reagent
(Tel-Test "B," Friendswood, TX), as specified by the manufacturer
and quantified by spectrophotometric analysis. Relative quantities of
mRNA for -actin and TNF- were determined by a coupled
semiquantitative RT-PCR and Southern blot analysis as detailed
previously (37) using the primers shown in Table I
and the following oligonucleotide
probes: 5'-GTACCACTGGCATCGTGATG-3' (-actin) and
5'-TCTTCTCGAACCCCGAGTGAC-3' (TNF-). In the other experiments, PCR
products were visualized by the incorporation of ethidium
bromide. The primers and annealing temperatures used for measuring
steady state levels of mRNA for GAPDH, TNF-, TLR2, TLR4, MD-2,
MyD88, TRAF-6, and IRAK-1 are shown in Table I. The optimal cycle
number for each gene was determined empirically and was defined as the
number of cycles that resulted in detectable PCR-amplified products
under nonsaturating conditions. Each cycle consisted of 1 min at
95°C, 1 min at a gene-specific annealing temperature, and a 2-min
primer extension at 72°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first series of experiments, we sought to establish
conditions under which endotoxin tolerance is reliably induced in human
monocytes. Human monocytes obtained by counterflow elutriation were
pretreated with medium or LPS for 18 h, washed, and restimulated
with LPS, followed by measurement of NF-B activation by EMSA, p38
MAPK phosphorylation by Western blot analysis, and TNF- gene
expression by RT-PCR. Prior incubation of human monocytes with 10 ng/ml
LPS resulted in a significant decrease in NF-B activation (Fig. 1A) and IB- degradation
(data not shown) in response to subsequent LPS challenge. Similarly,
LPS-mediated p38 MAPK phosphorylation was significantly suppressed in
cells pretreated with LPS compared with the response seen in
medium-pretreated cells, whereas total protein levels of p38 MAPK were
not affected (Fig. 1A). Significant inhibition of
LPS-induced NF-B activation and p38 MAPK phosphorylation in
LPS-tolerant monocytes was also observed at different time points after
LPS stimulation, i.e., 15 and 30 min for NF-B activation, and 15 and
45 min for p38 MAPK phosphorylation (data not shown). As detected by
RT-PCR, LPS rapidly increased steady state levels of TNF- mRNA in
medium-pretreated human monocytes. In contrast, prior exposure to LPS
markedly suppressed the ability of human monocytes to respond to LPS by
induction of TNF- gene expression (Figs. 1C and 2). Figs. 1C and 2 also demonstrate comparable levels of mRNA for
-actin and GAPDH housekeeping genes in cells subjected to LPS
restimulation or pretreatment. These data show that pretreatment of
human monocytes with LPS for 18 h effectively induces LPS
tolerance.
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Next, semiquantitative RT-PCR and Western blot analyses were
employed to examine mRNA and protein expression of various receptor and
intracellular signaling molecules involved in the TLR2 and TLR4
pathways. Fig. 2 demonstrates that LPS
markedly up-regulated TLR2 gene expression within 3–6 h in
medium-pretreated monocytes. TLR2 mRNA levels remained elevated
following 18 h of LPS pretreatment and were not influenced by LPS
in endotoxin-tolerant cells. Similar to the results obtained in mouse
macrophages (35), TLR4 mRNA levels were slightly
down-regulated by LPS after 3 h of restimulation, returned to
basal levels by 6 h, and were not further modulated by LPS
pretreatment or restimulation. In medium-pretreated cells, LPS modestly
up-regulated MD-2 and IRAK-1 gene expression, whereas MyD88 and TRAF-6
mRNA levels were unaffected (Fig. 2). Steady state levels of MD-2 mRNA
remained slightly increased in LPS-tolerant monocytes and were not
significantly changed by LPS restimulation. As shown in Fig. 2, normal
and LPS-tolerant human monocytes exhibited comparable basal and
LPS-induced expression patterns of mRNA for TRAF-6 and IRAK-1.
Consistent with the gene expression data, Western blot analysis of
whole cell extracts and membrane fractions prepared from LPS-tolerant
monocytes showed increased TLR2 protein levels compared with those
detected in medium-pretreated cells, while expression of TLR4, MyD88,
and TRAF-6 proteins was not affected (Fig. 3). FACS analysis of TLR2 cell surface
expression was then conducted using the anti-huTLR2 mAb TL2.1,
which recognizes huTLR2 in CHO cells stably transfected with huTLR2,
but not huTLR4 (30, 38). As shown in Fig. 4, LPS pretreatment of human monocytes
for 18 h modestly up-regulated TLR2 cell surface expression
compared with the expression levels seen in medium-pretreated cells.
FACS analysis of TLR4 cell surface expression in medium- and
LPS-pretreated human monocytes was also attempted using anti-TLR4
HTA 125 mAb that stains CHO cells which overexpress huTLR4, but not
huTLR2 (30). TLR4 cell surface expression in human
monocytes was undetectable by FACS using HTA 125 (data not shown),
which is probably due to relatively low TLR4 cell surface expression in
primary cells, as has been reported previously in mouse macrophages
(39). Taken together, these results indicate that LPS
tolerance induction increases TLR2 gene and protein expression and
slightly up-regulates MD-2 gene expression, but does not affect the
expression levels of TLR4, MyD88, TRAF-6, and IRAK-1 mRNA and TLR4,
MyD88, and TRAF6 proteins.
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Since LPS-tolerant and normal human monocytes exhibited comparable
total protein levels of major intracellular signaling intermediates
involved in the TLR4 pathway, we hypothesized that endotoxin tolerance
may interfere with protein-protein interactions among receptors and
adaptor molecules that play a crucial role in LPS signal transduction.
To address this question, LPS-induced complex formation between TLR4
and MyD88 was examined, since overexpression and knockout studies have
clearly established MyD88 as a key molecule for eliciting many
TLR4-mediated responses (20, 21, 40). Human monocytes were
pretreated with medium or LPS, washed, and restimulated with LPS over a
15-min time course. Thereafter, cells were lysed and TLR4 or MyD88
proteins were immunoprecipitated and subjected to Western blot
analysis. Medium- or LPS-pretreated cells showed comparable amounts of
total TLR4 and MyD88 proteins that were not affected by subsequent LPS
restimulation, consistent with our previous results obtained with whole
cellular extracts and membrane fractions. Coimmunoprecipitation
experiments in normal monocytes demonstrate rapid LPS-induced
TLR4-MyD88 complex formation, which was evident after 2 min, reached a
plateau within 5–15 min, and declined by 30 min (Fig. 5 and data not shown). In contrast,
LPS-tolerant cells exhibited markedly lower quantities of MyD88 in
association with TLR4, and LPS failed to increase the amount of MyD88
in the TLR4-MyD88 complex (Fig. 5). Overall, these data show a severe
impairment of LPS-induced TLR4/MyD88 association in LPS-tolerant human
monocytes.
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Li et al. (31) have previously reported suppressed
LPS-induced IRAK activation in the human THP-1 macrophage cell line
rendered endotoxin tolerant. However, they did not examine whether this
inhibition is secondary to a defect in the signal transduction cascade
upstream of IRAK. As IRAK-1 is downstream of MyD88 in the TLR4
signaling pathway (40, 41, 42), we hypothesized that disrupted
LPS-induced TLR4-MyD88 association seen in endotoxin-tolerant human
monocytes may lead to inhibition of IRAK-1 activation. To confirm and
extend the findings reported by Li et al. (31), we
examined IRAK-1 protein expression and kinase activity in normal and
LPS-tolerant cells. Western blot analysis of cellular extracts
immunoprecipitated with anti-IRAK Ab demonstrated the major IRAK-1
band with expected Mr of 85 kDa in both normal
and endotoxin-tolerant human monocytes (Fig. 6). In agreement with previously
published results (31, 43), LPS stimulation of
medium-pretreated human monocytes resulted in the appearance of an
additional IRAK-1 band with an Mr of 
120 kDa,
corresponding to a hyperphosphorylated form of IRAK-1 (p-IRAK-1; Fig. 6). The identification of these bands as p-IRAK-1 and IRAK-1 was also
confirmed by the detection of two bands with identical electrophoretic
mobilities in cellular extracts obtained from HEK 293T cells
transfected with a human IRAK-1 expression vector (data not
shown). As shown in Fig. 6B, stimulation of
medium-pretreated monocytes with LPS induced an elevation in IRAK-1
kinase activity detectable after 5 min of stimulation. IRAK-1 kinase
activity reached maximal levels within 5–10 min of LPS stimulation
(Fig. 6B), coinciding with the increase in levels of
p-IRAK-1 (Fig. 6A), and decreased to basal levels after 20
min of LPS pretreatment. Endotoxin-tolerant and medium-pretreated
monocytes exhibited similar amounts of the predominant 85-kDa IRAK-1
form (Fig. 6A). However, in contrast to normal cells,
LPS-tolerant monocytes showed very little, if any, induction of IRAK-1
phosphorylation and kinase activity in response to LPS stimulation
(Fig. 6, A and B).
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C,
LPS stimulation of medium-pretreated CHO/TLR4/MD-2 cells led to strong
induction of IRAK-1 kinase activity toward MBP with kinetics similar to
those observed in human monocytes. In contrast, very low levels of
IRAK-1-mediated MBP phosphorylation were seen in LPS-tolerant
CHO/TLR4/MD-2 cells that were not modulated by LPS restimulation (Fig. 6C). Taken together, these data suggest that the induction
of endotoxin tolerance correlates with disrupted recruitment of MyD88
by TLR4, resulting in impaired IRAK-1 activation, which, in turn, leads
to inhibition of MAPK induction, transcription factor activation, and
cytokine gene expression in response to LPS. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This paper demonstrates that LPS up-regulates steady state levels of TLR2 mRNA in human monocytes within 3–6 h, and levels remained elevated after long term (18-h) LPS pretreatment and were not further modulated by LPS. Consistent with the mRNA data, Western blot and FACS analyses showed an increase in TLR2 protein expression by LPS-tolerant cells, extending earlier data on LPS-induced up-regulation of TLR2 mRNA in mouse macrophages (35, 54) and supporting previous findings on the ability of LPS to increase TLR2 mRNA and protein expression in LPS-stimulated mouse macrophages (35), human monocytes (56), human microvessel endothelial cells (51), and mouse small intestinal epithelial cells (50). Our data on enhanced TLR2 cell surface expression in LPS-tolerant human monocytes are important for several reasons. First, the mechanisms of induction of cross-tolerance to TLR2 agonists by LPS remain largely unknown. This paper documents that induction of cross-tolerance to TLR2 agonists by prior exposure of cells to LPS seen in several models of tolerance induction cannot be attributed to down-regulation of TLR2 protein expression. Our current findings also substantiate our previous results obtained in CHO cells that express endogenous TLR4 and are stably transfected with human TLR2 (30). In this model, pretreatment of cells with LPS does not affect cell surface expression of transfected TLR2, which is the only functional TLR2, as CHO cells represent a natural TLR2 mutant (57), yet LPS induces cross-tolerance to TLR2 agonists (30). Secondly, this paper demonstrates the ability of a TLR4 agonist to up-regulate expression of a distinct TLR (TLR2), suggesting that cross-talk between different TLRs exists and may contribute to the capacity of the host to respond to additional microbial stimuli.
To gain further insight into mechanisms of LPS tolerance, we then examined mRNA and protein expression of intracellular signaling molecules involved in the TLR signaling pathway in normal and endotoxin-tolerant human monocytes. RT-PCR analysis showed similar steady state mRNA levels for MyD88, IRAK-1, and TRAF-6 following LPS stimulation of cells pretreated with medium or LPS. These data support and extend similar results obtained in mouse macrophages (35, 54). Likewise, as detected by Western blot analyses, no difference in the expression of MyD88 and TRAF-6 proteins was observed in normal and LPS-tolerant monocytes. LPS stimulation of medium-pretreated monocytes led to a rapid induction of IRAK-1 phosphorylation and kinase activity. In contrast, in LPS-tolerant cells IRAK-1 underwent very little, if any, phosphorylation in response to LPS stimulation and exhibited low levels of kinase activity that was not further up-regulated by LPS. While Li et al. (31) observed similar suppression of IRAK kinase activity in LPS-tolerant human THP-1 cells, they did not examine the effect of LPS tolerance on the expression of TLR4 protein. Addressing this question, our results show that inhibition of LPS-mediated IRAK-1 activation in LPS-tolerant human monocytes occurs under conditions where TLR4 protein expression is comparable in normal and LPS-tolerant cells. To substantiate further our results obtained in human monocytes, we employed CHO cells that were stably transfected with human TLR4 and MD-2. As reported previously (30), exposure of these cells to LPS induces a state of endotoxin tolerance without affecting expression of transfected TLR4 and MD-2 proteins. This paper extends these findings by showing that LPS tolerance induction in CHO/TLR4/MD-2 cells results in a significant decrease in IRAK-1 kinase activity in response to LPS restimulation. Thus, inhibition of LPS-mediated induction of IRAK-1 kinase activity occurs in LPS-tolerant CHO/TLR4/MD-2 cells even when TLR4 and MD-2 proteins are strongly overexpressed.
This paper demonstrates inhibited LPS-induced IRAK-1 activation in LPS-tolerant cells that showed comparable expression of TLR4, MyD88, and TRAF-6 proteins and lack of inhibition of MD-2 gene expression. Since IRAK-1 is downstream of TLR4/MD-2/MyD88 components in the TLR4 signaling pathway (40, 41, 42), we hypothesized that LPS tolerance dysregulates the association of TLR4 and MyD88. To address this question, we used co-IP to assess the amount of MyD88 complexed with TLR4 in normal and LPS-tolerant human monocytes in response to LPS restimulation. Similar to the results obtained with total cellular extracts, comparable expression of total TLR4 and MyD88 proteins was seen in normal and LPS-tolerant human monocytes. However, whereas LPS induced a rapid recruitment of MyD88 to TLR4 in medium-pretreated human monocytes, LPS-tolerant monocytes exhibited diminished association of MyD88 with TLR4 that was not up-regulated by subsequent LPS restimulation. To the best of our knowledge, this is the first report of disrupted TLR4/MyD88 association in LPS-tolerant human monocytes. Interaction between TLR4 and MyD88 is critical for IRAK-1 activation and subsequent engagement of TRAF-6, an evolutionary conserved intermediate in the Toll/IL-1 signal transduction pathway, and MAPK/ERK kinase kinase-1 (40, 41, 42, 58, 59, 60). MAPK/ERK kinase kinase-1, in turn, activates a number of MAPKs and transcription factors, leading to induction of gene expression and production of various cytokines and low m.w. mediators involved in innate immune response. Therefore, impaired LPS-induced recruitment of MyD88 to TLR4 in LPS-tolerant cells is likely to account for inhibited phosphorylation and activation of IRAK-1 and the suppressed downstream signaling observed in LPS-tolerant cells (29, 30, 31, 33, 35). Several examples of altered ligand-induced protein-protein interactions among intracellular intermediates of the TLR pathway in cells rendered tolerant to microbial products have been published, including severe inhibition of IRAK-1-MyD88 complex formation in LPS-tolerant THP-1 cells (31) and a block of IRAK-1 dissociation from TLR5 in cells rendered tolerant to flagellin (32). Our current finding that LPS-induced recruitment of MyD88 to TLR4 is inhibited in LPS-tolerant monocytes extends these earlier reports and supports the hypothesis that induction of tolerance to microbial products is the consequence of dysregulated protein-protein interaction among key intracellular intermediates involved in the TLR signaling pathway.
The mechanism by which LPS tolerance induction affects the interaction
of TLR4 with MyD88 remains unclear and several explanations are
plausible. First, TLR4 or MyD88 could undergo post-translational
modification in LPS-tolerant cells that could prevent conformational
changes necessary for TLR4-MyD88 association. An example of such a
modification among the TLR family has been recently demonstrated for
TLR2. Indeed, TLR2 stimulation with Staphylococcus aureus
induces a rapid and transient activation of the Rho GTPase, Rac1. The
recruitment of activated Rac1 and phosphatidylinositol 3-kinase to the
TLR2 cytosolic domain induces TLR2 tyrosine phosphorylation, which is
required for the assembly of a multiprotein complex composed of Rac1,
phosphatidylinositol 3-kinase, and Akt that mediate nuclear p65
trans-activation (61). It remains to be
elucidated whether TLR4 or MyD88 could also be subject to a similar
modification, which may be severely affected in LPS-tolerant cells.
Another important modification could be glycosylation of TLR4 and/or
MD-2, which is necessary for their ability to elicit LPS responses
(62, 63). Secondly, LPS tolerance induction may result in
the appearance of alternatively spliced forms of TLR4, MD-2, or MyD88
that could interfere with protein-protein interaction between
full-length TLR4 and MyD88 molecules. A recent publication reports the
identification of such a splice variant of MyD88, which can be induced
in monocytes upon LPS treatment and is defective in its ability to
induce IRAK phosphorylation and NF-B activation (64).
The splice form of MyD88 behaves as a dominant negative inhibitor of
IL-1- and LPS-, but not TNF-induced, NF-B activation
(64), which is highly reminiscent of our data on the LPS
induction of cross-tolerance to IL-1, but not to TNF (35).
Third, endotoxin-tolerant cells may up-regulate expression of
Toll-interacting protein (Tollip), a negative regulator of IL-1R/TLR
signaling (65, 66), which exists in a complex with IRAK-1
in unstimulated cells and coimmunoprecipitates with TLR2 and TLR4
(66). Although MyD88 is required for IRAK-1 activation
with subsequent IRAK-1-mediated phosphorylation of Tollip and release
of IRAK-1 to interact with TRAF-6 (65, 66), it is unknown
whether Tollip regulates association of MyD88 to TLR4. It is tempting
to speculate that increased expression of Tollip in LPS-tolerant cells
could inhibit the ability of MyD88 to be recruited to TLR4, leading to
decreased MyD88-mediated IRAK-1 activation.
The mechanisms by which MyD88 is recruited to TLR4 in normal cells in response to LPS are unclear. It is noteworthy that IRAK-M has very recently been reported to be a negative regulator of TLR signaling and has been suggested to act by inhibiting dissociation of IRAK-4 from MyD88 (67). IRAK-M has been implicated in endotoxin tolerance, as IRAK-M knockout macrophages were less susceptible to LPS tolerization at early time points (67), suggesting that up-regulation of IRAK-M expression could also contribute to the induction of LPS tolerance downstream of MyD88-TLR4 complex formation. However, it is also plausible that there may exist as yet uncharacterized analogous regulation of MyD88 recruitment to TLR4, which could be dysregulated in LPS-tolerant cells. Studies are in progress to define further the mechanism(s) by which LPS-induced TLR4-MyD88 association and IRAK-1 activation are inhibited in endotoxin-tolerant cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201-1559. E-mail address: svogel{at}som.umaryland.edu
3 Abbreviations used in this paper: TLR, Toll-like receptor; IP, immunoprecipitation; IRAK, IL1R-associated kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MyD88, myeloid differentiation factor 88; TIR, Toll/IL-1R;TIRAP, TIR adaptor protein; Tollip, Toll-interacting protein; TRAF, TNFR-associated factor.
4 A. E. Medvedev and S. N. Vogel. Overexpression of CD14, TLR4, and MD-2 in HEK 293 cells does not prevent induction of in vitro endotoxin tolerance. Submitted for publication.
Received for publication June 18, 2002. Accepted for publication August 20, 2002.
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C. del Fresno, F. Garcia-Rio, V. Gomez-Pina, A. Soares-Schanoski, I. Fernandez-Ruiz, T. Jurado, T. Kajiji, C. Shu, E. Marin, A. Gutierrez del Arroyo, et al. Potent Phagocytic Activity with Impaired Antigen Presentation Identifying Lipopolysaccharide-Tolerant Human Monocytes: Demonstration in Isolated Monocytes from Cystic Fibrosis Patients J. Immunol., May 15, 2009; 182(10): 6494 - 6507. [Abstract] [Full Text] [PDF]
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 M. Quigley, J. Martinez, X. Huang, and Y. Yang A critical role for direct TLR2-MyD88 signaling in CD8 T-cell clonal expansion and memory formation following vaccinia viral infection Blood, March 5, 2009; 113(10): 2256 - 2264. [Abstract] [Full Text] [PDF]
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 Y.-I. Kim, J.-E. Park, A. Martinez-Hernandez, and A.-K. Yi CpG DNA Prevents Liver Injury and Shock-mediated Death by Modulating Expression of Interleukin-1 Receptor-associated Kinases J. Biol. Chem., May 30, 2008; 283(22): 15258 - 15270. [Abstract] [Full Text] [PDF]
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 L. Frolova, P. Drastich, P. Rossmann, K. Klimesova, and H. Tlaskalova-Hogenova Expression of Toll-like Receptor 2 (TLR2), TLR4, and CD14 in Biopsy Samples of Patients With Inflammatory Bowel Diseases: Upregulated Expression of TLR2 in Terminal Ileum of Patients With Ulcerative Colitis J. Histochem. Cytochem., March 1, 2008; 56(3): 267 - 274. [Abstract] [Full Text] [PDF]
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W. Piao, C. Song, H. Chen, L. M. Wahl, K. A. Fitzgerald, L. A. O'Neill, and A. E. Medvedev Tyrosine Phosphorylation of MyD88 Adapter-like (Mal) Is Critical for Signal Transduction and Blocked in Endotoxin Tolerance J. Biol. Chem., February 8, 2008; 283(6): 3109 - 3119. [Abstract] [Full Text] [PDF]
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 K. P. Mollen, R. M. Levy, J. M. Prince, R. A. Hoffman, M. J. Scott, D. J. Kaczorowski, R. Vallabhaneni, Y. Vodovotz, and T. R. Billiar Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells J. Leukoc. Biol., January 1, 2008; 83(1): 80 - 88. [Abstract] [Full Text] [PDF]
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C. van 't Veer, P. S. van den Pangaart, M. A. D. van Zoelen, M. de Kruif, R. S. Birjmohun, E. S. Stroes, A. F. de Vos, and T. van der Poll Induction of IRAK-M Is Associated with Lipopolysaccharide Tolerance in a Human Endotoxemia Model J. Immunol., November 15, 2007; 179(10): 7110 - 7120. [Abstract] [Full Text] [PDF]
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H. Chen, M. J. Cowan, J. D. Hasday, S. N. Vogel, and A. E. Medvedev Tobacco Smoking Inhibits Expression of Proinflammatory Cytokines and Activation of IL-1R-Associated Kinase, p38, and NF-{kappa}B in Alveolar Macrophages Stimulated with TLR2 and TLR4 Agonists J. Immunol., November 1, 2007; 179(9): 6097 - 6106. [Abstract] [Full Text] [PDF]
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S. K. Biswas, P. Bist, M. K. Dhillon, T. Kajiji, C. del Fresno, M. Yamamoto, E. Lopez-Collazo, S. Akira, and V. Tergaonkar Role for MyD88-Independent, TRIF Pathway in Lipid A/TLR4-Induced Endotoxin Tolerance J. Immunol., September 15, 2007; 179(6): 4083 - 4092. [Abstract] [Full Text] [PDF]
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N. A. Kittan, A. Bergua, S. Haupt, N. Donhauser, P. Schuster, K. Korn, T. Harrer, and B. Schmidt Impaired Plasmacytoid Dendritic Cell Innate Immune Responses in Patients with Herpes Virus-Associated Acute Retinal Necrosis J. Immunol., September 15, 2007; 179(6): 4219 - 4230. [Abstract] [Full Text] [PDF]
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 Z. J. Roberts, N. Goutagny, P.-Y. Perera, H. Kato, H. Kumar, T. Kawai, S. Akira, R. Savan, D. van Echo, K. A. Fitzgerald, et al. The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis J. Exp. Med., July 9, 2007; 204(7): 1559 - 1569. [Abstract] [Full Text] [PDF]
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W.-H. Kwan, C. Boix, N. Gougelet, W. H. Fridman, and C. G. F. Mueller LPS induces rapid IL-10 release by M-CSF-conditioned tolerogenic dendritic cell precursors J. Leukoc. Biol., July 1, 2007; 82(1): 133 - 141. [Abstract] [Full Text] [PDF]
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 T. Sterzenbach, S. K. Lee, B. Brenneke, F. von Goetz, D. B. Schauer, J. G. Fox, S. Suerbaum, and C. Josenhans Inhibitory Effect of Enterohepatic Helicobacter hepaticus on Innate Immune Responses of Mouse Intestinal Epithelial Cells Infect. Immun., June 1, 2007; 75(6): 2717 - 2728. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, W. Piao, J. Shoenfelt, S. H. Rhee, H. Chen, S. Basu, L. M. Wahl, M. J. Fenton, and S. N. Vogel Role of TLR4 Tyrosine Phosphorylation in Signal Transduction and Endotoxin Tolerance J. Biol. Chem., June 1, 2007; 282(22): 16042 - 16053. [Abstract] [Full Text] [PDF]
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N. Satta, S. Dunoyer-Geindre, G. Reber, R. J. Fish, F. Boehlen, E. K. O. Kruithof, and P. de Moerloose The role of TLR2 in the inflammatory activation of mouse fibroblasts by human antiphospholipid antibodies Blood, February 15, 2007; 109(4): 1507 - 1514. [Abstract] [Full Text] [PDF]
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 C. del Fresno, L. Soler-Rangel, A. Soares-Schanoski, V. Gomez-Pina, M. C. Gonzalez-Leon, L. Gomez-Garcia, E. Mendoza-Barbera, A. Rodriguez-Rojas, F. Garcia, P. Fuentes-Prior, et al. Inflammatory responses associated with acute coronary syndrome up-regulate IRAK-M and induce endotoxin tolerance in circulating monocytes Innate Immunity, February 1, 2007; 13(1): 39 - 52. [Abstract] [PDF]
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K. Nakahira, H. P. Kim, X. H. Geng, A. Nakao, X. Wang, N. Murase, P. F. Drain, X. Wang, M. Sasidhar, E. G. Nabel, et al. Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts J. Exp. Med., October 2, 2006; 203(10): 2377 - 2389. [Abstract] [Full Text] [PDF]
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J. M. Buckley, J. H. Wang, and H. P. Redmond Cellular reprogramming by gram-positive bacterial components: a review J. Leukoc. Biol., October 1, 2006; 80(4): 731 - 741. [Abstract] [Full Text] [PDF]
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B. K. Yoza, J. Y.-Q. Hu, S. L. Cousart, L. M. Forrest, and C. E. McCall Induction of RelB Participates in Endotoxin Tolerance J. Immunol., September 15, 2006; 177(6): 4080 - 4085. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, I. Sabroe, J. D. Hasday, and S. N. Vogel Invited review: Tolerance to microbial TLR ligands: molecular mechanisms and relevance to disease Innate Immunity, June 1, 2006; 12(3): 133 - 150. [Abstract] [PDF]
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 L. Romics Jr, G. Szabo, J. C. Coffey, J. H. Wang, and H. P. Redmond The Emerging Role of Toll-Like Receptor Pathways in Surgical Diseases Arch Surg, June 1, 2006; 141(6): 595 - 601. [Abstract] [Full Text] [PDF]
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M. Lotz, D. Gutle, S. Walther, S. Menard, C. Bogdan, and M. W. Hornef Postnatal acquisition of endotoxin tolerance in intestinal epithelial cells J. Exp. Med., April 17, 2006; 203(4): 973 - 984. [Abstract] [Full Text] [PDF]
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C. H. Li, J. H. Wang, and H. P. Redmond Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation J. Leukoc. Biol., April 1, 2006; 79(4): 867 - 875. [Abstract] [Full Text] [PDF]
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K. L. Williams, J. D. Lich, J. A. Duncan, W. Reed, P. Rallabhandi, C. Moore, S. Kurtz, V. M. Coffield, M. A. Accavitti-Loper, L. Su, et al. The CATERPILLER Protein Monarch-1 Is an Antagonist of Toll-like Receptor-, Tumor Necrosis Factor {alpha}-, and Mycobacterium tuberculosis-induced Pro-inflammatory Signals J. Biol. Chem., December 2, 2005; 280(48): 39914 - 39924. [Abstract] [Full Text] [PDF]
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L. C. Parker, E. C. Jones, L. R. Prince, S. K. Dower, M. K. B. Whyte, and I. Sabroe Endotoxin tolerance induces selective alterations in neutrophil function J. Leukoc. Biol., December 1, 2005; 78(6): 1301 - 1305. [Abstract] [Full Text] [PDF]
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 W. Jiang, R. Sun, H. Wei, and Z. Tian Toll-like receptor 3 ligand attenuates LPS-induced liver injury by down-regulation of toll-like receptor 4 expression on macrophages PNAS, November 22, 2005; 102(47): 17077 - 17082. [Abstract] [Full Text] [PDF]
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 F. Gonzalez, J. Minium, N. S. Rote, and J. P. Kirwan Hyperglycemia Alters Tumor Necrosis Factor-{alpha} Release from Mononuclear Cells in Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5336 - 5342. [Abstract] [Full Text] [PDF]
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M. Wysocka, L. J. Montaner, and C. L. Karp Flt3 Ligand Treatment Reverses Endotoxin Tolerance-Related Immunoparalysis J. Immunol., June 1, 2005; 174(11): 7398 - 7402. [Abstract] [Full Text] [PDF]
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A. Lentschat, H. Karahashi, K. S. Michelsen, L. S. Thomas, W. Zhang, S. N. Vogel, and M. Arditi Mastoparan, a G Protein Agonist Peptide, Differentially Modulates TLR4- and TLR2-Mediated Signaling in Human Endothelial Cells and Murine Macrophages J. Immunol., April 1, 2005; 174(7): 4252 - 4261. [Abstract] [Full Text] [PDF]
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D. De Nardo, P. Masendycz, S. Ho, M. Cross, A. J. Fleetwood, E. C. Reynolds, J. A. Hamilton, and G. M. Scholz A Central Role for the Hsp90{middle dot}Cdc37 Molecular Chaperone Module in Interleukin-1 Receptor-associated-kinase-dependent Signaling by Toll-like Receptors J. Biol. Chem., March 18, 2005; 280(11): 9813 - 9822. [Abstract] [Full Text] [PDF]
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C. A. Ogden, J. D. Pound, B. K. Batth, S. Owens, I. Johannessen, K. Wood, and C. D. Gregory Enhanced Apoptotic Cell Clearance Capacity and B Cell Survival Factor Production by IL-10-Activated Macrophages: Implications for Burkitt's Lymphoma J. Immunol., March 1, 2005; 174(5): 3015 - 3023. [Abstract] [Full Text] [PDF]
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C. del Fresno, K. Otero, L. Gomez-Garcia, M. C. Gonzalez-Leon, L. Soler-Ranger, P. Fuentes-Prior, P. Escoll, R. Baos, L. Caveda, F. Garcia, et al. Tumor Cells Deactivate Human Monocytes by Up-Regulating IL-1 Receptor Associated Kinase-M Expression via CD44 and TLR4 J. Immunol., March 1, 2005; 174(5): 3032 - 3040. [Abstract] [Full Text] [PDF]
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H. S. Goodridge, F. A. Marshall, K. J. Else, K. M. Houston, C. Egan, L. Al-Riyami, F.-Y. Liew, W. Harnett, and M. M. Harnett Immunomodulation via Novel Use of TLR4 by the Filarial Nematode Phosphorylcholine-Containing Secreted Product, ES-62 J. Immunol., January 1, 2005; 174(1): 284 - 293. [Abstract] [Full Text] [PDF]
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M. Siedlar, M. Frankenberger, E. Benkhart, T. Espevik, M. Quirling, K. Brand, M. Zembala, and L. Ziegler-Heitbrock Tolerance Induced by the Lipopeptide Pam3Cys Is Due to Ablation of IL-1R-Associated Kinase-1 J. Immunol., August 15, 2004; 173(4): 2736 - 2745. [Abstract] [Full Text] [PDF]
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 J. Roman, J. D. Ritzenthaler, B. Boles, M. Lois, and S. Roser-Page Lipopolysaccharide induces expression of fibronectin {alpha}5{beta}1-integrin receptors in human monocytic cells in a protein kinase C-dependent fashion Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L239 - L249. [Abstract] [Full Text] [PDF]
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S. Noubir, Z. Hmama, and N. E. Reiner Dual Receptors and Distinct Pathways Mediate Interleukin-1 Receptor-associated Kinase Degradation in Response to Lipopolysaccharide: INVOLVEMENT OF CD14/TLR4, CR3, AND PHOSPHATIDYLINOSITOL 3-KINASE J. Biol. Chem., June 11, 2004; 279(24): 25189 - 25195. [Abstract] [Full Text] [PDF]
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S. L. McCoy, S. E. Kurtz, F. A. Hausman, D. R. Trune, R. M. Bennett, and S. H. Hefeneider Activation of RAW264.7 Macrophages by Bacterial DNA and Lipopolysaccharide Increases Cell Surface DNA Binding and Internalization J. Biol. Chem., April 23, 2004; 279(17): 17217 - 17223. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. N. Vogel, S. K. Dower, and I. Sabroe Toll-Like Receptor (TLR)2 and TLR4 Agonists Regulate CCR Expression in Human Monocytic Cells J. Immunol., April 15, 2004; 172(8): 4977 - 4986. [Abstract] [Full Text] [PDF]
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Hongkuan Fan and J. A. Cook Review: Molecular mechanisms of endotoxin tolerance Innate Immunity, April 1, 2004; 10(2): 71 - 84. [Abstract] [PDF]
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K. S. Kobayashi and R. A. Flavell Shielding the double-edged sword: negative regulation of the innate immune system J. Leukoc. Biol., March 1, 2004; 75(3): 428 - 433. [Abstract] [Full Text] [PDF]
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K. Nakayama, S. Okugawa, S. Yanagimoto, T. Kitazawa, K. Tsukada, M. Kawada, S. Kimura, K. Hirai, Y. Takagaki, and Y. Ota Involvement of IRAK-M in Peptidoglycan-induced Tolerance in Macrophages J. Biol. Chem., February 20, 2004; 279(8): 6629 - 6634. [Abstract] [Full Text] [PDF]
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G. Hajishengallis and R. J. Genco Downregulation of the DNA-Binding Activity of Nuclear Factor-{kappa}B p65 Subunit in Porphyromonas gingivalis Fimbria-Induced Tolerance Infect. Immun., February 1, 2004; 72(2): 1188 - 1191. [Abstract] [Full Text] [PDF]
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B. Mytar, M. Woloszyn, R. Szatanek, M. Baj-Krzyworzeka, M. Siedlar, I. Ruggiero, J. Wieckiewicz, and M. Zembala Tumor cell-induced deactivation of human monocytes J. Leukoc. Biol., December 1, 2003; 74(6): 1094 - 1101. [Abstract] [Full Text]
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A. E. Medvedev, A. Lentschat, D. B. Kuhns, J. C.G. Blanco, C. Salkowski, S. Zhang, M. Arditi, J. I. Gallin, and S. N. Vogel Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections J. Exp. Med., August 18, 2003; 198(4): 521 - 531. [Abstract] [Full Text] [PDF]
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I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
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U. Deiters, M. Gumenscheimer, C. Galanos, and P. F. Muhlradt Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses Infect. Immun., August 1, 2003; 71(8): 4456 - 4462. [Abstract] [Full Text] [PDF]
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C. Ropert, M. Closel, A. C. L. Chaves, and R. T. Gazzinelli Inhibition of a p38/Stress-Activated Protein Kinase-2-Dependent Phosphatase Restores Function of IL-1 Receptor-Associated Kinase-1 and Reverses Toll-Like Receptor 2- and 4-Dependent Tolerance of Macrophages J. Immunol., August 1, 2003; 171(3): 1456 - 1465. [Abstract] [Full Text] [PDF]
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T. Bartfai, M. M. Behrens, S. Gaidarova, J. Pemberton, A. Shivanyuk, and J. Rebek Jr A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses PNAS, June 24, 2003; 100(13): 7971 - 7976. [Abstract] [Full Text] [PDF]
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A. E. Medvedev and S. N. Vogel Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance Innate Immunity, February 1, 2003; 9(1): 60 - 64. [Abstract] [PDF]
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M. A. Dobrovolskaia, A. E. Medvedev, K. E. Thomas, N. Cuesta, V. Toshchakov, T. Ren, M. J. Cody, S. M. Michalek, N. R. Rice, and S. N. Vogel Induction of In Vitro Reprogramming by Toll-Like Receptor (TLR)2 and TLR4 Agonists in Murine Macrophages: Effects of TLR "Homotolerance" Versus "Heterotolerance" on NF-{kappa}B Signaling Pathway Components J. Immunol., January 1, 2003; 170(1): 508 - 519. [Abstract] [Full Text] [PDF]
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