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(Journal of Leukocyte Biology. 2001;70:821-829.)
© 2001 by Society for Leukocyte Biology

Effect of cross-tolerance between endotoxin and TNF-{alpha} or IL-1ß on cellular signaling and mediator production

Marcella Ferlito*,{dagger}, Olga G. Romanenko*, Sarah Ashton*, Francesco Squadrito{dagger}, Perry V. Halushka{ddagger} and James A. Cook*

* Departments of Physiology and Neuroscience and
{ddagger} Pharmacology and Medicine, Medical University of South Carolina, Charleston, and
{dagger} Institute of Pharmacology, Medical University of Messina, Italy

Correspondence: Prof. James A. Cook, Dept. of Physiology and Neuroscience, 167 Ashley Ave., Suite 617 Storm Eye Institute, Charleston, SC 29425. E-mail: cookja{at}musc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REGULATION OF MAP KINASES...
 DISCUSSION
 REFERENCES
 
Abstract: Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) {alpha} and interleukin (IL)-1ß. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1ß, and TNF-{alpha} stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) DNA binding of nuclear factor-{kappa}B (NF-{kappa}B), and mediator production were examined. In naive cells, LPS, TNF-{alpha}, and IL-1ß induced I{kappa}B{alpha} degradation, kinase phosphorylation, and NF-{kappa}B DNA binding. LPS stimulation induced production of TNF-{alpha} or TxB2 and degradation of IRAK. However, neither TNF-{alpha} nor IL-1ß induced IRAK degradation or stimulated TNF-{alpha} or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-{alpha} production. LPS pretreatment induced cross-tolerance to TNF-{alpha} or IL-1ß stimulation. Pretreatment with TNF-{alpha} induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1ß did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-{alpha} and TxB2 production. These data demonstrate that IL-1ß induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-{kappa}B activation.

Key Words: macrophage/monocyte • signal transduction • phosphorylation • MAP kinases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 REGULATION OF MAP KINASES...
 DISCUSSION
 REFERENCES
 
Endotoxin [lipopolysaccharide (LPS)] is the major component of the cell wall of gram-negative bacteria, and its release in the blood stream contributes to septic shock. The deleterious effects induced by LPS involve an immune and inflammatory response leading to release of different mediators including interleukin (IL)-1ß, tumor necrosis factor {alpha} (TNF-{alpha}), and arachidonic acid metabolites from various cell types, but predominantly by macrophages and monocytes [1 ].

LPS induction of pro- and anti-inflammatory molecules is mediated through activation of the glycosylphosphatidylinositol-linked protein CD14 receptor [2 ], Toll-like receptor (TLR) 4 [3 , 4 ], and intracellular signaling pathways. Through a series of post-receptor molecules, LPS induces phosphorylation and degradation of inhibitor protein {kappa}B{alpha} (I{kappa}B{alpha}) , which inhibits nuclear translocation of transcription nuclear factor (NF) {kappa}B (NF-{kappa}B) [5 ]. LPS also activates the mitogen-activated protein (MAP) kinases, including extracellular regulated kinase (ERK)1/2 [6 ], p38 kinase [7 , 8 ] and c-Jun N-terminal kinase (JNK) [9 ].

LPS is able to induce tolerance or desensitization to subsequent LPS challenge. Prior exposure to low concentrations of LPS in vitro or low-dose LPS challenge in vivo suppresses macrophage/monocyte proinflammatory mediator production and improves survival to otherwise lethal LPS shock [10 ]. Although LPS tolerance does not induce global suppression of mediators, a reduction in proinflammatory-cytokine production, e.g., TNF-{alpha} and thromboxane (Tx) B2, is observed during tolerance [11 ]. The decreased mediator release has been linked to altered signal transduction pathways. In tolerance, down-regulation of MAP kinases [6 , 8 ] and alterations of NF-{kappa}B DNA binding have been reported [12 ]. LPS tolerance can also confer cross-tolerance to a variety of other noxious stimuli, e.g., certain gram-positive bacteria [13 ] and ischemia reperfusion injury [14 ]. Cross-tolerance of LPS to TNF-{alpha} or IL-1ß also has been demonstrated by other investigators [15 16 17 18 19 ].

Recently, it has been reported that down-regulation of TLR4 expression occurs in murine peritoneal macrophages, which may be one of the molecular mechanisms of LPS tolerance [20 ]. Proteins that belong to the IL-1ß signal transduction pathway, such as myeloid differentiation factor 88 (MyD88), IL-1 receptor associated kinase (IRAK), TNF receptor-activated factor 6 and NF-{kappa}B-inducing kinase, are coupled to TLR4 and are involved in LPS activation of NF-{kappa}B and MAP kinases [21 22 23 ]. Also the TNF-{alpha} signal transduction pathway may converge, through activation of TRAF2, at the level of NF-{kappa}B-inducing kinase leading to nuclear translocation of NF-{kappa}B [24 , 25 ].

Because potential post-receptor convergent signal transduction pathways are activated in response to LPS, IL-1ß, and TNF-{alpha}, we hypothesized that down-regulation of this pathway is a primary mechanism for cross-tolerance between LPS and TNF-{alpha} or IL-1ß. Therefore, the aim of this study was to evaluate whether these inflammatory cytokines can induce cross-tolerance to LPS and vice versa in human promonocytic THP-1 cells. Specifically alterations of signal transduction pathways leading to degradation of IRAK and I{kappa}B{alpha}, or nuclear translocation of NF-{kappa}B, activation of MAP kinases and production of TNF-{alpha} and TxB2 were examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 REGULATION OF MAP KINASES...
 DISCUSSION
 REFERENCES
 
Cell line and experimental protocol
Human promonocytic THP-1 cells (American Type Culture Collection, Rockville, MD) were used in this study. The cells were grown in RPMI 1640 (Cellgro Mediatech Inc., Herndon, VA) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), 50 U/mL of penicillin, and 50 µg/mL of streptomycin (Cellgro Mediatech Inc.) in 150-cm2 tissue culture flasks and maintained at 37°C in 5% CO2, 95% incubator air. Cells were passed twice a week and were used between the passages 3 and 15. For all experiments, medium with 1% fetal calf serum, penicillin, and streptomycin was used.

Tolerance to LPS, TNF-{alpha}, and IL-1ß in human promonocyte THP-1 cells was examined. In the first series of experiments, the cells were rendered LPS tolerant by pretreatment with different LPS concentrations (10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and subsequently stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min. Tolerance was also induced by pretreatment with LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 18 h, and subsequent stimulation was with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min, 2 h, or 18 h. ERK and JNK phosphorylation, IRAK and I{kappa}B{alpha} degradation, NF-{kappa}B DNA binding, and TNF-{alpha} or TxB2 production were examined.

Cell lysate preparation
After appropriate stimulation, cells were centrifuged, and the pellet was kept on ice-cold RIPA lysis buffer [20 mM HEPES, pH 7.4, 1% Triton X-100, 50 mM NaCl, 1 mM EGTA, 5 mM ß-glycerophosphate, 30 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL of leupeptin, and 10 µg/mL of pepstatin A) for 15 min, sonicated for a few seconds, and then centrifuged for 10 min at 4°C at 10,000 g. An aliquot was taken for protein determination, and the remaining supernatant was stored at -20°C until Western blot analysis was performed.

Western blot analysis
For detection of ERK, JNK, IRAK, and I{kappa}B{alpha}, cellular lysates were added to Laemmli sample buffer [26 ] and boiled for 5 min. Subsequently, protein from each sample was loaded onto a sodium dodecyl sulfate-4–12% polyacrylamide gel, subjected to electrophoresis, and transferred onto a PVDF membrane. For immunodetection, membranes were washed with Tris-buffered saline-Tween 20 (TBS-T; 20 mM Tris, 500 mM NaCl, 0.1% Tween 20) and blocked in a 7% powdered-milk solution in TBS-T for 1 h. After three washes with TBS-T, membranes were incubated for 1 h or overnight with either one of the following polyclonal antibodies in TBS-T: anti-JNK, specific for the dual phosphorylated form on Thr183/tyr185 of JNK (1:1,000) (Promega, Madison, WI); anti-ERK specific for the dual phosphorylated form on Thr202/Tyr204 (1:1,000) (New England Biolabs, Inc., Beverly, MA); anti-I{kappa}B{alpha} (1:1000) (Cell Signaling, Beverly, MA); or anti-IRAK, recognizing IRAK at 80 kDa (1:1,000) (Upstate, Lake Placid, NY). Membranes were washed again before being incubated with a horseradish peroxidase-conjugated donkey anti rabbit-immunoglobulin (Ig) G antibody (1:4,000) (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) in blocking buffer for 1 h. After the last three washes with TBS-T (TBS-0.15% Tween-20 ), protein detection was visualized by incubation with ECL Plus detection reagents (Amersham Pharmacia Biotech, Inc.) for 5 min and development of the exposed enhanced-chemoluminescence hyperfilms (Amersham Pharmacia Biotech, Inc.).

Nuclear extraction
Nuclear extracts were isolated by a modified procedure of Dignam et al. [27 ]. Briefly, after treatment, cells were spun at 2,800 g for 10 min. The cell pellet was resuspended in 400 µL of hypotonic buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, and 10 µg/mL of the following protease inhibitors: leupeptin, aprotinin, and pepstatin], set on ice for 10 min, and vortexed for 4 s with 0.6% Nonidet-P40. After centrifugation at 2,800 g for 10 min at 4°C, the pellet was resuspended in 15 µL of buffer C (20 mM HEPES, pH 7.9, 1 M NaCl, 5% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/mL of protease inhibitor) and gently agitated on an orbital shaker at 4°C for 30 min. After centrifugation at 14,000 g for 10 min, an aliquot of each supernatant was taken for protein determination, and the remaining supernatant was stored at -70°C.

Electrophoretic mobility shift assay
Double-stranded consensus binding site oligonucleotide for NF-{kappa}B was obtained from Promega and was end-labeled with [{gamma}-32P]ATP (NEN Life Science Products, Boston, MA) using T4 polynucleotide kinase (Promega, Madison, WI). For binding reactions, 10 µg of nuclear extracts were incubated in 20 µL of total reaction mix (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 5% glycerol, 1 mM DTT, 250 µg/mL of bovine serum albumin) containing the nonspecific competitors pd(N)6 and poly(dI-dC)-poly(dI-dC) (Amersham Pharmacia Biotech). The labeled oligonucleotide was added, and the mixture was incubated for 20 min at room temperature. Samples were analyzed by 4% nondenaturing polyacrylamide gel electrophoresis, and the gel was dried and exposed to Hyperfilm ECL at -70°C. Antibodies against p50 and p65 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) subunits of NF-{kappa}B were used for the supershift analysis.

TNF-{alpha} assay
TNF-{alpha} was measured using an enzyme-linked immunosorbant assay. Briefly, anti-human TNF-{alpha} antibody (0.8 µg/mL) (R&D System Inc. Minneapolis, MN) was used to coat 96-well enhanced-binding plates overnight at 4°C. The plates were washed with PBS/Tween-20 and blocked for 2 h with blocking buffer containing 10% bovine serum albumin. Plates were washed, and standards and samples were added and incubated overnight at 4°C. After this washing, biotinylated anti-human TNF-{alpha} antibody (0.3 µg/mL) ((R&D System Inc.) was added to the wells, and plates were incubated for 1 h at room temperature. Plates were washed, streptavidin-peroxidase conjugate (1:2,000) (BioSourse International Inc., Camarillo, CA) was added, and plates were incubated for 45 min. Finally, after washing, the ABTS substrate solution containing 3% H2O2 was added, and plates were read at 415 nm within 1 h.

TxB2 assay
After treatment, cells were centrifuged, and supernatants were stored at -20°C. Samples were diluted 1:10 in buffer containing 0.1% polyvinylpyrolidine, 0.9% NaCl, 50 mM Tris base, 1.7 mM MgSO4, and 0.16 mM CaCl2 (pH 7.4) prior to radioimmunoassay. TxB2 was quantitated by radioimmunoassay as previously described [28 , 29 ].

Data analysis
Gels were analyzed by scanning densitometry, and analysis was performed on a Macintosh computer using the public domain National Institutes of Health (NIH; Bethesda, MD) Image program (available on the Internet at http://rsb.info.nih.gov/nih-image/). Statistical significance was determined by one-way analysis of variance with Fischer’s post-hoc correction using Statview software. A P value of <0.05 was considered significant. Data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
REGULATION OF MAP KINASES...
 DISCUSSION
 REFERENCES
 
I{kappa}B{alpha} degradation in naive and tolerant cells
THP-1 cells were rendered LPS tolerant by pretreatment with different LPS concentrations (10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and were then stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min. Whereas LPS, TNF-{alpha}, and IL-1ß stimulation induced evident I{kappa}B{alpha} degradation in naive cells (i.e., cells not pretreated with LPS), in LPS-pretreated cells with subsequent LPS, TNF-{alpha}, and IL-1ß, induced degradation of I{kappa}B{alpha} was suppressed (Fig. 1a 1b 1c ).



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  		Figure 1. Effect of pretreatment with different LPS concentrations on I{kappa}B{alpha} cellular content. THP-1 cells were pretreated (pretr.) with medium alone or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and then stimulated (stim.) with LPS (10 µg/mL) (a), TNF-{alpha} (10 ng/mL) (b), or IL-1ß (100 ng/mL) (c) for 40 min. Western blot analysis was carried out to detect I{kappa}B{alpha} cellular content as described in Materials and Methods. Results are representative of three independent experiments.

 
Cellular content of I{kappa}B{alpha} was quantitatively determined by scanning densitometry after stimulation of THP-1 cells with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min. A reduction (n =3; P<0.05) in cellular content of I{kappa}B{alpha} was induced by treatment with LPS (93.1±4.2%), TNF-{alpha} (94.1±3.5%), and IL-1ß (75.1±8.1%) compared with basal (Fig. 2a ).



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  		Figure 2. I{kappa}B{alpha} cellular content in LPS-, TNF-{alpha}-, or IL-1ß-pretreated cells. (a) THP-1 cells were incubated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40 min. (b) Cells were rendered LPS tolerant by pretreatment for 18 h with LPS (1 µg/mL) and subsequently stimulated with LPS (10 µg /mL), TNF-{alpha} (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40 min. (c) Cells were rendered TNF-{alpha} tolerant by pretreatment with TNF-{alpha} (100 ng/mL) for 18 h and then stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or medium alone (basal) for 40 min. (d) Cells were rendered IL-1ß tolerant by pretreatment with IL-1ß (100 ng/mL) for 18 h and subsequently stimulated with LPS (10 µg /mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40 min. Detected proteins were quantified by scanning densitometry, and results were expressed as percentages of basal value, which was arbitrarily assigned 100%. Data represent the mean ± SE from three to four independent experiments. *, P < 0.05 compared with basal of each group; #, P < 0.05 compared with stimulation in naive group.

 
THP-1 cells were rendered LPS tolerant by pretreatment for 18 h and then stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min (Fig. 2b) . Although some reduction in I{kappa}B{alpha} cellular content was observed after TNF-{alpha} (36.1±5.4%), LPS (39.5±9.2%) or IL-1ß (28.1±1.7%) stimulation, there was a significant decrease (n=3–4; P<0.05) in degradation of I{kappa}B{alpha} induced by these stimuli in tolerant cells compared with that in naive cells.

THP-1 cells were rendered TNF-{alpha} tolerant by pretreatment with TNF-{alpha} for 18 h followed by stimulation with either LPS (10 µg/mL) or TNF-{alpha} (10 ng/mL) for 40 min, and the I{kappa}B{alpha} content was measured. Stimulation with TNF-{alpha} resulted in only marginal degradation of I{kappa}B{alpha} (14.1±17%) which was significantly different (P<0.05) from that observed in naive cells (Fig. 2c) . In contrast, LPS stimulation in TNF-{alpha}-tolerant cells induced I{kappa}B{alpha} degradation similarly to that in naive cells.

When cells were pretreated with IL-1ß (Fig. 2d) and subsequently stimulated with LPS or IL-1ß, a pattern similar to the ones observed in TNF-{alpha}-pretreated cells was induced. I{kappa}B{alpha} was degraded by LPS stimulation, whereas in IL-1ß-pretreated cells, stimulation with IL-1ß did not result in degradation of I{kappa}B{alpha}.

NF-{kappa}B DNA binding
Because degradation of I{kappa}B{alpha} leads to nuclear translocation of NF-{kappa}B, we characterized NF-{kappa}B DNA binding using antibodies against the predominant proteins comprising NF-{kappa}B, i.e., RelA (or p65) and NF-{kappa}B1 (or p50). A concentration-dependent increase in NF-{kappa}B DNA binding was observed when cells were stimulated with LPS (0.01–10 µg/mL) (Fig. 3 ). Supershifts with antibody to the p50 or p65 subunits demonstrated that these NF-{kappa}B subunits were activated by LPS.



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  		Figure 3. Characterization of NF-{kappa}B DNA binding. A dose-response to LPS was examined by stimulating THP-1 cells with LPS (0.01 µg/mL to 10 µg/mL for 40 min). Electrophoretic mobility shift assay was performed as described in Materials and Methods. Characterization of NF-{kappa}B DNA binding was evaluated using anti-p50 and anti-p65 antibodies. The arrows show the supershift induced by anti-p50 and -p65 antibodies; n.s., nonspecific binding. Free probe is not shown.

 
Stimulation with LPS, TNF-{alpha}, and IL-1ß induced p65/p50-DNA binding in naive THP-1 cells (Fig. 4 ). LPS and TNF-{alpha} pretreatment for 18 h induced a substantial increase in p50/p50 homodimer-DNA binding which was not altered after stimulation with LPS, TNF-{alpha}, or IL-1ß. In contrast, IL-1ß pretreated cells did not show an increase in basal NF-{kappa}B-DNA binding, and LPS-induced DNA binding was comparable with that in naive cells although stimulation with IL-1ß did not result in DNA binding.



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  		Figure 4. NF-{kappa}B DNA binding in naive and tolerant cells. THP-1 cells were pretreated (pretr.) with medium alone (naive) or LPS (1 µg /mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 18 h and subsequently stimulated (stim) as is shown, for 40 min with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL). Electrophoretic mobility shift assay was carried out as described in Materials and Methods. The electrophoretic mobility shift assay shown is representative of three independent experiments.

 

   REGULATION OF MAP KINASES IN NAIVE AND TOLERANT CELLS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REGULATION OF MAP KINASES...
DISCUSSION
 REFERENCES
 
ERK
THP-1 cells were rendered LPS tolerant by pretreatment with different LPS concentrations (10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and then stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min. All three stimuli induced evident phosphorylation of ERK in naive cells, which was suppressed in LPS-tolerant groups in a concentration-dependent manner (Fig. 5a 5b 5c ).



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  		Figure 5. Effect of pretreatment with different LPS concentrations on ERK phosphorylation. THP-1 cells were pretreated (pretr.) with medium alone (naive) or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and then stimulated (stim.) with LPS (10 µg/mL) (a), TNF-{alpha} (10 ng/mL) (b), or IL-1ß (100 ng/mL) (c) for 40 min. Results are representative of three independent experiments for each group.

 
In subsequent studies the effect of LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) pretreatment followed by stimulation with LPS, TNF-{alpha}, or IL-1ß on ERK activation was determined. ERK was induced in naive cells after stimulation with all stimuli. When cells were rendered LPS- , TNF-{alpha}- , or IL-1ß-tolerant, no phosphorylation of ERK was observed after stimulation with the same agonist. LPS- and TNF-{alpha}-induced reciprocal cross-tolerance as evidenced by suppressed ERK phosphorylation (Fig. 6 ). In contrast, in IL-1ß-tolerant cells, ERK was activated by stimulation with LPS.



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  		Figure 6. ERK phosphorylation in LPS-, TNF-{alpha}-, or IL-1ß-pretreated cells. Cells were pretreated (pretr.) with LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (naive) for 18 h and subsequently stimulated (stim) as shown for 40 min with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL). Western blot was carried out as described in Materials and Methods. Results are representative of two independent experiments.

 
JNK
As with the studies of ERK, THP-1 cells were rendered LPS tolerant by pretreatment with different LPS concentrations and then stimulated with LPS, TNF-{alpha}, or IL-1ß. All of these stimuli induced strong phosphorylation of JNK in naive cells, which was suppressed in LPS-tolerant groups in a concentration-dependent manner (Fig. 7a 7b 7c ).



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  		Figure 7. Effect of pretreatment with different LPS concentrations on JNK phosphorylation. THP-1 cells were pretreated (pretr.) with medium alone (naive) or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and then stimulated (stim.) with LPS (10 µg/mL) (a), TNF-{alpha} (10 ng/mL) (b), or IL-1ß (100 ng/mL) (c) for 40 min. Results are representative of three independent experiments for each group.

 
In subsequent studies, the effect of LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) pretreatment followed by stimulation with LPS, TNF-{alpha}, or IL-1ß on JNK activation was determined. LPS, TNF-{alpha}, and IL-1ß stimulation induced JNK phosphorylation in comparison with naive basal level (Fig. 8 ). However the degree of activation with IL-1ß and TNF-{alpha} was less than with LPS. In LPS-, TNF-{alpha}-, and IL-1ß-tolerant cells, basal levels of JNK phosphorylation were significantly higher than in naive cells. The higher basal naive activation observed in the tolerant groups probably reflects some degree of residual activation of JNK after tolerance induction. However there was no further increase in JNK phosphorylation after stimulation with LPS or the cytokines. Although LPS-tolerant cells did not show an increase in JNK phosphorylation after LPS stimulation, in TNF-{alpha}- and IL-1ß-tolerant cells, LPS-induced JNK phosphorylation was not impaired.



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  		Figure 8. JNK phosphorylation in LPS-, TNF-{alpha}-, or IL-1ß-pretreated cells. JNK (46- and 54-kDa proteins) phosphorylation was in THP-1 cells. Cells were pretreated (pretr.) with LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (naive) for 18 h and subsequently stimulated (stim.) for 40 min with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL). Results are representative of two experiments.

 
IRAK degradation in naive and tolerant cells
THP-1 cells were stimulated with LPS, TNF-{alpha}, or IL-1ß for 40 min or 2 or 18 h, and cellular content of IRAK was observed. After 40 min of stimulation by all stimuli, no changes in IRAK cellular content were observed (Fig. 9a ). LPS stimulation induced IRAK degradation at 2 h, which persisted to 18 h. Stimulation with TNF-{alpha} or IL-1ß did not induce changes in cellular content of IRAK. However, when cells were rendered TNF-{alpha} or IL-1ß tolerant, the following LPS stimulation for 2 h induced IRAK degradation as in naive cells (Fig. 9b) .



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  		Figure 9. IRAK cellular content. (a) THP-1 cells were stimulated (stim.) with LPS (10 µg/mL), IL-1ß (100 ng/mL), or TNF-{alpha} (10 ng/mL) for 40 min, 2 h, or 18 h. (b) Cells were pretreated (pretr.) with IL-1ß (100 ng/mL) or TNF-{alpha} (10 ng/mL) for 18 h and subsequently stimulated for 2 h with LPS (10 µg/mL).

 
Mediator production in naive and tolerant cells
TNF-{alpha} (supernatant release)
THP-1 cells were rendered LPS tolerant by pretreatment with different LPS concentrations (1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and then stimulated with LPS (10 µg/mL) for another 18 h. Supernatant was collected and assayed for TNF-{alpha} production. LPS pretreatment induced a concentration-dependent decrease in TNF-{alpha} production (Fig. 10 ).



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  		Figure 10. Effect of pretreatment with different concentrations of LPS on TNF-{alpha} release in naive and tolerant cells. THP-1 cells were rendered LPS tolerant by pretreatment with LPS (1, 10, or 100 ng/mL or 1000 ng/mL) or medium alone (naive) for 18 h and then stimulated with LPS (10 µg/mL) for another 18 h. Data represent the means ± SE from three independent experiments. *, P < 0.05 compared with basal (naive group); #, P < 0.05 compared with LPS stimulation in naive group.

 
In later studies the effect of LPS or IL-1ß (100 ng/mL) pretreatment followed by stimulation with LPS or IL-1ß on TNF-{alpha} production was evaluated (Fig. 11 ). LPS (10 µg/mL) induced a significant TNF-{alpha} production [2,016±337 pg/mL (n=3)] compared with basal [23±7 pg/mL (n=3)], whereas IL-1ß did not induce TNF-{alpha}. LPS-pretreated cells showed a classic pattern of tolerance, as LPS stimulation failed to induce TNF-{alpha} production [172±39 pg/mL (n=3)]. IL-1ß did not stimulate TNF-{alpha} production in naive cells. It is important, however, that IL-1ß pretreatment induced a cross-tolerance to LPS-stimulated TNF-{alpha} production. Indeed, a significant decrease [P<0.05 (n=3)] in TNF-{alpha} production was observed after LPS stimulation [480±152 pg/mL (n=3)] in IL-1ß-pretreated cells compared with naive cells [2,016±337 pg/mL (n=3)].



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  		Figure 11. TNF-{alpha} release in LPS, TNF-{alpha} or IL-1ß pretreated cells. TNF-{alpha} production was evaluated in the supernatant of naive, LPS- or IL-1ß-tolerant THP-1 cells. Cells were pretreated (pretr.) with medium alone (naive), LPS (1 µg/mL), or IL-1ß (100 ng/mL) for 18 h and subsequently stimulated (stim.) with medium alone (basal), LPS (10 µg/mL), and IL-1ß (100 ng/mL) for 18 h. Data represent the means ± SE from three independent experiments. *, P < 0.05 compared with basal (naive) group; #, P < 0.05 compared with naive LPS stimulation.

 
Cell-associated TNF-{alpha}
Synthesis of TNF-{alpha} begins with a long amino acid propeptide sequence which is transferred to the plasma membrane as a trimeric protein [30 ]. TNF-{alpha} is released in its mature form from the plasma membrane by enzymatic cleavage [31 ]. Because release of TNF-{alpha} is subject to this complex process, we evaluated whether in LPS- or IL-1ß-tolerant cells an alteration of this process could occur. Cell-associated TNF-{alpha} was quantitated in tolerant cells after LPS or IL-1ß restimulation. As shown by Figure 12 , cellular concentrations of TNF-{alpha} were similar to ones measured in the supernatant of the corresponding groups. LPS induced a significant increase (P<0.05) in cell-associated TNF-{alpha} [2,143±556 pg/mL (n=3)] in naive cells compared with basal (62±3pg/mL). IL-1ß did not affect TNF-{alpha} production in naive cells. In the LPS- or IL-1ß-tolerant groups after restimulation with LPS, a significant decrease in TNF-{alpha} [353±72pg/mL vs. 866±237pg/mL, respectively (n=3)] was observed.



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  		Figure 12. Cell-associated TNF-{alpha} content. THP-1 cells were rendered LPS (1 µg/mL) or IL-1ß (100 ng/mL) tolerant by pretreatment (pretr.) with the stimuli or medium alone (naive) for 18 h and subsequently stimulated with medium alone (basal), LPS (10 µg/mL), or IL-1ß (100 ng/mL) for 24 h. TNF-{alpha} was assessed in the cell lysates as described in Materials and Methods. Data represent means ± SE from three independent experiments. *, P < 0.05 compared with basal (naive) group; #, P < 0.05 compared with LPS stimulation in naive group.

 
TxB2 production
THP-1 cells were rendered LPS tolerant by pretreatment with different LPS concentrations (1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and then stimulated with LPS (10 µg/mL) for another 18 h. Supernatant was collected and assayed for TxB2 production. LPS pretreatment induced a concentration-dependent decrease in TxB2 production (Fig. 13 ).



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  		Figure 13. Effect of pretreatment with different LPS concentrations on TxB2 production in naive and tolerant cells. THP-1 cells were rendered LPS tolerant by pretreatment with LPS (1, 10, or 100 ng/mL or 1000 ng/mL) or medium alone (naive) for 18 h and then stimulated with LPS (10 µg/mL) for another 18 h. Data represent means ± SE from three independent experiments. *, P < 0.05 compared with basal (naive) group; #, P < 0.05 compared with LPS stimulation in naive group.

 
In subsequent studies, TxB2 production was quantitated in naive cells and in cells pretreated with LPS, TNF-{alpha} or IL-1ß. Basal production of TxB2 was arbitrarily assigned the value of one (Fig. 14 ). LPS stimulation induced a significant increase in TxB2 production in naive cells (5.6-±1.2-fold; n=3; P<0.05) compared with basal values. However, neither TNF-{alpha} nor IL-1ß stimulated an increase in TxB2 in naive cells. Pretreatment with TNF-{alpha} or IL-1ß induced cross-tolerance to LPS restimulation. Diminished TxB2 synthesis was observed in TNF-{alpha}-, IL-1ß-, and LPS-tolerant groups after restimulation with LPS [TxB2 production was reduced 53.5±12.5%, 35.7±17%, and 62.5±5.3% (n=3), respectively].



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  		Figure 14. TxB2 production in LPS-, TNF-{alpha}-, or IL-1-ß-pretreated cells. TxB2 production was evaluated in the supernatant of THP-1 cells. Cells were pretreated (pretr.) with medium alone (naive), LPS (1 µg/mL), TNF-{alpha} (10 ng/mL), or IL-1ß (100 ng/mL) for 18 h and subsequently stimulated with LPS (10 µg/mL), TNF-{alpha} (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 18 h. TxB2 was assessed as described in Materials and Methods. Data represent means ± SE from three independent experiments. *, P < 0.05 compared with basal (naive) group; #, P < 0.05 compared with LPS stimulation in naive group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REGULATION OF MAP KINASES...
 DISCUSSION
REFERENCES
 
Our studies demonstrated that IL-1ß, TNF-{alpha}, and LPS activate signaling in naive THP-1 cells as evidenced by ERK and JNK phosphorylation, I{kappa}B{alpha} degradation and DNA p50/p65 NF-{kappa}B heterodimer binding. However, unlike LPS, neither TNF-{alpha} nor IL-1ß induced IRAK degradation, and these cytokines failed to induce measurable increases in TNF-{alpha} and TxB2 production. Pretreatment of TNF-{alpha}, IL-1ß, or LPS each induced homologous tolerance to restimulation with the same agonist. This was demonstrated by the decrease in ERK and JNK phosphorylation, I{kappa}B{alpha} degradation, and NF-{kappa}B activation. LPS induced a strong cross-tolerance to TNF-{alpha} and IL-1ß signaling events. Because this cross-tolerance was evident at LPS pretreatment concentrations as low as 10 and 100 ng/mL, it is not a consequence of the magnitude of the initial LPS stimulus. TNF-{alpha} induced some degree of cross-tolerance to LPS stimulation shown by decreases in ERK phosphorylation and TxB2 production. The major finding in our study was that IL-1ß tolerance did not produce cross-tolerance to any of the measured LPS-induced signaling events; nevertheless, IL-1ß tolerance strongly suppressed LPS-induced TxB2 and TNF-{alpha} synthesis.

Previous studies have shown that an ability of LPS and IL-1ß to induce cross-tolerance to one another was associated with suppression of activator protein-1 (AP-1) and NF-{kappa}B transactivation [18 ]. Decreased activation of these transcription factors could therefore provide a mechanism of suppressed LPS-induced TNF-{alpha} and TxB2 production observed in the present study with IL-1ß-tolerant cells. However, effects of IL-1ß pretreatment on blocking upstream signal transduction events were not evident in our study. The data demonstrated that IL-1ß pretreatment does not alter LPS-induced activation of MAP kinases, I{kappa}B{alpha} degradation, or NF-{kappa}B translocation. The latter suggests that alternative signaling pathways may exist which affect gene transactivation. Other studies have implicated an imprecise relationship between I{kappa}B{alpha} degradation and NF-{kappa}B DNA binding and transactivation [32 ]. Numerous other signaling pathways including protein kinase C, protein kinase A, phosphatidylcholine-specific phospholipase, sphingomyelinase, and tyrosine kinase have been implicated [33 34 35 ]. For example, IL-1ß activation of a NF-{kappa}B reporter gene in human alveolar epithelial cells was suppressed by a phospholipase C or protein kinase C inhibitor, but IL-1ß-induced I{kappa}B{alpha} degradation and NF-{kappa}B DNA binding were unaffected [36 ].

Because our data demonstrated that IL-1ß does not induce TNF-{alpha} production, TNF-{alpha} could not be implicated as an autocrine factor in inducing cross-tolerance to LPS. It has also been observed that TNF-{alpha} is not linked to LPS- or IL-1ß-induced cross-tolerance because TNF-{alpha} and LPS do not induce reciprocal tolerance in murine macrophages [18 ]. Also, macrophages from TNFR I/II knockout mice could be rendered LPS tolerant, and blocking endogenous TNF-{alpha} production with TNFR-Fc fusion protein did not alter tolerance induction . Our results demonstrated that TNF-{alpha} induces a degree of cross-tolerance to LPS in THP-1 cells. Thus murine macrophages and human THP-1 cells may differ in this respect. In THP-1 cells TNF-{alpha} tolerance suppressed LPS-induced ERK phosphorylation and induced an NF-{kappa}B-binding pattern consisting of increased p50 homodimers similar to LPS tolerance. TNF-{alpha} pretreatment also attenuated LPS-induced TxB2 synthesis. Our previous studies have demonstrated that TNF-{alpha} pretreatment in vivo results in cross-tolerance to LPS-induced lethality in rats and induces a macrophage functional phenotype typical of LPS tolerance [16 ].

LPS tolerance has been shown to suppress murine macrophage expression of TLR4 and this has been postulated as a mechanism of tolerance [37 ]. However the ability of LPS to induce cross-tolerance to TNF-{alpha}- and IL-1ß-induced signaling events, which occur independently of TLR4, argues that LPS tolerance alters postreceptor signaling mechanisms. The level at which LPS tolerance may be affecting the signaling cascade is uncertain. The ability of phorbol myristate acetate to restore the signaling responses in tolerant macrophages or monocytes shown by our studies [8 , 11 ] and others [38 ] suggests proximal signaling alterations. In a study by Li et al. [39 ], LPS-tolerant THP-1 cells were shown to have impaired signaling at the level of IRAK. This was evident from decreased LPS-induced IRAK association with MyD88, decreased IRAK phosphorylation, and decreased IRAK content. Our studies confirmed their observations that LPS induces IRAK degradation. IRAK was depleted at 2 h after LPS stimulation and remained depleted after 18 h, i.e., when the cells were LPS tolerant. Cellular content of MyD88 was not affected by the LPS pretreatment (data not shown). However, we demonstrated that neither TNF-{alpha} or IL-1ß stimulation nor tolerance affected the IRAK levels. Thus, it did not appear that IRAK depletion would be a limiting factor in cytokine-induced cross-tolerance to LPS. IL-1ß has been shown to induce IRAK degradation in other cell lines [40 ]. The observed lack of IL-1ß pretreatment on IRAK degradation in the present study is consistent with the observation of Li et.al. [39 ], who demonstrated that IL-1ß had no effect on IRAK protein levels or IRAK kinase activity in THP-1 cells.

In summary, these data demonstrate that LPS tolerance induces strong cross-tolerance to TNF-{alpha}- and IL-1ß-induced cell signaling events. TNF-{alpha} tolerance induced partial cross-tolerance to LPS-induced cell signaling and TxB2 production (Fig. 15 ). However, surprisingly, we found that IL-1ß-induced cross-tolerance to LPS was unique. IL-1ß pretreatment decreased LPS-stimulated TxB2 and TNF-{alpha} production (secreted and cell associated). However, IL-1ß pretreatment did not suppress LPS-induced ERK and JNK activation, I{kappa}B{alpha} degradation, or NF-{kappa}B-DNA binding. Therefore, these data demonstrate that IL-1ß is capable of inducing cross-tolerance to LPS-induced mediator production without altering LPS-induced activation of MAP kinase signaling pathways or NF-{kappa}B activation. It is possible that IL-1ß may affect more downstream signaling events that impact LPS-induced gene transactivation and/or posttranscriptional events leading to suppressed TxB2 and TNF-{alpha} production. Further studies examining signaling events and gene expression in LPS tolerance and cross-tolerance phenomena are merited.



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  		Figure 15. Schematic summary of key findings.

 


   ACKNOWLEDGEMENTS
 
This study was supported by NIH grant GM27673 and a Medical University of South Carolina postdoctoral fellowship.

Received September 11, 2000; revised June 12, 2001; accepted June 18, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REGULATION OF MAP KINASES...
 DISCUSSION
 REFERENCES
 

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