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The Journal of Immunology, 2003, 171: 1456-1465.
Copyright © 2003 by The American Association of Immunologists

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 1

Catherine Ropert2,*, Meire Closel*, Andréa C. L. Chaves* and Ricardo T. Gazzinelli*,{dagger}

* Centro de Pesquisas René Rachou, Fundaçao Oswaldo Cruz, and {dagger} Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pretreatment of macrophages with Toll-like receptor (TLR)2 or TLR4 agonists leads to a stage of cell hyporesponsiveness to a second stimulation with TLR agonists. This tolerance state is accompanied by the repression of IL-1 receptor-associated kinase-1, mitogen-activated protein kinases, and I{kappa}B phosphorylation and expression of genes encoding proinflammatory cytokines, like IL-1{beta} and TNF-{alpha}. In this report, we demonstrated that mucin-like glycoprotein (tGPI-mucin) of Trypanosoma cruzi trypomastigotes (TLR2 agonist) and LPS (TLR4 agonist) induce cross-tolerance in macrophages and we addressed the role of phosphatase activity in this process. Analysis of the kinetic of phosphatase activity induced by tGPI-mucin or LPS revealed maximum levels between 12 and 24 h, which correlate with the macrophage hyporesponsiveness stage. The addition of okadaic acid, an inhibitor of phosphatase activity, reversed macrophage hyporesponsiveness after exposure to either LPS or tGPI-mucin, allowing phosphorylation of IL-1R-associated kinase-1, mitogen-activated protein kinases, and I{kappa}B and leading to TNF-{alpha} gene transcription and cytokine production. Furthermore, pretreatment with either the specific p38/stress-activated protein kinase-2 inhibitor (SB203580) or the NF-{kappa}B translocation inhibitor (SN50) prevented the induction of phosphatase activity and hyporesponsiveness in macrophage, permitting cytokine production after restimulation with LPS. These results indicate a critical role of p38/stress-activated protein kinase-2 and NF-{kappa}B-dependent phosphatase in macrophage hyporesponsiveness induced by microbial products that activate TLR2 and TLR4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages respond to a wide range of microbial products leading to the induction of genes, among them cytokines (e.g., IL-6, IL-1, TNF-{alpha}, IL-12), required for the protective innate and acquired immunity (1, 2). Toll-like receptors (TLRs),3 which are expressed in the membrane of cells from the immune system, have been shown to be essential for the recognition and discrimination of specific conserved pattern of molecules derived from bacteria, protozoa, fungi, and viruses (3, 4, 5, 6, 7). Activation of TLRs results in stimulation of the innate immunity through well-described signaling pathway involving adaptor molecules like Myd-88, kinases such as mitogen-activated protein kinase (MAPKs, Refs. 8 and 9), and transcription factors that lead to the production of cytokines (reviewed in Ref. 10).

However, cytokine production must be tightly controlled; excessive production leads to amplified inflammatory responses and devastating illnesses. One major question open in this field is why TLRs are not continuously activated by the commensal microflora or during chronic infections with pathogens that contain TLR agonists (7). Several groups have attempted to model this phenomenon in vitro showing that prior exposure to LPS, the major constituent of the membrane from Gram-negative bacteria, significantly inhibits the ability of various cell types to respond to subsequent challenge with LPS (reviewed in Ref. 11). This phenomenon, known as endotoxin tolerance, incited a resurgence of interest in the mechanisms responsible for altered responsiveness to bacterial endotoxin. More recent experiments with bacterial DNA (12) or lipopeptides (13, 14, 15) indicate that down-regulation of macrophage responsiveness after stimulation is not restricted to LPS. Indeed, we have previously shown that GPI-anchored mucins like glycoproteins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucin), a TLR2 agonist (16, 17), induced tolerance to the TLR4 agonist, LPS (18).

Although, the molecular mechanisms of tolerance to microbial products have been extensively studied (19, 20, 21, 22), they are still largely unknown. Secretion of soluble mediators, changes in LPS receptor expression or function, and alterations in LPS-driven signaling pathways have all been implicated (23, 24, 25, 26, 27, 28, 29, 30). Many of the proposed mechanisms of tolerance would imply cross-desensitization of response to other bacterial products presumed to share the same intracellular pathway. The tolerance state is associated with suppression of signaling pathways that involve engagement of TLR and signaling molecules, such as MyD88, IL-1R-associated kinase-1 (IRAK-1), TNFR-associated factor-6 (21, 31, 32, 33, 34). This is accompanied by suppression of various genes and production of cytokines, such as TNF-{alpha}, IL-6, and IL-12 (35). Interestingly, expression of other mediators (e.g., IL-10 and NO) in LPS tolerant cells is not abrogated, suggesting that endotoxin tolerance does not inhibit all functions, but represents a reprogramming of cellular functions (35).

Interaction of TLR4 or TLR2 and coreceptors with LPS or tGPI-mucin triggers a multitude of signaling events including the phosphorylation of MAPKs and I{kappa}B (10, 18) leading to the synthesis of proinflammatory cytokines like TNF-{alpha} (reviewed in Refs. 36 , 37). The activation of MAPKs requires phosphorylation of conserved tyrosine and threonine residues by dual-specificity MAPK kinases, which in turn are activated in two serine residues by upstream MAPK kinase kinase. Because of the critical importance of MAPKs in cellular signaling the activity of the MAPKs is tightly regulated. Different classes of phosphatases, like protein-tyrosine phosphatase or serine/threonine protein phosphatase (PP), are involved in MAPKs inactivation (38, 39, 40). As phosphorylation of IRAK-1, MAPKs, and I{kappa}B is impeded in endotoxin tolerant cells, we investigated the possible role of TLR agonist-induced phosphatase in tolerized macrophages. In this report, using inhibitor of MAPKs activation or NF-{kappa}B translocation, we showed that p38/stress-activated protein kinase-2 (SAPK-2) and NF-{kappa}B play an important role in phosphatase induction, and consequently, in the desensitization process of macrophages by TLR2 and TLR4 agonists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Five- to six-week-old C57BL/6 and C3H/HeJ mice were maintained under standard conditions in the animal house of the Centro de Pesquisas René Rachou, Fundaçao Oswaldo Cruz, Belo Horizonte, Brazil. The TLR2 knockout (KO) mice were kindly provided by Dr. S. Akira from Osaka University, Japan, and maintained in specific pathogen-free conditions in the Laboratory of Nutrition and Gnotobiology at the Federal University of Minas Gerais, MG, Brazil.

Reagents and Abs

Reagents used were obtained from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. p38/SAPK-2 inhibitor SB203580, extracellular signal-related kinase (ERK)-1/ERK-2 inhibitor PD98059, and NF-{kappa}B translocation inhibitor SN50 and the control peptide SN50-M were purchased from Calbiochem (San Diego, CA). LPS from Escherichia coli O55:B5 was obtained from Sigma-Aldrich and purity evaluated through PAGE-SDS electrophoresis and silver staining. In addition, we confirmed the absence of cytokine production in response to LPS in macrophage from C3H/HeJ or activation in CHO cells that overexpressed TLR2 exposed to various concentrations of LPS. Abs were obtained from the following sources: anti-TNF-{alpha} and anti-IL-12 (p70) Duoset ELISA kits were purchased from R&D Systems (Minneapolis, MN); Abs against MAPK family members (i.e., ERK-1/ERK-2, c-Jun N-terminal kinase (JNK)/SAPKs and p38/SAPK-2), and I{kappa}B were obtained from New England Biolabs (Hertfordshire, U.K.); Ab against IRAK-1 was a generous gift from Drs. S. Sato and S. Akira from Osaka University, Japan.

Purification of T. cruzi-derived tGPI

The tGPI-mucin was isolated from tissue culture trypomastigotes as previously described (41), using sequential organic extraction followed by hydrophobic-interaction chromatography in octyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) and elution with a propan-1-ol gradient (5–60%). The tGPI-mucins were purified employing LPS-free reagents, the purity analyzed by Limulus amebocyte lysate assay (Charles River, Charleston, SC) and confirmed by the inability of these preparations to induce cytokine production in macrophages from TLR2 KO mice or activate CHO cells overexpressing CD14 and endogenous TLR4.

Murine macrophage preparation

Thioglycollate-elicited peritoneal macrophages were obtained from either C3H/HeJ, C57BL/6, or TLR2 KO mice by peritoneal washing. Adherent peritoneal macrophages were cultured in 96-well plates (2 x 105 cells/well) at 37°C/5% CO2 in DMEM (Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine, and 40 µg/ml of gentamicin. To study desensitization, macrophages were incubated in medium with tGPI-mucin or LPS for various period of time. Cells were then washed twice with PBS, and rechallenged with LPS or tGPI-mucin for 18 or 48 h to evaluate TNF-{alpha} or IL-12 production, respectively. To determine the involvement of the phosphatases in the tolerance induction, the cells were pretreated with okadaic acid (OA), a serine/threonine phosphatase inhibitor before or after the preexposure to LPS or tGPI-mucin. To determine the involvement of the MAPK or NF-{kappa}B in the tolerance induction, SB203580 (10 µM), PD98059 (40 µM), SN50 (18 µM) or control peptide SN50 M (18 µM) were added 30 min before the preexposure to LPS. After 24 h the cells were washed and challenged with LPS for 18 h to evaluate TNF-{alpha}. To investigate the capacity of OA treatment to induce TNF-{alpha} gene expression in tolerant cells, actinomycin 5 µM, cycloheximide 20 µg/ml, SN50 (18 µM) or control peptide SN50 M (18 µM) were added to cell culture 24 h after first LPS stimulation in the presence of OA (20 nM). After 30 min the cells were challenged with LPS for 8 h and TNF-{alpha} evaluated.

Cytokine measurement

TNF-{alpha} and IL-12 (p70) were quantified by ELISA using the Duoset kit from R&D in supernatants at 18 and 48 h after stimulation, respectively.

Cell lysate preparation

Peritoneal macrophages were cultured and stimulated with either LPS (50 ng/ml) and/or tGPI-mucin (2 nM) for 24 h. After washing, macrophages were challenged with LPS (100 ng/ml). At indicated time, cells were washed with PBS and lysed on ice in lysis buffer (20 mM Tris-acetate, pH 7.0, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 4 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM benzamidine, 0.1% v/v 2-ME). Lysates were scraped, collected into Eppendorf tubes, and centrifuged at 13,000 x g for 20 min at 4°C (42).

Electrophoresis and immunoblotting

Cell lysate samples were separated by 10%-acrylamide SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Bucks, U.K.). Membranes were blocked overnight at 4°C with PBS containing 5% (w/v) low fat milk and 0.1% Tween 20. Membranes were washed three times with PBS containing 0.1% Tween 20, then incubated with rabbit polyclonal Abs anti-phosphorylated MAPKs or transcription factors in PBS containing 5% (w/v) BSA and 0.1% Tween 20. After washing, the membranes were incubated with HRP-conjugated anti-rabbit Ab and assayed by the ECL chemiluminescent system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Immunoprecipitation and in vitro kinase assay

The cell lysate (107 cells for 800 µl) was treated with 5 µl anti-IRAK Ab for 3 h on a rotator at 4°C. A total of 50 µl of 50% slurry of prewashed protein A-agarose beads was then added to each sample followed by an additional incubation for 2 h at 4°C. The samples were spun briefly and washed in lysis buffer. The beads were then washed twice with kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM {beta}-glycerophosphate, 20 mM p-nitrophenylphosphate, 1 mM EDTA, 1 mM sodium orthovanadate, and 1 mM benzamidine). Each sample was incubated at 37°C for 30 min in 50 µl kinase buffer supplemented with 5 µM ATP and 1 µl {gamma}32-ATP. SDS sample buffer was added after incubation and the samples were subjected to SDS-PAGE (8% acrylamide) analysis.

Phosphatase activity

Cells were seeded in 24-well plates (2 x 106 cells/well) and cultured in the presence or absence of LPS or tGPI-mucin, treated or not with OA (20 nM), SB203580 (10 µM), PD98059 (40 µM), SN50 (18 µM) or SN50 M (18 µM). After an indicated period of incubation, cells were washed in PBS (one time), resuspended and disrupted in a cold buffer containing 50 mM Tris-HCl (pH 7.0 at 25°C), 0.1 mM EDTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin and 25 µg/ml PMSF. The lysate was centrifuged at 15,000 x g for 15 min at 4°C and total protein concentration measured. Total cellular PP activity was determined as previously described (43) by evaluating the capacity of cell lysates to hydrolyze the tyrosine analog p-nitrophenylphosphate. The assay was performed in 200 µl reaction mixture containing 10 mM p-nitrophenylphosphate, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 50 mM HEPES, pH 7.0 (obtained by addition of a 0.1 M Tris solution). Incubations were initiated by addition of 50 µl cell extracts and conducted at 37°C with gentle shaking and terminated after 60 min by the addition of 200 µl of 1 M NaOH following by the measure of the absorbance at 405 nm with a microplate reader to monitor PP activity. The results were expressed as OD 405 nm values per 10 µg protein.

RT-PCR analysis

To verify the induction of TNF-{alpha} mRNA expression, 24-wells culture plates containing 2 x 106 macrophages/well were incubated in the presence of LPS (50 ng/ml) and treated or not with OA (20 nM) for 24 h. After washing, the cells were restimulated with LPS (100 ng/ml). At indicated time, the medium was discarded and the cells were washed with PBS. Total RNA was extracted with TRIzol (Life Technologies) according to the manufacturer’s instructions. The cDNA synthesis was obtained in a final volume of 30 µl containing 200 U of Moloney murine leukemia virus reverse transcriptase (Pharmacia), 200 mM concentrations of each deoxynucleoside triphosphate (Promega, Madison, WI), 2.5 µl of buffer (Boehringer Mannheim, Indianapolis, IN), 240 pmol of oligo-dT10 (Boehringer Mannheim) per µl, 0.1 M DTT (Bio-Rad, Hercules CA), 1 U of the RNase inhibitor RNAsin (Promega) per µl, and 0.4 µg total RNA. The PCR was performed in 10 µl of reaction mixture containing 0.5 U of Taq DNA polymerase, 200 mM of the each deoxynucleoside triphosphate, 1.5 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 8.5), together with 10.0 and 5.0 pmol of the TNF-{alpha} and HPRT primers, respectively. The PCR program consisted of 30 cycles with the initial denaturation at 95°C for 5 min, annealing at 54°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The products were electrophoresed in polyacrylamide and developed by silver staining. The primer sequence used and the PCR product size are listed: HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-GATTCAACTTGCGCTCATCTTAGGC-3', 162 bp; TNF-{alpha}, 5'-TTCTCATTCCTGCTTGTGG-3' and 5'-GCTACAGGCTTGTCACTCG-3', 171 bp. For semiquantitative RT-PCR analysis, amplified products of TNF-{alpha} were quantified using specific bands of the housekeeping gene HPRT transcripts as a reference by densitometric scanning.

Statistics

Data are presented as means ± SEM. Statistical differences were determined by one-way ANOVA followed by Dunett’s multiple comparison test of all groups vs the control group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pretreatment with tGPI-mucin affects LPS-induced signaling pathway

As shown in Fig. 1A, pretreatment of macrophages from C3H/HeJ mice (which possess nonfunctional TLR4) with tGPI-mucin (2 nM) for 24 h induced a significant hyporesponsive state to a second tGPI-mucin stimulation, as measured by decreased TNF-{alpha} and IL-12 production. Unlike tGPI-mucin treatment, macrophages from C3H/HeJ mice treated for 24 h with LPS can still respond to further tGPI-mucin challenge and produce normal amounts of cytokine. When macrophages from TLR2 KO mice were pretreated with tGPI-mucin and subjected to a second stimulation with LPS, the cytokine production was unaffected compared with non-pretreated cells. In contrast, in the same macrophages LPS induced tolerance to itself. These observations indicate that cross-tolerance triggered by LPS and tGPI-mucin in macrophages is mediated by previous activation of TLR4 or TLR2, respectively.



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  		FIGURE 1. A, Macrophages from different mouse lineages were preincubated with medium, LPS (10 ng/ml), or tGPI-mucin (2 nM) for 24 h. After washing, the macrophages from C57BL/6 and TLR2 KO mice were challenged with LPS (50 ng/ml) and the macrophages from C3H/HeJ stimulated with tGPI-mucin (2 nM) for 24 and 48 h. The TNF-{alpha} (24 h) or IL-12 (48 h) concentrations in the culture supernatants were measured by ELISA. Data are the means of three independent experiments. *, p < 0.01, ** p < 0.05 vs control group (cells pretreated with medium). B, Comparison of LPS-mediated activation of the ERK-1/ERK-2, JNK/SAPK, p38/SAPK-2, and I{kappa}B in C57BL/6 macrophages pretreated with medium, LPS, or tGPI-mucin. Cells were pretreated for 24 h with either medium, LPS (50 ng/ml), or tGPI-mucin (2 nM). Subsequently, macrophages were restimulated for the indicated time points with 100 ng/ml of LPS. Concerning control cellular extract (preincubated with medium alone), macrophages were stimulated with LPS for a different time corresponding to maximum phosphorylation, i.e., pERK-1/ERK-2 (15 min), pJNK/SAPK (30 min), pp38/SAPK-2 (30 min), and pI{kappa}B (15 min). Cellular extracts were prepared and MAP kinase/I{kappa}B phosphorylation was examined by Western blot with Ab specific for the phosphorylated forms at different times post secondary stimulation. This figure is representative of two independent experiments that yielded similar results.

 
Then, we investigated whether the tolerance phenomenon affects phosphorylation of MAPKs and I{kappa}B in macrophages from C57BL/6 mice. The activation of three MAPKs was evidenced after 15 or 30 min of macrophage stimulation with LPS (Fig. 1B) in cells that did not receive any pretreatment. In contrast, MAPKs phosphorylation was mostly inhibited in macrophages pretreated with either LPS (50 ng/ml) or tGPI-mucin (2 nM) for 24 h following restimulation with LPS (100 ng/ml). Pretreatment with either LPS or tGPI-mucin also resulted in suppression of I{kappa}B{alpha} phosphorylation in response to a second stimulation with LPS (Fig. 1B) and resulted in nondegradation of I{kappa}B{alpha} (data not shown).

Induction of phosphatase activity correlates with LPS or tGPI-mucin induced tolerance state in macrophages

Initially, we sought to establish the kinetics under which microbial products induce maximal tolerance state. Macrophages were stimulated for indicated time in the presence of tGPI-mucin (2 nM) or LPS (10 ng/ml) and then restimulated after washing for an additional 24 h or 48 h with LPS (50 ng/ml) and assayed for cytokine production (Fig. 2A). We observed that, up to 6 h after LPS or tGPI-mucin pretreatment, TNF-{alpha} and IL-12 production was marginally affected in response to a second stimulation. In contrast, prolonged microbial pretreatment (i.e., 12 or 24 h) dramatically decreased cytokine production in macrophages restimulated with LPS. Interestingly, the highest level of phosphatase activity in macrophages was also to be found between 12 and 24 h after tGPI-mucin or LPS stimulation (Fig. 2B). Furthermore, we observed that LPS concentrations able to stimulate significant phosphatase activity corresponded to LPS concentrations (>=1 ng/ml) required to induce tolerance state (data not shown).



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  		FIGURE 2. A, Time-course of LPS tolerance and cross-tolerance between LPS and tGPI-mucin. Macrophages from C57BL/6 mice were preincubated with 10 ng/ml LPS (E. coli O55:B5) or tGPI-mucin (2 nM) for the indicated times. Then the cells were washed with medium and stimulated with 50 ng/ml LPS for 24 or 48 h. Levels of TNF-{alpha} and IL-12 were measured in the cell supernatants, 18 and 48 h later, respectively. Data are means obtained from three independent experiments. **, p < 0.01 vs respective control group (0 h of pretreatment). B, Time-course of LPS or tGPI-mucin-induced phosphatase activity. Macrophages from C57BL/6 mice were incubated with 100 ng/ml of LPS or 2 nM of tGPI-mucin for the indicated times and the phosphatase activity evaluated by colorimetric assays. This figure is representative of two independent experiments. C, The phosphatase activity is TLR dependent. Peritoneal macrophages from wild type, TLR2 KO, or C3H/HeJ mice were stimulated with LPS (100 ng/ml) or tGPI-mucin (2 nM) for 24 h and the phosphatase activity evaluated. The data represent means of two independent experiments. *, p < 0.05 vs control group (C57BL/6).

 
Next we determined the involvement of TLR2 or TLR4 in inducing phosphatase activity after macrophage stimulation by tGPI-mucin or LPS, respectively. Macrophages from C57BL/6 and TLR2 KO but not C3H/HeJ mice exhibited enhancement of phosphatase activity after 12 h of LPS treatment, reaching a maximum level at 24 h (Fig. 2C). tGPI-mucin induced significant enhancement of phosphatase activity in macrophages from C3H/HeJ and C57BL/6 mice, but not from TLR2 KO mice. Taken together these data indicate that tGPI-mucin or LPS-induced phosphatase activity is TLR-dependent and suggest that this is an important regulatory mechanism for controlling macrophage response to microbial stimulation.

Reversion of LPS- or tGPI-mucin-induced tolerance by phosphatase inhibitor

OA, a polyether compound that inhibits serine/threonine phosphatases PP type 1 and PP type 2A (44, 45), was found to be a potent inhibitor of the phosphatase activity induced by LPS (Fig. 3A). The phosphatase activity was completely abrogated when 20 nM OA was used. As shown in Fig. 3B, TNF-{alpha} production observed in response to a second LPS challenge in OA-treated cells indicated that OA can revert the tolerance state (Fig. 3B, line C). This effect is time dependent, and OA may be added to the cultured cells up to 8 h after the first stimulation to restore TNF-{alpha} release in response to a second LPS stimulation (Fig. 3B, line D and E). The most drastic effect was obtained when OA was added 1 h before LPS pretreatment, which resulted in 80% recovery of TNF-{alpha} production (Fig. 3B, line C).



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  		FIGURE 3. Reversion of LPS tolerance by OA. A, Inhibitory effect of OA on the LPS-induced phosphatase activity. Macrophages from C57BL/6 mice were pretreated with OA 1 h before the stimulation with LPS (100 ng/ml) or tGPI-mucin (2 nM) and the phosphatase activity evaluated at 24 h. The data are the means of two independent experiments. **, p < 0.01 vs control group (nontreated with OA). B, The reversion of LPS tolerance by OA is time dependent. The OA addition (20 nM) to the macrophages was performed at different times before and after the first stimulation with LPS (10 ng/ml), and after 24 h the cells were washed and restimulated with LPS (50 ng/ml) for 18 h. TNF-{alpha} release in the culture supernatants was measured by ELISA. The background levels of TNF-{alpha} in the supernatant of unstimulated macrophages cultured in absence of OA were always less than 200 pg/ml. Data are means of two independent experiments. *, p < 0.05, **, p < 0.01 vs control group (tolerized group nontreated with OA).

 
We next determined whether OA was capable of reverting the cross-desensitization observed between tGPI-mucin and LPS (Fig. 4). In these experiments, OA was added 1 hour before the pretreatment with LPS or tGPI-mucin. About 80% of the TNF-{alpha} production was recovered in the OA-treated cells prestimulated with LPS and challenged with LPS. The same efficiency of OA in reverting macrophage hyporesponsiveness was observed in macrophages tolerized with tGPI-mucin. This reversion was not specific to TNF-{alpha} because OA was also able to restore IL-12 production in a significant way (Fig. 4).



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  		FIGURE 4. Reversion of cross-tolerance between LPS and tGPI-mucin by OA. Macrophages from C57BL/6 mice were treated with OA 1 h before stimulation with LPS (10 ng/ml) or tGPI-mucin (2 nM). After 24 h the cells were washed and restimulated for 24 h with LPS (50 ng/ml) or tGPI-mucin (2 nM). TNF-{alpha} and IL-12 concentrations were evaluated in the culture supernatants at 18 and 48 h, respectively. *, p < 0.05, **, p < 0.01 vs control group (tolerized group nontreated with OA).

 
OA-induced TNF-{alpha} gene expression in tolerant cells

We sought to determine whether the effect of OA was due to an increase of cytokine mRNA stabilization after the first LPS stimulation or an induction of gene transcription in response to the second challenge. For this purpose, macrophages were previously treated or not with OA (20 nM) and stimulated for 24 h with LPS. After washing, cells were restimulated for various time with LPS and TNF-{alpha} mRNA levels evaluated by RT-PCR. As shown in the Fig. 5A, treatment with OA resulted in enhancement of TNF-{alpha} mRNA expression in LPS restimulated cells as compared with cells not treated with OA. Results demonstrated that after normalization for the HPRT signal, OA enhanced the TNF-{alpha} mRNA level 6.3 and 2.9 times after 0.5 and 2 h of LPS restimulation, as compared with macrophages not treated with OA. A basal level of TNF-{alpha} mRNA (time 0 h) was observed before LPS restimulation in OA treated cells, which is in agreement with the basal TNF-{alpha} release observed in OA-treated macrophages even in the absence of a second LPS challenge (Fig. 5A).



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  		FIGURE 5. OA treatment induced activation of TNF-{alpha} gene transcription in tolerant cells. A, TNF-{alpha} mRNA expression determined by RT-PCR. Macrophages were treated or not with OA (20 nM) and stimulated with LPS (50 ng/ml) for 24 h. After washing, the cells were restimulated with LPS (100 ng/ml) for the indicated times (0–8 h) and total RNA was isolated as described in Materials and Methods. RT-PCR products were resolved on polyacrylamide gel and developed by silver staining. The intensities of the different bands were quantitated using gene HPRT transcripts as a reference. Representative of three independent experiments that yielded similar results. B, Macrophages from C57BL/6 mice were treated or not with OA (20 nM) 1 h before stimulation with LPS (10 ng/ml). After 24 h the cells were washed and treated with the transcription inhibitor actinomycin D (ActD) at 5 µg/ml or the protein synthesis inhibitor cycloheximide (CHX) at 10 µg/ml or NF-{kappa}B translocation inhibitor SN50 (18 µM) or its control peptide SN50 M (18 µM), 30 min, before the restimulation with LPS (50 ng/ml) for 8 h. TNF-{alpha} concentration in the culture supernatants was measured by ELISA. *, p < 0.05, **, p < 0.01 vs control group (tolerant cells only treated with OA).

 
To confirm that restoration of TNF-{alpha} release in tolerant cells after OA treatment corresponded to mRNA transcription activation, we tested the effect of the transcription inhibitor actinomycin D (5 µg/ml), the protein synthesis inhibitor cycloheximide (20 µg/ml), and the inhibitor of NF-{kappa}B translocation SN50 (18 µM). Macrophages were treated with OA (20 nM) and simultaneously stimulated with LPS for 24 h (10 ng/ml). Cells were then washed and 30 min before LPS restimulation the different inhibitors added and TNF-{alpha} synthesis evaluated. As presented in Fig. 5B, actinomycin D was found to be a potent inhibitor of TNF-{alpha} synthesis, which demonstrated that transcription was required to detect cytokine production in response to the second LPS stimulation. Logically, the protein synthesis inhibitor cycloheximide affected the TNF-{alpha} release by macrophages in the same range of inhibition (~70%). Addition of SN50 also resulted in a significant decrease of TNF-{alpha} synthesis in OA-treated macrophages showing the involvement of NF-{kappa}B in the restoration of TNF-{alpha} production in tolerant macrophages treated with OA (Fig. 5B). Taken together these data strongly suggest that the restoration of TNF-{alpha} production induced by OA treatment in LPS tolerant macrophages is mostly due to new gene transcription in a NF-{kappa}B-dependent way.

Regulation of IRAK, MAPKs, and I{kappa}B phosphorylation by OA

We investigated whether OA was able to restore IRAK-1 phosphorylation and kinase activity in tolerant cells. As demonstrated in Fig. 6A, LPS-tolerant macrophages presented a significant decrease of IRAK-1 kinase activity after 30 min LPS restimulation. The addition of OA before the first LPS stimulation restored this kinase activity. Western blot analysis of cellular extracts immunoprecipitated with anti-IRAK Ab demonstrated an additional IRAK-1 band corresponding to a hyperphosphorylated form of IRAK-1 in LPS-stimulated macrophages (pIRAK-1; Fig. 6B). In confirmation of previous results (21, 33, 34), a prior exposure to LPS resulted in a marked decrease in the level of IRAK protein (Fig. 6B, middle panel). Importantly, OA addition to tolerant macrophages restored the level of active IRAK-1, which was similar or superior to that obtained in LPS-treated cells.



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  		FIGURE 6. OA treatment allowed IRAK-1 phosphorylation and kinase activity in LPS tolerant cells. A, Murine macrophages were treated or not with OA (20 nM) and stimulated or not with LPS (50 ng/ml). After 24 h, the cells were washed and restimulated or not with 100 ng/ml LPS for 30 min. Cellular extracts were prepared, and IRAK-1 was immunoprecipitated with anti-IRAK Ab plus protein A and IRAK-1 kinase activities were determined by in vitro kinase assay using IRAK-1 as substrate. Representative of two independent experiments that yielded similar results is shown. B, Macrophages were pretreated or not with OA (20 nM) and stimulated with 50 ng/ml LPS for 24 h, washed with PBS resuspended in fresh medium, and restimulated with 100 ng/ml LPS. Total protein extracts were prepared. The levels of IRAK-1 were detected through Western blot using anti-IRAK Ab. Representative of two independent experiments that yielded similar results is shown.

 
Because OA treatment restored the cytokine production in macrophages tolerized by microbial stimuli, we decided to investigate the effect of OA on the activation of the MAPKs (Fig. 7). Treatment with OA (20 nM) increased and prolonged the phosphorylated state of ERK-1/ERK-2, and more importantly, the OA treatment restored phosphorylation of the ERK-1/ERK-2 in response to second LPS stimulation in macrophages, indicating a reactivation of this signaling pathway. The OA effect on the phosphorylation state of JNK/SAPK was more moderate, but a return of JNK/SAPK activation was also observed after a second LPS stimulation. LPS prestimulated macrophages treated with OA only marginally recovered their capacity to phosphorylate p38/SAPK-2 upon second LPS stimulation. More surprisingly, OA inhibited the phosphorylation of the p38/SAPK-2 in response to primary LPS stimulation. It was also our interest to evaluate the impact of OA treatment on the I{kappa}B phosphorylation in tolerant macrophages. OA treatment decreased the phosphorylation state of I{kappa}B in macrophages primarily stimulated with LPS (Fig. 7). We also observed a partial restoration of I{kappa}B phosphorylation in response to a second stimulation with LPS in OA-treated cells. These results suggest an important role of an OA-sensitive phosphatase in controlling the activity of MAPKs and I{kappa}B.



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  		FIGURE 7. OA treatment allowed LPS mediated activation of the ERK-1/ERK-2, JNK/SAPKs, and I{kappa}B in LPS tolerized C57BL/6 macrophages. A, Time-course of ERK-1/ERK-2, JNK/SAPKs, p38/SAPK-2, and I{kappa}B phosphorylation in LPS-stimulated macrophages treated with OA (+OA) or not (-OA). Murine macrophages were treated or not with OA (20 nM) 1 h before the LPS stimulation (100 ng/ml). Cellular extracts were prepared and MAPKs and I{kappa}B phosphorylation was examined by Western blot with polyclonal Ab specific for the phosphorylated forms of the MAPKs and I{kappa}B at different times post stimulation. This is representative of two independent experiments. B, Time-course of ERK-1/ERK-2, JNK/SAPK, p38/SAPK-2, and I{kappa}B phosphorylation in LPS-tolerant macrophages treated with OA (+OA) or not (-OA). Murine macrophages were treated or not with OA (20 nM) 1 h before the LPS stimulation (50 ng/ml). After 24 h the cells were washed and restimulated with LPS (100 ng/ml). Cellular extracts were prepared, and MAPKs and I{kappa}B phosphorylation was examined by Western blot with polyclonal Ab specific for the phosphorylated forms of the MAPKs and I{kappa}B at different time post secondary stimulation. This is representative of two independent experiments that yielded similar results.

 
Activation of p38/SAPK-2 and NF-{kappa}B are necessary for TLR-agonist induced phosphatase activity

To investigate mechanisms of phosphatase activity up-regulation, macrophages were pretreated with specific inhibitors of ERK-1/ERK-2 (PD98059), p38/SAPK-2 (SB203580), and NF-{kappa}B translocation (SN50) followed by LPS stimulation for 24 h. After cell washing, phosphatase activity was measured. Pretreatment with SB203580 inhibited the induction of phosphatase activity in LPS stimulated macrophages (Fig. 8A) in a dose-dependent way, reaching a maximal activity at ~1 µM (Fig. 8B). In contrast blocking ERK-1/ERK-2 had no effect on the level of enzyme activity. Interestingly, inhibition of NF-{kappa}B translocation by SN50 dramatically decreased the induction of LPS-mediated phosphatase in a specific way, and the control peptide SN50 M was found to be inefficient.



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  		FIGURE 8. The p38/SAPK-2 pathway and NF-{kappa}B but not ERK-1/ERK-2 pathway are involved in the induction of phosphatase activity and tolerance in macrophages pretreated with LPS. A, Macrophages from C57BL/6 mice were pretreated with SB203580 (10 µM), PD98059 (40 µM), SN50 (18 µM), or control peptide SN50 M (18 µM) 30 min before LPS stimulation (100 ng/ml) and phosphatase activity evaluated at 24 h. The addition of SB203580, PD98059, or SN50 has no effect on the baseline level of phosphatase activity in cultures of unstimulated macrophages. The data are the means of two independent experiments. ** p < 0.01 vs control group (group stimulated with LPS but nontreated with the different inhibitors). B, Dose effect of SB203580 on phosphatase activity and tolerance in macrophages pretreated with LPS. Macrophages were preincubated with 0–10 µM SB203580 30 min before LPS exposure (100 ng/ml) and phosphatase activity evaluated at 24 h or macrophages were treated with 0–10 µM SB203580 30 min before LPS prestimulation (10 ng/ml) and 24 h later cells were washed, restimulated with LPS (50 ng/ml) for 18 h and TNF-{alpha} release evaluated. C, Macrophages from C57BL/6 mice were pretreated with SB203580 (10 µM), PD98059 (40 µM), SN50 (18 µM), or control peptide SN50 M (18 µM) 30 min before LPS preexposure (10 ng/ml) and 24 h later macrophages were restimulated with LPS (50 ng/ml) for 18 h. TNF-{alpha} release in the culture supernatants was measured by ELISA. The addition of SB203580, PD98059, or SN50 24 h before LPS has no effect on the production of TNF-{alpha} in macrophages compared with nontreated macrophages stimulated with LPS. The data are the means of two independent experiments. **, p < 0.01 vs control group (tolerized group nontreated).

 
To investigate the role of MAPKs and NF-{kappa}B in the tolerance phenomenon, we tested the impact of different inhibitors SB203580, PD98059, and SN50 on the restoration of TNF-{alpha} production in tolerant cells (Fig. 8C). The different inhibitors were added to cells 30 min before the first LPS stimulation (10 ng/ml), and after 24 h the cells were washed and restimulated with LPS (50 ng/ml). The same inhibitors SB203580 and SN50 that were able to inhibit LPS-induced phosphatase activity allowed the restoration of TNF-{alpha} release in response to a second LPS stimulation. Importantly, the SB203580 effect was dose-dependent and restored TNF-{alpha} production in tolerant macrophages with a 50% efficiency between 1 and 5 µM (Fig. 8B). In contrast, ERK-1/ERK-2 inhibitor did not alter the tolerance state of macrophages. Taken together, our data indicate the involvement of p38/SAPK-2 and NF-{kappa}B activation in the LPS-induced phosphatase activity, which plays a crucial role in the tolerant state of macrophages.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inhibition of inflammatory processes after a primary microbial stimulation may represent an important mechanism to limit excessive activation of the host immune system and self-destruction caused by a sustained local or systemic inflammation. Different in vivo studies of endotoxin tolerance have proposed a crucial role for macrophages in this phenomenon (35).

Mechanistic in vitro studies performed showed that pretreatment of macrophages with LPS results in down-regulation of the expression of some LPS-inducible cytokine genes, whereas expression of other LPS-inducible genes is unaffected or up-regulated (35). Inhibition of TNF-{alpha} secretion appears to be a common characteristic of the different models of endotoxin tolerance (25, 29, 46, 47, 48), whereas the decrease of others cytokines like IL-1 and IL-6 seems to be more controversial (49, 50, 51). In fact we show in this study that the cross-tolerance state induced by tGPI-mucin/LPS to subsequent LPS/tGPI-mucin challenge is accompanied by a decrease of TNF-{alpha} and IL-12 production. This tolerance state is accompanied by the inability of macrophages to phosphorylate IRAK-1, I{kappa}B, and MAPKs. In contrast, NO production was unaffected in tolerized macrophages (data not shown), suggesting that NO production could occur independently of the known downstream elements of the TLR pathway (52), i.e., MAPKs and NF-{kappa}B.

Some studies have asked whether endotoxin tolerance specifically changes the way that LPS interacts with cell surface receptors or membranes. LPS pretreatment of mouse macrophages has been reported to inhibit cell surface expression of the TLR4/MD2 signaling complex, suggesting that endotoxin tolerance occurs due to down-regulation of the LPS receptor (30). However this hypothesis seems unlikely, because it was demonstrated that CHO cells that overexpress TLR4 and MD2 can be rendered LPS-tolerant (28). Another study showed a similar expression level of TLR4 mRNA and protein in normal and LPS-tolerant human monocytes regardless of whether cells were restimulated with LPS (32). So it is more likely that the signaling molecules downstream of TLR receptor are targets of tolerance phenomenon.

In this study, we focused our interest on the control mechanism of the downstream elements of TLR pathway, like IRAK-1, MAPKs, and I{kappa}B, that are impeded to be phosphorylated in tolerant cells (22, 25, 32, 33, 34). We explored the role of phosphatases in the tolerance phenomenon and showed the induction of phosphatase activity by tGPI-mucin or LPS is TLR dependent. The timing of phosphatase induction by these TLR2 and TLR4 agonists correlates with the development of tolerance and cross-tolerance phenomena in our model (between 12 and 24 h).

Importantly, the phosphatase inhibitor OA was capable of restoring the cytokine (TNF-{alpha} and IL-12) production in tolerant cells. These findings are in agreement with those previously reported by Fernando et al. (53), indicating that it is possible to restore TNF-{alpha} synthesis in endotoxin-tolerant cells treated with OA. As a previous study has shown the capacity of OA to increase stabilization of cytokine mRNA and not to affect on transcription in the tolerant phenotype (54), we addressed the question of the mechanism of OA activity in our study. We showed that OA treatment induced TNF-{alpha} gene transcription activation in tolerant macrophages in a NF-{kappa}B-dependent way.

Based on its classical activity, we then sought to establish whether OA acted as inhibitor of the serine/threonine phosphatases PP type 1 or PP type 2A inhibitor (44, 45). Our results show that the maximal inhibitory activity of OA was reached at 20 nM suggesting that PP type 2A is the target enzyme for the OA activity detected in our system (55, 56). However, the TLR2/TLR4 agonists induced activity detected here was a tyrosine phosphatase activity, suggesting that the observed effect of OA may be indirect. In order, to investigate this possibility, we analyzed the effect of OA on phosphorylation of IRAK-1, MAPKs, and I{kappa}B during primary stimulation with LPS. Phosphorylation of IRAK-1 and JNK/SAPK were mostly unaffected. Interestingly, addition of OA during primary macrophage stimulation with LPS had a strong positive and negative effect on phosphorylation of ERK-1/ERK-2 and p38/SAPK-2, respectively. Because PP type 2A has been shown to be an important regulator of ERK-1/ERK-2 phosphorylation, we raise the hypothesis that OA reduces p38/SAPK-2 activation due to the strong activation of ERK-1/ERK-2 (57, 58). This cross-regulation between ERK-1/ERK-2 and p38/SAPK-2 (59) was also accompanied with the reduction of I{kappa}B activation, as observed by the reduced I{kappa}B phosphorylation in OA-treated macrophages stimulated with LPS.

Considering that p38/SAPK-2 and NF-{kappa}B have a primary role on induction of expression of various genes during macrophage activation via TLR agonists, we then investigated whether these signaling pathways were involved in induction of phosphatase activity by LPS. LPS-stimulated phosphatase induction seemed to be independent of ERK-1/ERK-2 activation and to be mediated at least in part by the activation of p38/SAPK-2 pathway, as treatment with SB203580 inhibited the phosphatase induction. Similarly, macrophage pretreatment with NF-{kappa}B translocation inhibitor (SN50) reduced LPS-induced phosphatase activity suggesting that NF-{kappa}B is involved in phosphatase regulation. The fundamental role of p38/SAPK-2 and NF-{kappa}B in the tolerance phenomenon was confirmed by the reversion of tolerance observed in macrophage pretreated with SB203580 and SN50. These data confirmed other studies illustrating the importance of p38/SAPK-2 in septic animals (60) or in monocytes LPS-hyporesponsiveness (61) through the capacity of p38/SAPK-2 inhibitors to revert the tolerance state.

We also investigated the impact of OA treatment during primary macrophage stimulation, and consequent inhibition of phosphatase induction by LPS, on phosphorylation of IRAK-1, MAPKs, and I{kappa}B in response to second LPS stimulation. As expected, no phosphorylation was observed when macrophages were prestimulated with LPS in the absence of OA. In contrast, when OA was added 1 hour before the first challenge with LPS, phosphorylation of IRAK-1 was restored to the normal levels. Consequently, significant phosphorylation of ERK-1/ERK-2, JNK/SAPK, and I{kappa}B was observed after the second LPS stimulation in cells pretreated with OA. In the case of p38/SAPK-2, we observed only discrete reversal of activation by OA pretreatment, at the earlier time points after rechallenge with LPS. These findings are consistent with the involvement of IRAK-1, MAPKs, and I{kappa}B on the induction of TNF-{alpha} production by macrophages and the capability of OA to restore the synthesis of this cytokine in tolerant macrophages (18, 31, 62, 63). Furthermore, these results suggest an emerging model where the restoration of phosphorylation of IRAK-1 seems to be crucial to revert MAPKs and I{kappa}B phosphorylation and consequently some functions of tolerant macrophages. Thus, once activated, IRAK-1 may be a self-limiting element of TLR signaling possibly protecting cells from prolonged and excessive activation.

Finally, a recent study has described a phosphatase activity induced in macrophages activated with LPS with a kinetic similar to the one described in this study. The gene encoding this phosphatase was cloned, sequenced, and characterized as phosphatase named MAPK phosphatase (M-KPM) (64), because its expression was associated with decreased phosphorylation of MAPKs. Importantly, the p38/SAPK-2 inhibitor SB203580 also inhibited the transcription of M-KPM induced by LPS. Thus, the tyrosine phosphatase activity measured in this study may be related to the M-KPM. However, its direct target and precise specificity remain to be investigated.

In conclusion, the model shown in Fig. 9 proposes that upon macrophage activation with TLR-2 or TLR-4 agonists, there is the induction of a phosphatase activity that coincides with macrophage hyporesponsiveness induced by microbial stimuli. The induction of this phosphatase activity is dependent on both p38/SAPK-2 phosphorylation and NF-{kappa}B translocation. Further, the increase in phosphatase activity is associated with an impaired ability of macrophages to phosphorylate IRAK-1, MAPKs, and I{kappa}B, as well as to produce TNF-{alpha} upon second stimulation with microbial products (Fig. 9A). As shown in Fig. 9B, our model suggests that treatment of macrophages with OA during primary stimulation with TLR2 or TLR4 agonists results in a inhibition of PP type 2A, up-regulation of ERK-1/ERK-2 activation, down-regulation of p38/SAPK-2, and reduction of I{kappa}B phosphorylation. These regulatory effects of the main signaling pathways triggered by TLRs result in blockade of phosphatase activity induced by TLR2/TLR4 agonists, leading to restoration of IRAK-1, MAPKs, and I{kappa}B phosphorylation and production of TNF-{alpha} upon second stimulation with microbial products.



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  		FIGURE 9. Conceptual model for the role of TLR2- or TLR4-induced phosphatase on tolerance of macrophage pretreated with tGPI-mucin or LPS. A, Interaction of LPS or tGPI-mucin with TLR4 or TLR2 induce in an early phase activation of IRAK-1 and downstream elements including MAPKs and I{kappa}B (a) responsible for the production of TNF-{alpha}. p38/SAPK-2 and NF-{kappa}B are also important for up-regulating phosphatase activity that will prevent IRAK-1, MAPKs and I{kappa}B upon a second stimulation with either TLR2 or TLR4 agonists (b). B, Hypothetical mechanism of tolerance reversion in macrophages treated with OA. OA, a PP2A inhibitor, induces hyperphosphorylation of ERK-1/ERK-2 (a) in macrophages leading to blockade of p38/SAPK-2 as well as I{kappa}B phosphorylation (a) and consequent down regulation of phosphatase activity (b) upon macrophage activation with either TLR2 or TLR4 agonists. The failure in inducing the phosphatase activity restores the ability of macrophages to phosphorylate IRAK-1, MAPKs, and I{kappa}B and to produce TNF-{alpha}.

 


   Acknowledgments
 
We thank Helia Cannizzaro for purified tGPI-mucin, Martin Olivier for providing OA, and Shintaro Sato and Shizuo Akira for providing the anti-IRAK-1 Ab. We are also grateful to Santuza R. Teixeira, Oscar Bruna-Romero, and Mauro M. Martins for critically reading this manuscript, and Martin Olivier for helpful discussions during the development of this study.


   Footnotes
 
1 This work was supported in part by the World Health Organization, A00477, Conselho Nacional de Desenvolvimento de Pesquisas Científica e Tecnológica, 470104/01-5, and Conselho Nacional de Desenvolvimento de Pesquisas Científica e Tecnológica, PADCT 62.0543/98-1. C.R. is a visiting scientist from Conselho Nacional de Desenvolvimento de Pesquisas Científica e Tecnológica, Fundaçao Oswaldo Cruz, and R.T.G. is a research fellow from Conselho Nacional de Desenvolvimento de Pesquisas Científica e Tecnológica, 522.056/95-4. Back

2 Address correspondence and reprint requests to Dr. Catherine Ropert, Laboratory of Immunopathology, Centro de Pesquisas René Rachou, Fundaçao Oswaldo Cruz, Avenida Augusto de Lima 1715, Barro Preto, 30190-002 Belo Horizonte, MG, Brazil. E-mail address: ropert{at}hotmail.com Back

3 Abbreviations used in this paper: TLR, Toll-like receptor; KO, knockout; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; M-KPM, MAPK phosphatase; tGPI-mucin, GPI-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes; IRAK, IL-1R-associated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; OA, okadaic acid; PP, protein phosphatase. Back

Received for publication June 18, 2002. Accepted for publication May 29, 2003.


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 Materials and Methods
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 Discussion
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