Cutting Edge: Endotoxin Tolerance in Mouse Peritoneal Macrophages Correlates with Down-Regulation of Surface Toll-Like Receptor 4 Expression

Cells and reagents

Peritoneal macrophages were isolated from C57BL/6J mice essentially as described. Briefly, mice were i.p. injected with 2 ml of 4% thioglycollate. After 3 days of injection, peritoneal exudate cells were isolated by washing the peritoneal cavity with ice-cold HBSS. These cells were incubated for 2 h, and adherent cells were used as peritoneal macrophages.

Phenol-extracted LPS (Escherichia coli O55:B5) was purchased from Sigma (St. Louis, MO). PE-conjugated Abs to IL-6 and IL-12 were purchased from PharMingen (San Diego, CA).

Intracellular staining of macrophages

Peritoneal macrophages were preincubated with 1, 10, or 100 ng/ml LPS for the indicated periods and washed with HBSS twice. Cells were stimulated with 10 ng/ml LPS in the presence of 10 μg/ml brefeldin A (Sigma) for 6 h. Cells were harvested and incubated with 4% paraformaldehyde. Then cells were incubated with PE-conjugated anti-cytokine Abs. Stained cells were analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, San Jose, CA).

Electrophoretic mobility shift assay and in vitro kinase assay

Peritoneal macrophages were incubated with 100 ng/ml LPS for the indicated periods and washed with HBSS. Cells were cultured with culture media alone for 1 h and then stimulated with 10 ng/ml LPS for 10 or 20 min. An electrophoretic mobility shift assay and in vitro kinase assay were performed as described previously (26).

Northern blot analysis

Peritoneal macrophages and RAW264.7 cells were incubated with 100 ng/ml LPS for the indicated periods. Total RNA was extracted with an RNeasy kit (Qiagen, Hilden, Germany). RNA (20 μg) was electrophoresed, transferred to nylon membrane, and hybridized with cDNA probe for mouse TLR4. The same membrane was stripped and rehybridized with GAPDH cDNA probe.

Establishment of a mAb to mouse TLR4

A rat was immunized with Ba/F3 cells expressing mouse TLR4 and MD-2 and used for hybridoma production. The MTS 510 mAb (rat IgG2a/k) that specifically reacted with the immunized transfectant but not with the original Ba/F3 line was selected for further analysis. The mAb was purified from ascites obtained from severe combined immunodeficient mice. Detailed characterization of the mAb will be described elsewhere (29).

Time-dependent and dose-dependent suppression of inflammatory cytokine production by pre-exposure to LPS

When mouse peritoneal macrophages were stimulated with 10 ng/ml LPS, these cells displayed a significant increase in production of inflammatory cytokines such as IL-12 and IL-6 (Fig. 1⇓). However, when the cells were preincubated with 100 ng/ml LPS for 1 h, IL-12 production was dramatically reduced (30.6 to 4.6% positive). When the cells were preincubated for 24 h, IL-12 production was almost completely blocked. Pre-exposure to LPS for 1 h also partially reduced production of IL-6 (15.1 to 8.8%). In addition, production of IL-6 reduced with the lapse of pre-exposure time, and production was severely reduced after 24 h of pre-exposure (0.8%). Thus, suppression of inflammatory cytokine production from LPS-pretreated macrophages was observed in a time-dependent manner.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Reduced production of IL-12 and IL-6 after pre-exposure to LPS. Peritoneal macrophages were preincubated with 100 ng/ml LPS for the indicated periods and then stimulated with 10 ng/ml LPS for 6 h (left). Productions of IL-12 (upper) and IL-6 (lower) were analyzed by flow cytometry. Peritoneal macrophages were preincubated with 1 or 10 ng/ml LPS for 24 h and then stimulated with 10 ng/ml LPS for 6 h (right). These data are representative of three independent experiments.

We further preincubated peritoneal macrophages with several doses of LPS for 24 h and analyzed production of inflammatory cytokines. As shown in Fig. 1⇑, 100 ng/ml LPS dramatically reduced the production of IL-12 and IL-6. However, when preincubated with 10 ng/ml, the reduction was partial, and further 1 ng/ml LPS did not cause significant reduction (Fig. 1⇑). These results suggest that suppression of inflammatory cytokine production was observed in a dose-dependent manner. Thus, when we used a system to detect intracellular cytokines, LPS tolerance does occur in a time-dependent and a dose-dependent manner.

Reduced activation of LPS-signaling cascade after exposure to LPS

The results from intracellular cytokine production indicate that a 24-h exposure to LPS results in almost complete LPS tolerance, but it is partial after a 3-h exposure. To assess LPS-induced activation of signaling molecules during LPS tolerance, we analyzed LPS-induced NF-κB activation by gel mobility shift assay (Fig. 2⇓A). In nontreated cells, DNA-binding activity of NF-κB transcription factors was slightly observed, and LPS stimulation induced a significant increase in their DNA-binding activity. When pretreated with LPS for 3 h, basal NF-κB activity was still observed; however, LPS-induced increase was not observed. In the 24-h pretreated cells, neither basal NF-κB activity nor LPS-induced activation was observed. Thus, LPS pre-exposure significantly reduced NF-κB DNA-binding activity.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Reduced activation of NF-κB and IRAK in LPS-tolerant macrophages. A, Peritoneal macrophages were incubated with or without 100 ng/ml LPS for 3 and 24 h. Cells were washed and then stimulated with 10 ng/ml LPS for 20 min. After the second stimulation, nuclear extracts were prepared and incubated with a specific probe containing NF-κB binding sites, and NF-κB activity was determined by a gel mobility shift assay. B, After the second stimulation for 10 min, cells were lysed and the lysates were immunoprecipitated with anti-IRAK-1 Ab. The kinase activity of IRAK was analyzed by an in vitro kinase assay (upper). The same lysates were immunoblotted with anti-IRAK-1 Ab (lower).

IRAK is known to be a downstream kinase of MyD88, which acts as an adaptor molecule in the LPS-signaling pathway (26). Stimulation with LPS for 10 min induced phosphorylation of IRAK in nontreated macrophages (Fig. 2⇑B). However, IRAK activation was not observed in cells pre-exposed to LPS for 3 and 24 h. Thus, LPS-induced IRAK activation was severely reduced in cells pre-exposed to LPS. We have previously shown that LPS-induced IRAK activation was not observed in both MyD88-deficient and TLR4-deficient mice (22, 26). Both strains of knockout mice are unresponsive to LPS. TLR4 knockout mice also displayed no LPS-induced NF-κB activation. These findings are quite similar to the data in the macrophages pre-exposed to LPS for 24 h. Therefore, we hypothesized that tolerant macrophages are affected in the MyD88-dependent pathway, which is essential for LPS responsiveness.

Decreased expression of TLR4 after exposure to LPS

We analyzed expression of TLR4, an essential signaling receptor for LPS. The former study demonstrated that LPS stimulation transiently reduced mRNA expression of TLR4 in the macrophage cell line RAW264.7 (7). We also obtained similar results in RAW264.7 cells. When cells were stimulated with 100 ng/ml LPS for 2.5 h, TLR4 mRNA expression was severely reduced; however, the expression returned to the original level after a 20-h stimulation (Fig. 3⇓A). When mouse peritoneal macrophages were stimulated with 100 ng/ml LPS, TLR4 mRNA expression was also transiently suppressed (Fig. 3⇓B). In both types of cells, TLR4 mRNA expression at 24-h LPS treatment was almost the same level as that of nontreated cells. Thus, the mRNA expression pattern of TLR4 seems not to correlate with LPS tolerance.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

TLR4 mRNA expression in macrophages after LPS treatment. A, RAW264.7 cells were stimulated with 100 ng/ml LPS for the indicated periods. Total RNA was extracted and subjected to Northern blot analysis using TLR4 cDNA probe. B, Peritoneal macrophages were stimulated with 100 ng/ml LPS for the indicated periods. Total RNA was extracted and subjected to Northern blot analysis using a TLR4 cDNA probe. The same membrane was rehybridized with a GAPDH cDNA probe.

We next analyzed surface expression of TLR4 using recently generated mAb MTS510. MTS510 was shown to detect the complex of murine TLR4 and MD-2 and inhibit LPS-induced NF-κB activation and TNF production (28, 29). Almost all nontreated macrophages were positive for this Ab (Fig. 4⇓A). The specificity of this Ab was confirmed by the staining of macrophages from TLR4 knockout mice. In TLR4 knockout mice, macrophages were negatively stained with this Ab, indicating that a positive staining by MTS510 reflects a surface TLR4 expression (Fig. 4⇓A). When macrophages were stimulated with 100 ng/ml LPS for 1 h, expression of TLR4 was partially decreased. In addition, the surface expression of TLR4 was gradually decreased as the culture time continued. At the 24-h culture period, the surface expression of TLR4 was severely reduced (Fig. 4⇓B). This was in sharp contrast with the surface expression of CD14, an LPS-binding receptor. The surface CD14 expression was not significantly changed by LPS treatment, although the 24-h culture induced a slight decrease. When the LPS concentration was decreased, the reduction of TLR4 expression was partial (Fig. 4⇓C). These results completely parallel with the production of inflammatory cytokine shown in Fig. 1⇑, indicating that LPS tolerance occurs mainly by suppression of surface TLR4 expression. We further analyzed TLR4 expression after treatment with other stimuli such as IL-1β and peptidoglycan (PGN), both of which activate the MyD88-dependent pathway via other receptors than TLR4. IL-1β and PGN are shown to utilize the IL-1R and TLR2 as the signaling receptor, respectively (22). Both stimulations for 24 h did not affect surface TLR4 expression, indicating that the reduced expression of surface TLR4 is induced only when macrophages are exposed to LPS, although all of these stimuli activate the same MyD88-dependent pathway (Fig. 4⇓D). Interestingly, reduction of surface TLR4 expression after LPS treatment was observed even in C3H/HeJ mice in a manner similar to that in C3H/HeN mice (Fig. 4⇓E). C3H/HeJ mice are shown to have a mutation in the Tlr4 gene, leading to the impaired LPS-mediated MyD88-dependent signaling pathway (7, 8, 20). These observations indicate that down-regulation of TLR4 expression occurs independently of the MyD88-dependent signaling pathway.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Reduced surface expression of TLR4 on peritoneal macrophages after LPS treatment. A, Peritoneal macrophages from wild-type and TLR4^−/− mice were incubated with biotinylated MTS510, followed by PE-conjugated streptavidin. B and C, Peritoneal macrophages from C57BL/6 mice were cultured with 100 ng/ml LPS for the indicated periods (B) or with the indicated concentrations of LPS for 24 h (C). Then cells were stained with MTS510 and analyzed by flow cytometry. These data are representative of three independent experiments. D, Peritoneal macrophages were cultured with 200 U/ml IL-1β, 10 μg/ml PGN, or 100 ng/ml LPS for 24 h. Then cells were analyzed for TLR4-MD2 expression. E, Peritoneal macrophages from C3H/HeJ or C3H/HeN mice were cultured with or without 1 μg/ml LPS for 1 h and analyzed for TLR4-MD2 expression by flow cytometry.

The LPS-signaling pathway has been intensively investigated for a long time and recently remarkable progress has been made. TLR4 is found to be an essential receptor for LPS signaling (7, 8, 20). MyD88 and TRAF6 are shown to be its key signaling molecules (26, 27). However, a molecular mechanism for LPS tolerance remains unclear. In the present study, we present one of the mechanisms for LPS tolerance by demonstrating that LPS tolerance in macrophages correlates with suppression of the surface TLR4 expression.

However, there seems to exist other mechanisms for LPS tolerance than down-regulation of TLR4 expression. Although intracellular production of inflammatory cytokines was not completely blocked during 1–12-h pre-exposure, secretion of these cytokines into the culture supernatants in these periods was not observed (our unpublished data). This suggests that LPS tolerance does not occur solely due to suppression of the surface expression of TLR4. There must be some modifications of intracellular transport or stability of newly synthesized proteins in the LPS-tolerant macrophages. Interestingly, the nuclear extract from the macrophages pretreated for 24 h did not show the basal NF-κB-binding activity that is observed in the nuclear extract from untreated macrophages. Thus, although down-regulation of TLR4 expression is responsible for LPS tolerance, other mechanisms along with it seem to operate toward LPS tolerance during LPS pretreatment.