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The Journal of Immunology, 2006, 177: 70-76.
Copyright © 2006 by The American Association of Immunologists

ERK and p38 MAPK Signaling Pathways Negatively Regulate CIITA Gene Expression in Dendritic Cells and Macrophages1

Yongxue Yao*, Qi Xu2,*, Myung-Ja Kwon*, Ranyia Matta{dagger}, Yusen Liu{dagger}, Soon-Cheol Hong* and Cheong-Hee Chang3,*

* Department of Microbiology and Immunology, Indiana University School of Medicine and Walther Oncology Center, Indianapolis, IN 46202; and {dagger} Children’s Research Institute, Children’s Hospital, Department of Pediatrics, and Integrated Biomedical Science Graduate Program, Ohio State University, Columbus, OH 43205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CIITA is a master regulator for MHC class II expression, but the signaling events that control CIITA expression remain poorly understood. In this study, we report that both constitutive and IFN-{gamma}-inducible expression of CIITA in mouse bone marrow-derived dendritic cells (DC) and macrophages, respectively, are regulated by MAPK signals. In DC, the inhibitory effect of LPS on CIITA expression was prevented by MyD88 deficiency or pharmacological MAPK inhibitors specific for MEK (U0126) and p38 (SB203580), but not JNK (SP600125). In macrophages, LPS inhibited IFN-{gamma}-inducible CIITA and MHC class II expression without affecting expression of IFN regulatory factor-1 and MHC class I. Blocking ERK and p38 by MAPK inhibitors not only rescued LPS-mediated inhibition, but also augmented IFN-{gamma} induction of CIITA. Moreover, the induction of CIITA by IFN-{gamma} was enhanced by overexpressing MAPK phosphatase-1 that inactivates MAPK. Conversely, CIITA expression was attenuated in the absence of MAPK phosphatase-1. The down-regulation of CIITA gene expression by ERK and p38 was at least partly due to decreased histone acetylation of the CIITA promoter. Our study indicates that both MAPK and phosphatase play an important role for CIITA regulation in DC and macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class II molecules, pivotal for the adaptive immune system, are constitutively expressed in B cells and dendritic cells (DC),4 and inducible by IFN-{gamma} in macrophages (1, 2). Cell type-specific as well as IFN-{gamma}-inducible expression of MHC class II requires CIITA, a non-DNA-binding coactivator (3, 4, 5, 6, 7). Because CIITA is a critical regulator for MHC class II expression, effort has been put forth to study the molecular mechanisms that control its expression. CIITA expression is regulated mainly at the level of transcription, although it can be further modulated by changes in mRNA and protein stability (3, 8). Transcription of CIITA is driven by at least three distinct promoters known as pI, pIII, and pIV (9). All three promoters were reported to be inducible by IFN-{gamma} in macrophages, and both pI and pIII were found to be active in DC (10, 11). In contrast, pIII is exclusively used by B cells and plasmacytoid DC (9, 12), while pIV is essential for driving IFN-{gamma}-inducible CIITA expression in nonhemopoietic cells such as fibroblasts and thymic epithelial cells (13, 14). Activation of pIV by IFN-{gamma} depends on both STAT1 and IFN regulatory factor (IRF)-1 (15). IRF-1 expression is also controlled by STAT1, which explains the delayed kinetics of CIITA expression upon IFN-{gamma} stimulation relative to the rapid induction of other genes that are controlled only by STAT1 (16).

The TLR4 ligand LPS activates NF-{kappa}B and MAPK family members, including ERK, p38, and JNK (17). The MAPK family plays a crucial role in mediating the induction of proinflammatory cytokines (17, 18). Inactivation of MAPK is primarily achieved by MAPK phosphatases (MKP), including MKP-1, which is LPS inducible and thus acts as a negative feedback for LPS signaling (19, 20). In the absence of MKP-1, both DC and macrophages produce elevated levels of cytokines and, as a consequence, MKP-1-deficient mice are highly susceptible to LPS and undergo septic shock due to multiple organ failure (21).

LPS can trigger downstream signaling by both MyD88-dependent and -independent pathways (17, 22). In the absence of MyD88, LPS can still activate NF-{kappa}B and MAPK albeit with delayed kinetics (23). However, DC from MyD88-deficient mice failed to produce inflammatory cytokines upon LPS stimulation, indicating that cytokine production requires MyD88-mediated signaling. Interestingly, MyD88–/– DC retain the ability to up-regulate costimulatory and MHC molecules on the cell surface, which is Toll/IL-1 recptor domain-containing adaptor-inducing IFN-beta dependent (24). This increase in MHC class II on the cell surface results from changes in the intracellular localization and the increase in the stability of pre-existing MHC class II proteins (25). In contrast, de novo MHC class II synthesis is shut down in LPS-treated cells as a result of transcriptional inactivation of the CIITA gene (11). However, signaling pathways responsible for this process are not well defined.

In the present study, we demonstrate that LPS down-regulated CIITA expression in DC via MyD88-dependent signaling pathway that involves ERK and p38 MAPK. Similarly, LPS prevented IFN-{gamma}-inducible CIITA and MHC class II expression in macrophages. We also found that IFN-{gamma} itself activated ERK and p38 MAPK in macrophages, and the induction of CIITA by IFN-{gamma} was enhanced by overexpressing MKP-1, but was attenuated in the absence of MKP-1. The down-regulation of IFN-{gamma}-inducible CIITA by LPS was at least partly due to decreased histone acetylation of the CIITA promoter. Taken together, MAPK and MKP-1 play an important role for CIITA gene regulation in professional APC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Murine rGM-CSF, rIL-4, and rIFN-{gamma} were purchased from BD Pharmingen, and rM-CSF was obtained from R&D Systems. LPS (Escherichia coli O55:B5 serotype) and actinomycin D were obtained from Sigma-Aldrich. MAPK inhibitors U0126 and SB203580 were from Promega; SP600125 was from Sigma-Aldrich.

Mice and cells

C57BL/6 mice were purchased from The Jackson Laboratory. MyD88–/– and MKP-1–/– mice were previously described (21, 26). All mice were maintained under specific pathogen-free conditions at the Indiana University School of Medicine and the Ohio State University Columbus Children’s Research Institute animal facilities.

Bone marrow (BM)-derived DC (BMDC) were prepared, as previously described (27). In brief, total BM cells depleted of RBC, T, and B cells were cultured 5 days in RPMI 1640 supplemented with 5% FBS and 10 ng/ml murine rGM-CSF and rIL-4. DC were replated at 1 x 106 cells/ml and treated under the condition indicated. LPS stimulation was performed using 1 µg/ml LPS for 24 h.

BM-derived macrophages were generated similarly as BMDC, except that cells were cultured for 10 days in the presence of 10 ng/ml murine rM-CSF. Cells were stimulated by 10 ng/ml IFN-{gamma} with or without 100 ng/ml LPS for 6 h. Resident peritoneal macrophages were isolated by peritoneal lavage and stimulated with 5 ng/ml IFN-{gamma} for 24 h.

RAW264.7 cells were maintained in DMEM supplemented with 10% FBS. The stable RAW264.7 clones expressing either MKP-1 (PC39) or its control empty vector (D2) were previously described and maintained in medium containing 400 µg/ml G418 (28).

FACS analysis

Abs used for flow cytometry, MHC class I (H-2Kd, clone SF1-1.1), and MHC class II (I-Ek, clone 14-4-4S) were obtained from BD Biosciences. Flow cytometric analysis was performed using FACSCalibur and analyzed using CellQuest software (BD Biosciences).

RT-PCR and quantitative real-time PCR

Total RNA preparation, cDNA synthesis, and PCR were conducted, as described (6). The following primers were used: total CIITA (5'-GCATGCCCGAACCTGCGCTGA-3' and 5'-GGCCATCTTGGGCCTCTAGCT-3'), MHC class II (I-E{alpha}d; 5'-CCAGAAGTCATGGGCTATCA-3' and 5'-GGCTCCTTGTCGGCGTTCTA-3'), and IRF-1 (5'-GGAAGTGAAGGATCAGAGTAGG-3' and 5'-GAGTCCATATTCTTCATCTCC-3'). Primers for amplifying hypoxanthine guanine phosphoribosyltransferase (HPRT) were described previously (29).

Quantitative real-time PCR was performed by the comparative threshold cycle ({Delta}CT) method and normalized to GAPDH. The primers used for types I, III, and IV CIITA, IL-10, and GAPDH were as described (10, 27). The primers used for total CIITA were 5'-CGTGCAGACCCAGAGGCT-3' and 5'-GGAAGATCCTTGGCTGCATC-3', for MHC class II (I-A{alpha}b) were 5'-CTGTCTGGATGCTTCCTGAGTTT-3' and 5'-CAGCTATGTTTTGCAGTCCACC-3', and for IRF-7 were 5'-CGCACAGTGCTACAGGCAGT-3' and 5'-TGTACAGGAACACGCATCTGG-3'.

Western blot

Cells were lysed with 1x SDS sample buffer. Protein was separated by 10% SDS-PAGE and electroblotted onto Hybond nitrocellulose membrane. The membrane was then incubated with anti-phospho-STAT1 (Tyr701) Ab, anti-phospho-ERK1/2 Ab, anti-phospho-p38 (Cell Signaling Technology), or anti-IRF-1 Ab (Santa Cruz Biotechnology), and developed by using the Amersham ECL system, according to the manufacturer’s instructions. As a loading control, the blot was stripped and reprobed with anti-ERK1 Ab (Santa Cruz Biotechnology).

Transient transfection and luciferase assays

The pIV-driven luciferase plasmid was constructed by subcloning 1.1 kb (–1046 ~ +58 bp) of the pIV promoter of the mouse CIITA gene into the pGL2 basic (Promega). A total of 107 RAW246.7 cells was transiently transfected with 8 µg of the pIV-luciferase reporter plasmid using the Lipofectamine Plus reagents (Invitrogen Life Technologies). Four hours after transfection, cells were divided and rested overnight before receiving different treatments for 8 h. Cell lysates were prepared and used for luciferase assays, as previously described (30). Relative luciferase activity was normalized by protein concentration.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed essentially according to Upstate Biotechnology’s protocol and as described previously (31). In brief, 1 x 107 cells were treated 10 min with 1% formaldehyde to cross-link DNA-binding proteins to the DNA and lysed in SDS-containing buffer. Cell extracts were sonicated to sheer DNA to ~500 bp and immunoprecipitated overnight with Ab specific for acetylated histone H4 (Upstate Biotechnology). The recovered protein-nucleic acid complexes were incubated 4 h with 0.4 M sodium chloride at 65°C to reverse cross-links. Purified DNA fragments were amplified 30 cycles using PCR and analyzed on 1.5% agarose gels. Immunoprecipitations with normal rabbit serum served as a negative control, and PCR for the proximal promoter of the mouse HPRT gene was used as an internal control. The primers used for the type IV CIITA promoter were 5'-AGCAAACTTGGGTTGCATGT-3' and 5'-TCCTGGCAGCTATCTCACAA-3'. The primers used for the HPRT promoter were as described (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPS down-regulates CIITA via MyD88 and MAPK signaling pathways in BMDC

LPS is known to down-regulate CIITA gene expression in DC (11). To elucidate the signaling pathways responsible for this down-regulation, we first examined the role of MyD88. BM cells from wild-type (WT) or MyD88–/– mice were cultured with GM-CSF for 5 days, followed by stimulation with LPS for 24 h. BMDC from MyD88–/– mice expressed slightly lower levels of both CIITA and MHC class II mRNA before LPS stimulation (Fig. 1A). However, unlike DC from control mice, LPS was unable to down-regulate CIITA and MHC class II mRNA expression in MyD88–/– DC (Fig. 1A). Consistent with a previous report that MyD88 is essential for cytokine production (17), the IL-10 gene was not induced in MyD88–/– DC upon LPS stimulation (Fig. 1A). In contrast, induction of IRF-7 gene expression by LPS was not affected by MyD88 deficiency (Fig. 1A). Therefore, similar to cytokine gene regulation, MyD88 is a critical signaling intermediate delivering the inhibitory effect of LPS on CIITA and MHC class II expression in DC.


Figure 1
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  		FIGURE 1. LPS down-regulates CIITA expression via MyD88 and MAPK signaling pathways in BMDC. A, BMDC from C57BL/6 WT and MyD88–/– mice were stimulated by LPS for 24 h. Quantitative real-time PCR was performed to assess the amount of CIITA, MHC class II, IRF-7, and IL-10 mRNA. B, BMDC from C57BL/6 mice were pretreated with indicated inhibitors (10 µM) for 30 min, followed by overnight LPS stimulation. Quantitative real-time PCR was performed to measure CIITA mRNA. The relative mRNA levels were normalized to the GAPDH gene. C, Quantification of the three types of CIITA mRNA levels relative to GAPDH. Data are means ± SE of at least three independent experiments. DM, DMSO; U, U0126; SB, SB203580; SP, SP600125.

 
It is well known that MAPK signaling plays a crucial role in mediating MyD88-dependent induction of cytokines (17, 18). To determine whether MAPK signaling is responsible for CIITA down-regulation by LPS, we used pharmacological reagents that inhibit MAPK pathways with different specificity: U0126, SB203580, and SP600125, selectively inhibiting MEK1/2, p38, and JNK, respectively. BMDC were pretreated with each inhibitor for 30 min, followed by LPS stimulation for overnight. U0126 and SB203580, but not SP600125 treatment blocked the inhibitory effect of LPS (Fig. 1B), suggesting that LPS down-regulates CIITA via the ERK and p38 signaling pathways. DC express three different types of CIITA mRNA (11), and therefore, we wanted to determine whether MAPK pathways regulate all CIITA mRNA species. Consistent with the published data (11), type I and III CIITA were the major form in DC (Fig. 1C). Quantification of each type of CIITA mRNA revealed that LPS suppressed all types of CIITA, but at a different degree. The amount of type I mRNA was greatly diminished by LPS, but the type III and IV levels were less affected (Fig. 1C). In addition, type I CIITA expression was partially restored, whereas both type III and IV CIITA were completely recovered by U0126 or SB203580 treatment (Fig. 1C), suggesting that type I CIITA expression is regulated by an additional signaling.

ERK and p38 MAPK are responsible for down-regulation of IFN-{gamma}-inducible CIITA expression by LPS in macrophages

LPS also prevents IFN-{gamma}-inducible MHC class II expression in macrophages, but molecular mechanism(s) underlying this inhibitory effect has not been defined (32). It is possible that, similar to what occurred in DC, activation of MAPK signaling in response to LPS inhibits the CIITA induction by IFN-{gamma} and subsequent MHC class II expression. We tested this hypothesis by treating RAW246.7 cells with IFN-{gamma} alone or IFN-{gamma} together with LPS for 24 h. As shown in Fig. 2A, IFN-{gamma}-inducible expression of MHC class II on the cell surface was reduced in the presence of LPS. This inhibitory effect was not global because there was little difference in MHC class I expression with or without LPS (Fig. 2A). The reduction of MHC class II expression on the cell surface correlated with the loss of MHC class II mRNA, presumably due to the absence of CIITA gene expression in LPS-treated cells (Fig. 2B). However, the expression of IRF-1, another IFN-{gamma}-inducible gene (15), was not altered by LPS. It has been shown that the predominant forms of CIITA mRNA in IFN-{gamma}-treated macrophages are type I and IV CIITA (10). Therefore, we assessed the level of each type of CIITA mRNA in RAW264.7 cells treated by IFN-{gamma} alone or IFN-{gamma} with LPS. Similar to DC shown in Fig. 1C, LPS inhibited the expression of all three types of CIITA (Fig. 2C).


Figure 2
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  		FIGURE 2. LPS prevents IFN-{gamma}-inducible expression of CIITA and MHC class II in macrophages. RAW264.7 cells were stimulated by IFN-{gamma} with or without LPS for 24 h. A, MHC class II and class I expression was analyzed by flow cytometry. B, RT-PCR was performed to measure MHC class II, CIITA, and IRF-1 mRNA. C, Expression of different CIITA isoforms. The relative mRNA levels were normalized to the GAPDH gene. The mRNA level of each isoform expressed in IFN-{gamma}-treated cells was set at 1. Data are representative (A and B) or means ± SE (C) of three independent experiments.

 
To determine whether ERK and p38 MAPK also play a role in CIITA inhibition by LPS in macrophages, RAW264.7 cells were pretreated with MAPK inhibitors for 30 min and then subjected to stimulation by IFN-{gamma} in the presence or absence of LPS for 6 h. Two interesting observations emerged. First, unlike their effect in DC, neither U0126 nor SB203580 alone completely restored CIITA expression in the presence of LPS (Fig. 3A). These data indicate that blocking any single MAPK pathway is not sufficient to prevent the inhibitory effect of LPS in macrophages. Second, U0126 and, to a less extent, SB203580 enhanced CIITA expression when the inhibitor was added together with IFN-{gamma} (Fig. 3A). Because we were not able to maintain CIITA expression by blocking a single MAPK pathway, we next asked whether preventing two pathways simultaneously could rescue LPS-mediated inhibition. Indeed, a combination of U0126 and SB203580 completely abolished the inhibitory effect of LPS on CIITA expression without affecting IRF-1 expression (Fig. 3B). Quantification of the different types of CIITA mRNA showed that either U0126 or SB203580 enhanced the expression of all three CIITA types in IFN-{gamma}-treated cells, but was not sufficient to prevent LPS-mediated inhibition (Fig. 3C; note that the absolute levels of type III CIITA were so low that the bars are barely visible). However, U0126 and SB203580 together completely reversed LPS-mediated inhibition of type III and IV CIITA expression, but only partially rescued type I CIITA inhibition by LPS (Fig. 3C).


Figure 3
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  		FIGURE 3. Inhibition of ERK and p38 restores IFN-{gamma}-inducible CIITA expression in LPS-treated macrophages. A, RAW264.7 cells were pretreated with indicated inhibitors (10 µM) for 30 min, followed by IFN-{gamma} stimulation with or without LPS for 6 h. Total CIITA mRNA levels were determined by quantitative real-time PCR. B, RAW264.7 cells were treated as indicated for 6 h. RT-PCR was performed to detect CIITA and IRF-1 mRNA expression. C, RAW264.7 cells were treated as indicated. Expression of different CIITA isoforms was determined by quantitative real-time PCR, and the levels were compared with that of GAPDH. D, BM macrophages from C57BL/6 mice were pretreated with indicated inhibitors (10 µM) for 30 min, followed by IFN-{gamma} stimulation with or without LPS for 6 h. Type IV CIITA mRNA levels were measured by quantitative real-time PCR. The relative mRNA levels were normalized to the GAPDH gene. Data are means ± SE (A, C, and D) or representative (B) of three independent experiments. DM, DMSO; U, U0126; SB, SB203580; SP, SP600125.

 
To determine whether macrophages prepared from mice show a similar response to LPS as observed in a macrophage cell line, we generated BM-derived macrophages and treated them the same way as RAW264.7. Because the type IV was affected mostly in RAW264.7 cells, we assessed the amount of type IV CIITA. Consistent with the data from RAW264.7, both U0126 and SB203580 alone enhanced IFN-{gamma}-inducible expression of type IV CIITA. Moreover, the two inhibitors together prevented the down-regulation of type IV CIITA expression by LPS (Fig. 3D).

MKP-1 regulates CIITA expression in both macrophages and DC

The effect of MAPK inhibitors suggests that ERK and p38 MAPK signaling is a negative regulator of CIITA gene expression. If so, MKP-1 would have an opposite effect on CIITA expression because MKP-1 inactivates MAPK (19, 20). We tested this hypothesis by two approaches: overexpression and deficiency of MKP-1. We first compared CIITA and MHC class II expression between RAW264.7 cells stably expressing MKP-1 and those harboring an empty vector (28). As shown in Fig. 4A, overexpression of MKP-1 in RAW264.7 cells, designated as PC39, augmented MHC class II expression on the cell surface as well as mRNA upon IFN-{gamma} treatment (Fig. 4, A and B). As expected, CIITA expression was also enhanced by MKP-1 overexpression (Fig. 4B). Again, IRF-1 expression was not affected by MKP-1 overexpression (Fig. 4B). These data suggest that MKP-1 prevents the activation of MAPK, which in turn allows maintaining CIITA and MHC class II induction by IFN-{gamma}. If this were the case, the deficiency of MKP-1 would show an opposite effect. To test this, we prepared macrophages resident in the peritoneal cavity from control and MKP-1–/– mice and stimulated them with IFN-{gamma}. As shown in Fig. 4C, CIITA and MHC class II induction by IFN-{gamma} was dramatically attenuated in MKP-1–/– cells. We also examined BMDC from control and MKP-1–/– mice and found that the basal level of CIITA and MHC class II mRNA was slightly reduced in MKP-1–/– DC (Fig. 4D). More importantly, the inhibitory effect of LPS treatment was greater in MKP-1–/– DC than that in MKP-1+/+ cells, presumably due to the sustained MAPK activity in the absence of MKP-1. Together, both MKP-1 and MAPK are important regulators for CIITA and MHC class II gene expression in DC and macrophages.


Figure 4
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  		FIGURE 4. MKP-1 regulates CIITA expression in both macrophages and BMDC. A and B, RAW264.7 cells overexpressing MKP-1 (PC39) or its control vector (D2) were treated with IFN-{gamma} for 24 h. Cell surface MHC class II expression was detected by flow cytometry (A). RT-PCR was performed to determine MHC class II, CIITA, and IRF-1 mRNA levels (B). C and D, Resident peritoneal macrophages (C) and BMDC (D) from WT and MKP-1–/– mice were treated with IFN-{gamma} (C) or LPS (D), respectively, for 24 h. MHC class II and total CIITA mRNA levels were determined by quantitative real-time PCR. The relative mRNA levels were normalized to the GAPDH gene. *, Numbers indicate the fold decrease relative to the untreated control. Data are representative (A and B) or means ± SE (C and D) of three independent experiments.

 
ERK and p38 MAPK suppress CIITA expression by decreasing histone acetylation

There are at least two possibilities that account for the inhibitory effect of ERK and p38 on CIITA expression in DC and macrophages: decreased mRNA stability or transcription. To distinguish these possibilities, we first measured CIITA mRNA t1/2 and found that CIITA mRNA t1/2 was comparable with or without LPS or MAPK inhibitors in DC (Fig. 5A, left panel). Similarly, MEK and p38 inhibitors had little effect on CIITA mRNA stability in RAW264.7 cells (Fig. 5A, right panel). We next tested the effects of LPS and MAPK inhibitors on CIITA promoter activity. RAW264.7 cells were transiently transfected with the luciferase reporter driven by the type IV CIITA promoter. The same pool of transfected cells was then divided and received different treatments. As shown in Fig. 5B, pIV activity was enhanced by IFN-{gamma} treatment. However, unlike the endogenous CIITA gene, the transfected CIITA promoter was not affected by the presence of LPS or MAPK inhibitors (Fig. 5B). In addition, when transfected cells were pretreated with LPS for 4 h before IFN-{gamma} treatment, the pIV promoter activity was properly induced by IFN-{gamma} (data not shown). These data suggest that the pIV promoter used in this assay does not contain the LPS-responsive element, and that LPS may act on chromatin remodeling of the CIITA locus rather than affecting specific transcription factor(s).


Figure 5
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  		FIGURE 5. ERK and p38 MAPK regulate histone acetylation of the CIITA promoter. A, BMDC (left panel) and RAW264.7 cells (right panel) were pretreated with U0126 and/or SB203580 (10 µM) for 30 min, followed by LPS treatment for overnight or IFN-{gamma} treatment for 4 h, respectively. Actinomycin D (ActD, 5 µg/ml) was then added to stop RNA synthesis. The remaining CIITA mRNA levels were measured by quantitative real-time PCR at the indicated time points. B, RAW264.7 cells were transfected with the type IV CIITA promoter-driven luciferase reporter, pretreated with indicated inhibitors (10 µM) for 30 min, stimulated by IFN-{gamma} with or without LPS for 8 h, and harvested to assess luciferase activity. Relative luciferase activity was normalized by protein concentrations. C, RAW264.7 cells were pretreated with U0126 and SB203580 (10 µM) for 30 min, followed by IFN-{gamma} treatment with or without LPS for indicated time points. Total cell lysate was used for immunoblot to detect ERK1/2, p38, STAT1 activation, and IRF-1 expression. D, RAW264.7 cells were pretreated with U0126 and SB203580 (10 µM) for 30 min, followed by IFN-{gamma} treatment with or without LPS for 3 h. ChIP was performed using Abs specific for the acetylated histone H4 or normal rabbit serum. Purified DNA fragments were amplified using primers specific for the type IV CIITA or the HPRT promoter. Data are means ± SE (A and B) or representative (C and D) of at least three independent experiments. DM, DMSO; U, U0126; SB, SB203580.

 
Because activation of the pIV depends on STAT1 phosphorylation and IRF-1 induction, we assessed the status of STAT1 and IRF-1 in RAW264.7 cells under different conditions. As expected, STAT1 was phosphorylated upon IFN-{gamma} stimulation and remained phosphorylated in the presence of LPS and/or MAPK inhibitors (Fig. 5C). Similarly, IRF-1 expression was induced within 1 h and the level was increased after 2 h of IFN-{gamma} treatment. Again, the amount of IRF-1 was comparable under all conditions (Fig. 5C). On the contrary, phosphorylation of ERK and p38 was enhanced by IFN-{gamma} or LPS at a different degree and was abolished when cells were treated with MAPK inhibitors, indicating that each treatment worked properly (Fig. 5C). Therefore, these data showed that neither IRF-1 synthesis nor tyrosine phosphorylation of STAT1 is under a direct control of LPS signaling and that these two cannot account for down-regulation of CIITA gene expression.

LPS is reported to down-regulate MHC class II expression in DC by transcriptional silencing of CIITA via histone deacetylation of the entire CIITA locus (11). Therefore, we tested whether the inhibitory effect of LPS on CIITA expression in macrophages is also contributed by chromatin remodeling of the CIITA locus. Consistent with the previous study (16), histone acetylation of the type IV promoter was enhanced in IFN-{gamma}-treated cells, but decreased in cells that received LPS (Fig. 5D, compare lanes 2 and 3). However, blocking ERK and p38 signaling pathways completely restored histone acetylation (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The role of CIITA in regulating MHC class II expression has been greatly appreciated, but the signaling events governing its expression are not well understood. In the present study, we showed that both constitutive and IFN-{gamma}-inducible CIITA expression in DC and macrophages, respectively, are negatively regulated by ERK and p38 MAPK signals.

The cross-talk between LPS and IFN-{gamma} signaling pathways has long been recognized. IFN-{gamma} primes macrophages for more rapid and heightened responses to LPS. Conversely, LPS pretreatment is able to promote the IFN-{gamma} signaling pathway through triggering type I IFN production (33). However, the effects of LPS and IFN-{gamma} on MHC class II expression are rather complex. Sicher et al. (32) reported that LPS inhibits MHC class II when added simultaneously with IFN-{gamma}, while it augments MHC class II expression when added after IFN-{gamma}. They also showed that the LPS augmentation of MHC class II occurs at a posttranscriptional level. Interestingly, the increased density of cell surface MHC class II during DC maturation by LPS also occurs at a posttranscriptional level (25). In contrast, de novo MHC class II synthesis is shut down in LPS-treated DC as a result of transcriptional inactivation of the CIITA gene via histone deacetylation (11). Our data indicated that LPS exerts the same effect on CIITA and MHC class II gene expression in macrophages. We also have observed that splenic B cells express lower levels of CIITA upon LPS stimulation (data not shown). Therefore, it appears that all professional APC down-regulate their MHC class II expression by turning off CIITA transcription when they are treated with LPS.

In addition to LPS, other TLR agonists are reported to modulate IFN-{gamma} signaling and to regulate IFN-{gamma}-inducible CIITA and MHC class II expression (34, 35, 36). CpG induces suppressor of cytokine signaling (SOCS) proteins to inhibit JAK-STAT signaling, while a 19-kDa lipoprotein produced by Mycobacterium tuberculosis inhibits CIITA independent of SOCS-1 and STAT1 activation. We observed that LPS did not affect JAK-STAT signaling and SOCS-1 expression in RAW264.7 cells (Fig. 5C and data not shown). Therefore, different TLR agonists seem to use different strategies to interfere with CIITA expression.

Recent studies have established that, in addition to the well-known classical JAK-STAT pathway, IFN-{gamma} activates multiple signaling cascades, including MAPK (37). The JAK-STAT signaling can be amplified by p38 through STAT1 phosphorylation on serine 727 (38). ERK activates C/EBP-dependent gene transcription in response to IFN-{gamma} (39). However, our data suggest that ERK and p38 MAPK not only mediate CIITA inhibition by LPS, but also act as a negative feedback for CIITA induction by IFN-{gamma}. This is supported by the fact that IFN-{gamma}-inducible CIITA expression was augmented by MEK and p38 inhibitors and by overexpressing MKP-1, but was attenuated by MKP-1 deficiency. In contrast, an opposite effect of MAPK inhibitors on IFN-{gamma}-inducible CIITA expression was reported in human astroglioma cell lines (40). This discrepancy could be explained by species and/or cell type specificity. Indeed, we have observed that IFN-{gamma} activates MAPK in a cell-type specific manner: it activated ERK and p38 in RAW264.7 cells, but not in NIH3T3 fibroblasts (Fig. 5C and data not shown), and MEK and/or p38 inhibitors had no effect on IFN-{gamma}-inducible CIITA expression in NIH3T3 cells (data not shown). In addition, our data indicate that CIITA is regulated differently in DC and macrophages. First, in DC, LPS suppressed type I CIITA more dramatically than type III or IV CIITA, whereas LPS prevented the induction of all three types of IFN-{gamma}-inducible CIITA in macrophages (Figs. 1C and 2C). Second, MEK or p38 inhibitor alone completely rescued LPS-mediated repression of type III and IV CIITA in DC. However, a combination of MEK and p38 inhibitors was required to prevent type III and IV CIITA inhibition by LPS in macrophages, suggesting a cooperation between ERK and p38 MAPK pathways (Figs. 1C and 3C). We speculate that these differences may be due to the additional IFN-{gamma} signaling component in macrophages. The outcome of CIITA expression is most likely the result of a cross-regulation between MAPK and IFN-{gamma} signaling pathways in macrophages. In contrast, CIITA expression in DC does not involve IFN-{gamma} signaling, and therefore the effect of LPS/MAPK is not the same between the two cell types. Lastly, the regulation of type I CIITA seems to be different from type III and IV CIITA because the level of type I CIITA was partially restored by MEK and/or p38 inhibitors in both DC and macrophages (Figs. 1C and 3C), suggesting that other signaling pathways, in addition to ERK and p38 MAPK, are most likely involved in the regulation of type I CIITA expression.

Blocking ERK and/or p38 signaling pathways rescued LPS-inhibited CIITA mRNA expression in DC and augmented IFN-{gamma}-inducible CIITA mRNA expression in macrophages without affecting its mRNA stability, suggesting of transcriptional regulation. Yet, in a transient transfection assay, neither LPS nor MAPK pathway inhibitors altered the IFN-{gamma}-inducible activity of the CIITA pIV promoter that contains the IFN-{gamma} activation site, E box, and IRF element, all of which are required for its activation (3). This finding is not surprising because LPS did not have a global inhibitory effect on IFN-{gamma}-inducible genes. LPS selectively inhibited IFN-{gamma}-inducible CIITA and MHC class II expression, but had no effect on IRF-1 and MHC class I expression, indicating that JAK-STAT signaling is intact. Our ChIP data revealed that LPS prevents IFN-{gamma}-induced CIITA expression in macrophages and down-regulates constitutive CIITA expression in DC through a same mechanism, that is, by decreasing histone acetylation at the chromatin of the CIITA promoter.

In conclusion, our current studies demonstrate that CIITA expression is negatively regulated by ERK and p38 MAPK signals in DC and macrophages. Although the underlying mechanisms by which ERK and p38 MAPK signals regulate the CIITA locus need to be investigated, it is likely that these signals affect chromatin remodeling of the CIITA locus rather than specific transcription factor(s) based on the published report and our current study. A better understanding of signaling events will provide insights into the molecular mechanisms underlying not only LPS-mediated reduction, but also IFN-{gamma}-mediated induction of the CIITA gene expression.


   Acknowledgments
 
We thank Dr. Shizuo Akira for providing the MyD88–/– mice, and Bristol-Myers Squibb Pharmaceutical Research Institute for providing the MKP-1–/– mice. We also thank Brian McCarthy for technical help and Dr. Wei Li for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants AI56097 (to C.-H.C.), DE13988 (to S.-C.H.), and AI57798 (to Y.L.), and American Heart Association Postdoctoral Fellowship Award 0520111z (to Y.Y.). Back

2 Current address: MedImmune Vaccine, 297 North Bernardo Avenue, Mountain View, CA 94043. Back

3 Address correspondence and reprint requests to Dr. Cheong-Hee Chang, Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, 950 West Walnut Street R2-302, Indianapolis, IN 46202-5188. E-mail address: chechang{at}iupui.edu Back

4 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; BMDC, BM-derived DC; ChIP, chromatin immunoprecipitation; HPRT, hypoxanthine guanine phosphoribosyltransferase; IRF, IFN regulatory factor; MKP, MAPK phosphatase; SOCS, suppressor of cytokine signaling; WT, wild type. Back

Received for publication December 20, 2005. Accepted for publication April 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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