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The Journal of Immunology, 2007, 179, 5378 -5386
Copyright © 2007 by The American Association of Immunologists, Inc.
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The C/EBPbeta Isoform 34-kDa LAP Is Responsible for NF-IL-6-Mediated Gene Induction in Activated Macrophages, but Is Not Essential for Intracellular Bacteria Killing1

Satoshi Uematsu*, Tsuneyasu Kaisho{ddagger}, Takashi Tanaka{ddagger}, Makoto Matsumoto§, Megumi Yamakami, Hiroko Omori, Masahiro Yamamoto*, Tamotsu Yoshimori and Shizuo Akira2,*,{dagger}

* Department of Host Defense, Research Institute for Microbial Diseases, Osaka University and {dagger} Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Osaka, Japan; {ddagger} Laboratory for Host Defense, Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Kanagawa, Japan; § Department of Immunology and Medical Zoology, Hyogo College of Medicine, Nishinomiya, Japan; and Department of Cell Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The C/ebpb gene is translated into three different protein isoforms, two transcriptional activating proteins (38-kDa Full and 34-kDa liver-enriched transcriptional activation protein (LAP)) and one transcriptional inhibitory protein, by alternative use of different AUG initiation codons within the same open reading frame. The isoform 34-kDa LAP is thought to be the most transcriptionally active form of C/EBPbeta in macrophages. To assess the function of the 34-kDa LAP in vivo, we generated knock-in mice, in which methionine 20 of C/EBPbeta, the start site for the 34-kDa LAP is replaced with an alanine. The expression of the 34-kDa LAP was abolished in C/ebpbM20A/M20A mice. The induction of C/EBPbeta target genes, such as inflammatory cytokines, chemokines, prostanoid synthetase, and antimicrobial peptides, was abolished in C/ebpbM20A/M20A macrophages, and C/ebpbM20A/M20A mice were susceptible to Listeria monocytogenes infection. Furthermore, the heat-killed Propionibacterium acnes-induced Th1 response, granuloma formation, and LPS shock were severely impaired. Nevertheless, impairment of intracellular bacteria killing, which is the most prominent phenotype in C/EBPbeta-deficient mice, was not observed in C/ebpbM20A/M20A mice. Collectively, we demonstrated that 34-kDa LAP is responsible for NF-IL6-mediated gene induction, but not essential for intracellular bacteria killing in activated macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nuclear factor-IL6 was originally identified as a protein that binds to the IL-1 response element of the human IL-6 gene (1). The cDNA of NF-IL6 exhibits homology with C/EBP, which belongs to the larger family of basic leucine zipper transcription factors (2). NF-IL6 has been reported by other groups under the names LAP (3), AGP/EBP (4), IL-6DBP (5), rNFIL-6 (6), C/EBPbeta (7), and CRP2 (8). C/EBPbeta (NF-IL6) exhibits low translational activity unless activated by inflammatory stimuli, which induce phosphorylation of C/EBPbeta and augment its transcriptional activity (9, 10, 11). C/EBPbeta is dramatically induced during macrophage differentiation and C/EBPbeta-binding motifs are found in the functional regulatory regions of genes specifically induced in activated macrophages, such as the those encoding IL-6, IL-1{alpha}, IL-8, TNF-{alpha}, G-CSF, NO synthase, and lysozyme (2, 12, 13). Overexpression of C/EBPbeta protein indicated that IL-6, macrophage chemoattractant protein 1, MIP-1{alpha}, MIP-1beta, osteopontin, CD14, and lysozyme are target genes of C/EBPbeta in several hemopoietic cell lines (14, 15, 16). Thus, C/EBPbeta has been shown to be necessary for the coordinate expression of a group of macrophage-specific genes and for the acquisition of the macrophage phenotype (17). Meanwhile, C/EBPbeta-deficient mice were generated and analyzed extensively. C/EBPbeta-deficient mice were highly susceptible to infections with microorganisms such as Listeria monocytogenes, Salmonella typhimurium, and Candida albicans (18, 19). C/EBPbeta-deficient peritoneal macrophages were defective in the intracellular killing of L. monocytogenes and displayed impaired tumoricidal and tumoristatic activity (18). The induction of some LPS-inducible genes, such as G-CSF (18), Mincle (20), and mPGES-1 (21), was impaired in C/EBPbeta-deficient macrophages, suggesting that C/EBPbeta is a crucial mediator of inflammation in activated macrophages.

The C/ebpb gene is intronless (Fig. 1A) and produces several proteins that have specific transcriptional regulatory functions (22). This is accomplished by alternative use of different AUG initiation codons within the same open reading frame (Fig. 1B). The presence of a small open reading frame 5' to the second AUG initiation codon of C/ebpb mRNA is likely to be essential for the leaky ribosome-scanning mechanism that causes some ribosomes to ignore the first AUG codon and start translation at internal AUGs. The C/ebpb gene is translated into three different protein isoforms; the liver-enriched transcriptional activating proteins (LAPs),3 38 kDa (Full) and 34 kDa (LAP), which function as activators of transcription, and the liver-enriched transcription inhibitory protein (LIP; 20 kDa), which lacks most of the transactivation domain and, therefore, acts as a dominant-negative transcriptional repressor (22) (Fig. 1B). LIP not only binds to the C/EBP consensus sequence with a higher affinity than the LAPs, but also forms heterodimers with LAP to inhibit transactivation at substoichiometric ratios (22). The ratio of these isoforms varies depending on the cell type and developmental stage and can be altered to activation dominance by IL-6 or other extracellular signals such as retinoic acid (22, 23). Therefore, the ratio of LIP:LAP is an important determinant of overall C/EBPbeta function.


Figure 1
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  		FIGURE 1. C/EBPbeta isoforms. A, DNA sequences of the gene encoding C/EBPbeta. The ATG start sites of the three protein isoforms are underlined. B, Map of the translation start sites in the C/EBPbeta mRNA. The positions of the AUG initiation sites in each mRNA are indicated. The nucleotide number indicates the position of the A in each initiation codon. C, thioglycolate-elicited peritoneal macrophages from wild-type and C/EBPbeta-deficient mice were cultured with or without 1 µg/ml LPS for 12 h, followed by Western blot analysis with an Ab specific for C/EBPbeta. Equal loading was controlled by reprobing the membrane with an Ab to total ERK.

 
Although the importance and uniqueness of C/EBPbeta isoforms have been shown in many studies, the in vivo functions of these proteins have not been fully elucidated. Because all of the isoforms are diminished in C/EBPbeta-deficient mice, it has been quite difficult to examine the function of each isoform in vivo. To ascertain the role of the 34-kDa LAP, we generated C/ebpbM20A/M20A knock-in mice by gene targeting. The expression of the 34-kDa LAP was abolished in C/ebpbM20A/M20A mice. In this study, we demonstrated that the 34-kDa LAP is responsible for NF-IL6-mediated gene induction in activated macrophages, but is not essential for intracellular bacteria killing.


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

C/EBPbeta-deficient mice have been described previously (18). All animal experiments were performed under an experimental protocol approved by the Ethics Review Committee for Animal Experimentation of Research Institute for Microbial Diseases (Osaka University, Osaka, Japan).

Cells, reagents, and bacteria

Preparation of peritoneal macrophages was described previously (18). LPS from Salmonella minnesota Re-595, prepared using a phenol-chloroform-petroleum ether extraction procedure, was purchased from Sigma-Aldrich. Thioglycolate broth (Brewer’s formula) was purchased from Difco. IFN-{gamma} was obtained from Genzyme Techne. Restriction and DNA modification enzymes were products of Toyoba. L. monocytogenes was described previously (18). Heat-killed Propionibacterium acnes was provided by H. Tsutsui (Hyogo College of Medicine, Nishinomiya, Japan).

Western blot analysis

Peritoneal macrophages (1 x 105) were stimulated with LPS plus IFN-{gamma} for the indicated periods. The cells were then lysed in a lysis buffer containing 1.0% Nonidet P-40, 150 mM NaCl, 20 mM Tris-Cl (pH 7.5), 5 mM EDTA, and a protease inhibitor mixture (Roche). Whole cell lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with blocking buffer containing 5.0% skim milk. Membranes were incubated with the indicated Abs and then visualized using an ECL system (NEN Life Science Product). Anti-C/EBPbeta (C-19; sc-150) and anti-ERK1 (K23; SC-94) Abs were obtained from Santa Cruz Biotechnology.

Generation of C/ebpbM20A/M20A knock-in mice

An 11-kb genomic fragment spanning from 8.5 kb upstream of the C/ebpb transcription initiation site to 2.3 kb 3' of the end of the exon was subcloned into a pUC18 plasmid vector. Site-directed mutagenesis was performed to change methionine 20 to alanine within the C/ebpb exon. A targeting vector was constructed by replacing the C/ebpb exon with the C/ebpbM20A/M20A exon and pMC1-neo (Stratagene); this vector contained a HSV-thymidine kinase cassette at the 5' end of the vector. The targeting vector was linearized with SalI and electroporated into E14.1 embryonic stem (ES) cells. The clones resistant to G418 and gancyclovir were screened for homologous recombination by PCR and confirmed by Southern blot analysis using the probe indicated in Fig. 2A. Chimeric mice were generated by microinjection of the targeted ES clones into C57BL/6 blastocysts. Male chimeric mice were bred with C57BL/6 females to produce heterozygous mice. Heterozygous mice were interbred to obtain homozygotes.


Figure 2
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  		FIGURE 2. Generation of C/ebpbM20A/M20A mice. A, The structure of the C/ebpb gene, the knock-in vector, and the predicted mutated allele. Closed box denotes the coding exon. The knock-in vector contained mutant mouse C/ebpb (C/ebpbM20A/M20A) cDNA. B, Southern blot analysis of offspring from heterozygote intercrosses. Genomic DNA was extracted from mouse tails, digested with HincII, electrophoresed, and hybridized with the radiolabeled probe indicated in A. Southern blotting resulted in the detection of a single 3.2-kb band from wild-type mice (+/+), a 4.4-kb band from homozygous mice (–/–), and both bands from heterozygous mice (+/–). C, The sequence of the C/ebpb exon in the tail genome of C/ebpbM20A/M20A mice. ATG for methionine 20 was replaced with GCG. D, Thioglycolate-elicited peritoneal macrophages from wild-type and C/ebpbM20A/M20A mice were cultured with or without 1 µg/ml LPS for 12 h followed by Western blot analysis with Abs specific for the indicated molecules.

 
Northern blot analysis

Total RNA was extracted from peritoneal macrophages (5 x 106) using TRIzol reagent (Invitrogen Life Technologies). Total RNA (5 µg) was electrophoresed, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA probes specific for Arg1, Ptges, Cxcl1, and Il12a, as described previously (21). The same membrane was rehybridized with a cDNA specific for Actb.

Measurement of proinflammatory cytokine concentrations

To evaluate the production of cytokines by macrophages in vitro, thioglycolate-elicited peritoneal cells were seeded onto 96-well-plates (2 x 105 cells/well) and stimulated with the indicated reagents for 24 h. The concentrations of TNF-{alpha}, IL-6, IL-12p40, IL-12p70, and G-CSF were measured by ELISA according to the manufacturer’s instructions (Genzyme for TNF-{alpha} and IL-12 p40, BD Biosciences for IL-12p70, and R&D Systems for IL-6 and G-CSF).

Microarray analysis

Peritoneal macrophages collected from wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A knock-in mice were treated with or without LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 12 h. Total RNA was extracted using an RNeasy kit (Qiagen); dsDNA was synthesized from 10 µg of total RNA using the SuperScript Choice System (Invitrogen Life Technologies) primed with T7-(dT) 24-mer primers. These cDNAs were used to prepare biotin-labeled cRNA by an in vitro transcription reaction performed using T7 RNA polymerase in the presence of biotinylated ribonucleotides according to the manufacturer’s protocol (Enzo Diagnostics). The cRNA product was purified using an RNeasy kit (Qiagen), fragmented, and hybridized to Affymetrix mouse expression array U74Av2 microarray chips according to the manufacturer’s protocol (Affymetrix). The hybridized chips were stained, washed, and scanned using a GeneArray Scanner (Affymetrix). Signal scaling was used for all probe sets and target intensity was adjusted to 500 points with GeneChip Operating software 1.0 (Affymetrix). Comparative analysis between genes was conducted on GeneSpring software version 6.0 (Silicongenetics). The signals were clustered by Pearson correlation. Complete microarray data have been deposited in the Gene Expression Omnibus database (accession code GSE7649).

In vitro killing assay

Peritoneal macrophages were plated on a 24-well plate at 1 x 106 cells/well and cultured with LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 20 h. After washing with PBS, 1 ml of RPMI 1640 plus 10% normal mouse serum without antibiotics was added to a 1 x 107 CFU/ml culture of L. monocytogenes. At the 30-min time point, wells were washed, 1 ml of 0.05% Triton X-100 was added per well, and the cells were lysed (time 0). Other wells were incubated with RPMI 1640 plus 10% FCS and 5 µg/ml gentamicin for 30 min to kill bacteria that were not phagocytized, washed with PBS, and incubated with RPMI 1640 plus 10% FCS for 5.5 h (time 6 h). These wells were washed and then lysed with 0.05% Triton X-100. The lysates were plated on tryptic soy agar. Listeria killing was determined by counting the number of CFU per well.

Fluorescence microscopy

Immunostaining of cells was performed as previously described (24). The primary Ab was rat monoclonal anti-mouse LAMP-1, which was used at 10 µg/ml (clone 1D4B; BD Biosciences). The secondary Ab was Alexa Fluor 594-conjugated anti-rat IgG (Molecular Probes) diluted 1/200. To label bacterial and cellular DNA, cells were stained with 1 µg/ml 4',6-diamino-2-phenylindole (Promega) in PBS. All fluorescence micrographs are confocal images acquired using a LSM510 laser-scanning microscope (Zeiss).

Electron microscopy

Peritoneal macrophages were cultured on 12-mm Cellocate (Eppendorf) in 24-well culture plates with complete medium containing LPS (100 ng/ml) plus IFN-{gamma} (100 U/ml) for 20 h, and then infected with L. monocytogenes for 30 min at a bacteria:macrophage ratio of 10:1 (18). Cells were then incubated with RPMI 1640 plus 10% FCS and 5 µg/ml gentamicin for 30 min to kill not phagocytized bacteria, washed with PBS, and incubated with RPMI 1640 plus 10% FCS for 1 h (total 2 h). Macrophages were harvested and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.4) or 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h. Conventional electron microscopy was performed as previously described (24). All sections were observed using a H7600 electron microscope (Hitachi).

In vivo induction of Th1 responses

Mice were injected i.p. with 200 µg of heat-killed P. acnes to induce Th1 responses in vivo. Seven days after injection, splenic CD4+ T cells were purified and stimulated on immobilized anti-CD3-coated plates for 24 h as described previously (25). The concentration of IFN-{gamma} was measured by ELISA according to the manufacturer’s instructions (Genzyme).

Histologic analysis

Frozen livers embedded in OCT compound (Sakura Finetek) were sliced into 5-µm-thick sections, fixed with 1% paraformaldehyde, and stained with Mayer’s H&E. Sections of livers were observed under a microscope (Olympus BX51).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of C/ebpbM20A/M20A knock-in mice

In peritoneal macrophages, all of the isoforms of C/EBPbeta are slightly expressed, but these are dramatically induced by LPS stimulation (Fig. 1C). Since the Kozak sequence of the second C/EBPbeta AUG codon is more optimum than that of the first one, the 34-kDa LAP is predominantly produced. The 34-kDa LAP is thought to be the most transcriptionally active form of C/EBPbeta (26). To elucidate the physiological role of the 34-kDa LAP, we generated knock-in mice in which methionine 20, the start site for the 34-kDa LAP, is replaced with an alanine. The murine C/ebpb gene consists of one exon. We constructed a targeting vector to replace the C/ebpb exon with C/ebpbM20A/M20A along with a neomycin resistance gene cassette (Fig. 2A). A HSV-thymidine kinase cassette for negative selection was inserted upstream of the homology arms in the C/ebpbM20A/M20A construct. Three correctly targeted ES clones were microinjected into C57BL/6 blastocysts to generate chimeric mice. Chimeric mice were crossed with female C57BL/6 mice, and transmission of the mutated allele was monitored by Southern blot analysis (Fig. 2B). Heterozygote mice were then interbred to produce offspring carrying homozygous alleles of the mutated C/ebpb gene. C/ebpbM20A/M20A mice were born at the expected Mendelian ratio and showed no developmental abnormalities. To confirm the replacement of the mutated C/ebpb gene in C/ebpbM20A/M20A mice, we checked the sequence of the C/ebpb exon in the tail genome of C/ebpbM20A/M20A mice. As shown in Fig. 2C, ATG corresponding to methionine 20 was replaced with GCG. We next investigated by Western blot analysis the expression of the 34-kDa LAP in peritoneal macrophages either stimulated with 100 ng/ml LPS for 8 h or unstimulated. The expression of the 34-kDa LAP protein was diminished in peritoneal macrophages from C/ebpbM20A/M20A mice (Fig. 2D).

Impaired cytokine production in C/ebpbM20A/M20A macrophages

Many genes encoding macrophage inflammatory mediators, such as cytokines and chemokines, carry C/EBPbeta sites on their promoters. It is reported that G-CSF induction after LPS stimulation was severely impaired in C/EBPbeta-deficient macrophages (18), whereas IL-12p40 was up-regulated in the absence of C/EBPbeta (27). We examined the production of TNF-{alpha}, IL-6, IL-12p40, and G-CSF after LPS stimulation, with or without IFN-{gamma}, in C/ebpbM20A/M20A peritoneal macrophages, using ELISA (Fig. 3). LPS-mediated G-CSF production was severely impaired in C/ebpbM20A/M20A macrophages. Furthermore, IL-12p40 was overproduced in C/ebpbM20A/M20A macrophages compared with wild-type cells. However, C/ebpbM20A/M20A macrophages produced the same amount of TNF-{alpha} and IL-6 as wild-type cells. Taken together, these findings suggest that C/ebpbM20A/M20A macrophages have a similar phenotype to C/EBPbeta-deficient macrophages in terms of LPS-mediated proinflammatory cytokine production.


Figure 3
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  		FIGURE 3. Cytokine production in C/ebpbM20A/M20A macrophages. Thioglycolate-elicited peritoneal macrophages from wild-type mice and C/ebpbM20A/M20A mice were cultured with either medium only, LPS (100 ng/ml), IFN-{gamma} (50 U/ml), or LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 24 h. The cytokine levels in culture supernatants were measured. Data are means ± SD of triplicate samples of a representative from three independent experiments.

 
LAP is essential for C/EBPbeta-mediated gene induction

We next examined the induction of various inflammatory mediators, which are known to be target genes of C/EBPbeta, by Northern blot analysis (Refs. 21 and 27 ; Fig. 4). The induction of Ptges, Cxcl13, and Il12a mRNAs was severely impaired in C/ebpbM20A/M20A macrophages stimulated with LPS plus IFN-{gamma}. The induction of Arg1 mRNA was also impaired in C/ebpbM20A/M20A cells, albeit the impairment was less than that observed in C/EBPbeta-deficient mice. In addition to proinflammatory cytokine production, the phenotype of C/ebpbM20A/M20A mice was similar to that of C/EBPbeta-deficient mice in terms of the induction of inflammatory mediators.


Figure 4
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  		FIGURE 4. Induction of C/EBPbeta target genes in C/ebpbM20A/M20A macrophages. Thioglycolate-elicited peritoneal macrophages from wild-type mice, C/EBPbeta-deficient mice, and C/ebpbM20A/M20A mice were cultured with LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for the indicated periods and analyzed for the expression of Arg1, Ptges, Cxcl13, and Il12a by Northern blotting.

 
To comprehensively examine gene induction in response to proinflammatory stimuli, we stimulated wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A peritoneal macrophages with LPS plus IFN-{gamma} for 4 h, prepared RNA samples, and performed cDNA microarray analyses (Fig. 5). We identified several transcripts that were markedly up-regulated in wild-type macrophages at 4 h after stimulation, whereas these genes were not induced in C/EBPbeta-deficient cells. They include genes associated with host defense, such as cytokines (Il1a), chemokines (Cxcl13), prostanoid synthetase (Ptges), formyl peptide receptors (Fpr1and Fpr-rs), a class A scavenger receptor (Marco), and secretary antimicrobial peptides (S100a8, Lcn2, Slpi). In addition, genes encoding antioxidative enzymes (Prdx6 and Sod2), branched chain {alpha}-ketoacid dehydrogenase (Bckdhb), the structural protein of caveolae (Cav1), an endogenous ligand for growth hormone secretagogue receptor (Ghrl), cell surface glycoprotein (Cd38 and BstI), cationic acid transporter (Slc7a1), C-type lectin (Clec4e), plasminogen activator inhibitor (Serpinb2), acute phase protein (Saa3), microsomal glutathione transferase (Mgst1), and arginase (Arg1 and Arg2) were up-regulated by LPS plus IFN-{gamma} stimulation. The induction of these C/EBPbeta target genes was severely impaired in C/ebpbM20A/M20A macrophages. Thus, C/EBPbeta is critically involved in the induction of a variety of genes in activated macrophages, and most of these are regulated by the 34-kDa LAP.


Figure 5
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  		FIGURE 5. Gene induction profiles in C/ebpbM20A/M20A macrophages. Thioglycolate-elicited peritoneal macrophages from wild-type mice, C/EBPbeta-deficient mice, and C/ebpbM20A/M20A mice were cultured with LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 12 h, and their expression profiles were analyzed using cDNA microarrays. Data are representative of two independent experiments.

 
Normal bactericidal activity in C/ebpbM20A/M20A macrophages

C/EBPbeta-deficient mice showed high susceptibility to L. monocytogenes infection due to severely impaired listericidal activity in macrophages (18). Therefore, we examined whether there was any difference in listericidal activity among wild-type, C/EBPbeta-deficient and C/ebpbM20A/M20A macrophages using an in vitro killing assay (Fig. 6A). When phagocytized by untreated resident macrophages, Listeria readily escape from the phagosome and subsequently replicate in the cytoplasm (28). By contrast, IFN-{gamma}-treated macrophages prevent phagosomal escape and confine the bacteria to the phagosome (29). For maximal activation, peritoneal macrophages from wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A mice were treated with LPS plus IFN-{gamma} for 20 h. Activated macrophages were infected with L. monocytogenes for 0.5 h and intracellular killing of bacteria over a period of 6 h was determined by a colony assay. Consistent with a previous report (18), the number of bacteria increased about 2-fold over 6 h in C/EBPbeta-deficient macrophages, while macrophages from wild-type mice killed Listeria efficiently (Fig. 6A, left panel). Surprisingly, the rate of killing bacteria was almost identical between wild-type and C/ebpbM20A/M20A macrophages (Fig. 6A, right panel). We next examined bacterial uptakes in macrophages. Macrophages from wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A mice were activated with LPS plus IFN-{gamma} for 20 h, infected with L. monocytogenes for 0.5 h, and the number of bacteria phagocytized by macrophages were determined by a colony assay. Macrophages from wild-type mice, C/EBPbeta-deficient mice, and C/ebpbM20A/M20A mice phagocytized Listeria to a similar extent, suggesting that there was no apparent difference in bacterial uptakes among these cells (Fig. 6B). We also examined macrophages from wild-type mice, C/EBPbeta-deficient mice, and C/ebpbM20A/M20A mice at 6 h after Listeria infection using confocal microscopy. This analysis identified that many intracellular bacteria were increased in C/EBPbeta-deficient macrophages, but not in wild-type or C/ebpbM20A/M20A macrophages (Fig. 6C). Furthermore, LPS plus IFN-{gamma}-treated peritoneal macrophages from wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A mice were infected with L. monocytogenes for 2 h, then fixed, sectioned, and examined by electron microscopy (Fig. 6D). In macrophages from both wild-type and C/ebpbM20A/M20A mice, the majority of bacteria were in phagocytic vacuoles (Fig. 6, D and E). By contrast, in C/EBPbeta-deficient macrophages, most bacteria were found in the cytoplasm and some were in the process of division (Fig. 6, D and E). In sharp contrast to C/EBPbeta-deficient mice, the Listeria killing defect was not observed in C/ebpbM20A/M20A mice.


Figure 6
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  		FIGURE 6. Normal bactericidal activity in C/ebpbM20A/M20A macrophages. A, Bactericidal assay of activated macrophages. Thioglycolate-elicited peritoneal macrophages from C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their littermates were stimulated with LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 20 h, and intracellular Listeria killing was determined. The intracellular killing of L. monocytogenes is indicated as a percentage of phagocytized bacteria. B, Uptake of Listeria in activated macrophages. Peritoneal macrophages from C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their littermates were stimulated with LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 20 h, and infected with L. monocytogenes for 30 min. Cells were lysed with 0.05% Triton X-100 and the numbers of bacteria phagocytized by macrophages were determined by plating the lysates on tryptic soy agar. C, Confocal microscopic images of peritoneal macrophages from wild-type mice (left panel), C/EBPbeta-deficient mice (middle panel), and C/ebpbM20A/M20A mice (right panel) at 6 h after infection of cells with L. monocytogenes. After fixation, lysosomes and nuclei were stained using an Ab to LAMP-1 (red) and 4',6-diamino-2-phenylindole (blue), respectively. Thirty cells in each mouse were scored (n = 6/ group). Bar, 10 µm. D, Representative electron micrographs of peritoneal macrophages from wild-type mice (left panel), C/EBPbeta-deficient mice (middle panel), and C/ebpbM20A/M20A mice (right panel) at 2 h after infection with L. monocytogenes. Black arrows, L. monocytogenes. Although L. monocytogenes was present in phagosomes in both wild-type and C/ebpbM20A/M20A macrophages, L. monocytogenes were found in the cytoplasms of C/EBPbeta-deficient macrophages. The micrograph shows L. monocytogenes in the process of division (middle panel). Original magnifications were as follows: x4000 (wild-type macrophage), x5000 (C/EBPbeta-deficient macrophage), and x4000 (C/ebpbM20A/M20A macrophage). Forty cells were scored in each mouse (n = 3/each group). E, Intracellular location of L. monocytogenes in peritoneal macrophages from wild-type, C/EBPbeta-deficient, and C/ebpbM20A/M20A mice. Wild-type macrophages, C/EBPbeta-deficient macrophages, and C/ebpbM20A/M20A macrophages were observed using electron microscopy at 2 h after infection of L. monocytogenes. Cells that contain bacteria in endosome, cytoplasm, and the process of division are scored. Forty cells were counted in each mouse (n = 3/each group). F, C/EBPbeta-deficient mice (n = 5), C/ebpbM20A/M20A mice (n = 5), and their wild-type littermates (n = 5) were infected with 1 x 105 (left panel) or 1 x 103 (right panel) L. monocytogenes, and the survival of the mice was examined. KO, Knockout.

 
We next investigated whether the 34-kDa LAP plays a role in protecting against in vivo bacterial infection. C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their wild-type littermates were infected with 1 x 105 CFU of L. monocytogenes. All control mice survived, whereas C/EBPbeta-deficient mice died within 3 days and C/ebpbM20A/M20A mice died within 6 days (Fig. 6F, left panel). When infected with an extremely low dose (1 x 103 CFU), only C/EBPbeta-deficient mice died within 4 days after the challenge with Listeria. Thus, C/ebpbM20A/M20A mice were susceptible to infection by L. monocytogenes, although their phenotype was milder than that of C/EBPbeta-deficient mice (Fig. 6F, right panel).

Impaired Th1 response and granuloma formation in C/ebpbM20A/M20A mice

Transcription of the IL-12 p35 gene is positively regulated by C/EBPbeta (27) and the 34-kDa LAP was essential for its induction (Fig. 4). Because IL-12 is bioactive only as a heterodimer (p70) composed of p40 and p35 subunits, we examined the production of IL-12p70 in response to LPS plus IFN-{gamma} stimulation in C/EBPbeta-deficient and C/ebpbM20A/M20A peritoneal macrophages using ELISA. IL-12p70 production was impaired in both C/EBPbeta-deficient and C/ebpbM20A/M20A macrophages (Fig. 7A). In addition to the impaired production of bioactive IL-12p70, overproduction of IL-12p40 was observed in C/EBPbeta-deficient and C/ebpbM20A/M20A macrophages (Fig. 3). The p40 homodimer is known to act as a receptor antagonist (30). We investigated the role of C/EBPbeta and the 34-kDa LAP in in vivo Th1 responses by using an established model of P. acnes. P. acnes is suspected to be a causative bacteria for human sarcoidosis, and its cell wall components show strong immunoadjuvant activities, which induce Th1 responses and granuloma formation in the liver. C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their wild-type littermates were i.p. injected with P. acnes. Seven days after injection, CD4+ T cells were purified from splenocytes and stimulated with immobilized anti-CD3. Elevated levels of IFN-{gamma} were observed in wild-type mice, whereas C/EBPbeta-deficient cells produced a much smaller amount of IFN-{gamma} (Fig. 7B). Similar to C/EBPbeta-deficient cells, IFN-{gamma} production from C/ebpbM20A/M20A CD4+ T cells was severely impaired. To examine the roles of C/EBPbeta and the 34-kDa LAP in granuloma formation in vivo, C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their wild-type littermates were i.p. injected with P. acnes. Granuloma formation was evident in the livers of wild-type mice on day 7 after injection, whereas it was hardly seen in those of both C/EBPbeta-deficient mice and C/ebpbM20A/M20A mice (Fig. 7C). LPS injection into P. acnes-primed mice stimulates dendritic cells and macrophages to produce a large amount of proinflammatory cytokines, such as TNF-{alpha} and IFN-{gamma}, which causes lethal endotoxin shock in vivo (31, 32, 33). We next examined the role of C/EBPbeta and the 34-kDa LAP in LPS-induced endotoxin shock. On day 7 after injection of 200 µg of heat-killed P. acnes, 5 µg of LPS was injected into C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their wild-type littermates to induce lethal endotoxin shock. P. acnes-primed wild-type mice were sensitive to LPS-induced endotoxin shock, and all mice died within 5 h. By contrast, C/EBPbeta-deficient mice and C/ebpbM20A/M20A mice were strongly resistant and survived (Fig. 7D). We further examined the production of TNF-{alpha} and IFN-{gamma} in C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their wild-type littermates. Shortly after LPS injection (2 h), these cytokines were detected in the sera of control wild-type mice, whereas only small amounts of these cytokines were produced in the sera of C/EBPbeta-deficient mice and C/ebpbM20A/M20A mice (Fig. 7E). These results suggested that the 34-kDa LAP takes a major role in C/EBPbeta-mediated immune responses, such as the Th1 response, granuloma formation, and endotoxin shock.


Figure 7
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  		FIGURE 7. Impaired Th1 response and granuloma formation in C/ebpbM20A/M20A mice. A, Thioglycolate-elicited peritoneal macrophages from wild-type mice, C/EBPbeta-deficient mice, and C/ebpbM20A/M20A mice were cultured with either medium only or LPS (100 ng/ml) plus IFN-{gamma} (50 U/ml) for 24 h. The IL-12p70 levels in culture supernatants were measured. Data are means ± SD of triplicate samples of a representative from three independent experiments. B, Th1 responses induced by P. acnes C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their littermates were i.p. injected with 200 µg of heat-killed P. acnes. Seven days after injection, CD4+ T cells from the spleen were stimulated with anti-CD3 for 48 h. The concentrations of IFN-{gamma} in the culture supernatants were measured using ELISA. Data are means ± SD of triplicate samples of a representative from three independent experiments. C, Granuloma formation in the livers of C/EBPbeta-deficient mice, C/ebpbM20A/M20A mice, and their littermates. Livers were taken on day 7 after injection with 200 µg of heat-killed P. acnes and sections were stained with H & E. Three sections were randomly selected from each mouse (n = 5/group) and five areas per section were scored. Arrows, Granulomas. Original magnifications: upper panels, x100; lower panels, x400. Bar, 100 µm. D, On day 7 after injection of 200 µg of heat-killed P. acnes, 5 µg of LPS was injected into C/EBPbeta-deficient mice (n = 5), C/ebpbM20A/M20A mice (n = 5), and their littermates (n = 5) to induce lethal endotoxin shock. The survival of the mice was monitored. E, The serum levels of TNF-{alpha} and IFN-{gamma} were measured by ELISA in heat-killed P. acnes-primed mice at 2 h after LPS injection (5 µg). Values represent means ± SD (n = 5/group). Data are representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study is the first attempt to assess the function of a C/EBPbeta isoform in vivo. We clearly demonstrated that the 34-kDa LAP is responsible for the induction of C/EBPbeta target genes in activated macrophages by generating C/ebpbM20A/M20A mice. The 34-kDa LAP was essential for the production of proinflammatory cytokines such as G-CSF and IL-12 from activated macrophages (Figs. 3, 4, and 7A). In addition, DNA microarray analysis revealed that the 34-kDa LAP mainly regulates the induction of a variety of C/EBPbeta target genes in macrophages when stimulated with LPS plus IFN-{gamma} (Fig. 5). Some of these target genes have been reported to be critically involved in host defense. mPGES-1 (Ptges) is markedly induced by proinflammatory stimuli and synthesizes a large amount of PGE2, which plays an important role in inflammation (21, 34). The formyl peptide receptors (Fpr1 and Fpr-rs) respond not only to formyl peptides derived from bacterial and mitochondrial proteins, but also to endogenous ligands. Activated formyl peptide receptors induce chemotaxis, arachidonic acid production, superoxide production, phagocytosis, and release of proteolytic enzymes (35). Marco, which is a class A scavenger receptor molecule, is important for the removal of pathogens from the body (36). MRP8 (S100a8), a calcium-binding protein is a proinflammatory mediator in acute and chronic inflammation (37). Secreted lipocalin 2 (Lcn2) sequesters the iron-laden siderophore of bacteria and limits bacterial growth by inhibiting their iron uptake. Lipocalin 2-deficient mice showed susceptibility to Escherichia coli infection (38). Although secretory leucoprotease inhibitor was initially identified as a serine protease inhibitor, it has been shown to possess an ability to protect the host from infection and injury (39). A recent report also showed that secretory leucoprotease inhibitor regulates Ig class switching (40). Consistent with the impaired induction of these crucial immune mediators, C/ebpbM20A/M20A mice were susceptible to high-dose infection with L. monocytogenes (105 CFU).

IL-12 is a key proinflammatory cytokine that bridges both innate and adaptive immunity. IL-12 is bioactive only as a heterodimer (p70) composed of p40 and p35 subunits. IL-12p70 production was severely impaired in both C/EBPbeta-deficient macrophages and C/ebpbM20A/M20A macrophages (Fig. 7A). IL-12 acts mostly on NK and T cells by binding to a receptor composed of IL-12Rbeta1 and IL-12Rbeta2 (41). The important role of IL-12 is related to the efficient induction of IFN-{gamma} production in these cells (42, 43, 44). IL-12 also enhances the proliferation and cytolytic activity of NK cells and T cells and promotes Th1 cell differentiation, thus driving cell-mediated immunity (45). Previously, Screpanti et al. (19) reported that C/EBPbeta-deficient mice showed increased susceptibility to C. albicans infection and an altered Th response. In C/ebpbM20A/M20A mice, the heat-killed P. acnes-induced Th1 response, granuloma formation, and LPS shock were severely impaired, suggesting that the 34-kDa LAP is crucial for not only innate immune responses to infection, but also induction of humoral and cellular immunity. Nevertheless, impaired intracellular bacteria killing, which is the most prominent phenotype in C/EBPbeta-deficient mice, was not observed at all in C/ebpbM20A/M20A mice. Since we assumed that the 34-kDa LAP is responsible for C/EBPbeta-mediated intracellular bacteria killing as a major active isoform, this result was quite unexpected and surprising. C/ebpbM20A/M20A mice showed a milder phenotype in terms of listerial infection compared with C/EBPbeta-deficient mice, which died following infection with an extremely low dose (1 x 103 CFU) of Listeria (Fig. 6E). These divergent results of listerial infection may be due to normal bactericidal activity in C/ebpbM20A/M20A macrophages.

Besides the normal intracellular bacteria killing, we found some discrepancy in phenotypes between C/ebpbM20A/M20A mice and C/EBPbeta-deficient mice. C/ebpbM20A/M20A mice were born at the expected Mendelian ratio, although C/EBPbeta-deficient mice were obtained from heterozygous parents at a frequency lower than the expected Mendelian ratio (18, 19). Although female mice lacking C/EBPbeta are sterile as a result of defects in ovarian granulosa cell differentiation (46), we can normally obtain pups from homozygous parents of C/ebpbM20A/M20A mice. Mammary gland development and differentiation are impaired in C/EBPbeta-deficient mice (47). However, these phenotypes were not observed in C/ebpbM20A/M20A mice (data not shown). Furthermore, C/EBPbeta-deficient mice look skinny because of impaired adipogenesis (48), whereas C/ebpbM20A/M20A mice do not show such an appearance. Thus, the development and differentiation defects in multiple tissues of C/EBPbeta-deficient mice were not observed in C/ebpbM20A/M20A mice, suggesting that the 34-kDa LAP is not essential for C/EBPbeta-mediated development and differentiation. Other isoforms may be responsible for bactericidal activity in macrophages, fetus development, fertility, and functional differentiation of mammary glands and adipogenesis. To clarify the mechanism of the killing defect in C/EBPbeta-deficient macrophages, it is necessary to analyze the other isoforms of C/EBPbeta, Full, and LIP, in vivo, in the near future.


   Acknowledgments
 
We are grateful to Dr. Tsutsui for providing heat-killed P. acnes. We thank Drs. T. Kawai, K. J. Ishii, S. Sato, C. Coban, O. Takeuchi, and K. Takeda for discussion; N. Kitagaki for technical assistance; and M. Hashimoto for secretarial assistance. We also thank Dr. Daisuke Okuzai of DNA-Chip Development Center for Infectious Diseases (Research Institute for Microbial Diseases, Osaka University) for technical advice.


   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 by grants from Special Coordination Funds, the Ministry of Education, Culture, Sports, Science and Technology, and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Back

2 Address correspondence and reprint requests to Dr. Shizuo Akira, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, Japan. E-mail address: sakira{at}biken.osaka-u.ac.jp Back

3 Abbreviations used in this paper: LAP, liver-enriched transcriptional protein; LIP, liver-enriched transcriptional inhibitory protein; ES, embryonic stem. Back

Received for publication April 23, 2007. Accepted for publication August 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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