C5a-regulated CCAAT/Enhancer-binding Proteins β and δ Are Essential in Fcγ Receptor-mediated Inflammatory Cytokine and Chemokine Production in Macrophages*

Background: The nuclear molecules responsible for FcγR-mediated inflammation remain unknown. Results: C/EBPβ and -δ are activated by IgG IC, which plays a critical role in TNF-α, MIP-2, and MIP-1α production in macrophages. Conclusion: C/EBPβ and -δ play a key role in FcγR-mediated induction of inflammatory mediators. Significance: The finding encourages further investigation of the role of C/EBPβ and -δ in IC diseases. CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPβ and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPβ and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPβ and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPβ and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPβ and/or -δ, we demonstrate that C/EBPβ and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPβ and -δ activities. Collectively, these data suggest that C/EBPβ and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.

The Fc␥ receptors (Fc␥Rs) 2 are a family of cell surface molecules that bind the Fc portion of IgG immunoglobulins. Different cell types bear different sets of Fc␥Rs, and their specificities for various antibody classes thus determine which types of cells will be engaged in given responses. Four types of Fc␥Rs are identified in mice: Fc␥RI, Fc␥RIIB, Fc␥RIII, and the newest member, Fc␥RIV. Fc␥RI, Fc␥RIII, and Fc␥RIV are activating receptors, all of which associate with FcR common ␥-chain, whereas Fc␥RIIB is an inhibitory member. The corresponding human Fc␥Rs are Fc␥RIA, Fc␥RIIA, Fc␥RIIIA (activating), and Fc␥IIB (inhibitory) receptors. Human Fc␥RIA and mouse Fc␥RI have high affinity for IgG. Based on the sequence similarity, human Fc␥RIIA is most closely related to mouse Fc␥RIII, and human Fc␥RIIIA seems to be the orthologue of mouse Fc␥RIV (1). However, human Fc␥RIIA has an immunoreceptor tyrosine-based activation motif in its cytosolic domain, which is not present in mouse Fc␥RIII and does not require ␥-chain for functionality (1). Macrophages, the sentinels of innate immunity, play key roles in inflammation. Activation of macrophages by Fc␥Rs triggers a wide variety of cellular responses (1,2). In addition to phagocytosis, one of the major cellular responses in macrophages initiated by IgG immune complex (IC)-mediated Fc␥R cross-linking is the activation of genes encoding chemokines and cytokines important in inflammation. Although the production of these mediators is mainly controlled at the transcriptional level in response to various inflammatory stimuli, the key nuclear molecules that mediate Fc␥R signaling in macrophages remain unknown. Several transcription factors, such as NF-B, AP-1, CCAAT/enhancer-binding protein ␤ (C/EBP␤), and Stat3, are activated in macrophages/monocytes following IgG IC stimulation (3,4). Studies using pharmacological inhibitors suggested that NF-B activation was involved in the expression of several chemokines, including macrophage inflammatory protein-1␣ and ␤ (MIP-1␣ and MIP-1␤), upon cross-linking of Fc␥R (3,5). However, these lines of evidence are either indirect or correlative. Therefore, additional studies are necessary to elucidate the molecular mechanisms whereby the expression of inflammatory mediators is induced by Fc␥Rs.
C/EBP␣, -␤, -␦, -⑀, -␥, and -comprise a family of basic region-leucine zipper (bZIP) transcription factors that dimerize through a leucine zipper motif and bind to DNA through an adjacent basic region. C/EBP␤ and -␦ have been implicated in the regulation of inflammatory mediators as well as other gene products associated with the activation of macrophages and the acute phase inflammatory response (6). Two major forms of C/EBP␤ are translated from the same messenger RNA, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) (7). The roles of C/EBP family members in regulating inflammation have also been studied using knock-out mice. Interestingly, the LPS stimulation of peritoneal macrophages from C/EBP␤-deficient mice led to normal induction of several inflammatory cytokines, including IL-6 and TNF-␣, with the exception of G-CSF, Mincle, and mPGES-1 (8 -11), although macrophages from these mice demonstrate defective intracellular bacteria killing. Similarly, C/EBP␦-deficient macrophages show nominal defects in IL-6 and TNF-␣ production in response to several TLR ligands (12). In contrast, the absence of both C/EBP␤ and -␦ results in a significant decrease in the TLR ligand-induced production of both IL-6 and TNF-␣ (12). In another study, Gorgoni et al. (13) found that LPS-induced expression of IL-1␤, TNF-␣, IL-6, and inducible nitric-oxide synthase is partially impaired in C/EBP␤deficient macrophages and that expression of IL-12 p35 is completely defective. However, IL-12 p40, RANTES, and MIP1-␤ are more efficiently induced in response to IFN-␥ and LPS in the absence of C/EBP␤. These results raise the possibility that the redundant expression of multiple C/EBP isoforms as well as differences in C/EBP homo-/heterodimer occupancy in specific gene promoters may account for the differential effects of C/EBP␤ and -␦ deficiency. Thus, the role of C/EBP␤ and -␦ in regulating transcription of the different inflammation-regulated mediators warrants further investigation. Furthermore, whether and how C/EBP␤ and -␦ are involved in the Fc␥Rinduced inflammatory response remains unclear, and the signals that mediate the activation of C/EBP␤ and -␦ are unknown. In this report, we demonstrate that both C/EBP␤ and -␦ are induced by IgG IC in macrophages. Furthermore, activation of C/EBP␤ and -␦ in macrophages is mediated, to a large extent, by Fc␥Rs. We provide evidence that C/EBP␤ and -␦ are key transcription factors that regulate the Fc␥R-mediated induction of inflammatory cytokine and chemokines in macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in the C/EBP activation and mediator production induced by IgG IC. Interestingly, we show that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBP activity.
Luciferase Assay-Transient transfections were performed with 1.5 ϫ 10 5 cells plated in 12-well plates by using 0.5 g of DNA and 1.5 l of Fugene6 transfection reagent (Roche Applied Science) in 50 l of Opti-MEM I medium (Invitrogen). 24 h after transfection, the cells were incubated either with or without 100 g/ml IgG IC for 5 h. Cell lysates were subjected to luciferase activity analysis by using the Dual-Luciferase reporter assay system (Promega, Madison, WI). IgG IC were formed by the addition to anti-BSA of BSA at the point of antigen equivalence as described previously (17).
siRNA Transfection-Transient siRNA transfections were performed by transfecting 2 ϫ 10 6 RAW264.7 cells with control siRNA or C/EBP␤/␦ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using the Amaxa nucleofector kit V. 12 h later, the cells were treated with or without 100 g/ml IgG IC for 5 h. Supernatants were collected for ELISA analysis.
Peritoneal Macrophage Isolation and Culture-8 -9-Weekold specific pathogen-free male C57BL/6 were obtained from Jackson Laboratories. FcR␥-and Fc␥RII-deficient mice were obtained from Taconic. C5aR knock-out mice were kindly provided by Craig Gerard (Harvard Medical School). C/EBP␤ Ϫ/Ϫ and C/EBP␦ Ϫ/Ϫ mice have been described previously (15,16). Mouse peritoneal macrophages were isolated by using the thioglycollate method. Briefly, mice were injected intraperitoneally with 1.5 ml of 2.4% sterile thioglycollate dissolved in double distilled H 2 O. Peritoneal macrophages were obtained by instillation and aspiration of 10 ml of PBS 4 days after thioglycollate administration. The macrophages were maintained in DMEM supplemented with 5% FBS and allowed to adhere for 2 h, followed by 100 g/ml IgG IC treatment for the indicated time periods. The supernatants were harvested and subjected to ELISA analysis. The purity of the cell suspension was ϳ99%, which was determined by staining of the peritoneal cell suspension with the HEMA 3 Stain Set (Fisher).
ELISA-Macrophages were stimulated by IgG IC for the indicated time. The supernatants were centrifuged at 3000 rpm for 5 min, and the cell-free supernatants were harvested for TNF-␣, MIP-2, and MIP-1␣ measurements according to the manufacturer's protocols.
RNA Isolation and Detection of mRNA by Semiquantitative RT-PCR-Total RNAs were extracted from cells with TRIzol (Invitrogen) according to the manufacturer's procedure. After isolation, total cellular RNA was incubated with RQ1 RNasefree DNase (Promega, Madison, WI) to remove contaminating DNA. 2 g of total RNA was submitted to reverse transcription by using Superscript II RNase H reverse transcriptase (Invitrogen). PCR was performed with the following primers: for C/EBP␤, 5Ј primer (5Ј-CAA GCT GAG CGA CGA GTA CA-3Ј) and 3Ј primer (5Ј-AGC TGC TCC ACC TTC TTC TG-3Ј); for C/EBP␦, 5Ј primer (5Ј-CGC AGA CAG TGG TGA GCT T-3Ј) and 3Ј primer (5Ј-CTT CTG CTG CAT CTC CTG GT-3Ј); for GAPDH, 5Ј primer (5Ј-GCC TCG TCT CAT AGA CAA GAT G-3Ј) and 3Ј primer (5Ј-CAG TAG ACT CCA CGA CAT AC-3Ј). After a "hot start" for 5 min at 94°C, 28 -33 cycles were used for amplification with a melting temperature of 94°C, an annealing temperature of 60°C, and an extending temperature of 72°C, each for 1 min, followed by a final extension at 72°C for 8 min. PCR was performed using different cycle numbers for all primers, to ensure that DNA was detected within the linear part of the amplifying curves for both primers.
Western Blot Analysis-RAW264.7 cells were lysed in cold radioimmune precipitation assay buffer. Samples containing 80 g of protein were electrophoresed in a 12% polyacrylamide gel and then transferred to a PVDF membrane. Membranes were incubated with rabbit anti-C/EBP␤ antibody (Santa Cruz Biotechnology, Inc.), rabbit anti-C/EBP␦ antibody (Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-p38 MAPK antibody (Cell Signaling), rabbit anti-phospho-p44/42 MAPK antibody (Cell Signaling), rabbit anti-p38 MAPK antibody (Cell Signaling), rabbit anti-p44/42 antibody (Cell Signaling), and rabbit anti-GAPDH antibody (Cell Signaling), respectively. After three washes in TBST, the membranes were incubated with a 1:5000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare). The membrane was developed by the enhanced chemiluminescence technique according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL).
Statistical Analysis-All values were expressed as the mean Ϯ S.E. Significance was assigned where p Ͻ 0.05. Data sets were analyzed using Student's t test or one-way analysis of variance, with individual group means being compared with the Student-Newman-Keuls multiple comparison test.

IgG IC Induces Expression of C/EBP␤ and -␦ in Macrophages-
To determine if C/EBP activities are induced by IgG IC, we first examined the DNA binding activity of C/EBPs in RAW264.7 macrophages. As shown in Fig. 1A, RAW264.7 cells were challenged with IgG IC for different time periods, and extracted nuclear proteins were subjected to EMSA. The results show that strong C/EBP DNA binding activity was induced after IgG IC stimulation (Fig. 1A). To determine which C/EBP family members are induced by IgG IC, supershift assays were performed. As shown in Fig. 1B, there was a low but detectable level of C/EBP␤ binding activity in nuclear extract from control-treated RAW264.7 cells. The basal level of C/EBP␦ binding was minimal; however, DNA binding activities of both C/EBP␤ and -␦ were dramatically enhanced by IgG IC stimulation.
We further examined the IgG IC-induced C/EBP activation in transient transfections using 2XC/EBP-Luc, a promoter-reporter that contains two copies of a canonical C/EBP binding site, and expression vectors for C/EB␤ and/or C/EBP␦. These transfections were carried out with and without IgG IC treatment. Consistent with the results from EMSA, IgG IC stimulation alone (without transfection of a C/EBP expression vector) increased luciferase activity 4-fold compared with the control group (Fig. 1C). IgG IC treatment of C/EBP␤, C/EBP␦, and C/EBP␤ plus C/EBP␦ transfectants induced luciferase expression 14-, 10-, and 19-fold, respectively, over the control value. Interestingly, overexpression of C/EBP␤, C/EBP␦, and C/EBP␤ plus C/EBP␦ alone (i.e. in the absence of IC treatment) stimulated luciferase activity 4-, 2.6-, and 6.2-fold, respectively; this is significantly less than the values observed in the presence of IgG IC. These data suggest that IgG IC signaling may stimulate C/EBP transcriptional activity in addition to inducing expression of the endogenous proteins. Moreover, C/EBP␤ and -␦ together were more potent activators than either protein alone, indicating that C/EBP␤ and -␦ are preferential dimerization partners, C/EBP␤/␦ heterodimers have stronger DNA binding activity than homodimers, or both.
We next examined whether IgG IC induces expression of C/EBP␤ and -␦ at the mRNA level. RT-PCR showed that C/EBP␤ and -␦ mRNAs were highly induced in RAW264.7 cells following a 24-h treatment with IgG IC (Fig. 1D). We then sought to determine whether this induction resulted in increased abundance of C/EBP␤ and -␦ proteins in RAW264.7 cells. The Western blot analysis of proteins isolated over a time course of IgG IC treatment revealed a time-dependent increase in the abundance of both C/EBP␤ (mainly LAP) and C/EBP␦ proteins (Fig. 1E). These data demonstrate that increased abundance of C/EBP␤ and -␦ is coincident with their increased DNA binding activity in RAW264.7 cells.

FIGURE 1. IgG immune complexes induce C/EBP␤ and -␦ expression and increase C/EBP DNA binding activity in macrophages.
A, RAW264.7 cells were stimulated with 100 g/ml immune complexes for the times indicated. Nuclear proteins were harvested and subjected to EMSA to measure C/EBP DNA binding activity. B, RAW264.7 cells were treated or left untreated with 100 g/ml IgG immune complexes for 4 h. The nuclear extracts were harvested for gel supershift assay to identify which C/EBP family member DNA binding activity was regulated by IgG immune complex treatment. N, ␣, ␤, ␦, ⑀, and ␥, normal rabbit IgG, anti-C/EBP␣ antibody, anti-C/EBP␤ antibody, anti-C/EBP␦ antibody, anti-C/EBP⑀ antibody, and anti-C/EBP␥ antibody, respectively. The arrows indicate C/EBP complex and supershift species. C, RAW264.7 cells were transiently transfected with a total of 0.5 g of the indicated DNA. 24 h after transfection, the cells were treated with 100 g/ml IgG immune complex for 5 h. Cell lysates were used to perform a luciferase activity assay. Luminometer values were normalized for expression from a co-transfected thymidine kinase reporter gene. The data were expressed as means of three experiments Ϯ S.E. (error bars). RAW264.7 cells were stimulated with 100 g/ml IgG immune complexes for different time periods. Then total cellular RNA was isolated for RT-PCR with primers for C/EBP␤, C/EBP␦, and GAPDH, respectively (D). The level of GAPDH is shown at the bottom as a loading control. E, the total proteins were extracted to conduct Western blot using rabbit anti-C/EBP␤ antibody, rabbit anti-C/EBP␦ antibody, and rabbit anti-GAPDH antibody, respectively. The level of GAPDH is shown at the bottom as a loading control.

C/EBP␤ and -␦ Are Critical Regulators of Cytokine and
Chemokine Expression in IgG IC-stimulated Macrophages-C/EBP␤ and -␦ play important roles in regulating inflammatory responses in many different cells, including macrophages (12, 13, 18 -21). However, whether they are involved in macrophage inflammatory responses after Fc␥R cross-linking is unknown. TNF-␣ is an early proinflammatory cytokine secreted by macrophages during inflammation. Both MIP-2 and MIP-1␣ were initially identified as monokines secreted from LPSstimulated RAW264.7 cells (22)(23)(24)(25). MIP-2 belongs to the C-X-C chemokine subfamily and has been shown to possess potent chemotactic activity for neutrophils, whereas MIP-1␣, as a member of the C-C chemokine subfamily, has been shown to chemoattract leukocytes of the monocyte lineage (22,23,26,27). Therefore, we sought to determine the effect of C/EBP␤ and -␦ deficiency on the expression of TNF-␣, MIP-2, and MIP-1␣ from IgG-IC-stimulated macrophages.
NF-B plays a central role in coordinating immune and inflammatory responses. To test its involvement in IgG ICinduced inflammation, RAW264.7 cells were treated with a NF-B inhibitor, BAY 11-7082 (supplemental Fig. 2). The data showed that upon IgG IC stimulation, the levels of all three mediators were down-regulated by BAY 11-7082, with a significant inhibitory effect on TNF-␣ and MIP-1␣ production, suggesting that NF-B also plays an important regulatory role in IgG IC-induced cytokine and chemokine production.
Fc␥Rs Trigger C/EBP-mediated Cytokine and Chemokine Production in IC-stimulated Macrophages-Fc␥Rs exert both activating and inhibitory effects on inflammatory responses, and the cellular threshold for inflammation is dependent on the ratio of the opposing signaling Fc␥Rs. Because both activating and inhibitory Fc␥Rs are expressed on macrophages (1, 28), we sought to determine the role of Fc␥Rs in IgG IC-induced inflammation in macrophages. Using peritoneal macrophages from FcR ␥-chain knock-out mice, we first examined the influence of conventional activating Fc␥Rs on IgG IC-induced cytokine and chemokine production. As shown in Fig. 4, A-C, FcR ␥-chain mutation resulted in a significantly decreased production of TNF-␣ (ϳ57%), MIP-2 (ϳ38%), and MIP-1␣ (ϳ45%) in IgG IC-treated macrophages compared with their wild type counterparts. In contrast, Fc␥RII deficiency resulted in an approximately 2-fold increase of TNF-␣, MIP-2, and MIP-1␣ production (supplemental Fig. 3). Because our data suggest that C/EBP␤ and -␦ may play important roles in cytokine and chemokine expression in IgG IC-stimulated macrophages (Figs. 2 and 3), we next examined the effect of Fc␥Rs deficiency on C/EBP␤ and -␦ activation. We found that knock-out of activating Fc␥Rs suppressed induction of C/EBP DNA binding activity (Fig. 4D). Furthermore, inhibitory Fc␥RII deficiency elevated C/EBP DNA binding activity (mainly LAP/LIP and LAP/␥ heterodimers) (data not shown). In summary, these findings suggest that activating Fc␥Rs play an important role in IgG IC-induced C/EBP␤ and -␦ induction, leading to production of TNF-␣, MIP-2, and MIP-1␣, whereas the inhibitory Fc␥RII has an opposite effect.

ERK1/2 and p38 MAPK Are Involved in IgG IC-induced C/EBP␤ and -␦ Activation and Subsequent Cytokine/Chemokine Production-Previous studies have shown that
Fc␥R crosslinking on macrophages/monocytes activates the extracellular signal-regulated kinases (ERK1/2, p44/42) and p38 MAPK signaling pathways (29,30). However, whether ERK and p38 signaling pathways function as transducers connecting Fc␥R stimulation to C/EBP␤ and -␦ activation remains unclear. To investigate this possibility, we first evaluated the MAPK pathways in IgG IC-stimulated macrophages. As shown in Fig. 5A and 6A, IgG IC treatment led to the phosphorylation of both ERK1/2 and p38 MAPK in a time-dependent manner. We next evaluated the influence of these phosphorylated MAPK on C/EBP␤ and -␦ activation by using specific pharmacological inhibitors for ERK1/2 and p38 MAPK. We observed that phosphorylation of ERK1/2 and p38 MAPK was significantly inhibited by U0126 and p38 MAPK inhibitor VIII, respectively (Figs. 5A and 6A). Further, U0126 and p38 MAPK inhibitor VIII markedly suppressed C/EBP DNA binding activity induced by IgG IC (Figs. 5B and 6B).
The effect of MAPK inhibitors on IgG IC-induced NF-B activation was also examined. EMSA showed that none of these inhibitors reduced NF-B DNA binding activity (Figs. 5C and   6C). Interestingly, U0126 slightly elevated IgG IC-induced NF-B activation (Fig. 5C). To determine whether MAPK activation is involved in IgG IC-induced cytokine and chemokine production (which are regulated by C/EBP␤ and -␦), we evaluated the effect of U0126 and p38 MAPK inhibitor VIII on TNF-␣, MIP-2, and MIP-1␣ secretion from RAW264.7 macro-FIGURE 3. C/EBP␤ and -␦ are critical regulators of cytokine and chemokine production in IgG IC-stimulated macrophages. A, primary peritoneal macrophages obtained from wild type were treated or left untreated with 100 g/ml IgG immune complexes for 4 h. The nuclear extracts were harvested for a gel supershift assay to identify which C/EBP family member DNA binding activity was regulated by IgG immune complex treatment. N, ␣, ␤, ␦, ⑀, and ␥, normal rabbit IgG, anti-C/EBP␣ antibody, anti-C/EBP␤ antibody, anti-C/EBP␦ antibody, anti-C/EBP⑀ antibody, and anti-C/EBP␥ antibody, respectively. The arrows indicate C/EBP binding bands and supershift bands. B-D and E-G, primary peritoneal macrophages obtained from corresponding wild type, C/EBP␤ knock-out (B-D), and C/EBP␦ knock-out (E-G) mice were treated with 100 g/ml IgG immune complex for different time periods, and supernatants were subjected to ELISA analysis for TNF-␣, MIP-2, and MIP-1␣ production. Data are presented as mean Ϯ S.E. (error bars) (n ϭ 6).
C5a Enhances IgG IC-stimulated Cytokine and Chemokine Production by Elevating C/EBPs but Not NF-B DNA Binding Activity-C5a has been shown to synergistically enhance ICinduced TNF-␣ and MIP-2 production in alveolar macrophages (31,32). Thus, we examined the role of C5a in the activation of peritoneal macrophages treated with IgG IC. As shown in Fig. 7, A-C, the addition of C5a significantly increased IgG IC-induced TNF-␣, MIP-2, and MIP-1␣ production, whereas stimulation with C5a alone had no effect on these inflammatory mediators. C5a plays a critical role in regulating the Fc␥R III/II pair to connect complement and Fc␥R pathways during IC-associated inflammation (31). Furthermore, C5a can be generated by IC-stimulated macrophages (33). There-fore, we determined if C5a signaling plays a role in activation of C/EBPs and NF-B, which may lead to the increased production of TNF-␣, MIP-2, and MIP-1␣. Reporter assays demonstrated that IgG IC-induced C/EBP activity, but not NF-B activity, was further increased by the addition of C5a (Fig. 7, D and E). Moreover, IgG IC-stimulated C/EBP DNA binding was significantly reduced in peritoneal macrophages from C5aR-deficient mice compared with WT controls (Fig.  7F). In contrast, C5aR deficiency had no effect on NF-B DNA-binding activity (Fig. 7G), consistent with the reporter data. Thus, C5a appears to have a specific role in activating C/EBPs.
To further address the underlying mechanisms whereby C5a enhances IgG IC-induced cytokine and chemokine production, we explored the influence of C5a on MAPK pathways. As shown in Fig. 7, H and I, C5a treatment further increased both phospho-p38 and phospho-p44/42 levels induced by IgG IC in RAW264.7 cells. Taken together, these data indicate that C5a enhances IgG IC-induced cytokine and chemokine production by elevating phospho-p38 and phospho-p44/42 levels, which lead to increased C/EBP␤ and -␦ activities.

DISCUSSION
Macrophage Fc␥R activation plays a central role in the immune defense system (1, 2). However, the signaling pathways from Fc␥Rs to the nucleus remain largely unknown. In the current study, we provide the evidence that C/EBP␤ and -␦ are key transcription factors that regulate Fc␥R-mediated induction of inflammatory cytokine and chemokines in macrophages. To our knowledge, this is the first report showing that C/EBPs are key regulators of immune complex-induced inflammatory responses.
Increasing evidence suggests an important role for C/EBPs, such as C/EBP␤ and -␦, in inflammation (12,34,35). For exam-ple, C/EBP␤ has been shown to be an effector in the induction of acute phase and inflammatory genes responsive to LPS, IL-1, or IL-6 (36, 37). C/EBP␦ has been less well characterized than C/EBP␤. However, similarly to C/EBP␤, C/EBP␦ has also been implicated in regulation of the acute phase and inflammatory responses (38,39). Interestingly, a recent study demonstrates a critical function for C/EBP␦ in a regulatory circuit that discriminates between transient and persistent TLR4 stimulation (34). In another recent study, Maitra et al. (21) show that C/EBP␦ is

. ERK1/2 is involved in IgG IC-induced C/EBP␤ and -␦ activation and subsequent cytokine/chemokine production in macrophages.
A, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of ERK1/2 inhibitor, U0126 (10 M), for the indicated time periods. Total proteins were subjected to Western blot by using rabbit anti-phospho-p44/42 (p-p44/42) antibody and rabbit anti-p44/42 antibody, respectively. B and C, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of ERK1/2 inhibitor, U0126 (10 M), for 4 h. The nuclear proteins were subjected to EMSA for C/EBP DNA binding (B) and NF-B DNA binding (C). D-F, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of ERK1/2 inhibitor, U0126 (10 M), for 5 h, and supernatants were subjected to ELISA analysis for TNF-␣ (D), MIP-2 (E), and MIP-1␣ (F) production. Data are presented as mean Ϯ S.E. (error bars) (n ϭ 10).

Immune Complex Activation of C/EBP␤ and -␦ in Macrophages
the key mediator to initiate low dose endotoxin-induced inflammation. Functional C/EBP binding sites have been identified in the promoter regions of the TNF-␣, MIP-2, and MIP-1␣ (3, 40 -42). For example, C/EBP␤ has been shown to play an important role in the regulation of the TNF-␣ gene in myelomonocytic cells (40). Furthermore, serial and site-directed deletion mutants of MIP-2 luciferase reporter genes demonstrate that the binding sites for both C/EBP␤ and NF-B are essential for the activity of the MIP-2 promoter in response to nitric oxide (41). In addition, a recent study suggests that C/EBP␤ and NF-B are both involved in the IL-1␤-responsive up-regulation of MIP-1␣ genes in chondrocytes (42). Using monocytic cells, Fernández et al. (3) recently showed that crosslinking of Fc␥R induced C/EBP␤ DNA binding to its binding

. p38 MAPK is involved in IgG IC-induced C/EBP␤ and -␦ activation and subsequent cytokine/chemokine production in macrophages.
A, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of p38 MAPK inhibitor VII (10 M) for the indicated time periods. Total proteins were subjected to Western blot by using rabbit anti-phospho-p38 (p-p38) antibody and rabbit anti-p38 antibody, respectively. B and C, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of p38 MAPK inhibitor VII (10 M) for 4 h. The nuclear proteins were subjected to EMSA for C/EBP DNA binding (B) and NF-B DNA binding (C). D-F, RAW264.7 cells were treated with 100 g/ml IgG immune complexes in the presence or absence of p38 MAPK inhibitor VII (10 M) for 5 h, and supernatants were subjected to ELISA analysis for TNF-␣ (D), MIP-2 (E), and MIP-1␣ (F) production. Data are presented as mean Ϯ S.E. (error bars) (n ϭ 10).  7). D and E, RAW264.7 cells were transiently transfected with a total of 0.5 g of DNA. 24 h after transfection, the cells were treated with 10 nM recombinant human C5a, 100 g/ml IgG immune complexes, or a combination of both stimuli for 4 h. Cell lysates were used to perform the luciferase activity assay. Luminometer values were normalized for expression from a co-transfected thymidine kinase reporter gene. The data were expressed as means of three experiments Ϯ S.E. (error bars). F and G, peritoneal macrophages obtained from wild type and C5aR-deficient mice were treated with or without 100 g/ml IgG IC for 4 h, and nuclear proteins were subjected to EMSA for C/EBP (F) or NF-B (G) DNA binding activity. H and I, RAW264.7 cells were treated with 100 g/ml IgG immune complex or IgG immune complex plus 10 nM rhC5a for 4 h. Total proteins were extracted and subjected to Western blot by using rabbit anti-phospho-p44/42 (p-p44/42) antibody, rabbit anti-p44/42 antibody, and rabbit anti-GAPDH (H) and using rabbit anti-phospho-p38 (p-p38) antibody, rabbit anti-p38 antibody, and rabbit anti-GAPDH (I).
site in the MIP-1␣ promoter, which suggests the possible involvement of C/EBP␤ in IC-induced MIP-1␣ expression. However, whether and to what extent C/EBP␤ and -␦ contribute to Fc␥R-mediated inflammatory mediator production has been unclear. Our study shows that C/EBP␣ and -␤ are expressed in unstimulated macrophages, and IgG IC stimulation up-regulates C/EBP␤ and -␦ activities. Using an siRNA-mediated knockdown approach and mice deficient for C/EBP␤ or/and -␦, our results clearly demonstrate that C/EBP␤ and -␦ play critical roles in the production of TNF-␣, MIP-2, and MIP-1␣ in IgG IC-stimulated macrophages. Interestingly, the functions of C/EBP␤ and -␦ seem to be partially redundant, although lack of either protein has a significant effect on TNF-␣, MIP-2, and MIP-1␣ production. This could also indicate the importance of C/EBP␤ and -␦ heterodimer occupancy in regulating these promoters. This is further supported by data showing that co-expression of C/EBP␤-and C/EBP␦-expressing vectors stimulated a C/EBP-driven luciferase reporter significantly more strongly than either C/EBP␤ or C/EBP␦ alone (Fig. 1C). Furthermore, because a relatively low level of DNA binding for C/EBP␦ contributes a vigorous induction of TNF-␣, MIP-2, and MIP-1␣, it is tempting to speculate that C/EBP␦ may be more effective than C/EBP␤ in supporting the IgG IC-induced transcription of TNF-␣, MIP-2, and MIP-1␣ genes.
Although the primary mechanism of C/EBP␤ regulation within inflammatory responses appears to be post-transcriptional (43), C/EBP␤ mRNA levels are also induced by inflammatory stimuli, including LPS (36). On the other hand, unlike C/EBP␤, the primary mechanism of C/EBP␦ regulation within the inflammatory responses is transcriptional (38,43). For example, LPS, IL-1, and IL-6 all can induce C/EBP␦ at the mRNA level (37). Here we show for the first time that IgG IC induces C/EBP␤ and -␦ expression at both mRNA and protein levels. Furthermore, the DNA binding activities of both C/EBP␤ and -␦ are increased upon Fc␥R activation, although whether this is due solely to increased expression of the proteins versus post-translational regulation of C/EBP protein activity is unknown and requires further investigation.
Using MAPK inhibitors, previous studies have shown that activation of MAPK is necessary for the Fc␥R-dependent induction of TNF-␣ expression in monocytes (44). Furthermore, Fc␥R ligation of monocytes/macrophages leads to both ERK1/2 and p38 activation (45,46). In addition, Song et al. (47) demonstrated that in microglia, the Ras/MEK/ERK pathway was necessary and sufficient for IC-induced MIP-1␣ expression. Thus, our finding that IgG IC treatment leads to the phosphorylation of ERK1/2 and p38 MAPK is consistent with these previous reports. Importantly, our results suggest that both ERK and p38 MAPK pathways are involved in IgG IC-induced C/EBP␤ and -␦ activation, thus stimulating cytokine and chemokine production in macrophages. Interestingly, neither ERK nor p38 MAPK affects NF-B DNA binding activity, suggesting that other signaling pathways are involved in its activation by IgG IC. This is consistent with a previous report showing that a MEK inhibitor failed to affect IC-induced p65 nuclear translocation in microglia (47). In contrast, other studies have suggested that MAPK activation is necessary for Fc␥R-depen-dent activation of NF-B in monocytes (48 -50). Collectively, however, these data do not exclude the requirement for NF-B activation in Fc␥R-mediated inflammation. In the current study, IgG IC-stimulated production of TNF-␣, MIP-2, and MIP-1␣ is inhibited by a NF-B inhibitor with a significant effect observed on TNF-␣ and MIP-1␣ levels (supplemental Fig. 2). These data suggest that NF-B may play important but differential regulatory roles in Fc␥R-mediated inflammatory mediator production.
Products of macrophages play a major role in events leading to tissue injury. TNF-␣, other cytokines, and chemokines, such as MIP-2 and MIP-1␣, secreted by macrophages have been shown to modulate the cell signaling cascades for the production of other proinflammatory and anti-inflammatory mediators during inflammation. The coordinate expression of activating and inhibitory Fc␥Rs on macrophages and other cells thus ensures the homeostasis of IC-induced inflammatory responses. For example, genetic deletion of FcR ␥-chain, which leads to the loss of cell surface expression and functional inactivation of all three activating Fc␥Rs, results in dramatically impaired inflammatory responses associated with IC formation (1,28). In contrast, enhanced macrophage responses were observed in Fc␥RII knock-out mice (1,28). Using macrophages from mice lacking FcR ␥-chain (FcR␥), we demonstrated that activation of C/EBP␤ and -␦ in macrophages by IgG IC stimulation is mediated, to a large extent, by activating Fc␥Rs. Furthermore, we showed an enhanced C/EBP DNA binding activity in macrophages from Fc␥RII-deficient mice (data not shown). These data further suggest that activation of C/EBP␤ and -␦ may function as a pivotal regulatory mechanism of IgG IC-associated responses.
Perhaps one of the most interesting results reported here is that C5a enhances IgG IC-induced cytokine and chemokine production by elevating C/EBPs but not NF-B DNA binding activities. However, the exact mechanism whereby C5a signals control C/EBP activation remains an important open question. Several proinflammatory cytokines and mediators, such as TNF, interferon-␥, and LPS have been shown to up-regulate activating Fc␥R (1,28,51). Recent studies have shown that C5a causes induction of Fc␥RIII and suppression of Fc␥RII on both alveolar macrophages and RAW264.7 cells (31,52,53). Furthermore, genetic ablation of C5aR expression completely abolished this regulation of Fc␥Rs. These studies provide definite evidence that C5a plays a critical role in regulating the Fc␥R III/II pair to connect complement and Fc␥R pathways during IC-associated inflammation. Moreover, our current data suggest that the enhancement by C5a of C/EBP activity may be mediated by increased phospho-p38 and phospho-p44/42 levels. Interestingly, our finding that C5a/C5aR has no effect on IgG IC-induced NF-B activation suggests that the C5a/C5aR pathway may have a specific role in C/EBP activation.
In summary, we report here that Fc␥R-mediated activation of C/EBP␤ and -␦ leads to cytokine and chemokine production from IgG IC-stimulated macrophages, and both MAPKs and C5a signal pathways are involved in C/EBP activation. These data support an important regulatory role of C/EBP␤ and -␦ in immune complex-associated inflammatory responses. Under-standing the underlying roles of various transcription factors in regulating the network of inflammatory system may be a crucial step for the development of new therapeutic drugs for treatment of immune complex diseases.