Characterization of the Murine IgG Fc Receptor III and IIB Gene Promoters

The two low affinity IgG Fc receptors (FcγR), FcγRIII and FcγRIIB, are coexpressed on myeloid effector cells, and their genes, as reported here, are positively and negatively regulated by both C5a and interferon-γ through different signaling mechanisms. Two 48- and 43-bp sequences (C5a-inductive region (CIR) and C5a-suppressive region (CSR)) in the FcγRIII and FcγRIIB 5′-flanking regions that are necessary for C5a induction and suppression, respectively, are defined. Sequence analysis of the CIR and CSR, which localize apart from the interferon-γ-responsive regions in each gene, revealed the presence of a novel element that differs by two nucleotides between FcγRIII and FcγRIIB. Mutation analysis of the CIR and CSR showed that this small difference determines inverse responsiveness in an FcγR gene context-dependent manner. Our study suggests that C5a uses similar DNA motifs (defined as GTGAXXTCCA) in both pathways of transcriptional induction and suppression of FcγRIII and FcγRIIB.

Activated complement component C5a is a pleiotropic molecule that regulates the activity of many cell types, with a broad range of biological functions in the immune system (1). C5a binds to at least two seven-transmembrane domain receptors, the C5a anaphylatoxin receptor (C5aR 2 ; CD88) and C5L2, expressed on a variety of immune cells, including circulating leukocytes, mast cells, basophils, macrophages, and many others. C5aR-dependent activation of these cells by C5a results in inflammatory mediator release and granule secretion, which in turn alter vascular permeability, induce smooth muscle contraction, and promote inflammatory cell migration (2). It is well established that this C5a-triggered cascade of events contributes to the pathogenesis of various diseases in humans, including myocardial ischemia/reperfusion injury and respiratory distress syndrome (3)(4)(5). In addition, genetic deletion of C5aR is very effective in preventing inflammation in animal models of erosive arthritis, anti-phospholipid syndrome, and antibodydependent type II autoimmunity (6 -8).
Complement activation occurs through multiple pathways (classical, alternative, and lectin-binding) in the circulation, each of which produces C5a. Interestingly, C5a is also formed within the extravascular tissue compartments through activation of resident innate immune cells such as tissue macrophages and requires the presence of receptors for the Fc portion of IgG (Fc␥R) (reviewed in Ref. 9). The Fc␥R exert their function through paired expression of activating (Fc␥RI, Fc␥RIII, and Fc␥RIV) and inhibitory (Fc␥RIIB) receptors (reviewed in Refs. 10 and 11). Compelling evidence suggests that the ratio of the opposing signaling Fc␥R is critical in setting the cellular thresholds for the pathogenic activity of autoantibodies (reviewed in Refs. 10 -12) and that C5a regulates this ratio, thus amplifying inflammatory Fc␥R responses in autoimmunity (8,13).
Although C5a and Fc␥R likely cooperate in the context of immunological diseases, the molecular mechanisms of transcriptional regulation of C5a-responsive genes, such as those encoding Fc␥RIII and Fc␥RIIB, remain to be elucidated. Fc␥RIII expression on myeloid cells is enhanced by exposure to C5a, and this reflects an increase in the rate of Fc␥RIII and FcR␥ transcription (8,13). In contrast, C5a rapidly down-regulates Fc␥RIIB both in vitro and in vivo (13,14). Fc␥RIII and Fc␥RIIB thus represent a prototypic C5a-responsive gene pair whose simultaneous induction and suppression result in augmentation of the innate immune response.
In this work, we present the first characterization of cis-acting DNA elements in the Fc␥RIII and Fc␥RIIB 5Ј-flanking regions that are responsible for gene activation and inhibition by C5a and compare the data with those obtained for interferon-␥ (IFN-␥), a lymphokine that has previously been established to induce and suppress gene transcription of the same two low affinity Fc␥R (15). We found that inverse Fc␥R gene regulation differs between C5a and IFN-␥ with respect to time kinetics, signaling requirements, and promoter localizations. Two positive and negative acting regions, termed the C5a-inductive region (CIR) and the C5a-suppressive region (CSR), are shown to be responsible for Fc␥RIII induction and Fc␥RIIB suppression. Both the CIR and CSR contain homologous GTGAXXTCCA sequences, indicating that similar DNA motifs are involved in the transcriptional activation and inhibition of the functionally distinct Fc␥R. * The work was supported by Deutsche Forschungsgemeinschaft Grant SFB587 (to J. E. G.). 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
Plasmid Construction-Mouse Fc␥RIII and Fc␥RIIB gene promoter fragments were amplified by PCR using Fc␥RIII and Fc␥RIIB genomic clones (kindly provided by Dr. Toshiyuki Takai, Department of Experimental Immunology, Tohoku University, Sendai, Japan) as templates and subcloned into the NheI and HindIII sites of the luciferase reporter vector pGL3-Basic (Promega Corp.). The PCR primers used for cloning of the Fc␥RIIB promoter (positions Ϫ729 to ϩ585) were 5Ј-ATAGCTAGCGGCCTCAGAAG-AGC-3Ј (sense) and 5Ј-CTTAAGCTTGTCGACGGATCA-GTAGGAAC-3Ј (antisense). The PCR primers used for cloning of the Fc␥RIII promoter (positions Ϫ1117 to ϩ18) were 5Ј-CTTAAGCTTGCTAGCCTTGATAGGTATC-3Ј (sense) and 5Ј-CGCAAGCTTAAAGTCATTCAAGAAC-3Ј (antisense). 5Ј-and 3Ј-end deletions were introduced by standard PCR mutagenesis. Point mutations were introduced using the QuikChange mutagenesis protocol (Stratagene, La Jolla, CA): GTGAAGTCCA to GTGAGTTCCA for the negative C5a-responsive region of Fc␥RIIB and GTGAGTTCCA to GTGAAGTCCA for the positive C5aresponsive region of Fc␥RIII. All constructs were confirmed by direct DNA sequencing.
Transient Transfection and Luciferase Reporter Gene Assays-All Fc␥RIII and Fc␥RIIB promoter-reporter plasmids (0.7 g) were cotransfected with 0.3 g of the reference plasmid pRL-CMV into 5 ϫ 10 5 RAW 264.7 cells in 12-well plates using 1.6 l of Lipofectamine TM (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. The cells were recovered after 24 h, cultured for 24 h in 1% fetal calf serum-containing RPMI 1640 medium, and treated with 4.6 nM C5a (50 ng of recombinant human C5a) or 300 units/ml recombinant mouse IFN-␥ or left unstimulated for an additional 0.5-4 h. In some experiments, cells were pretreated for 1 h with pertussis toxin (200 ng/ml) to block G i -dependent C5aR activity (14). In additional inhibition experiments, the following reagents were used: PP2 (10 M; inhibitor of Srk kinases), PI3K␥ inhibitor (10 nM), and Akt inhibitor IV (1.25 M). The cells were then lysed and measured for luciferase activities using the Dual-Luciferase reporter assay system (Promega Corp.). The firefly luciferase activity was normalized to Renilla luciferase activity to yield the relative promoter activity.
Statistical Analysis-Data for comparison of mean values among samples were analyzed by a two-sided unpaired Student's t test.

Different Time Kinetics and Signaling Pathways in the Inverse Gene Regulation of Fc␥RIIB and Fc␥RIII by IFN-␥ and C5a-To
compare the promoter regions of murine Fc␥RIII and Fc␥RIIB, two reporter plasmids were constructed using Fc␥RIII upstream sequences from positions Ϫ1117 to ϩ18 and Fc␥RIIB upstream sequences from positions Ϫ729 to ϩ585 cloned in front of the promoterless luciferase gene. These two constructs, Fc␥RIII p(Ϫ1117)Luc and Fc␥RIIB p(Ϫ729)Luc, were transfected into alveolar (MH-S) and peritoneal (RAW 264.7) macrophage cell lines of mouse origin. The representative experiments in RAW 264.7 cells are shown in Fig. 1. In both RAW 264.7 and MH-S cells, Fc␥RIII p(Ϫ1117)Luc was induced by C5a and IFN-␥, whereas Fc␥RIIB p(Ϫ729)Luc was reduced by each stimulus. Optimal responses of enhanced versus suppressed Fc␥RIII and Fc␥RIIB promoter activities in transfected cells were obtained by stimulating them with C5a for 1-2 h and with IFN-␥ for 4 h (Fig. 1). These data indicate that both C5a and IFN-␥ activate, by different kinetics, inverse Fc␥R regulation in macrophages, thus confirming previous findings in Fc␥R mRNA and protein detection assays (13)(14)(15).
C5a and IFN-␥ were also analyzed for their signaling requirements in the regulation of Fc␥RIII p(Ϫ1117)Luc and Fc␥RIIB p(Ϫ729)Luc promoter activities. In RAW 264.7 cells, the simultaneous positive and negative Fc␥RIII and Fc␥RIIB regulation by C5a, but not by IFN-␥ (which, in contrast to C5a, was inhibited by the Src kinase inhibitor PP2), was sensitive to pharmacological inhibition with pertussis toxin (a general inhibitor of G i proteins) and the PI3K␥-and Akt-specific inhibitors (Fig. 2). These results indicate that Fc␥R regulation by C5a involves the same G i -PI3K␥-Akt signaling complex that is activated by C5aR for triggering inflammatory cell migration and suppression of interleukin-12/23 production (14,16). They also suggest that IFN-␥ and C5a control inverse Fc␥R expression through different pathways.
Localization of the Positive CIR in the Fc␥RIII Promoter-Various 5Ј-deletion mutants of Fc␥RIII p(Ϫ1117/ϩ18)Luc were tested for their ability to respond to C5a in comparison with IFN-␥. In RAW 264.7 cells, the property of induction by IFN-␥ was constantly seen for all deletion mutants containing at least 92 bp of 5Ј-flanking sequence and the first 18 bp of the 5UT1/S1 exon (Fig. 3). In contrast to IFN-␥, however, induction by C5a, which was significantly detectable in Fc␥RIII p(Ϫ1117)Luc, was lost when an additional 570 bp of 5Ј-flanking sequence was deleted (Fig. 3A). These results suggest that the promoter elements that confer IFN-␥ induction lie within a sequence from positions Ϫ92 to ϩ18, the previously established region that constitutes the basal promoter of mouse Fc␥RIII (17,18). They also suggest that the C5a-responsive DNA elements localize to more upstream sequences in a region between positions Ϫ1117 and Ϫ548. As shown in Fig. 3B, functional characterization of additional deletion mutants identified C5a responsiveness within a 48-bp sequence at positions Ϫ808 to Ϫ761 in the Fc␥RIII gene promoter, which we have termed the CIR.
Localization of the Negative CSR in the Fc␥RIIB Promoter-In both Bal17 B cells and P388D1 macrophages, it has been shown that a sequence at positions ϩ75 to ϩ585 is required to drive full reporter gene activity in stable Fc␥RIIB promoter transfectants (19). This observation, combined with the finding that Fc␥RIIB p(Ϫ729/ϩ585)Luc but not Fc␥RIIB p(Ϫ729/ϩ75)Luc responded to C5a and IFN-␥, prompted us to examine the presence of regulatory elements in region ϩ75 to ϩ585 that may contribute to Fc␥RIIB suppression. Several 3Ј-deletion mutants of Fc␥RIIB p(Ϫ729/ϩ585)Luc were analyzed for their ability to negatively respond to C5a in comparison with IFN-␥. Suppression of Fc␥RIIB gene activity by C5a was seen in p(Ϫ729/ ϩ585)Luc and p(Ϫ729/ϩ484)Luc, but not in the p(Ϫ729/ ϩ442)Luc deletion mutant (Fig. 4), thus indicating that C5a suppression is contained within the 43-bp sequence at positions ϩ442 to ϩ484, which we have termed the CSR. In contrast to C5a, suppression by IFN-␥ remained detectable in

C5a-mediated Inverse Fc␥R Gene Regulation
DECEMBER 28, 2007 • VOLUME 282 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 37909 p(Ϫ729/ϩ442)Luc and the additional Fc␥RIIB p(Ϫ729/ ϩ394)Luc deletion mutant (Fig. 4). These results suggest that the regulatory elements that confer transcriptional repression by IFN-␥ are differently located compared with those that mediate Fc␥RIIB suppression by C5a.
A Two-nucleotide Difference in the CIR and CSR Determines Induction Versus Suppression of Fc␥RIII and Fc␥RIIB by C5a-Sequence analysis of the CSR of Fc␥RIIB showed no obvious homology to previously defined consensus sites of known transcription factors. Comparison with the CIR, however, revealed the existence of a DNA motif (GTGAAGTCCA; here named the C5a homology element (CHE)) in the Fc␥RIII CSR that is similar to the GTGAGTTCCA sequence of the Fc␥RIIB CIR with a two-nucleotide difference (Fig. 5A). To examine whether this difference in the two CHE determines the opposite functionalities of the CSR and CIR, the Fc␥RIII-related CHE was introduced into the Fc␥RIIB gene and vice versa, creating Fc␥RIIB p(CHE-RIII)Luc and Fc␥RIII p(CHE-RIIB)Luc. When transfected into RAW 264.7 cells, both constructs achieved almost normal levels of constitutive expression, but lost any C5a responsiveness (Fig. 5B), indicating that an intact GTGAXYTCCA CHE motif is critical for both activation and repression by C5a. In addition, it appears that the two CHE act in an Fc␥R gene context-specific manner because they are active in transferring neither induction nor suppression from one gene to the other.

DISCUSSION
Our analysis of the murine Fc␥RIII and Fc␥RIIB gene regulatory regions suggests different mechanisms of transcriptional control by IFN-␥ and C5a. Within the promoter of the Fc␥RIII gene, we have confirmed that the 92 bp upstream from the transcription initiation site are sufficient to drive reporter gene activity in macrophage cell lines and to mediate PP2-sensitive IFN-␥ activation. Furthermore, we have defined a distal region responsible for C5a induction as the 48 bp at position Ϫ808 to Ϫ761, called the CIR. The CIR acts as a transcriptional activator in the presence of C5a, but not IFN-␥. The 5Ј-flanking region of Fc␥RIIB contains, in addition to upstream sequences from the start of transcription, two noncoding 5UT1 and 5UT2 exons. Constitutive Fc␥RIIB activity in macrophages depends on intronic sequences downstream of the 5UT2 exon. We define the region responsible for C5a suppression, the CSR, as the 43 bp within this intronic domain, from positions ϩ442 to ϩ484. Moreover, the CSR localizes apart from the negative IFN-␥responsive region, and transcriptional repression of Fc␥RIIB by C5a involves the G i -PI3K␥-Akt pathway of C5aR signaling.
Previous work on the intracellular signaling mechanisms of C5aR suggested that, in addition to the G i -PI3K␥-Akt cascade, other kinase pathways that lead to activation of NF-B, cAMP-responsive element-binding protein (CREB), and STAT3 (signal transducer and activator of transcription 3) can be activated by C5aR (20 -23). Although the CSR acts as a transcriptional repressor in the presence of C5a, it appears not to involve binding of NF-B, CREB, and STAT3. This is suggested by the lack of any consensus sites for these tran-scription factors within the CSR. Examination of the CSR reveals, however, the presence of a DNA motif that is similar to the CHE in the CIR of Fc␥RIII. For the Fc␥RIIB and Fc␥RIII promoters, we have found that a single two-nucle-

C5a-mediated Inverse Fc␥R Gene Regulation
DECEMBER 28, 2007 • VOLUME 282 • NUMBER 52 otide difference in the two CHE is responsible for the selectivity of induction versus suppression by C5a.
Recent studies elucidating the synergy by which complement and Fc␥R trigger inflammatory processes and immunopathological events provide us with a better understanding of the role of C5a as a critical amplifier of Fc␥R-mediated responses through induction of Fc␥RIII and suppression of Fc␥RIIB (7-9, 13, 14, 24 -27). The results from our study suggest that C5a uses similar DNA motifs (defined as GTGAXXTCCA, the CHE) in both pathways of transcriptional induction and suppression of Fc␥RIII and Fc␥RIIB. To our knowledge, this is the first work that examines the molecular events of both the positive and negative gene regulatory capacity of C5a at the promoter level. It may thus form the basis for future work on the isolation and characterization of the relevant CHE-binding factors that, as indicated here, are likely not related to the NF-B and CREB transcription factors.