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Originally published online as doi:10.1189/jlb.1007676 on March 11, 2008

Published online before print March 11, 2008
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(Journal of Leukocyte Biology. 2008;83:1431-1439.)
© 2008 by Society for Leukocyte Biology

TGF-β1 and IFN-{gamma} stimulate mouse macrophages to express BAFF via different signaling pathways

Hyun-A Kim*, Seong-Hyun Jeon*, Goo-Young Seo*, Jae-Bong Park{dagger} and Pyeung-Hyeun Kim*,{ddagger},1

* Department of Molecular Bioscience, School of Bioscience and Biotechnology, and
{ddagger} Vascular System Research Center, Kangwon National University, Chunchon, Republic of Korea; and
{dagger} Department of Biochemistry, College of Medicine, Hallym University, Chunchon, Republic of Korea

1Correspondence: Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chunchon 200-701, Republic of Korea. E-mail: phkim{at}kangwon.ac.kr


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ABSTRACT
 
B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF-β1 and IFN-{gamma} induce BAFF expression to see if TGF-β1 and IFN-{gamma} regulate B cell differentiation via macrophages. We found that TGF-β1 stimulated mouse macrophages to express BAFF and that a typical TGF-β signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad-binding elements. Importantly, TGF-β1 enhanced the expression of membrane-bound and soluble forms of BAFF. IFN-{gamma} further augmented TGF-β1-induced BAFF expression. IFN-{gamma} caused phosphorylation of CREB, and overexpression of CREB increased IFN-{gamma}-induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1{alpha} (a well-known transducing molecule of IFN-{gamma}) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN-{gamma}. Taken together, these results demonstrate that TGF-β1 and IFN-{gamma} up-regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

Key Words: Smad • CREB • promoter


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INTRODUCTION
 
It is generally accepted that Th cells have the most effect on B cell maturation and differentiation once the B cells recognize protein antigens. However, it is increasingly clear that APCs directly affect B cells. Thus, dendritic cells (DCs) enhance B cell proliferation and differentiation [1 2 3 ], and macrophages also regulate B cell responses [4 , 5 ]. B cell-activating factor belonging to the TNF family (BAFF; also known as TALL-1, THANK, BLyS, and zTNF4), derived from macrophages and DCs, is thought to play a key role in such effects [6 ]. It is involved in cell survival and maturation, germinal center formation, antibody production, and class-switching recombination (CSR) [7 8 9 ]. Activation-induced deaminase (AID) is expressed during CSR, and it has been demonstrated that BAFF induces AID expression [10 11 12 ]. In addition, transgenic mice overexpressing BAFF have increased numbers of peripheral B cells and elevated serum Ig levels and develop symptoms of autoimmune disease [7 , 13 , 14 ]. Conversely, BAFF-deficient mice display severely impaired B cell maturation beyond the immature, transitional stage with significant defects in peripheral B cell numbers and antibody responses [15 16 17 ].

IFN-{gamma} is known to stimulate BAFF synthesis by monocytes, macrophages, DCs, and neutrophils [10 , 18 , 19 ], and IL-10, IFN-{alpha}, and bacterial components such as LPS and peptidoglycan also enhance BAFF expression [5 , 10 , 18 ]. On the other hand, TGF-β, a multifunctional peptide, promotes switching to IgA and IgG2b in mouse and human B cells [20 21 22 ]. We have demonstrated that Smad3/4, Runt-related transcription factor 3 (Runx3), and p300 are important mediators of TGF-β-induced germline-{alpha} and -{gamma}2b transcription and of the subsequent IgA and IgG2b CSR [23 24 25 ]. However, it is not known if the stimulatory action of TGF-β1 on macrophages is implicated in B cell activation and differentiation [26 , 27 ].

In this study, we explored the effects of TGF-β1 along with IFN-{gamma} on BAFF expression by mouse macrophages to see if TGF-β1 and IFN-{gamma} regulate B cell differentiation indirectly by influencing macrophages. We found that TGF-β1 and IFN-{gamma} stimulated macrophages to express BAFF. In essence, TGF-β1 induced BAFF expression via Smad3/4, and protein kinase A (PKA) and CREB were major mediators of the BAFF expression induced by IFN-{gamma}.


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MATERIALS AND METHODS
 
Reagent
TGF-β1 and IFN-{gamma} were purchased from R & D Systems (Minneapolis, MN, USA). 2,2'-Azinobis-(3-ethylbenzthiazoline sulphonic acid) (ABTS), H89, LPS, AG490, and forskolin were from Sigma Chemical Co. (St. Louis, MO, USA). The antibodies used in BAFF ELISA and FACS were purchased from Alexis Biosystems (San Diego, CA, USA).

Mice
BALB/c mice were purchased from Orient Co. Ltd. (Gyeonggi-do, Korea) and maintained on an 8:16-h light:dark cycle in an animal environmental control chamber (Myung Jin Inst. Co., Korea). They were fed Purina Laboratory Rodent Chow 5001 ad libitum. Mice that were 8–12 weeks old were used in this study. Animal care was in accordance with the institutional guidelines of Kangwon National University (Korea).

Cell culture
The murine macrophage cell line RAW264.7 was cultured in DMEM (2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) plus 10% FBS (HyClone Labs, Logan, UT, USA) in a humidified CO2 incubator. Bone marrow stem cells were isolated from BALB/c mouse femurs and cultured with 10 ng/ml M-CSF (R & D Systems) for 7 days. Thereafter, expression of the macrophage surface marker CD11b was detected by using mouse anti-CD11b mAb (Dinona Inc., Seoul, Korea)—{approx}90% when analyzed by FACS. In the proliferation assay, RAW 264.7 cells were incubated with TGF-β1 (1 ng/ml), and cell proliferation was measured with the Cell Counting Kit-8 reagent (Dojindo Laboratories, Tokyo, Japan).

ELISA for mouse BAFF
Cells were cultured in various conditions, and supernatant BAFF levels were determined by ELISA. Briefly, anti-mouse BAFF antibody (2 µg/ml) was added to 96-well U-bottom polyvinyl microplates. After incubation overnight at 4°C, the plates were washed and blocked with 1% gelatin for 1 h. Supernatant samples (50 µl) or standard protein (mouse recombinant BAFF) diluted in 0.5% gelatin were added to the wells. After incubation for 1 h at 37°C, the plates were washed again, and 2 µg/ml biotinylated anti-mouse BAFF antibody was added for 1 h at 37°C. The plates were then washed and incubated with streptavidin-HRP (R & D Systems) for a further hour. After washing, 0.2 mM ABTS was added to the wells, and 10 min later, the colorimetric reaction was measured at 405 nm with a VERSAmax ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

Flow cytometry
Cultured cells were washed with HBSS and resuspended in 0.01 M PBS at a density of 1 x 106 cells/ml. Anti-mouse BAFF antibody was added to the cell suspension and the cells were placed at 4°C for 30 min. After washing, they were incubated with PE-conjugated anti-rat IgG antibody (Becton Dickinson, San Jose, CA, USA) at 4°C for 30 min, washed three times with 0.01 M PBS, and resuspended in 0.01 M PBS–1% formalin. Cytofluorometric analysis was carried out with a FACScan (Becton Dickinson, Mountain View, CA, USA).

RT-PCR
RNA preparation, RT, and PCR were performed as described previously [23 ]. Primers for PCR were synthesized by Bioneer Corp. (Seoul, Korea). The primers for mouse BAFF were: forward primer, 5'-GCC GCC ATT CTC AAC ATG AT-3', and reverse primer, 5'-TTA GGG CAC CAA AGA AGG TG-3'. Primers spanning the mouse BAFF gene amplified the two reported mRNA forms: BAFF and {Delta}BAFF, as the expected 468 and 409 bp products, respectively. PCR products were separated on a 2% agarose gel and photographed. Band intensities were quantified using Scion Image software (Scion Corp., Frederick, MD, USA).

Plasmid construction
A mouse BAFF promoter DNA fragment (–703~+210, 912 bp) was amplified from mouse spleen genomic DNA by PCR. The PCR primers were based on the sequences in GenBank (Accession No. NT_039455). It was subcloned into pGL3 (Promega, Madison, WI, USA) using KpnI and BglII restriction enzyme sites, and this BAFF promoter reporter was designated pBAFF. MatInspector software (Genomatix Software, Munich, Germany) and TFSEARCH, Version 1.3 (Computational Biology Research Center, Japan), were used to identify putative transcription factor-binding motifs. Mutations were introduced into the putative Smad-binding elements (SBEs) of pBAFF using a QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The mammalian expression vectors Smad3, Smad4, and Smad7, generously provided by Dr. Masahiro Kawabata (Department of Biochemistry, The Cancer Institute, Tokyo, Japan), were subcloned into N-terminal Flag-tagged pcDNA3. pCMV2-CREB (a rat CREB expression plasmid) was obtained from Dr. Paul R. Dobner (University of Massachusetts Medical School, Worcester, MA, USA). Dominant-negative (DN)-CREB with its serine 133 phosphorylation site mutated to alanine was made as described previously [28 ]. The expression plasmid containing the cDNA for Stat1 and DN-Stat1, which were subcloned into plasmid RC/CMV, was generously provided by Dr. James Darnell Jr. (Laboratory of Molecular Cell Biology, The Rockefeller University, New York, NY, USA).

Transfection and luciferase assay
RAW264.7 cells were transfected using GeneSHUTTLE-20 according to the manufacturer’s protocol (Qbiogene, Irvine, CA, USA). Reporter plasmids were cotransfected with expression plasmids and pCMVβ-galactosidase (pCMVβ-gal; Stratagene), and luciferase and β-gal assays were performed as described [23 ].

Cell lysis and immunoblotting
For Western blot analysis, total cell lysates were subjected to SDS-PAGE under reducing conditions, and proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Specific immunodetection was carried out by incubation with anti-CREB antibody (Cell Signaling Technology, Beverly, MA, USA), antiphospho-CREB antibody (Cell Signaling Technology), or anti-Flag antibody (Sigma Chemical Co.), followed by peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL, USA) or goat anti-rabbit IgG (Pierce). The presence of specific proteins was revealed by chemiluminescence assays with a Supersignal detection kit (Pierce).

Immunofluorescence
RAW 264.7 cells were grown on round coverslips and fixed with 3.7% paraformaldehyde in 0.01 M PBS for 30 min. They were permeabilized with 0.2% Triton X-100 in 0.01 M PBS for 15 min and incubated with a blocking solution (2% BSA in 0.01 M PBS). They were then incubated with anti-BAFF antibody (Alexis Biosystems) for 2 h at room temperature and further with FITC-conjugated anti-rat IgG (Pierce) for 30 min in the blocking solution. The stained cells were mounted on glass slides with Gel/Mount and observed with a laser-scanning confocal microscope (Fluoview, FV-300, Olympus, Melville, NY, USA).

Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed using a ChIP assay kit (Upstate Biotechnology, Inc., Lake Placid, NY, USA). RAW264.7 cells (1x106) were fixed with 1% formaldehyde, washed, resuspended in lysis buffer, and sonicated. After removing cell debris by centrifugation, the supernatant was diluted tenfold with ChIP dilution buffer and precleared with a salmon sperm DNA/Protein A agarose–50% slurry. The supernatant fraction was transferred to a fresh tube with 10 µg/ml anti-Smad3 antibody (Zymed Laboratories Inc., San Francisco, CA, USA) and incubated overnight at 4°C. Salmon sperm DNA/Protein A agarose–50% slurry (60 µl) was added to the immune complexes, and after incubation for 1 h at 4°C, the supernatant was discarded. The histone-DNA cross-links were reversed by digestion with 2 µl 10 mg/ml proteinase K, and the DNA was extracted, dissolved in 20 µl Tris/EDTA buffer, and subjected to PCR. The primer sequences were the following: pBAFF promoter region, forward, 5'-CCT TCC AGA CCA GGA AAG AC-3', and reverse, 5'-GTG CTT GAG TCT GAA CTG CAT-3', and the products were resolved by electrophoresis on 2% agarose gels.

Preparation of oligonucleotide probes
The sequences of the upper strands of the double-stranded oligonucleotides used in EMSAs are given below. SBE1 probe (–691 to –662): 5'-CCA GCC AGC CTT CCA GAC CAG GAA AGA CTA-3'; SBE2 probe (–633 to –604): 5'-CCA AGC CCA GGC ACA GAC TGA GGA CAT CCT-3'; SBE3 probe (–304 to –273): 5'-CCA AAT GCA GTT CAG ACT CAA GCA CTG AGC-3'. 32P-end-labeled oligonucleotides were prepared using the Gel Shift assay system (Promega).

EMSA
DNA binding reactions were performed in 20 µl reaction volumes containing 50 fmol end-labeled dsDNA probe and 2 µg nuclear extract in the buffer of the Gel Shift assay system (Promega). The reaction mixtures were incubated at room temperature for 30 min and then loaded onto 6% native polyacrylamide gels and run in 0.5x Tris–boric acid–EDTA buffer at 130 V for 4 h. For competition experiments, cold probe (a tenfold molar excess of unlabeled oligonucleotide) was added to the complete mixture with the probe added last.

Statistical analysis
Statistical differences between experimental groups were determined by ANOVAs, and values of P < 0.01 by unpaired two-tailed Student’s t-test were considered significant.


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RESULTS
 
Effects of TGF-β1 on the expression of BAFF by mouse macrophages
Macrophages are involved in B cell activation and differentiation [4 , 6 ], and BAFF is one of the important factors expressed by macrophages [5 , 10 , 18 , 29 ]. Ig isotype switching is a critical event in B cell differentiation, in which TGF-β1 acts as a powerful switching factor for the IgA and IgG2b mouse isotypes [22 , 30 ]. However, it is not known if the action of TGF-β1 on macrophages is associated with B cell activation and differentiation. To explore the possibility that TGF-β1 regulates BAFF expression in mouse macrophages, we examined the effect of TGF-β1 on levels of endogenous BAFF transcripts in parallel to that of IFN-{gamma}, which is known to induce BAFF expression in macrophages [10 , 18 ]. As shown in Figure 1A , TGF-β1 increased the level of BAFF mRNA as much as did IFN-{gamma} in the macrophage cell line. In contrast, neither LPS (a general macrophage activator) nor IL-2 has this effect. TGF-β1 (1 ng/ml) was optimal, and BAFF transcripts were detectable by 6 h after stimulation. BAFF exists in a secreted and a membrane-bound form [29 , 31 ]. TGF-β1 was found to increase the production of BAFF by macrophages for at least 72 h (Fig. 1B , left panel) in the absence of any substantial effect on cell proliferation (Fig. 1B , right panel), indicating that it actually regulates macrophage BAFF gene expression. TGF-β1 also stimulated BAFF expression at the mRNA and protein levels in primary bone marrow-derived macrophages (Fig. 1C) .


Figure 1
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  		Figure 1. TGF-β1 stimulates mouse macrophages to express BAFF. (A) Effect of TGF-β1 on BAFF transcripts in mouse macrophages. Effects of various potential stimuli on BAFF transcription by mouse macrophages (left panel). A mouse macrophage cell line, RAW264.7, was incubated with TGF-β1 (1 ng/ml), LPS (10 µg/ml), IFN-{gamma} (10 ng/ml), or IL-2 (100 IU/ml) for 24 h. Dose response of BAFF transcription to TGF-β1 (middle panel). RAW264.7 was treated with TGF-β1 (0.04, 0.2, 1, 5 ng/ml) for 24 h. Effect of TGF-β1 on BAFF transcripts as a function of time (right panel). TGF-β1 (1 ng/ml) was added to cultures for the indicated times. BAFF mRNA levels were determined by RT-PCR. Fold increases represent relative amounts of BAFF DNA normalized with the expression of β-actin cDNA using Scion Image Analysis [National Institutes of Health (NIH) software]. (B) Effect of TGF-β1 on BAFF secretion. RAW264.7 cells were treated TGF-β1 (1 ng/ml), and soluble BAFF was measured by ELISA (left panel). Effects of TGF-β1 on the proliferation of mouse macrophages (right panel). RAW264.7 cells were incubated with TGF-β1 (1 ng/ml), and cell proliferation was assessed using a Cell Counting Kit-8 (Dojindo Laboratories). Data are means of triplicate samples ± SEM. (C) Effect of TGF-β1 on BAFF expression by bone marrow-derived macrophages (M{phi}). Freshly isolated bone marrow stem cells were differentiated as described in Materials and Methods and incubated with TGF-β1 (1 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR (left panel). For PCR, cDNAs from each sample were prepared to 1:1, 1:3, and 1:9 dilutions. BAFF secretion was measured by ELISA. Data are means of triplicate samples ± SEM;*, statistical significance when compared with media controls (P<0.01).

Effects of overexpressed Smad3 and Smad4 on TGF-β1-induced BAFF expression
As Smad3 and Smad4 are well-known intermediates in the TGF-β signaling pathway [32 33 34 ], we asked whether they mediated the BAFF expression. Overexpression of Smad3/4 enhanced the TGF-β1-induced BAFF mRNA expression, and overexpression of a DN-Smad3 abrogated BAFF transcription in a dose-dependent manner (Fig. 2A ). Overexpression of Smad3/4 also enhanced TGF-β1-induced BAFF secretion (Fig. 2B) . In addition, we examined the effect of TGF-β1 on the expression of membrane-bound BAFF by FACS and found that it progressively increased the frequency of BAFF-expressing cells as well as the intensity of their fluorescence (Table 1 ). In contrast, in the presence of TGF-β1, overexpression of Smad3/4 only marginally increased the frequency of BAFF-expressing cells but markedly increased the fluorescence intensity of the BAFF-expressing cells, indicating that the transfected Smad3/4 acted specifically on cells already committed to make BAFF. Finally, DN-Smad3 completely abolished the increase in frequency of BAFF-expressing cells promoted by TGF-β1 and the increase in their fluorescence intensity. Taken together, these results show that Smad3 and Smad4 are critical mediators of TGF-β1-induced BAFF expression in mouse macrophages.


Figure 2
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  		Figure 2. Smad3/4 mediates TGF-β1-induced BAFF expression in mouse macrophages. (A) RAW264.7 cells (1x106) were transfected with the expression plasmids Smad3/4 (each 1 µg) or DN-Smad3 (1, 3, 9 µg) and stimulated with TGF-β1 (1 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR. Fold increases represent relative BAFF DNA levels normalized with the expression of β-actin cDNA by Scion Image Analysis software. (B) BAFF secretion by Smad3/4-transfected RAW264.7 was measured by ELISA after 72 h incubation with TGF-β1. Data are means of triplicate samples ± SEM; *, P < 0.01.


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  		Table 1. Effects of TGF-β1 and Smad3/4 on Surface BAFF Expressiona

Construction and analysis of mouse BAFF promoters
To elucidate BAFF transcriptional regulation by TGF-β1 and Smad3/4, we constructed a mouse BAFF promoter reporter, pBAFF. There are at least three putative SBEs (see Fig. 3B ) within the putative BAFF promoter region (TFSEARCH, Version. 1.3, and MatInspector, Version 3.0, Genomatix Software). We first examined the effect of overexpressed Smad3/4 on pBAFF reporter activity in RAW264.7. As shown in Figure 3A , TGF-β1 stimulated reporter activity approximately twofold, and this effect was augmented by overexpressed Smad3/4. On the other hand, overexpression of Smad7, a TGF-β-inducible antagonist of TGF-β signaling [35 ], led to complete abolition of Smad3/4-mediated promoter activity.


Figure 3
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  		Figure 3. Role of putative SBEs in mouse BAFF promoter activity. (A) Effects of TGF-β1 and Smad on promoter activity. DNA segments –703 to +210 were cloned into pGL3-basic vector as described in Materials and Methods and designated pBAFF. RAW264.7 cells (1x106) were transiently cotransfected with pBAFF (1 µg) and expression vectors coding for Smad3/4 and Smad7 (1 µg each). Cells were incubated with TGF-β1 (1 ng/ml), and luciferase activity was determined 24 h later. RLA, Relative luciferase activity. (B) Effect of mutant BAFF reporters (pBAFF-mS1, -2, and -3). SBE, Putative SBE; mSBE, mutated SBE. Transfection efficiency was normalized by β-gal activity. Data are average luciferase (LUC) activities of three independent transfections with SEM (bars); *, P < 0.01; **, P < 0.05.

To determine which of the three potential SBEs are important for promoter activity, we substituted each of the CAGAC elements (Fig. 3B) . The mutant reporters (pBAFF-mS1, pBAFF-mS2, and pBAFF-mS3) did not respond to TGF-β1 and Smad3/4, indicating that each of these putative SBEs is indispensable for TGF-β1-induced BAFF promoter activity. We investigated binding of Smad3 to the SBEs by ChIP assays (Fig. 4A ). TGF-β1 markedly increased binding of Smad3 to the promoter, and the binding was further enhanced by overexpression of Smad3/4. To distinguish which SBEs bind Smad3, we investigated the binding of Smad3 to three probes, each containing a different SBE by EMSA. TGF-β1 treatment stimulated the formation of DNA–Smad3 complexes with all three probes, although binding to the second probe was relatively weak, and all of the complexes were eliminated by preincubation with a tenfold excess of cold probe (Fig. 4C) . Similar results were obtained when nuclear extracts were tested with the three probes (Fig. 4D) . These results reveal that all three putative SBEs are required for optimal TGF-β1-induced BAFF promoter activity.


Figure 4
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  		Figure 4. Smad3 specifically binds to the BAFF promoter under the influence of TGF-β1. (A) ChIP assay for Smad3 binding. Primer pairs to amplify a 404-bp segment (–683~–280 bp) encompassing SBE1, SBE2, and SBE3 were: forward, 5'-CCTTCCAGACCAGGAAAGACT-3', and reverse, 5'-GTGCTTGAGTCTGAACTGCAT-3'. RAW264.7 cells (1x106) were transfected with the expression vectors coding for Smad3/4 (each 1 µg) and cultured with TGF-β1 (1 ng/ml) for 12 h. Anti-Smad3 antibody was used to detect probe-bound Smad3. For PCR, cDNAs from each sample were prepared at 1:1 and 1:5 dilutions. (B) Western blotting (WB) to detect the expression of transfected Smad3. RAW264.7 cells (1x106) were transfected with the expression vectors coding for Smad3/4 (each 1 µg) and cultured with TGF-β1 (1 ng/ml) for 12 h. Anti-Smad3 antibody was used to detect Smad3. (C) EMSA showing complexes formed with Smad3 and pBAFF probes containing SBE1 (–691 to –662), SBE2 (–633 to –604), or SBE3 (–304 to –273). (D) EMSA showing complexes formed with a TGF-β1-induced, Smad3/4-transfected nuclear extract and pBAFF probes containing SBE1, SBE2, or SBE3. Nuclear extracts from Smad3/4-transfected RAW264.7 cells were prepared after 12 h incubation with TGF-β1.

TGF-β1 and IFN-{gamma} in combination increase BAFF expression by mouse macrophages
It has been demonstrated that IFN-{gamma} induces BAFF expression in macrophages and DCs [10 , 18 ], as also shown in Figure 1A . As the signaling pathways of TGF-β1 and IFN-{gamma} are thought to be different, we were interested in their combined effect on BAFF gene expression. In macrophage cell line and normal bone marrow-derived macrophages, stimulation of endogenous BAFF transcription by the two cytokines proved to be greater than by either on its own (Fig. 5A ), and the same effect was obtained for promoter activity (Fig. 5B) . There was parallel increase of BAFF protein in the cytoplasm (Fig. 5C) and at the secretion cell supernatants (Fig. 5D) . To investigate how IFN-{gamma} induces BAFF expression in mouse macrophages, we explored the possible involvement of Stat1, a key intermediate in IFN-{gamma}-induced gene expression [36 37 38 ]. Unexpectedly, overexpressed Stat1{alpha} hardly affected endogenous BAFF transcription (Fig. 6A ), and the same was true for promoter activity (Fig. 6B , left panel). Moreover, neither overexpression of DN-Stat1{alpha} nor AG490 (a JAK inhibitor) affected IFN-{gamma}-induced BAFF promoter activity (Fig. 6B , right panel). Apparently, the JAK/Stat1 pathway is not involved in IFN-{gamma}-induced BAFF transcription.


Figure 5
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  		Figure 5. TGF-β1 and IFN-{gamma} in combination increase BAFF expression in mouse macrophages. (A) Effects of TGF-β1 and IFN-{gamma} on endogenous levels of BAFF transcripts. RAW264.7 cells or bone marrow-derived macrophages were incubated with TGF-β1 (1 ng/ml) and IFN-{gamma} (10 ng/ml) for 24 h. Total RNA was extracted, and levels of BAFF mRNA were determined by RT-PCR. Fold increases represent relative amounts of BAFF DNA normalized with the expression of β-actin cDNA using Scion Image Analysis (NIH software). (B) Effects of TGF-β1 and IFN-{gamma} on BAFF promoter activity. RAW264.7 cells were transfected with the pBAFF reporter (1 µg) and cultured with TGF-β1 (1 ng/ml) and IFN-{gamma} (10 ng/ml) for 24 h. Promoter activity was measured using the luciferase reporter assay, and transfection efficiency was normalized by β-gal activity. Data are average luciferase activities of three independent transfections with SEM (bars). (C) Localization of BAFF protein by confocal laser microscopy. RAW264.7 cells were cultured with TGF-β1 (1 ng/ml) and IFN-{gamma} (10 ng/ml) for 24 h. Anti-BAFF antibody was used to detect cytoplasmic BAFF. (D) Effect of TGF-β1 and IFN-{gamma} on BAFF secretion. Cells were cultured with TGF-β1 (1 ng/ml) and IFN-{gamma} (10 ng/ml) for 72 h, culture supernatants were harvested, and levels of BAFF were measured by ELISA. Data are means of triplicate samples ± SEM; *, P < 0.01.


Figure 6
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  		Figure 6. CREB mediates IFN-{gamma}-induced BAFF expression by mouse macrophages. (A) Effects of overexpressed Stat1{alpha} on the expression of endogenous BAFF transcripts. RAW264.7 cells were transiently transfected with Stat1{alpha} (1 µg) and incubated with IFN-{gamma} (10 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR. Fold increase represents relative amounts of BAFF DNA normalized with the β-actin cDNA. (B) Effects of overexpressed Stat1{alpha}, DN-Stat1{alpha}, and AG490 on BAFF promoter activity. RAW264.7 cells were transiently cotransfected with pBAFF and expression vectors coding for Flag-Stat1{alpha} and Flag-DN-Stat1{alpha} (each 1 µg). Cells were then cultured with IFN-{gamma} (10 ng/ml) for 24 h. Cells were preincubated with AG490 (25 µM) for 1 h. Transcriptional activity was measured using the luciferase reporter assay, and transfection efficiency was normalized with β-gal activity (left panel). Data are average luciferase activities of three independent transfections with SEM (bars). Western blotting to detect the expression of transfected Stat1{alpha} and DN-Stat1{alpha} using anti-Flag antibody (right panel). Not shown here, AG490 (25 µM) successfully inhibited the IL-4-induced, Stat-mediated target gene expression [39 ], and overexpression of DN-Stat1{alpha} (1 µg) markedly restored the Ig germline-{alpha} promoter activity, which was repressed by IFN-{gamma} through Stat1. (C) IFN-{gamma} induces CREB phosphorylation via PKA. RAW264.7 cells were preincubated with H89 (10 µM) for 1 h and incubated with IFN-{gamma} (10 ng/ml) and forskolin (10 µM). Phosphorylated CREB (p-CREB) and total CREB (t-CREB) were detected by Western blotting. (D) H89 and DN-CREB abrogate IFN-{gamma}-induced, CREB-mediated BAFF promoter activity. RAW264.7 cells were transiently cotransfected with pBAFF and CREB or DN-CREB (each 1 µg). They were then treated with H89 (10 µM) for 1 h when it was required and incubated with IFN-{gamma} (10 ng/ml) for 24 h. Data are average luciferase activities of three independent transfections with SEM (bars); *, P < 0.01.

As IFN-{gamma} activates the PKA/CREB signaling pathway in murine peritoneal macrophages [40 ], we examined this pathway in our system and detected IFN-{gamma}-activated CREB phosphorylation (Fig. 6C) . Forskolin, a PKA activator, which stimulates the phosphorylation of CREB [41 ], was included as a positive control and also stimulated CREB phosphorylation, and preincubation with H89, a PKA inhibitor, inhibited CREB phosphorylation. Moreover, as shown in Figure 6D , overexpression of CREB increased IFN-{gamma}-induced BAFF promoter activity twofold, and DN-CREB abrogated this effect. Taken together, these results suggest that IFN-{gamma} stimulates BAFF expression mainly through the PKA–CREB pathway. We are currently analyzing CREB-binding elements, i.e., CREs within the cloned BAFF promoter sequences.

Thus far, we have demonstrated that TGF-β1 and IFN-{gamma} stimulate mouse macrophages to express BAFF, mainly via Smad3/4 in the first case and PKA/CREB in the second. Cytokines often cross-talk during their signal transduction. Thus, we assessed the roles of Smad3/4 and PKA/CREB in BAFF expression in the presence of both cytokines. Overexpressed DN-Smad3 and H89 each only partially abrogated the combined effect of the two cytokines on promoter activity (Fig. 7 ). Meanwhile, overexpression of DN-Smad3 together with H89 treatment completely eliminated the promoter activity. These results confirm that TGF-β1 and IFN-{gamma} stimulate BAFF expression via different signaling pathways.


Figure 7
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  		Figure 7. Effects of DN-Smad3 and H89 on BAFF promoter activity under the influence of TGF-β1 and IFN-{gamma}. RAW264.7 cells were transiently cotransfected with pBAFF (1 µg) and the expression vector coding for DN-Smad3 (1 µg). They were then treated with H89 for 1 h when it was required and incubated with TGF-β1 (1 ng/ml) and IFN-{gamma} (10 ng/ml) for 24 h. Transcriptional activity was measured using the luciferase reporter assay, and transfection efficiency was normalized with β-gal activity. Data are average luciferase activities of three independent transfections with SEM (bars); *, P < 0.01.


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DISCUSSION
 
Although BAFF is believed to be the most important macrophage-derived B cell-activating factor [5 , 6 ], the pathway involved in BAFF expression is not clear. In the present study, we demonstrate that TGF-β1 stimulates mouse macrophages to produce BAFF and that a typical TGF-β signaling pathway is involved [42 ]. Smad3 and Smad4 promoted BAFF expression, and Smad7 inhibited it in the mouse macrophage cell line, and we showed that Smad3 binds three putative SBEs in the promoter and that these elements are indispensable for promoter activity. More importantly, normal bone marrow-derived macrophages also responded to TGF-β1. It is thus clear that TGF-β1 is an important stimulant of BAFF expression by mouse macrophages and that Smad3/4 is mainly responsible for mediating the TGF-β1-induced BAFF expression.

It is known that the DNA-binding specificity of Smad proteins is relatively low. Thus, the individual Smad proteins must cooperate with other DNA-binding proteins to elicit specific transcriptional responses [43 , 44 ]. In this context, we have demonstrated that Runx3 synergizes with Smad3/4 to induce Ig germline-{alpha} transcription, leading to IgA isotype switching [24 ]. In addition, hypoxia-inducible factor-1{alpha} cooperates with Smad3/4 to mediate TGF-β1-induced vascular endothelial growth factor transcription in mouse macrophages [45 ]. We have tested the possible involvement of Runx3, activating transcription factor 2, AP-1, specificity protein 1, and NF-{kappa}B in Smad3/4-mediated BAFF promoter activity, as these DNA-binding proteins have been reported to be involved in Smad3-mediated TGF-β target gene expression [44 , 46 47 48 49 50 51 ], and in particular, NF-{kappa}B is involved in BAFF transcriptional activation in EBV-infected and malignant human B cells [52 , 53 ]. However, none of them had much effect on promoter activity (data not shown).

We found that TGF-β1 and IFN-{gamma} increased the expression of membrane-bound and soluble forms of BAFF. Further, Smad3/4 augmented the expression of both forms in macrophages stimulated with TGF-β1. These results suggest that expression of the two BAFF forms is coordinately regulated. In this regard, it is noteworthy that overexpression of Smad3/4 greatly increased the amount of membrane-bound BAFF in cells already committed to make it by exposure to TGF-β1. What is the physiological meaning of this phenomenon? It has been proposed that soluble BAFF is derived from the membrane-bound form in human mononuclear cells [18 ]. We suggest therefore that TGF-β1 first induces the production of membrane-bound BAFF, and the soluble form is then derived from it. BAFF is known to induce AID and Ig CSR in B cells [11 , 12 ], and we found that the soluble form of BAFF derived from TGF-β1-activated macrophages actually increases AID expression in mouse B cells (Supplemental Fig. 1). These results suggest that TGF-β1 contributes indirectly to B cell Ig isotype switching by inducing macrophages to produce BAFF.

Our findings extend an earlier study of the effect of IFN-{gamma} on BAFF expression, in which IFN-{gamma} enhanced membrane-bound and soluble forms of BAFF in normal blood monocytes [18 ]. It is well known that IFN-{gamma} responses are mainly mediated via Jak–Stat and sequence elements (IFN-{gamma}-activated sequence) in the promoters of IFN-{gamma} target genes [36 37 38 ]. However, we observed that JAK and Stat1 were not involved in the IFN-{gamma}-induced BAFF expression and that PKA/CREB was the dominant pathway. Consistent with these results, it has been reported that IFN-{gamma} activates the cAMP/PKA/CREB signaling pathway in murine peritoneal macrophages [40 ]. In fact, there is now considerable evidence for Stat1-independent pathways in IFN-{gamma} signaling [54 55 56 ]. In this context, our finding that TGF-β1 and IFN-{gamma} synergized in stimulating BAFF expression warrants a comment. TGF-β signaling involves phosphorylation of CREB [57 ], and CREB cooperates with Smads to mediate TGF-β-induced germline Ig-{alpha} promoter activity [58 ]. Thus, cross-talk between the two cytokines may occur at multiple levels during BAFF gene expression.

In conclusion, TGF-β1 and IFN-{gamma} are well-known cytokines that induce isotype-switching recombination of IgA and IgG2a, respectively, in mouse B cells [59 60 61 62 63 ]. It is generally accepted that activated Th cells produce these cytokines and that they in turn directly modulate B cells. Nonetheless, there is accumulating evidence that macrophages affect B cell proliferation and differentiation [4 5 6 , 10 ]. We have shown that TGF-β1 and IFN-{gamma} stimulate mouse macrophages to produce BAFF, which can activate B cells to express AID [10 11 12 ], and BAFF null and BAFF-receptor null mice mount poor T-dependent and T-independent antibody responses [11 , 16 , 64 ]. Therefore, our in vitro studies raise the possibility that TGF-β1 and IFN-{gamma} cause macrophages to produce BAFF and so, have an important effect on Ig isotype switching in vivo.


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ACKNOWLEDGEMENTS
 
This work was supported by a Basic Research Program grant (No. R01-2006-000-11127-0) from the Korea Science and Engineering Foundation and by the second stage of the Brain Korea 21 program. It was carried out in facilities of the Vascular System Research Center and Institute of Bioscience and Biotechnology at Kangwon National University.

Received October 8, 2007; revised January 4, 2008; accepted February 18, 2008.


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