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The Journal of Immunology, 2004, 173: 6211-6219.
Copyright © 2004 by The American Association of Immunologists

Differential Gene Expression Modulated by the Cytoplasmic Domain of Fc{gamma}RIa (CD64) {alpha}-Chain1

Hongwei Qin*, Jeffrey C. Edberg*, Andrew W. Gibson*, Grier P. Page{dagger}, Lihong Teng* and Robert P. Kimberly2,*

* Department of Medicine, and {dagger} Section on Statistical Genetics, Department of Biostatistics, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytoplasmic domain (CY) of the ligand-binding {alpha}-chain of the {gamma}-chain-associated FcRs can modulate receptor function such as phagocytosis, endocytosis, and intracellular trafficking of receptor-Ag complexes. To assess the potential role of the CY domain of human Fc{gamma}RIa (CD64) {alpha}-chain in the transcriptional regulation of receptor-induced gene expression, we developed stably transfected murine macrophage cell lines expressing a full-length or a CY deletion mutant (tail-less) of human Fc{gamma}RIa to analyze gene expression in response to receptor-specific cross-linking. Using the Affymetrix murine genome U74Av2 GeneChip array, we observed >100 candidate genes having ≥2-fold difference expression at 1.5 and 3 h after stimulation. Focusing on several immunologically related genes, we confirmed differential expression of M-CSF, macrophage inhibitory cytokine-1, leukocyte-specific protein 1, MIP-2, and IL-1R antagonist by RT-PCR and RNase protection assays. Analysis of mRNA stability indicated that the differential regulation of gene expression by the CY of the CD64 {alpha}-chain is at the level of gene transcription. Our results indicate that the CY of the CD64 {alpha}-chain modulates transcriptional activity induced by receptor-specific engagement in macrophages and provides a framework for understanding distinct expression profiles elicited by different Fc {gamma}-chain-associated receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Structurally distinct receptors for each class of Ig bind the Fc region of their respective ligands with a high degree of specificity (1, 2, 3). The Fc{gamma}R are a multigene family that includes Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16). These receptor families differ in receptor structure, cell distribution, and affinity for IgG (1, 4, 5, 6, 7). They are expressed by most hemopoietic cells, including phagocytes, lymphocytes and platelets, and mediate functions such as Ab-dependent cell-mediated cytotoxicity, endocytosis and phagocytosis of immune complex, Ag presentation, and cytokine production (4, 5, 7, 8). Their expression can be regulated by certain inflammatory cytokines such as IFN-{gamma}, G-CSF, IL-10, and IL-4 (4, 5, 7).

The FcR {gamma}-chain, initially described as a subunit of the Fc{epsilon}RI receptor complex, is capable of forming multichain complexes with the ligand-binding {alpha}-chain of several FcRs including Fc{gamma}RIa, Fc{gamma}RIIIa, Fc{alpha}RIa, and Fc{epsilon}RI (3, 7, 9). In all of these receptor complexes, the {gamma}-chain with its ITAM is thought to be necessary for receptor function, and little attention has been given to possible functional roles for the {alpha}-chains of these receptor complexes beyond ligand binding. However, for Fc{alpha}RI, the {alpha}-chain is expressed both in the presence and absence of an associated {gamma}-chain, and a role for the {alpha}-chain in recycling bound and internalized IgA back to the cell surface and away from lysosomal degradation has been suggested (3, 10). Although natural expression of Fc{gamma}RIa is restricted to cells of the myeloid lineage, a role for the {alpha}-chain in facilitating Ag processing and presentation was suggested in studies with Fc{gamma}R null B cells transfected with Fc{gamma}RIa (11). Within a myeloid environment, we have shown that the cytoplasmic domain of Fc{gamma}RIa is critical for Fc{gamma}RIa-induced secretion of IL-6 (12, 13).

Fc{gamma}RIa, a receptor with high affinity for IgG, has been proposed as a potential therapeutic effector target receptor in malignancies (5, 14, 15). Targeting of tumors to Fc{gamma}RI with bispecific mAbs can facilitate tumor killing via Fc{gamma}RI-expressing macrophages, and humanized bispecific reagents targeting human (h)3 Fc{gamma}RIa are currently in clinical trials (5, 16, 17, 18). Because of the precedent that the {alpha}-chain of Fc{gamma}RIa can regulate the secretion of IL-6, we have hypothesized that the {alpha}-chain will be important in regulating the transcriptional potential of the Fc{gamma}RIa {alpha}-chain/{gamma}-chain complex. We have assessed Fc{gamma}RIa-induced gene expression in stably transfected murine macrophage expressing a wild-type (WT) or an {alpha}-chain CY domain mutant of hFc{gamma}RIa lacking the entire CY domain (tail-less (TL)). We have found that the presence or absence of the CY domain of the Fc{gamma}RIa {alpha}-chain alters the transcription of a number of immunologically important cytokines/chemokines. These data demonstrate that the Fc{gamma}RIa {alpha}-chain modulates receptor-induced transcriptional regulation of gene expression in macrophages and provide a model for studying the biological significance of the CY domain of other {gamma}-chain-associated FcR {alpha}-chains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

The murine macrophage cell line, P388D1 (American Type Culture Collection, Manassas, VA), was stably transfected with a cDNA-encoding hFc{gamma}RIa (WT) or a mutant form of Fc{gamma}RIa lacking the cytoplasmic domain tail (TL) and were maintained as adherent cultures in commercial RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) with the lowest available endotoxin levels as we have previously described (12, 13, 19). F(ab')2 and FITC-conjugated IgG of the anti-Fc{gamma}RIa mAbs 22.2 were obtained from Medarex (Annandale, NJ). Mouse F(ab')2 and F(ab')2 goat anti-mouse IgG (G{alpha}M) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents were from Sigma-Aldrich (St. Louis, MO). Quantitative hFc{gamma}RI expression was matched for cells expressing the WT and the TL by fluorescence-activated cell sorting using anti-Fc{gamma}RI mAb 22.2-FITC (Medarex).

Flow cytometry

Aliquots of cells at 5 x 106 cell/ml were incubated with saturating concentrations of FITC-conjugated primary mAb for 30 min at 4°C followed by two washes as we have described (12). After washing, the cells were analyzed immediately for immunofluorescence using a FACScan (BD Immunocytometry Systems, San Jose, CA).

Immunoprecipitation analysis

Fc{gamma}RI was immunoprecipitated from transfected lines using mAb 22.2 or mIgG1 as a negative control prebound to protein-G Sepharose (Amersham Bioscience, Piscataway, NJ). Cells were lysed in PBS containing 1% digitonin (Wako Biochemicals, Waco, TX) and inhibitors (EDTA/pepstatin/aprotinin/sodium orthovanadate/pefabloc). Immunoprecipitates or whole cell lysates were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked with 10% nonfat milk followed by incubation with either polyclonal anti-{gamma}-chain Ab (12) or anti-GAPDH mAb (AbCam, Cambridge, MA). Blots were washed three times with PBS-0.1% Tween 20 and bound mAb, or Ab was detected with HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Following three more washes, bound Ab was detected using ECL (Amersham Biosciences) according to the manufacturer’s directions.

Receptor-specific cell activation

Transfected P388D1 cells were incubated with a saturating concentration of anti-Fc{gamma}RI mAb 22.2 F(ab')2 or control F(ab')2 for 30 min at 4°C. Following two washes, cells were plated onto wells with surface absorbed F(ab')2 G{alpha}M for varying periods of time to achieve receptor-specific cross-linking as we have previously described (12, 13).

RNA isolation and microarray analysis

Affymetrix murine genome U74Av2 (MG-U74Av2; Affymetrix, Santa Clara, CA) arrays were used to study the differential gene expression profiles modulated by the cytoplasmic domain of Fc{gamma}RIa (CD64) {alpha}-chain. MG-U74Av2 represents ~6000 sequences in the mouse UniGene database (Build74) (www.ncbi.nlm.nih.gov) that have been functionally characterized in addition to ~6000 expressed sequence tag clusters. Total RNA was isolated from 5 x 106-stimulated cells with TRIzol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies). Double-stranded cDNA was generated by linear amplification of the RNA using an oligo(dT)-T7 primer and reverse transcriptase. Subsequently, biotin-labeled complementary RNA (cRNA) was synthesized by in vitro transcription and then broken into 50–200 base fragments for more uniform hybridization kinetics. Before hybridizing to the MG-U74Av2 arrays, Affymetrix test arrays were used to determine the quality of the hybridization target. RNA sample preparation, array hybridization, array washing, and scanning were performed following the protocols in the Affymetrix GeneChip Expression Analysis Technical manual. Initial data extraction, paired comparisons for fold change determination, and data filter were performed using the Affymetrix Microarray Suite (4.01) and Data Mining Tool.

RT-PCR and RNase protection assay (RPA)

Differential expression of selected genes between the WT and TL Fc{gamma}RI-activated P388D1 cells were initially confirmed by RT-PCR analysis. First strand cDNA synthesis was conducted in a total volume of 10 µl using SUPERSCRIPT One-Step RT-PCR System (Invitrogen Life Technologies) using 2 µg of total RNA. The primers used to amplify murine IL-6, IL-1{beta}, M-CSF, macrophage inhibitory cytokine-1 (MIC-1), leukocyte-specific protein 1 (LSP1), MIP-2, and IL-1R antagonist (IL-1Ra) are listed in Table I. Thirty-five cycles of PCR amplification were performed in a 50-µl reaction volume containing 1x reaction buffer (1.5 mM MgCl2, 200 µM dNTPs, 50 pmol of each primer, and 2.5 U of TaqDNA polymerase; Invitrogen Life Technologies) using a PerkinElmer Gene Amp PCR System 9600 (PerkinElmer, Norwalk, CT). Each cycle consisted of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min.


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  		Table I. Primers for RT-PCR

 
Differential gene expression was quantitated by RPA. For IL-6, M-CSF, MIC-1, LSP1, and MIP-2 mRNA expression, a 419-bp fragment corresponding to 181–600 nt of murine IL-6 cDNA, a 610-bp fragment corresponding to 458–1047 nt of murine M-CSF cDNA, a 322-bp fragment corresponding to 1066–1387 nt of murine MIC-1 cDNA, a 394-bp fragment corresponding to 153–526 nt of murine LSP1 cDNA, a 288-bp fragment corresponding to 64–332 nt of murine MIP-2 cDNA, and a 212-bp fragment corresponding to 223–434 nt of murine GAPDH cDNA (from RT-PCR products) were each subcloned in the HindIII/PstI polylinker site of the pGEM3Z vector (Promega, Madison, WI). The constructs were linearized by HindIII and used to generate radio labeled antisense RNA probes. The riboprobes for murine IL-6, M-CSF, MIC-1, LSP1, MIP-2, and GAPDH prepared from in vitro transcription with T7 polymerase are 467, 658, 370, 442, 380, and 270 nt, respectively. A total of 15 µg of total RNA from transfected P388D1 cells was hybridized with M-CSF, MIC-1, LSP1, MIP-2, and GAPDH riboprobes (50 x 103 cpm) at 42°C overnight in 20 µl of 40 mM PIPES (pH 6.4), 80% deionized formamide, 400 mM NaOAc, and 1 mM EDTA. The hybridized mixture was then treated with RNase A/T1 (1/200 dilution in 200 µl of the RNase digestion buffer) at room temperature for 1 h and separated by gel electrophoresis (5% denaturing (8 M urea) polyacrylamide gel), and the gels were analyzed by autoradiography. To detect IL-1{beta} and IL-1Ra mRNA expression, the RPA was performed using 20 µg of total RNA with the RiboQuant multiprobe RNase assay system using mCK-2b kits (BD Pharmingen, San Diego, CA) following the manufacturer’s specification as previously described (20). Protected RNA fragments were quantitated using Cyclone Storage Phosphor Screens (PerkinElmer, Boston, MA). Expression of mRNA was normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA was used as a control gene, as its levels are not affected by Fc{gamma}R-specific engagement.

mRNA stability analysis

The stability of mRNA was assessed by established methods (20). WT and TL stably transfected cell lines were incubated with or without cross-linking of hFc{gamma}RIa for 2 h to reach the highest expression of IL-6, M-CSF, MIC-1, and LSP1 mRNA, after which actinomycin-D (5 µg/ml) was added for an additional 8 h. RNA was isolated at the indicated time points and analyzed for IL-6, M-CSF, MIC-1, LSP1, and GAPDH mRNA levels by RPA.

Quantitation cytokine secretion

Levels of murine IL-6 or murine IL-1Ra were quantitated by ELISA according to the manufacturer’s instructions (BioSource International, Camarillo, CA or Pierce, Rockford, IL). Cells were stimulated in a receptor-specific manner as described above and supernatants were harvested for cytokine determinations.

EMSA analysis

Nuclear and cytoplasmic extracts were obtained from cells with or without stimulation by the modified method as previously described (21). Protein content was determined in all extracts using the Bio-Rad dye reagent assay (Hercules, CA). An equal amount (5 µg) of nuclear protein from each sample and NF-{kappa}B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; purchased from Promega, Madison WI) were used for EMSA following the manufacturer’s instructions as previously described (21). Anti-human p50 and p65 Abs for supershifts were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Western blots

Blots were prepared using 50 µg of cytoplasmic extracts as previously described (20, 21). I{kappa}B{alpha} affinity-purified rabbit polyclonal Ab to the C-terminal of hI{kappa}B{alpha} was obtained from Santa Cruz Biotechnology.

Statistical analysis

Data from three independent microarray studies were initially analyzed using the Affymetrix GeneChip Analysis Suite V4.01 (Affymetrix) to identify genes with a ≥2-fold change between the WT and TL Fc{gamma}RI activated P3388D1 cells. Additional statistical evaluation of the differences in WT and TL Fc{gamma}RI expression of genes was performed by two-tailed Student’s t test with a Bonferroni correction (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The CY of Fc{gamma}RIa {alpha}-chain transcriptionally regulates IL-6 expression

We have previously reported that deletion of the Fc{gamma}RIa CY abrogates Fc{gamma}RIa-specific induction of IL-6 protein secretion without affecting Fc{gamma}RIa-induced secretion of IL-1{beta} (12). Deletion of the CY domain does not alter association with the {gamma}-chain (see below) nor does it alter tyrosine phosphorylation of the {gamma}-chain in response to receptor-specific cross-linking (12, 13). We initially sought to determine whether the differential Fc{gamma}RIa-induced IL-6 protein secretion is due to differential IL-6 transcription. Using two independent parental P388D1 cell lines, we stably transfected either the full length hFc{gamma}RIa (WT) or the cytoplasmic domain-lacking mutant form of Fc{gamma}RIa (TL) and derived three WT-expressing lines and three TL-expressing lines. Expression of the WT and TL constructs was comparable (Fig. 1A), and association with the endogenous {gamma}-chain was not altered (Fig. 1B) in these lines (12, 13). In the TL-expressing lines, we confirmed the lack of IL-6 protein production 16 h after stimulation (results not shown).



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  		FIGURE 1. Expression and association with the endogenous {gamma}-chain of human WT and TL Fc{gamma}RIa on the surface of stably transfected P388D1 cells. A, Cells were incubated with a saturating concentration of the anti-human Fc{gamma}RIa mAb 22.2-FITC and analyzed by flow cytometry. B, Coimmunoprecipitation of murine {gamma}-chain with both WT and TL human Fc{gamma}RIa. Cells were lysed in buffer containing 1% digitonin as described in Materials and Methods. Human Fc{gamma}RIa was immunoprecipitated with the anti-Fc{gamma}RIa mAb 22.2 bound to protein G-agarose. As a negative control, a murine IgG1 myeloma was used in the immunoprecipitation. The {gamma}-chain was detected with a polyclonal anti-serum prepared against a C'-terminal peptide (12 ). As a loading control, identical amounts of whole cell lysates were run on a second gel and probed with an Ab against murine GAPDH.

 
Using the WT and TL expressing P388D1 cells, we quantitated IL-6 mRNA expression after receptor-specific stimulation. As a control, levels of IL-1{beta} mRNA were also assessed and were identical after cross-linking of WT and TL Fc{gamma}RI (Fig. 2). GAPDH mRNA levels were used to normalize mRNA levels. In contrast, elevated levels of IL-6 mRNA were only observed after cross-linking of WT Fc{gamma}RIa (Fig. 2). A time-dependent increase in IL-6 mRNA was observed by both qualitative RT-PCR and quantitative RPA. Kinetics analysis of IL-6 mRNA demonstrated a rise in mRNA ~1 h with a peak at ~2 h after receptor-specific stimulation (Fig. 2C). Little to no increase in IL-6 mRNA was observed after cross-linking of the TL Fc{gamma}RIa.



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  		FIGURE 2. The differential regulation of IL-6 and IL-1{beta} mRNA expression by the CY of Fc{gamma}RIa {alpha}-chain. A, WT and TL human Fc{gamma}RIa stably transfected P388D1 cells were incubated with or without specific cross-linking using mAb 22.2 F(ab') 2 and G{alpha}M F(ab')2 for different time points (1–6 h). Total RNA was isolated and expression of IL-6, IL-1{beta}, and GAPDH mRNA detected by RT-PCR. B, The RNA from cells stimulated under the same experimental protocol described in A were analyzed by RPA and quantitated by the Cyclone Storage Phosphor Screen (PerkinElmer) (C). Results are representative of three separate experiments.

 
To exclude a differential effect of receptor-specific stimulation on mRNA stability, we assessed the half-life of IL-6 mRNA after treatment with actinomycin-D (20). RNA was isolated from actinomycin-D-treated cells at the indicated time points and analyzed for IL-6 and GAPDH mRNA levels by RPA. Fig. 3 demonstrates comparable rates of IL-6 message degradation in both the WT and TL cell lines. Together with the RPA results, these data demonstrate that WT Fc{gamma}RI transcriptionally regulates IL-6 production and that the CY domain of the Fc{gamma}RIa {alpha}-chain plays an essential role in this process.



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  		FIGURE 3. Truncation of the CY of Fc{gamma}RIa {alpha}-chain does not affect the stability of the IL-6 message. WT and TL stably transfected P388D1 cells were stimulated with Fc{gamma}RIa-specific cross-linking using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2 for 2 h, then actinomycin D (ACT-D; 5 µg/ml) was added, cells were harvested at the indicated times (1–8 h), and RNA was subjected to RPA. IL-6 mRNA values were controlled for GAPDH hybridization within each sample and normalized to the IL-6 mRNA at time 0 before addition of ACT-D. Results are representative of three separate experiments.

 
Microarray analysis of differential gene expression induced by CY of Fc{gamma}RIa {alpha}-chain

Based on our observations with IL-6, we hypothesized that the CY of the ligand binding {alpha}-chain might transcriptionally regulate the expression of other immunologically related genes. Accordingly, we performed experiments at two time points (1.5 and 3 h after receptor-specific cross-linking) to screen for genes differentially regulated by the WT and TL receptors. To identify target genes putatively regulated by the CY of Fc{gamma}RIa {alpha}-chain, we analyzed the hybridization data to identify genes with a hybridization signal with ≥2-fold difference in signal intensity between the WT and TL cell lines in three independent experiments and with a statistically significant differences (regular t test with a Bonferroni correction; p < 0.05) as described (22). Based on these criteria, we identified five genes which are immunologically related to the functions of macrophages (Table II). Expression of M-CSF, MIC-1, and LSP1 was greater after stimulation of WT-Fc{gamma}RI relative to the TL-Fc{gamma}RI, while expression of MIP-2 and IL-1Ra were lower after stimulation of WT-Fc{gamma}RI relative to the TL-Fc{gamma}RI (Table II).


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  		Table II. Genes selected from GeneChip analysis

 
Confirmation of differential gene expression by RT-PCR and RPA analysis

Initially, we used RT-PCR to confirm Fc{gamma}RI induced changes in mRNA expression and to screen for relative kinetics of mRNA expression for each of the five genes identified by the microarray analysis. By semiquantitative RT-PCR, using the primers specified in Table I, we confirmed our microarray analysis and demonstrated that the specific cross-linking of Fc{gamma}RIa stimulates the M-CSF, MIC-1, and LSP1 more potently in WT than in TL while IL-1Ra and MIP-2 mRNA expression was greater in TL than in WT (Fig. 4). Analysis of time course showed induction of expression of these genes by 1 h and a peak within 2–3 h after stimulation.



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  		FIGURE 4. Expression of M-CSF, MIC-1, LSP1, IL-1Ra, and MIP-2 mRNA by RT-PCR in stably transfected P388D1 cells. Stably transfected P388D1 cells were incubated with or without cross-linking of human Fc{gamma}RIa using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2 for different times (1–6 h). RNA was isolated and analyzed by RT-PCR using specific primers for M-CSF, MIC-1, LSP1, IL-1Ra, MIP-2, and GAPDH mRNA. GAPDH was used as a housekeeping gene and is not altered by receptor activation. Results are representative of three independent experiments.

 
To quantitate the differences in expression of these genes, we quantitated mRNA levels in an RNase protection assay. Fig. 5, A and B, show the time course of M-CSF, MIC-1, and LSP1 mRNA induction after cross-linking of hFc{gamma}RIa in WT and TL stable transfectants. The kinetics of M-CSF, MIC-1, and LSP1 were comparable to the RT-PCR results with optimal expression levels at ~2 h after specific stimulation (Figs. 4 and 5A). The induction of M-CSF, MIC-1, and LSP1 mRNA was significantly higher after stimulation with WT relative to stimulation with TL. Similarly, the kinetics of expression of MIP-2 peaked at ~2 h after TL-specific stimulation with little induction of expression after WT stimulation (Fig. 6A). The time course of IL-1Ra mRNA induction after cross-linking of TL showed a more delayed peak of mRNA expression at ~6 h with more potent induction in TL compared with WT. Taken together, these data indicated that the CY of hFc{gamma}RIa {alpha}-chain differentially regulates gene expression in macrophages with induction of IL-6, M-CSF, MIC-1, and LSP1 requiring the CY domain while the CY domain inhibits expression of IL-1Ra and MIP-2.



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  		FIGURE 5. Expression of M-CSF, MIC-1, and LSP1 mRNA by RPA in stably transfected P388D1 cells. Stably transfected P388D1 cells were incubated with or without cross-linking of human Fc{gamma}RIa using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2 for 1, 2, 3, and 6 h. RNA was isolated and analyzed by RPA for M-CSF, MIC-1, LSP1, and GAPDH mRNA. GAPDH was used as a housekeeping gene and is not altered by receptor activation. Results are representative of three independent experiments.

 


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  		FIGURE 6. Expression of IL-1Ra and MIP-2 mRNA by RPA in stably transfected P388D1 cells. Stably transfected P388D1 cells were incubated with or without cross-linking of human Fc{gamma}RIa using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2 for 1, 2, 3, and 6 h. RNA was isolated and analyzed by RPA for M-CSF, MIC-1, LSP1 and GAPDH mRNA. GAPDH was used as a housekeeping gene and is not altered by receptor activation. Results are representative of three independent experiments.

 
mRNA stability of M-CSF, MIC-1, and LSP1

We considered the possibility that the differences in quantitative mRNA levels observed in P388D1 cells after stimulation through WT or TL Fc{gamma}RI were due to altered mRNA stability. Our data with IL-6 demonstrated no difference in the rate of IL-6 mRNA degradation, and we extended our mRNA stability analysis to include M-CSF, MIC-1, and LSP1. WT and TL transfectants were incubated with or without cross-linking of hFc{gamma}RIa for 2 h for maximal mRNA expression and actinomycin-D (5 µg/ml) was added for an additional 8 h. RNA was isolated at the indicated time points and analyzed for M-CSF, MIC-1, LSP1, and GAPDH mRNA levels by RPA. As shown in Fig. 7, mRNA levels for M-CSF, MIC-1, and LSP1 were greater after stimulation with WT relative to TL and the rate of mRNA degradation for each gene was identical between the WT and TL lines over the 8-h time period. These results indicate that the Fc{gamma}RIa CY is altering quantitative mRNA production at the transcriptional level.



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  		FIGURE 7. Truncation of CY of Fc{gamma}RIa {alpha}-chain does not affect the stability of the M-CSF, MIC-1, and LSP1 message. A, WT and TL stably transfected P388D1 cells were stimulated with Fc{gamma}RIa-specific cross-linking using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2 for 2 h, then ACT-D (5 µg/ml) was added, cells were harvested at the indicated times (1–8 h), and the RNA was subjected to RPA. M-CSF, MIC-1, and LSP1 mRNA values were corrected for GAPDH hybridization within each sample and normalized at time 0. Values are representative of three independent experiments.

 
Fc{gamma}RIa CY domain results in altered protein production

We have demonstrated that in the case IL-6, where the CY domain of the {alpha}-chain enables IL-6 transcription, there is altered protein production (see above and Ref.12). IL-1Ra mRNA levels are regulated differently in that the presence of the CY domain of Fc{gamma}RI blocks the production of IL-1Ra mRNA. To determine whether this transcriptional alteration results in altered protein production, we measured IL-1Ra protein secretion from WT- and TL-stimulated P388D1 cells. After 16 h of stimulation, there was no significant difference in IL-1Ra protein secretion from the WT-stimulated cells. However, there was a significant >2.5-fold increase in IL-1Ra protein secretion from the TL stimulated cells (p < 0.0025; Fig. 8). These data establish that transcriptional changes induced by the presence or absence of the Fc{gamma}RI CY domain can result in demonstrable alterations in protein secretion.



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  		FIGURE 8. IL-1Ra protein secretion from WT- and TL-stimulated P388D1 cells. Cells transfected with either the full length wild-type hFc{gamma}RI (WT) or the cytoplasmic domain lacking hFc{gamma}RI (TL) were stimulated in a receptor-specific manner as described in Materials and Methods. After 16 h of stimulation, supernatants were harvested and IL-1Ra protein was quantitated by ELISA.

 
The CY of Fc{gamma}RIa {alpha}-chain alters Fc{gamma}RIa-induced NF-{kappa}B activation

Cross-linking of activating Fc{gamma}R, including the {gamma}-chain-associated receptors, is known to induce the of activation of the p42-MAP/ERK kinase resulting in NF-{kappa}B-mediated transcriptional regulation (23, 24, 25). To establish the role of the CY domain of Fc{gamma}RI in the regulation of transcription factor activity, we quantitated NF-{kappa}B activity in WT and TL-transfected cells after receptor-specific stimulation of Fc{gamma}RIa. An EMSA demonstrated receptor-specific stimulation of NF-{kappa}B by both WT and TL Fc{gamma}RI (Fig. 9A) with maximum activation between 15 and 30 min after stimulation. Of interest is the apparent greater level of binding in extracts from WT-stimulated cells relative to TL stimulated cells. The addition of 100x and 200x unlabeled NF-{kappa}B inhibited the binding of the labeled NF-{kappa}B probe and nonspecific-unlabeled SP1 probe did not alter the binding of the labeled NF-{kappa}B probe demonstrating the specificity of the gel shift band. Further confirmation of the specificity of the binding was shown by the supershifting of the band with mAbs against the p50 and/or p65 subunits (Fig. 9A).



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  		FIGURE 9. Cross-linking of hFc{gamma}RIa stimulates NF{kappa}B DNA-binding activity and I{kappa}B{alpha} degradation in stably transfected P388D1 cells. A, WT and TL stably transfected P388D1 cells were stimulated with Fc{gamma}RIa-specific cross-linking using mAb 22.2 F(ab')2 and G{alpha}M F(ab')2, cells were harvested at the indicated times (15 min to 2 h), and nuclear extraction was subjected to EMSA for NF-{kappa}B activity. B, Cytoplasmic extracts at the indicated times (15–60 min) were subjected to Western blot for I{kappa}B{alpha} degradation. Data represent three independent experiments.

 
The common pathway for NF-{kappa}B activation is via phosphorylation and degradation of I{kappa}B{alpha} (26, 27). Cytoplasmic extracts obtained from WT- and TL-transfected cells at the indicated times after Fc{gamma}RIa cross-linking were evaluated for degradation of I{kappa}B{alpha}. More rapid degradation of cytoplasmic I{kappa}B{alpha} was apparent in WT-stimulated cells relative to TL-stimulated cells (Fig. 9B) which correlates with the level of activation of NF-{kappa}B (Fig. 9A). Taken together, these data demonstrate that the CY of Fc{gamma}RIa functionally regulates the activation of NF-{kappa}B induced by Fc{gamma}RIa-specific stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that the CY of the CD64 {alpha}-chain plays an important role in endocytosis, phagocytosis, induction of the secretion of IL-6, regulation of kinase activation and internalization, and Ag processing by Fc{gamma}RI (11, 12, 28). Furthermore, deletion of the CY domain of Fc{gamma}RI does not alter association with the {gamma}-chain, nor does it alter tyrosine phosphorylation of the {gamma}-chain in response to receptor-specific cross-linking (12, 13). More recently, the recognition that the serine phosphorylation status of the CY modulates the activation-induced tyrosine phosphorylation of common FcR {gamma}-chain (13) has raised the possibility that the CY of CD64 {alpha}-chain might modulate receptor-induced gene transcription and provide the basis for unique activation-induced expression profiles for each {gamma}-chain-associated Fc{gamma}R. Using a combination of an oligonucleotide-based microarray, RT-PCR and quantitative RNase protection assays, we have identified several immunologically important cytokines/chemokines whose expression is regulated by the presence or absence of the CY domain of the CD64 {alpha}-chain.

The basis for the differential regulation of mRNA levels for these cytokines/chemokines is transcriptional as mRNA stability experiments indicated identical degradation rates for the mRNA species. Although not comprising the whole set of genes potentially modulated by the CD64 CY, these six genes (IL-6, M-CSF, MIC-1, LSP1, MIP-2, and IL-1Ra) establish that the CY of CD64 {alpha}-chain modulates the transcriptional activity induced by receptor-specific engagement in macrophages. Furthermore, because the CY sequence of each of FcR {gamma}-chain-associated receptor is unique, the potential for distinct transcriptional profiles initiated by each receptor is clearly evident. Indeed, a distinct role for murine Fc{gamma}RI in the production of IL-10 has been proposed in macrophages (29, 30, 31).

The specific mechanisms for differential modulation of gene expression by the CY of the CD64 {alpha}-chain are currently unknown. Myeloid FcR cross-linking is known to induce multiple signaling pathways resulting in the activation of transcription factor activity (23, 24, 25, 32, 33). Similarly, Fc{gamma}R engagement can also negatively regulate transcriptional activity induced through other cell surface receptors (30, 34). Among the genes that were transcriptionally regulated in this study, we could not identify common regulatory elements in up-regulated genes that might explain the differential regulation compared with the down-regulated genes. Transcription elements typical of early to intermediate response genes are present in these genes and there is no clear pattern of transcription factor binding motifs that can characterize the promoter of the genes that are up-regulated or down-regulated by the presence of the CY domain of CD64. It is likely that regulation of transcription of these genes is complex and will have many interacting pathways similar to other genes such as IL-4-induced IL-1Ra expression mediated by STAT6 (35, 36), and IL-10 inhibition of IL-12 production (31, 37). Nonetheless, our studies do establish the clear precedent that the CY domain of CD64 regulates the activity of a transcription factor (NF-{kappa}B).

The functional impact of the CY domain of CD64 is not restricted to transcriptional regulation. Lack of the CY domain converts receptor-specific phagocytosis by CD64 from a Ca2+-independent to a Ca2+-sensitive process, suggesting a role of CY in the recruitment of Ca2+-independent signaling elements (13). Several cytoskeletal proteins interact with the cytoplasmic domain of CD64, and specific kinases, phosphatases, and/or adaptor molecules that modulate serine phosphorylation of the Fc{gamma}RIa {alpha}-chain may inhibit early receptor-initiated tyrosine phosphorylation events (13). Overall, a functional interaction between the CY of the {alpha}-chain and FcR {gamma}-chain leading to downstream modulation of transcription factors, including NF-{kappa}B (Fig. 9) which is involved in regulation of many proteins in inflammation, would seem plausible. Dissection of the specific signaling elements involved remains for future study.

In therapeutic targeting of FcR, the possibility that the {gamma}-chain-associated FcR may be functionally distinct has not received much attention. However, among knockout models, there are suggestions of distinct receptor functions. For example, the Fc{gamma}RIIIa (CD16) knockout mouse is protected from some forms of autoimmunity, while the Fc{gamma}RIa (CD64) knockout mouse displays altered Ab responses due to changes in Ag presentation mediated by uptake of Ab-targeted Ag (38, 39). These studies suggest a distinct role for Fc{gamma}RI, including in endocytosis of monomeric IgG, in phagocytosis of immune complexes, in macrophage-based Ab-dependent cell-mediated cytotoxicity, and in immune complex-dependent Ag presentation to primed T cells. Such differences could be important in the biological effects, or side-effects, of Ig-based therapeutics (15). Furthermore, the occurrence of nonsynonymous single nucleotide polymorphisms in the {alpha}-chain of Fc{gamma}RI (CD64) (40) is particularly exciting because Fc{gamma}RIa is located on chromosome 1q21.2, a region of linkage to systemic lupus (41, 42, 43, 44). Differential gene expression by each genetically unique FcR {alpha}-chain would provide a framework to consider the contribution of these single nucleotide polymorphisms to disease predisposition, and for exploring the functional properties of each common Fc {gamma}-chain-associated receptor and the roles that they play in regulating immune responses.


   Acknowledgments
 
We thank Dr. Bradley Yoder for his assistance in the initial microarray studies.


   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 National Institutes of Health Grants R01 AR42476, R01 AR33062, and P01 AR49084. Back

2 Address correspondence and reprint requests to Dr. Robert P. Kimberly, University of Alabama, 1900 University Boulevard, Tinsley Harrison Tower 429, Birmingham, AL 35294-0006. E-mail address: rpk{at}uab.edu Back

3 Abbreviations used in this paper: h, human; WT, wild type; TL, tail-less form of hFc{gamma}RIa; CY, cytoplasmic domain; G{alpha}M, goat anti-mouse IgG; IL-1Ra, IL-1R antagonist; LSP1, leukocyte-specific protein 1; MIC-1, macrophage inhibitory cytokine-1; RPA, RNase protection assay. Back

Received for publication April 14, 2004. Accepted for publication August 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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