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Originally published online as doi:10.1189/jlb.0904532 on February 9, 2005

Published online before print February 9, 2005
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(Journal of Leukocyte Biology. 2005;77:767-776.)
© 2005 by Society for Leukocyte Biology

Cytokine-mediated regulation of activating and inhibitory Fc{gamma} receptors in human monocytes

Yi Liu, Emi Masuda, Marissa C. Blank, Kyriakos A. Kirou, Xiaoni Gao, Mee-Soon Park and Luminita Pricop1

Research Division, Hospital for Special Surgery and Department of Medicine and Graduate Program in Immunology, Weill Medical College of Cornell University, New York, New York

1 Correspondence: Hospital for Special Surgery, Weill Medical College of Cornell University, 535 East 70th Street, New York, NY 10021. E-mail: PRICOPL{at}HSS.EDU


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fc{gamma} receptors (Fc{gamma}R) trigger inflammatory reactions in response to immunoglobulin-opsonized pathogens and antigen-antibody complexes. The coordinate expression of activating and inhibitory Fc{gamma}R ensures the homeostasis of immune complex-driven inflammatory responses. In this study, we used antibodies with preferential binding for activating Fc{gamma}RIIa and inhibitory Fc{gamma}RIIb receptors to investigate the expression and regulation of Fc{gamma}RII isoforms in human monocytes. Cross-linking of Fc{gamma}RIIa triggered phagocytosis and cytokine production. Cross-linking of Fc{gamma}RIIb was associated with phosphorylation of the immunoreceptor tyrosine-based inhibitory motif and with a marked reduction in monocyte effector functions. Our study revealed that tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-10, and IL-13 altered the transcriptional activity of the Fc{gamma}RIIB promoter in transfected cell lines and skewed the balance of activating versus inhibitory Fc{gamma}R in human monocytes. TNF-{alpha} decreased the expression of inhibitory Fc{gamma}RIIb. IL-10 up-regulated all classes of Fc{gamma}R and induced alternative activation in monocytes, an effect that was synergistic with that of TNF-{alpha}. In contrast, IL-4 and IL-13, in combination with TNF-{alpha}, decreased the expression of activating Fc{gamma}R and markedly down-regulated Fc{gamma}R-mediated function. Our findings suggest that the cytokine milieu can induce changes in the relative expression of Fc{gamma}R with opposing function and thus, may regulate the amplitude of Fc{gamma}R-mediated uptake and inflammation.

Key Words: phagocytes • hypersensitivity • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the course of inflammation, important components of the immune response are initiated upon the interaction of phagocytes with opsonized pathogens and soluble immune complexes [1 , 2 ]. Complex cytokine networks alter monocyte functions during antimicrobial and autoimmune responses [3 4 5 ]. In acute infections, proinflammatory cytokines transiently up-regulate monocyte functions, such as phagocytosis and release of inflammatory mediators. These effects are rapidly down-regulated by anti-inflammatory mechanisms and end upon clearance of the infectious agent. In chronic inflammation, the production of inflammatory cytokines fails to cease, resulting in sustained changes in receptor expression, augmented monocyte effector functions, and sometimes tissue injury.

In immune complex-mediated reactions, the amplitude of inflammatory responses is believed to depend on the ratio of activating and inhibitory Fc{gamma} receptors (Fc{gamma}R) [6 ]. Three classes of FcR (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII) are expressed in monocytes [7 ]. Fc{gamma}RI and Fc{gamma}RIII are activating receptors that trigger phagocytosis and release of inflammatory mediators. Within the Fc{gamma}RII family, Fc{gamma}RIIa and Fc{gamma}RIIb carry out divergent functions. Fc{gamma}RIIa, receptors for which no mouse ortholog has been described, contains immunoreceptor tyrosine-activating motifs (ITAM), mediating positive signaling, resulting in internalization of immune complexes and initiation of inflammatory responses. Inhibitory Fc{gamma}RIIb contains an immunoreceptor tyrosine-based inhibitory motif (ITIM), which mediates negative signaling [8 ]. Experimental evidence suggested that cross-linking of ITIM-bearing Fc{gamma}RIIb inhibited responses triggered by ITAM-bearing Fc{gamma}R [9 , 10 ].

Targeted deletions of activating and inhibitory Fc{gamma}R isoforms have verified the involvement of Fc{gamma}R in the development of hypersensitivity reactions. Deficiency in activating Fc{gamma}R was associated with diminished antibody- and immune complex-mediated reactions [11 12 13 14 ]. Conversely, mice deficient in inhibitory Fc{gamma}R developed severe hypersensitivity reactions [15 16 17 ]. Although an important role for Fc{gamma}R in immune regulation and host defense is acknowledged, the factors that modulate the relative expression of Fc{gamma}R with opposing function remain poorly characterized. Moreover, only limited data are available regarding the regulation of Fc{gamma}RIIB gene expression [18 , 19 ].

We undertook the study of activating and inhibitory Fc{gamma}R in human monocytes and examined their regulation and function. Here, we characterize antibodies that interacted preferentially with Fc{gamma}RIIb and inhibited cytokine production and phagocytosis of antibody-opsonized erythrocytes (E) in monocytes. We detected alterations in the ratio of ITAM- and ITIM-bearing Fc{gamma}R, mediated by a panel of cytokines produced during inflammatory responses. Our study revealed that tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-10, and IL-13 altered the transcriptional activity of the Fc{gamma}RIIB promoter. TNF-{alpha} and IL-10 determined an activating Fc{gamma}R phenotype through two distinct mechanisms: TNF-{alpha} down-regulated the expression of inhibitory Fc{gamma}RIIb, and IL-10 up-regulated all activating Fc{gamma}R. Fc{gamma}R-mediated function was decreased by IL-4 and IL-13, which skewed the Fc{gamma}R balance toward an inhibitory phenotype. Differential regulation of Fc{gamma}R function by cytokine imbalances could create functional deficiencies similar to those seen in Fc{gamma}R knockout mice. Our results extend these observations to human biology. Furthermore, our findings constitute a basis for developing and testing new strategies for Fc{gamma}RIIb-mediated modulation of effector functions in human monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
The human melanoma cell line A375 [American Type Culture Collection (ATCC), Manassas, VA], transfected with Fc{gamma}RIIA and Fc{gamma}RIIB2 cDNAs provided by Catherine Sautes-Fridman (INSERM U255, Université Paris IV, Paris, France), was described in detail before [20 ]. The Raji B cell line was purchased from ATCC. B cell separation was performed from fresh peripheral blood with RosetteSep antibody cocktail (StemCell Technologies, Vancouver, British Columbia, Canada), following the manufacturer’s instructions. Mononuclear cells were separated from peripheral blood of healthy donors by Ficoll-Hypaque density gradient centrifugation. Monocytes were purified using CD14-positive magnetic selection (StemCell Technologies) following the manufacturer’s instructions [20 ]. Monocyte purity was between 96% and 99%, as determined by fluorescein-activated cell sorter analysis using fluorescein isothiocyanate (FITC)-CD14 monoclonal antibodies (mAb). Monocytes (6x106cells/well) were cultured in six-well plates in RPMI-1640 medium supplemented with 10% ultra-low immunoglobulin G (IgG) fetal bovine serum (Gibco-BRL, Grand Island, NY). When indicated, cytokines (R&D Systems, Minneapolis, MN) were added to culture media at the following concentration: TNF-{alpha} (20 ng/ml), IL-10 (50 ng/ml), IL-13 (50 ng/ml), and IL-4 (100 ng/ml). Monocytes were cultured for 18 h for evaluation of modulation of RNA transcripts and 42 h for evaluation of phagocytic function.

Reporter gene construct and luciferase assays
Polymerase chain reaction (PCR) primers containing recognition sites for KpnI in the forward primer (5'-GCGCGGTACCGCCATCCTGACATACCTCCTT-3') and XhoI in the reverse primer (5'-GCGCCTCGAGCACTCCCTGGAGCGACGTGGC-3') were used to amplify a 578-bp fragment of the Fc{gamma}RIIB 5'-flanking sequence. PCR conditions included the following: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 25 cycles. After restriction enzyme digestion, the products were directionally ligated using a rapid DNA ligation kit (Roche, Indianapolis, IN) into the pGL3-enhancer vector (Promega, Madison, WI). The resulting construct carried luciferase reporter genes under the control of the Fc{gamma}RIIB promoter and a simian virus 40 enhancer to augment expression levels. Plasmids were purified using QIAprep Spin miniprep columns (Qiagen, Valencia, CA), and the construct was verified by automated sequencing.

U937 cells (ATCC) were cultured in RPMI, supplemented with 10% heat-inactivated fetal calf serum (FCS), nonessential amino acids, and penicillin (50 U/ml)/streptomycin (50 µg/ml). U937 cells (4x105) were washed, resuspended in OPTIMEM medium (Life Technologies, Gaithersburg, MD), and transfected with 1 µg reporter plasmid plus 0.3 µg control plasmid encoding the renilla luciferase gene using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 6 h, an equal volume of 20% complete medium, supplemented with TNF-{alpha} (20 ng/ml), IL-10 (50 ng/ml), or IL-13 (50 ng/ml), was added to the cells. After an additional 24 h of culture, cells were harvested, and luciferase activity in cell lysates was determined by luminescence spectroscopy.

Real-time PCR evaluation of RNA transcripts
Total RNA was isolated with the RNeasy kit (Qiagen). cDNA was synthesized from 1 µg total RNA with random hexamers (Invitrogen). Real-time PCR was carried out with the SYBR Green PCR supermix (Perkin Elmer, Foster City, CA) and the iQ Multi-Color Real-Time PCR detection system (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. The PCR reaction consisted of 40 cycles at 94°C for 30 sec and 54°C for 30 sec. The following primer pairs were used for amplification: Fc{gamma}RIIA forward 5'-GACTACGGATACCCAAATGTC-3' and Fc{gamma}RIIA reverse 5'-AAGCCAGCAGCAGCAAAA-3', resulting in an 86-bp amplicon; Fc{gamma}RIIB2 forward 5'-GGGATGATTGTGGCTGTG-3' and Fc{gamma}RIIB2 reverse 5'-ATTAGTGGGATTGGCTGAA-3', resulting in a 106-bp amplicon; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5'-CAACGGATTTGGTCGTATT-3' and GAPDH reverse 5'-GATGGCAACAATATCCACTT-3'. During amplification, absorption readings measured the relative amount of amplicon produced in each cycle. These data were used to make a relative determination of gene expression under each experimental condition. All PCR assays were triplicated, and the data were pooled. The amplified products were verified by melting curves and by direct sequencing of Fc{gamma}RIIA and Fc{gamma}RIIB2 PCR products and comparison with Genebank sequences. Sequencing was performed by Dye terminator reaction using capillary slab gel electrophoresis.

Evaluation of phenotypic markers by flow cytometry
CD14-FITC and CD19-FITC were purchased from PharMingen (San Diego, CA). Unlabeled and fluorescein (FITC)-labeled anti-Fc{gamma}RI mAb (22.2), anti-Fc{gamma}RII mAb (IV.3), and anti-Fc{gamma}RIII mAb (3G8) were purchased from Medarex (Princeton, NJ). Intact and F(ab')2 fragments of 7.3 mAb as well as 7.3-FITC were obtained from Research Diagnostics (Flanders, NJ). Where indicated, cells were incubated with biotinylated 7.3 F(ab')2, IV.3 Fab, purified mouse IgG2b, or IgG1 and incubated at room temperatures for 15 min, followed by phycoerythrin-conjugated streptavidin and goat anti-mouse (GAM) IgG.

Immunofluorescence microscopy
Purified human monocytes were cytospun at 800 rpm for 5 min with cytospin 2 (Shandon Scientific, Runcorn, UK). Slides were fixed with freezing acetone for 5 min at –20°C and permeabilized with 0.1% saponin for 10 min. After blocking with phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and 0.1% saponin for 15 min, the slides were stained with 7.3-FITC and unlabeled polyclonal antibodies specific for the intracellular domain of human Fc{gamma}RIIa and Fc{gamma}RIIb, respectively (kindly provided by Catherine Sautes-Fridman) [21 , 22 ]. Staining with the rabbit anti-human Fc{gamma}RIIa polyclonal antibody 260 (1:60 dilution) and the rabbit anti-human Fc{gamma}RIIb/IC polyclonal antibody (1:60 dilution) was performed for 1 h at room temperature. Slides were washed 4x in PBS + 0.05% Tween 20 for 10 min and treated with goat anti-rabbit IgG (H+L) conjugated with Alexa 546 (Sigma Chemical Co., St. Louis, MO) at 1: 500 dilution for 30 min at room temperature. The slides were then washed 4x and mounted with Fluoromount-G (Sourthern Biotechnology Associates, Birmingham, AL). Images were collected using a Leica LSM 510 laser confocal microscope in the mode of sequential excitation of FITC and rhodamine dyes to exclude crossover of their fluorescence. Confocal images were then analyzed with a Zeiss LSM 5 image browser (Version 3.1.0.99).

Immunoprecipitation and Western blotting analysis
Freshly separated CD14+ human monocytes were cultured overnight at 37°C. Cells were resuspended at 2 x 107 in 50 µl buffer containing 125 mM NaCl, 5 mM KCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 5 mM glucose, pH 7.35, incubated with 5 µg/ml 7.3 F(ab')2 at room temperature for 30 min, then washed, and resuspended in the same buffer supplemented with 1.09 mM CaCl2 and 1.62 mM MgCl2. Signaling was initiated by cross-linking the surface-bound 7.3 mAb with GAM IgG F(ab')2 (30 µg/ml) at 37°C for varying times. Cells were then washed once with ice-cold buffer, pelleted, and solubilized in lysis buffer [1% Nonidet P-40 (NP-40), 10% glycerol, 70 mM NaCl, 50 mM NaF, 16 mM Na2HPO4, 4 mM NaH2PO4, 5 mM EDTA, 0.4 mM Na3VO4, 10 µg/ml each aprotinin, leupeptin, soybean trypsin inhibitor, and pepstatin A, and 500 µg/ml pefabloc, pH 7.4] for 1 h at 4°C. The lysates were centrifuged at 16,000 g for 10 min at 4°C, and the supernatants were immunoprecipitated using anti-human Fc{gamma}RII antibody (FLI8.26) and protein G-Sepharose beads (15 µl) for 16 h at 4°C. The immune complexes were washed four times in PBS plus 1% NP-40, mixed with 2x Laemmli buffer plus 2-mercaptoethanol, heated for 5 min at 95°C, and separated on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were electrophoretically transferred to nitrocellulose membranes, blocked in PBS, supplemented with 5% BSA, analyzed for phosphotyrosine content with 1 µg/ml 4G10 mAb (Upstate Biotechnology, Lake Placid, NY) and 1 µg/ml PY-20 mAb (BD Transduction Laboratories, Franklin Lakes, NJ), and overlayed for assessment of protein specificity with anti-human Fc{gamma}RIIb antibody (Fc{gamma}RIIB/IC) [20 ]. Proteins were visualized following incubation with peroxidase-conjugated second antibodies using the enhanced chemiluminiscent detection (Amersham, Piscataway, NJ) and Kodak X-Omat radiographic film (Eastman Kodak, Rochester, NY).

Quantitation of Fc{gamma}R-mediated phagocytosis by flow cytometry
Phagocytosis of E labeled with the lipophilic fluorescent dye PKH26 (Sigma Chemical Co.), coupled to specific anti-Fc{gamma}R mAb, was performed as described previously [23 ]. Anti-Fc{gamma}RI, 22.2 F(ab')2, anti-Fc{gamma}RII, IV.3 Fab, 7.3 F(ab')2, anti-Fc{gamma}RIII 3G8 F(ab')2, and human IgG1 were biotinylated with EZlink-sulfoNHS-LC-biotin (Sigma Chemical Co.) and coupled to bovine E [24 ]. Fc{gamma}R-specific probes were labeled with PKH26, washed, and resuspended in RPMI 1640 plus 20% FCS to a final concentration of 1 x 108 E/ml. Monocytes (5x105cells/ml) were incubated with Fc{gamma}R-specific probes (1.25x107 E/ml) at 37°C for 15 min, followed by lysis of noninternalized probes. The capacity of monocytes to internalize target particles was measured by flow cytometry using a FACScan (Becton Dickinson Immunocytometry Systems, Palo Alto, CA). PKH26 fluorescence was detected in the FL2 channel and displayed on a logarithmic scale. Data analysis was done using Cell Quest software (Becton Dickinson, San Jose, CA). The phagocytic index (PI) was calculated by multiplying the percentage of cells that internalized PKH26-labeled E by the mean fluorescence intensity (MFI) of internalized E/100 phagocytes.

Production of cytokines by monocytes after Fc{gamma}R-mediated phagocytosis
E probes were prepared as described above without PKH26 labeling. Monocytes (5x106/ml) were placed in wells with Fc{gamma}R-specific probes at an effector:target concentration of 1:25 at 37°C for 18 h. Cell-free supernatants were collected, and samples were frozen at –70°C. Production of TNF-{alpha}, IL-6, and IL-1ß by monocytes after Fc{gamma}R-mediated phagocytosis was determined by sandwich enzyme-linked immunosorbent assay (ELISA). Anticytokine antibodies were purchased from R&D Systems. Streptavidin alkaline-phosphatase (R&D Systems) and phosphatase substrate p-nitrophenol phosphate (Sigma-Aldrich, St. Louis, MO) were used as a read-out system. Optical density values were determined spectrophotometrically at 405 nm.

Statistical analysis
Data are displayed as mean ± SEM. The effects of different treatments were compared using paired Student’s t-test (two-tailed) or Mann-Whitney rank sum test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Decreased production of cytokines and phagocytosis after ligation of Fc{gamma}RIIb in monocytes
The production of cytokines following interaction of immune complexes with Fc{gamma}R is believed to be an essential host defense mechanism. We sought to test the ability of different Fc{gamma}R-specific probes to induce cytokine production in monocytes. We measured the production of TNF-{alpha}, IL-6, and IL-1ß by ELISA after incubation of monocytes with probes opsonized with mAb interacting with Fc{gamma}RI (clone 22.2) and Fc{gamma}RII (clones IV.3 and 7.3). Phagocytosis of 22.2 probes and IV.3 probes induced the production of TNF-{alpha}, IL-6, and IL-1ß in five independent donors (Fig. 1A 1B 1C ). It is interesting that the production of all three cytokines was nearly absent following incubation of monocytes with 7.3 probes (Fig. 1A 1B 1C) , although IV.3-FITC and 7.3-FITC mAb bound to the membrane of monocytes with similar MFI (Fig. 1D) . These results raised the possibility that ligation of Fc{gamma}RII by IV.3 and 7.3 probes could have distinct reactivities for the activating Fc{gamma}RIIa and inhibitory Fc{gamma}RIIb isoforms.



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  		Figure 1. Differences in cytokine production by human monocytes mediated by different anti-Fc{gamma}R mAb. Production of TNF-{alpha} (A), IL-6 (B), and IL-1ß (C) was measured by ELISA in culture supernatants of monocytes after Fc{gamma}R-specific phagocytosis. Probes opsonized with 22.2 F(ab')2, IV.3 Fab, and 7.3 F(ab')2 were used for internalization via Fc{gamma}RI and Fc{gamma}RII. (D) Fc{gamma}R binding was assessed by direct staining with 22.2-FITC, IV.3-FITC, and 7.3-FITC and flow cytometric analysis.

 
To investigate potential differences in phagocytosis mediated by IV.3 and 7.3 mAb, we measured uptake of IV.3 probes and 7.3 probes by human monocytes. Purified monocytes phagocytosed IV.3 probes efficiently (Fig. 2 , upper panel). Incubation of human primary monocytes with 7.3 probes showed a marked reduction in Fc{gamma}RII-mediated phagocytic function (Fig. 2 , lower panel). Surface expression of Fc{gamma}RII evaluated by flow cytometry showed similar binding of IV.3 and 7.3 mAb (Fig. 2) . These results indicated that in spite of similar binding, Fc{gamma}RII probes generated with IV.3 and 7.3 mAb induce different functional effects in human monocytes.



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  		Figure 2. Different capacities of two Fc{gamma}RII-specific mAb to mediate phagocytosis in monocytes. PI of human monocytes internalizing IV.3 probes and 7.3 probes are indicated. MFI of IV.3-FITC and 7.3-FITC binding in monocytes detected by flow cytometry is shown in right panels. SSC, Saline sodium citrate.

 
The dynamic interplay of activating and inhibitory Fc{gamma}R in human monocytes remains unelucidated. Conformational differences between Fc{gamma}RIIa and Fc{gamma}RIIb are a result of six amino acid changes and an additional glycosylation site present in the second extracellular domain of Fc{gamma}RIIb [25 ]. We tested the ability of the two clones of anti-Fc{gamma}RII mAb, IV.3 and 7.3, to sense conformational differences between Fc{gamma}RIIa and Fc{gamma}RIIb by analyzing their binding to recombinant Fc{gamma}RIIa and Fc{gamma}RIIb in transfected A375 cell lines (Fig. 3A ) [20 , 26 ]. Preferential binding of IV.3 mAb to Fc{gamma}RIIa and of 7.3 mAb to Fc{gamma}RIIb has been reported previously [22 , 27 ]. Indeed, IV.3 mAb showed high binding to Fc{gamma}RIIa-expressing A375 cells, and binding to Fc{gamma}RIIb-expressing A375 cells was minimal (Fig. 3A , upper panels). mAb 7.3 displayed increased binding to Fc{gamma}RIIb-expressing A375 cells compared with Fc{gamma}RIIa-expressing A375 cells (Fig. 3A , lower panels). Staining of Fc{gamma}RIIb in Raji B cells and human peripheral blood B cells showed higher intensity with 7.3 mAb compared with IV.3 mAb (data not shown). Blocking studies indicated that preincubation with 7.3 mAb inhibited IV.3 binding, and IV.3 pretreatment only moderately decreased the binding of 7.3 mAb to Fc{gamma}RIIb expressed in B cells (Fig. 3B) .



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  		Figure 3. Reactivity of anti-Fc{gamma}RII IV.3 and 7.3 with Fc{gamma}RIIa and Fc{gamma}RIIb isoforms. (A) Anti-Fc{gamma}RII mAb IV.3 and 7.3 displayed distinct reactivies with Fc{gamma}RIIA- and Fc{gamma}RIIB-transfected A375 cells. Representative histograms of 7.3 F(ab')2, IV.3 Fab, and mouse IgG2b and IgG1 isotype control (Ctr) staining in Fc{gamma}RIIA- or Fc{gamma}RIIB-transfected A375 cells (left panels). Direct staining with IV.3-FITC and 7.3-FITC is expressed as MFI (right panel). (B) Percent blocking of IV.3-FITC and 7.3-FITC binding by unlabeled 7.3 mAb (solid bars) and IV.3 mAb (open bars) in Raji cells and CD19+ human peripheral B cells. Cells were incubated with unlabeled 7.3 or IV.3 for 30 min at 4°C, washed 3x with PBS, and subsequently stained with FITC-conjugated IV.3 or 7.3 mAb. (C) Colocalization of 7.3-FITC (green) with rabbit polyclonal antibodies generated against the intracellular domains of human Fc{gamma}RIIa (C I) and Fc{gamma}RIIb (C II) followed by Alexa 546-conjugated goat anti-rabbit IgG (red). Arrows indicate the colocalization (yellow). Images were collected under 63x original magnification. (D) Tyrosine phosphorylation of the cytoplasmic domain of Fc{gamma}RIIb following cross-linking with 7.3 mAb. CD14+ human monocytes were incubated with medium (lane 1) and with 7.3 F(ab')2and GAM F(ab')2 for 1 and 5 min (lanes 2 and 3). Cells were lysed, immunoprecipitated with FLI8.26 mAb, immunoblotted with antiphosphotyrosine antibodies 4G10 plus PY-20 (upper blot), and overlayed with Fc{gamma}RIIB/IC antibodies (lower blot). Representative blot from five different experiments is shown.

 
Antibodies raised against the intracellular domain of Fc{gamma}RIIa (260) and Fc{gamma}RIIb (anti-Fc{gamma}RIIB/IC) [28 ] were used in confocal microscopy to analyze colocalization with 7.3 mAb staining (Fig. 3C) . Experiments performed with human monocytes showed intense colocalization of 7.3 mAb with anti-Fc{gamma}RIIB/IC antibodies (Fig. 3C I) compared with less colocalization of 7.3 mAb with anti-Fc{gamma}RIIa antibodies 260 (Fig. 3C II).

Fc{gamma}RIIb molecules contain an ITIM motif (AENTITYSSL) in their intracellular domain, which upon cross-linking of Fc{gamma}RIIb, becomes tyrosine-phosphorylated, binds the Src homology 2 (SH2)-containing inositol phosphatase, and initiates inhibitory signaling [29 30 31 ]. We investigated whether 7.3 mAb could trigger tyrosine phosphorylation of the intracellular domain of Fc{gamma}RIIb in monocytes. Cells were treated with medium as control (Fig. 3D , lane 1) and with 7.3 F(ab')2 followed by cross-linking with GAM F(ab')2 for 1 and 5 min at 37°C (Fig. 3D , lanes 2 and 3). Cell lysates were immunoprecipitated with anti-Fc{gamma}RII mAb (FLI8.26) and separated on SDS-PAGE. Immunoblotting with antiphosphotyrosine antibodies showed phosphorylation of an ~35-kDa protein, which reacted with specific antibodies for the intracellular domain Fc{gamma}RIIb (Fc{gamma}RIIB/IC), indicating that 7.3 mAb could mediate signaling via Fc{gamma}RIIb [20 ].

Taken together, these results indicated the preferential reactivity of mAb IV.3 with Fc{gamma}RIIa (Fc{gamma}RIIahigh+Fc{gamma}RIIblow) and that of mAb 7.3 with Fc{gamma}RIIb (Fc{gamma}RIIalow+Fc{gamma}RIIbhigh). Based on their distinct reactivity, IV.3 and 7.3 mAb were tested for their ability to mediate Fc{gamma}RIIa and Fc{gamma}RIIb functions in human monocytes.

Regulation of activating and inhibitory Fc{gamma}R in human monocytes by TNF-{alpha}, IL-10, and IL-13
Numerous groups have confirmed the ability of cytokines to alter Fc{gamma}R expression and function. Interferon-{gamma} (IFN-{gamma}) is an inducer of Fc{gamma}RI expression, and IL-4 decreased the expression of activating receptors Fc{gamma}RI and Fc{gamma}RIII [32 , 33 ]. We and others have reported the regulation of inhibitory Fc{gamma}RIIb receptors by T helper 1 (Th1) and Th2 cytokines, and IFN-{gamma} acts as a down-regulator and IL-4 as an up-regulator of Fc{gamma}RIIb expression [20 , 34 ]. These observations put forward the hypothesis that cytokines differentially regulate activating and inhibitory Fc{gamma}R, leading to alterations in their ratio.

The differential regulation of the Fc{gamma}RIIA and Fc{gamma}RIIB gene transcription seems to be operated, at least in part, at the promoter level. There is no homology in the 5'-flanking sequence of the human Fc{gamma}RIIA and Fc{gamma}RIIB genes [35 , 36 ]. We investigated the effect of TNF-{alpha}, IL-10, and IL-13 on the transcriptional activity of the Fc{gamma}RIIB promoter. We generated a 579-bp construct of the human Fc{gamma}RIIB promoter, which upon transfection into U937 cells, showed promoter activity in luciferase assays (Fig. 4 ). There was a significant decrease (29%±8.9, P<0.05) in luciferase activity of the Fc{gamma}RIIB promoter after treatment of U937 cells with TNF-{alpha} as compared with medium (Fig. 4) . In contrast, luciferase activity of the Fc{gamma}RIIB promoter construct was increased in U937 treated with IL-10 (208%±86.9, P<0.05) and to a lesser extent, with IL-13 (130.1%±17.6, P<0.05). Altogether, the results demonstrated the ability of TNF-{alpha}, IL-10, and IL-13 to modulate the transactivation of the human Fc{gamma}RIIB promoter in U937 cells.



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  		Figure 4. Regulation of Fc{gamma}RIIB promoter activity by cytokines. U937 cells were transfected with Fc{gamma}RIIB promoter plasmid plus the renilla luciferase gene as control. After 4 h, an equal volume of complete medium or medium supplemented with TNF-{alpha}, IL-10, or IL-13 was added to the cells. After an additional 48 h of culture, luciferase activity in U937 cell lysates was determined by luminescence spectroscopy. Results represent means ± SEM of firefly luciferase counts normalized to renilla luciferase of six to eight independent transfections.

 
Next, we investigated whether TNF-{alpha}, IL-10, and IL-13 affected the balance of activating and inhibitory Fc{gamma}R in primary monocytes. We analyzed the expression of Fc{gamma}RIIA and Fc{gamma}RIIB RNA transcripts by real-time PCR and verified the respective products by sequencing. Modulation of Fc{gamma}RII receptor expression and phagocytic function was evaluated with IV.3 and 7.3 mAb and corresponding probes by flow cytometric assays [20 , 23 ].

TNF-{alpha} plays a prominent role in inflammatory conditions. TNF-{alpha}-mediated activation of monocyte functions is important in host defense and autoimmunity [3 ]. Our results revealed that TNF-{alpha} treatment decreased the expression of Fc{gamma}RIIB RNA transcripts and was associated with reduced surface binding of 7.3-FITC in monocytes (Fig. 5A and 5B ).



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  		Figure 5. Regulation of activating and inhibitory Fc{gamma}R in monocytes by TNF-{alpha}, IL-10, and IL-13. (A) Expression of Fc{gamma}RIIA and Fc{gamma}RIIB RNA transcripts following culture of monocytes for 18 h with TNF-{alpha}, IL-10, and IL-13 was determined by real-time PCR and was normalized to medium (set to 100). Values represent mean ± SEM of 11–21 experiments. (B) Membrane expression of Fc{gamma}RII isoforms was determined by direct staining with IV.3-FITC and 7.3-FITC following culture of monocytes for 24 h with TNF-{alpha}, IL-10, and IL-13. (C) Membrane expression of Fc{gamma}RI and Fc{gamma}RIII was assessed by direct staining with 22.2-FITC and 3G8-FITC following culture conditions described above. Results represent mean ± SEM of three to 13 experiments. For all experiments, significant differences are marked (*) for P< 0.01.

 
IL-10 is a potent deactivator of monocytes and is believed to limit the intensity of immune and inflammatory reactions [37 ]. IL-10 was predicted to reduce tissue injury and abort autoimmune reactions. However, the benefit of IL-10 treatment in antibody-mediated autoimmune diseases is controversial. We examined the effect of IL-10 on the expression of activating and inhibitory Fc{gamma}R in human monocytes. Treatment of monocytes with IL-10 up-regulated the expression of Fc{gamma}RIIA and Fc{gamma}RIIB RNA transcripts and increased the binding of IV.3 and 7.3 mAb. Expression of activating receptors Fc{gamma}RI (22.2) and Fc{gamma}RIII (3G8) was also up-regulated by IL-10, as reported by others (Fig. 5C) [38 ].

The effect of IL-13 on the expression of activating and inhibitory Fc{gamma}R in monocytes was also studied. IL-13 decreased the expression of Fc{gamma}RIIA at RNA and protein level (Fig. 5A and 5B) and reduced surface expression of Fc{gamma}RI and Fc{gamma}RIII (Fig. 5C) . IL-13 induced a shift in the ratio of Fc{gamma}R by down-regulating all activating Fc{gamma}R isoforms.

We investigated whether the observed changes in RNA transcript and surface receptor expression were accompanied by alterations in Fc{gamma}R phagocytic function. Results of Fc{gamma}R-specific phagocytosis after culture of monocytes with medium and medium supplemented with TNF-{alpha}, IL-10, IL-13, and IL-4 are shown in Figure 6 . For all treatments, phagocytosis mediated by 7.3 probes was significantly lower compared with IV.3-mediated phagocytosis (77–98% inhibition, P<0.001; Fig. 6A ).



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  		Figure 6. Modulation of Fc{gamma}R-mediated phagocytic function by TNF-{alpha}, IL-10, IL-13, and IL-4. (A) Fc{gamma}RII-mediated internalization of IV.3 probes and 7.3 probes was measured following culture of monocytes for 48 h with TNF-{alpha}, IL-10, IL-13, IL-4, and medium as control. Significant differences between internalization of IV.3 probes and 7.3 probes are marked (**) for P < 0.001. (B) Phagocytosis mediated by Fc{gamma}RI and Fc{gamma}RIII was determined with 22.2 and 3G8 probes, respectively, under each culture condition. Results represent mean ± SEM of four to 20 experiments. Significant differences between treatments and control are marked (*) for P< 0.01.

 
It is interesting that IL-10 was the most potent inducer of Fc{gamma}R-mediated phagocytosis. IL-10 induced a marked increase in Fc{gamma}RIIa-mediated phagocytosis (IV.3 probes) compared with monocytes cultured in medium (Fig. 6A) . IL-10 also increased phagocytosis mediated by Fc{gamma}RI and Fc{gamma}RIII (Fig. 6B) , indicating the ability of IL-10 to enhance phagocytosis mediated by activating Fc{gamma}R in monocytes.

In contrast to the IL-10-mediated activating effect, IL-13 and IL-4 moderately decreased phagocytosis mediated by 22.2 (Fc{gamma}RI), IV.3 (Fc{gamma}RIIahigh+Fc{gamma}RIIblow), and 3G8 (Fc{gamma}RIII; Fig. 6A and B ).

Regulation of Fc{gamma}R function mediated by TNF-{alpha} in combination with anti-inflammatory cytokines
The combined action of pro- and anti-inflammatory cytokines is thought to maintain immune homeostasis. At sites of inflammation, increased production of TNF-{alpha} is often accompanied by increased levels of IL-10, IL-13, and occasionally, by IL-4. We investigated the combined effect of TNF-{alpha} plus IL-10, TNF-{alpha} plus IL-4, and TNF-{alpha} plus IL-13 on Fc{gamma}R-mediated function.

Treatment of monocyte with TNF-{alpha} + IL-10 increased expression of Fc{gamma}RIIA RNA transcripts compared with TNF-{alpha} treatment (data not shown). Binding of IV.3-FITC was increased, and binding of 7.3-FITC was unaffected (Fig. 7A ). These changes were associated with increased uptake of IV.3 probes following TNF-{alpha} + IL-10 treatment (Fig. 7B) . Expression and phagocytosis of Fc{gamma}RI (22.2) were also up-regulated by TNF-{alpha} + IL-10 (Fig. 7C and 7D) .



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  		Figure 7. Regulation of Fc{gamma}R expression and phagocytic function by combinations of cytokines. (A) Membrane expression was determined by direct staining with IV.3-FITC and 7.3-FITC following culture of monocytes for 24 h with medium, TNF-{alpha}, TNF-{alpha} + IL-10, TNF-{alpha} + IL-4, and TNF-{alpha} + IL-13. (B) Phagocytosis of IV.3 probes and 7.3 probes was measured following culture of monocytes for 48 h with medium, TNF-{alpha}, TNF-{alpha} + IL-10, TNF-{alpha} + IL-4, and TNF-{alpha} + IL-13. (C) Expression of Fc{gamma}RI and Fc{gamma}RIII on monocytes was determined by direct staining with 22.2-FITC and 3G8-FITC following culture conditions described above. (D) Phagocytosis mediated by Fc{gamma}RI (22.2 probes) and Fc{gamma}RIII (3G8 probes) was determined under the same culture conditions as above. Values represent mean ± SEM of four to 16 experiments, and significant differences between TNF-{alpha} plus IL-10, TNF-{alpha} plus IL-13, and TNF-{alpha} plus IL-4 compared with TNF-{alpha} alone are marked (*) for P< 0.001.

 
Although TNF-{alpha} and IL-10 had a synergistic and additive effect, resulting in a potent induction of Fc{gamma}R phagocytic function (Figs. 7 and 8 F 8G 8H 8I 8J ), TNF-{alpha}, in combination with IL-4 or IL-13, had the opposite effect. Treatment of monocytes with TNF-{alpha} + IL-13 (Figs. 7 and 8K 8L 8M 8N 8O) down-regulated the phagocytic function mediated through Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII compared with TNF-{alpha} treatment (Figs. 7 and 8A 8B 8C 8D 8E) .



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  		Figure 8. Modulation of Fc{gamma}R-mediated phagocytosis by TNF-{alpha}, TNF-{alpha} + IL-10, and TNF-{alpha} + IL-13. Dot-plots indicate PKH26 fluorescence in FL2 (y-axis) versus SSC (x-axis) in a representative experiment. PI of monocytes internalizing IV.3 probes (A, F, K), 7.3 probes (B, G, L), 22.2 probes (Fc{gamma}RI; C, H, M), 3G8 probes (Fc{gamma}RIII; D, I, N), and human (h)IgG1 probes (E, J, O) following culture with TNF-{alpha} (left panels), TNF-{alpha} + IL-10 (middle panels), and TNF-{alpha} + IL-13 (right panels).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Monocytes are producers and targets of cytokines, which can act in an autocrine or paracrine manner. Monocyte functions are amplified by the activity of proinflammatory cytokines, and efficient counter-action by anti-inflammatory cytokines is believed to diminish effector functions, reducing the inflammatory potential of activated monocytes. More recently, the concept of alternative activation of mononuclear phagocytes by IL-4, IL-13, and IL-10 has been put forward [39 40 41 ].

A major role for Fc{gamma}R in the initiation and propagation of inflammatory reactions is well established (reviewed in refs. [2 , 42 ]). Functional deficiency in Fc{gamma}RI and Fc{gamma}RIII, as observed in {gamma}-chain knockout mice, is associated with diminished inflammation in response to IgG immune complexes. Severe inflammation was observed following deletion of inhibitory Fc{gamma}RIIb. At sites of immune-complex deposition, monocytes develop an inflammatory phenotype. We investigated a panel of cytokines, known to induce classical or alternative activation in monocytes, for their ability to change the ratio of activating and inhibitory Fc{gamma}R.

The pivotal role of TNF-{alpha} has been established for several autoimmune and inflammatory conditions. The basis for the TNF-{alpha} pathogenic action is the induction of other proinflammatory cytokines [43 ]. We observed that TNF-{alpha} down-regulated the Fc{gamma}RIIB promoter activity in U937 cells and decreased the expression of Fc{gamma}RIIb in human monocytes. This finding represents a novel component in the mechanism of TNF-mediated activation of monocytes. A decrease in inhibitory Fc{gamma}RIIb function induced by TNF-{alpha} may contribute to the amplification of inflammation.

Efforts to interrupt inflammatory pathways include the administration of IL-10, a cytokine synthesis inhibitory factor. The immunoregulatory effects of IL-10 were based on its ability to suppress monocyte and T helper cytokine production [44 ]. Yet, IL-10 is known to induce differentiation of monocytes into macrophages and to mediate up-regulation of Fc{gamma}R [45 ]. We investigated this mechanism in greater detail by assessing the IL-10 regulation of activating and inhibitory Fc{gamma}R. IL-10 treatment increased the luciferase activity driven by the Fc{gamma}RIIB promoter in U937 cells and induced the expression of Fc{gamma}RIIb in human monocytes. In addition, IL-10 determined a marked up-regulation of all ITAM-bearing Fc{gamma}Rs, resulting in a net activating phenotype. In combination with TNF-{alpha}, IL-10 lost its ability to up-regulate inhibitory Fc{gamma}R and determined a further increase in Fc{gamma}RIIa phagocytosis, suggesting a synergistic activating effect of TNF-{alpha} and IL-10 on Fc{gamma}R function.

Analogous studies have evaluated the up-regulation of Fc{gamma}R function by IL-10 treatment in patients with rheumatoid arthritis (RA), a disease associated with elevated levels of TNF-{alpha}. Paradoxically, IL-10-primed RA monocytes produced more TNF-{alpha} following stimulation with immune complexes [45 ]. We observed alterations in IL-10 signaling after FcR ligation, which abolished the IL-10-dependent suppression of cytokine production [46 ]. The results of the present study suggest that an imbalance in Fc{gamma}R, as a result of the combined action of TNF-{alpha} and IL-10, could further potentiate the inflammatory responses mediated by activated macrophages.

It has been proposed that the relative ratio of ITAM- and ITIM-containing Fc{gamma}R determines their net functional effect. We identified cytokines that could adjust the responsiveness of monocytes to immune complex stimulation and may restore the physiologic balance of Fc{gamma}R. IL-4 and IL-13 share many structural and functional similarities and have well-defined, anti-inflammatory properties [47 , 48 ]. Treatment with IL-4 or IL-13 decreased the expression of activating Fc{gamma}R, resulting in a higher ratio of FcR mediating inhibition versus activation. Furthermore, the effect of IL-4 and IL-13 was evident in combination with TNF-{alpha}, resulting in a marked decrease in Fc{gamma}R-mediated phagocytosis. This observation could be of particular relevance for conditions such as autoimmune hemolytic anemia and immune thrombocytopenic purpura, in which phagocytes ingest the antibody-coated E and platelets via an Fc{gamma}R-mediated mechanism [49 , 50 ].

We propose that the cytokine modulation of activating and inhibitory Fc{gamma}R during inflammation is a pivotal regulatory mechanism of efferent immune responses. The mechanism involved in the divergent functional effect of TNF-{alpha} in combination with IL-10 and IL-13 is currently under investigation. In inflammatory conditions, a systematic increase in activating Fc{gamma}R and/or a decline in the expression levels of inhibitory Fc{gamma}RIIb could mimic the phenotype of the Fc{gamma}RIIB-deficient mice. Combinations of cytokines, such as TNF-{alpha} and IL-10, which shift the balance of Fc{gamma}R toward activation, may be beneficial for host defense but could increase susceptibility to autoimmunity. In contrast, IL-4 and IL-13, in combination with TNF-{alpha}, markedly decreased Fc{gamma}R-mediated monocyte functions, an effect that needs to be investigated further for potential therapeutic use.

The results of our study suggest the possibility to down-regulate effector functions by cross-linking Fc{gamma}RIIb in human monocytes. Preferential ligation of Fc{gamma}RIIb with 7.3 mAb was associated with a marked decrease in phagocytosis and cytokine production, indicating that Fc{gamma}RIIb could constitute a target for modulation of monocyte functions. The characterization of reagents that interact preferentially with activating and inhibitory Fc{gamma}R enables further dissection of mechanisms involved in the regulation of Fc{gamma}R in human cells and the gain of knowledge regarding Fc{gamma}R dysfunction at sites of inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Arthritis Foundation, the SLE Foundation, and the National Institutes of Health, AR47106 and AR49765 (to L. P.). We thank the members of the Research Division of the Hospital for Special Surgery for helpful suggestions and their scientific input during the execution of this project.

Received September 17, 2004; revised January 18, 2005; accepted January 19, 2005.


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