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(Journal of Leukocyte Biology. 2002;72:657-667.)
© 2002 by Society for Leukocyte Biology

Fc{gamma}R cross-linking mediates NF-{kappa}B activation, reduced antigen presentation capacity, and decreased IL-12 production in monocytes without modulation of myeloid dendritic cell development

Yvonne Drechsler, Sangeeta Chavan, Donna Catalano, Pranoti Mandrekar and Gyongyi Szabo

Department of Medicine, University of Massachusetts Medical Center, Worcester, MA

Correspondence: Gyongyi Szabo, M.D., Ph.D., Associate Professor, Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School, NRB Floor 2, 364 Plantation Street, Worcester, MA 01605. E-mail: gyongyi.szabo{at}umassmed.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of monocytes (MO) through receptors for the Fc region of immunoglobulin G (Fc{gamma}R) activates a variety of responses, including phagocytosis, antibody (Ab)-dependent cellular cytotoxicity, and production of cytokines. We previously reported that the MO subpopulation that expresses Fc{gamma}R in high density produces high levels of tumor necrosis factor {alpha} (TNF-{alpha}) compared with Fc{gamma}R-negative MO. Here, we show that cross-linking MO via Fc{gamma}RI or Fc{gamma}RII but not via Fc{gamma}RIII activates nuclear regulatory factor-{kappa}B (NF-{kappa}B), a transcription factor involved in regulation of TNF-{alpha}. NF-{kappa}B activation peaked at 2.75 h after Fc{gamma}RI cross-linking, involved p65 and p50 (heterodimer) and not c-rel-containing NF-{kappa}B complexes, and was mediated via I{kappa}B degradation. Cross-linking Fc{gamma}RI, -II, as well as -III inhibited interleukin (IL)-12 (p70) production in MO, whether stimulated with Staphylococcal enterotoxin B (P<0.02) or lipopolysaccharide (P<0.02). Inhibition of IL-12 by Fc{gamma}R cross-linking was not mediated by TNF-{alpha}, as the presence of an anti-TNF-{alpha} Ab could not restore the reduced IL-12 production. Decreased IL-12 production correlated with reduced antigen presentation capacity (P<0.01) in the Fc{gamma}R-cross-linked MO. Blood MO can give rise to myeloid dendritic cells (DC). Fc{gamma}R cross-linking did not modulate in vitro maturation of MO to fully functional myeloid DC. Allostimulatory capacity in mixed leukocyte reaction and DC marker expression (CDla, CD80, CD86) was not different between control and Fc{gamma}RI cross-linked DC. These results suggest that signals mediated via Fc{gamma}RI, -II, and -III have overlapping yet distinct effects on MO, which are likely to be involved in the fine-tuning of the immune responses to various stimuli.

Key Words: Fc{gamma}RI (CD64) • Fc{gamma}RII (C32) • Fc{gamma}RIII (C16) • TNF-{alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptors for the Fc region of the immunoglobulin G (IgG; Fc{gamma}R), members of the Ig superfamily, include three different types on human blood monocytes (MO): the high-affinity, Fc{gamma}RI (CD64), which preferentially binds monomeric IgG, and Fc{gamma}RII (CD32) and Fc{gamma}RIII (CD16), which bind aggregated IgG [1 , 2 ]. In MO/macrophages, Fc{gamma}R ligation induces a number of responses, including phagocytosis of IgG-coated particles, antibody (Ab)-dependent cytotoxicity, and release of mediators including tumor necrosis factor {alpha} (TNF-{alpha}) and MO chemoattractant protein-1 (MCP-1) [1 , 3 ]. Fc{gamma}R ligation occurs under a variety of physiologic conditions such as opsonized particle binding, phagocytosis, and therapy with intravenous Ig or RhO immune globulin. Although multiple reports documented immunoregulatory effects of intravenous Ig therapy (ivIG), the mechanisms for its beneficial effects are yet to be understood [4 , 5 ]. Likely targets of ivIG therapy are Fc{gamma}R-expressing immune cells, particularly MO, which can be regulated via ligation/clustering of Fc{gamma}R [5 ].

Although regulation on MO and macrophage activation via Fc{gamma}R ligation has been extensively studied in murine macrophages, the effects of Fc{gamma}R cross-linking in human MO are less well-characterized. We and others have previously shown that Fc{gamma}R cross-linking activation of the Fc{gamma}R-positive MO subpopulation results in high levels of TNF-{alpha}, prostaglandin E2 (PGE2), and procoagulant activity production [6 , 7 ]. In contrast, MO expressing no or very low density Fc{gamma}R (Fc{gamma}R-negative MO) produce more plasminogen activator and are more potent antigen-presenting cells (APC) compared with Fc{gamma}R-positive MO [8 , 9 ].

Immune complexes may bind to any of the Fc{gamma}R and trigger TNF-{alpha} production in MO. TNF-{alpha} plays a key role not only in the initiation and progression of an inflammatory response but also in inhibition of interleukin (IL)-12 production [10 , 11 ]. TNF-{alpha}-deficient mice developed a vigorous inflammatory response with high levels of serum IL-12 leading to death when injected with heat-inactivated C. parvum [10 ]. It has been suggested that TNF-{alpha} plays an important role to limit the extent and duration of the inflammatory response by down-regulating IL-12 and thereby interferon-{gamma} (IFN-{gamma}) production. To investigate the relationship between TNF-{alpha} and IL-12 production, we examined the effect of cross-linking Fc{gamma}R on MO in the absence or presence of anti-TNF-{alpha} Ab.

Nuclear factor-{kappa}B (NF-{kappa}B) is an inducible transcription activator involved in the expression of a variety of genes encoding immunoreceptors, cell-adhesion molecules, and cytokines, including TNF-{alpha} [12 ]. NF-{kappa}B plays a pivotal role in cells of the immune system, as it is rapidly activated by many signals, including mitogens, lipopolysaccharides (LPS), and cytokines. The active DNA-binding form of NF-{kappa}B is a heterodimer, which is commonly formed by p50 and p65 proteins [13 ]. In untreated cells, NF-{kappa}B is sequestered in the cytoplasm bound to its inhibitors, I{kappa}B proteins [14 ]. Cell exposure to inducing conditions causes I{kappa}B degradation by the 26S proteasome pathway, allowing NF-{kappa}B translocation to the nucleus [15 , 16 ]. To investigate the mechanisms of MO activation via Fc{gamma}R, we analyzed the activation of NF-{kappa}B (TNF-{alpha} consensus sequence) using antibodies against Fc{gamma}I, -II, and -III. We found a selective induction of NF-{kappa}B by Fc{gamma}RI and -II but not by Fc{gamma}RIII cross-linking.

In murine bone marrow-derived macrophages, Fc{gamma}R ligation suppresses IL-12 induction [17 ]. Immune complexes were also shown to inhibit IL-12 secretion by human MO [18 ], suggesting that Fc{gamma}RII and Fc{gamma}RIII ligation is a potential mechanism for regulation of IL-12 production in blood MO. IL-12 is important in antigen-specific T cell proliferation, initiation of type 1 immune response, and T helper cell type 1 (Th1) CD4+ proliferation [19 ]. IL-12 acts as a proinflammatory cytokine and as an immunomodulator and therefore bridges innate and adaptive immune responses [19 ]. Thus, we investigated whether independently cross-linking the Fc{gamma}RI, -II, or -III results in attenuation of IL-12 production in human MO. We found that reduced IL-12 production in Fc{gamma}R-cross-linked MO correlated with decreased antigen presentation capacity compared with non-Fc{gamma}R-cross-linked MO.

Blood MO give rise to myeloid dendritic cells (DC) upon antigenic or cytokine stimulation in vitro and in vivo [20 ]. Maturation to myeloid DC involves well-characterized, morphologic, phenotypic, and functional changes, including superior accessory cell and antigen presentation function [21 ]. Decrease or loss of surface receptors, such as CD14 and Fc{gamma}R, typically associated with "phagocytic" and inflammatory MO functions, also occurs during DC maturation [22 ]. Cell stimulation signals provided to MO in the early phase of DC maturation may promote or inhibit full DC maturation [23 ]. Thus, signals mediated via Fc{gamma}R cross-linking of MO may also modulate subsequent DC development. Our results show that cross-linking MO via Fc{gamma}RI prior to DC maturation results in no modulation in functional maturation of myeloid DC in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
RPMI 1640, Iscove’s modified Dulbecco’s media, and Medium 199 were obtained from JRH Biosciences (Lenexa, KS), and fetal bovine serum (FBS) was from Hyclone (Logan, UT). Endotoxin contamination was less than 8 pg/ml in the culture media and FBS. Human (h) recombinant IFN-{gamma} (Escherichia coli) was obtained from Collaborative Research Inc. (Bedford, MA). Staphylococcal enterotoxin B (SEB) was obtained from Sigma Chemical Co. (St. Louis, MO), and LPS (E. coli strain 0111:B4) was from Difco Laboratories (Detroit, MI). Isotypic control IgG1 rat Ab was obtained from Biosource International, Inc. (Camarillo, CA). Fc Ab were obtained from MEDAREX (Princeton, NJ), IgG-Fab was from Sigma Chemical Co., and NF-{kappa}B oligonucleotide was from Promega (Madison, WI).

Blood donors
Peripheral blood was taken by venipuncture from healthy volunteers (laboratory and hospital staff of the University of Massachusetts Medical School, Worcester), aged 20–58 years, using 10 U/ml heparin as an anticoagulant. This study was approved by the Institutional Human Subjects Committee, and informed consent was obtained from each blood donor.

Separation of MO and Fc{gamma}R cross-linking, and isolation of Fc{gamma}R-positive and Fc{gamma}R-negative MO subpopulations
MO were isolated from human peripheral blood from Ficoll-Hypaque-purified mononuclear cell preparations by adherence, as previously described [8 , 9 , 24 ]. Briefly, Ficoll-Hypaque density-separated mononuclear cells were washed in ice-cold Hanks’ balanced saline solution, supplemented with 3% FBS, and then depleted of T cells by rosetting with neuraminidase-treated sheep red blood cells. After 2 h adherence, there was less than 5% total contaminating lymphocytes in the adherent MO, as determined by CD3, CD56, and CD19 staining by fluorescein-activated cell sorter (FACS) analysis.

After overnight resting, antibodies against Fc{gamma}RI, -II, or -III (MEDAREX) were added to the MO at 4°C for 30 min. Cells were washed in complete medium, and 20 µl of the second Ab (IgG-Fab fragment) was added at 37°C for different periods of time (0.5–24 h). For NF-{kappa}B analysis, cells were washed in ice-cold phosphate-buffered saline without Ca2+/Mg2+, followed by nuclear and cytoplasmic extraction. For TNF-{alpha} and IL-12 measurements, cells were stimulated for 24 h, supernatants were collected, and subsequently, enzyme-linked immunosorbent assay (ELISA) was performed.

Where indicated, MO were stimulated via cross-linking cell surface Fc{gamma}R [6 , 8 ] via rosetting MO with anti-RhO(D) human Ig-treated (RhoGAM, Ortho Diagnostic System, Raritan, NJ) or human O+, RhO(D)+ erythrocytes (Selectogen, Ortho Diagnostic System). This method cross-links all three Fc{gamma}R but primarily Fc{gamma}RI [6 , 8 ]. Erythrocytes were lysed after 30 min rosetting, and MO were further stimulated as indicated. In selected experiments, the Fc{gamma}R-positive and -negative MO subpopulations were isolated using Ficoll-Hypaque gradient centrifugation as described before [6 7 8 9 ]. In the 12 normal blood donors used for the present experiments, the ratio of MO subpopulations was 59 ± 7% Fc{gamma}R+ and 41 ± 7% Fc{gamma}R- MO.

In the antigen presentation experiments, 4 x 105 Fc{gamma}R+ or Fc{gamma}R- MO were cultured with 2 LF/ml tetanus toxoid overnight, then washed vigorously, and recombined with 2 x 106 syngeneic T cells, which were isolated by E-rosetting as described before [9 ]. Antigen-specific T cell proliferation was determined by 3H-thymidine incorporation during the last 18 h of the 6-day proliferation assay as described before [9 ].

Generation of myeloid DC and quantitation of allostimulatory activity
MO with or without Fc{gamma}RI cross-linking were cultured in six-well plates in RPMI containing 10% FBS, 0.1% ß-mercaptoethanol, granulocyte macrophage-colony stimulating factor (GM-CSF; 100 U/ml), and IL-4 (200 U/ml; Peprotech, Rocky Hill, NJ) as described before [22 ]. Mixed lymphocyte reactions (MLRs) were established in 96-well plates using allogeneic-purified T lymphocytes as described before [20 , 24 ]. On day 7, DC were stained with fluorescein isothiocyanate-conjugated antibodies against CD1a, CD80, CD86 (BD Pharmingen, San Diego, CA), and isotypic controls, according to the manufacturer’s instructions, and cells were analyzed on a FACS analysis (Becton Dickinson, San Jose, CA).

Extraction, electrophoretic gel mobility shift assay (EMSA), and Western blots
Nuclear and cytoplasmic extracts were obtained from cells with or without stimulation by the modified method of Dignam et al. [25 ], as previously described [26 ]. Protein content was determined in all extracts using the Biorad Dye Reagent assay. An equal amount (5 µg) of protein from each sample and a TNF-{alpha} consensus NF-{kappa}B oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') were used for EMSA as previously described [26 , 27 ]. Anti-human p50 and p65 antibodies for supershifts were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blots, using 10 µg cytoplasmic extracts, were performed as previously described [26 ]. I{kappa}B{alpha} affinity-purified rabbit polyclonal Ab to the C-terminal of human I{kappa}B{alpha} was obtained from Santa Cruz Biotechnology.

Cytokine measurements
Cell-free MO supernatants were tested for TNF-{alpha}, IL-12 p70, p40, plus p70 levels in highly specific ELISA assays, respectively (Endogen, Cambridge, MA). The IL-12 p70 ELISA was highly sensitive, with 1 pg/ml as the lower detection limit.

Statistical analysis
Individual differences in monokine production have been demonstrated, particularly in TNF-{alpha} responses as linked to human leukocyte antigen class II haplotype [8 , 24 ]. The levels of monokine production varied in our experiments from individual to individual; thus, statistical significance between the appropriate groups was calculated from all of the experiments done with the same experimental protocol using the Wilcoxon signed-rank, nonparametric data analysis. Data are presented as mean values of monokine-response levels or as results from representative experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cross-linking Fc{gamma}RI and -II but not Fc{gamma}RIII activates transcription factor NF-{kappa}B
Previous work from our laboratory showed that the MO subpopulation expressing Fc{gamma}R in high density can be induced for increased production of TNF-{alpha} via Fc{gamma}R cross-linking [8 , 26 ]. Increased NF-{kappa}B activation was shown in THP-1 cells after the low-affinity Fc{gamma}R activation with insoluble aggregates of human IgG [28 ]. Here, we sought to investigate the role of NF-{kappa}B in human blood MO activation via the different Fc{gamma}R. Fc{gamma}R were cross-linked using specific monoclonal antibodies (mAb) as primary Ab against Fc{gamma}RI, -II, and -III (MEDAREX) and an IgG-Fab fragment as secondary Ab, as described in Materials and Methods. EMSA demonstrated activation of NF-{kappa}B (consensus sequence for TNF-{alpha}) after cross-linking MO with antibodies against Fc{gamma}RI and -II, evidenced by increased NF-{kappa}B DNA binding (Fig. 1A ). Cross-linking Fc{gamma}RIII induced no activation of NF-{kappa}B in MO. Induction of NF-{kappa}B by Fc{gamma}R cross-linking was specific, as addition of control IgG without the cross-linking Ab induced no NF-{kappa}B activation (Fig. 1B) .



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  		Figure 1. Cross-linking Fc{gamma}RI and -II but not Fc{gamma}RIII induces NF-{kappa}B activation in MO. Fc{gamma}R were cross-linked using specific antibodies (10 µg/ml) as primary Ab directed against Fc{gamma}RI, -II, or -III (0.5 h) as described in Materials and Methods. IgGFAB (20 µg/ml) was used as a secondary, cross-linking Ab (2 h). Unstimulated (unst) MO were included for control. (A) Equal amounts of protein (5 µg) per stimulation group were evaluated in the EMSA as described in Materials and Methods. Bottom graph shows quantitation of DNA binding determined by using the NIH Image Analyzer program. One of four experiments with the same results is shown. (B) Nuclear extracts (5 µg) from unstimulated (unst), Fc{gamma}RI cross-linked (Fc{gamma}RI), or control IgG-stimulated MO were evaluated in EMSA. A 20-fold excess of the unlabeled NF-{kappa}B was included as a cold competitor (comp). One of three experiments with the same results is shown.

 
Activation of NF-{kappa}B on Fc{gamma}RI cross-linking is time-dependent with a maximum activation after 2.5–3 h of cross-linking
Next, the kinetics of NF-{kappa}B activation was studied after Fc{gamma}RI cross-linking. The primary Ab against Fc{gamma}RI was applied to MO for 0.5 h followed by different periods of incubation with the secondary Ab (Fab fragment). The gel shift assay shows increased NF-{kappa}B activation over time, with a maximum activation at 2.75 h, followed by a decrease of activation up to 24 h (Fig. 2A ). Stimulation of the cells with SEB after cross-linking with Fc{gamma}RI for 0.5 h further enhanced NF-{kappa}B activation. However, NF-{kappa}B activation was not enhanced further by SEB stimulation in MO previously Fc{gamma}RI-cross-linked for 2.75 h (Fig. 2B) .



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  		Figure 2. Kinetics of NF-{kappa}B activation by Fc{gamma}RI cross-linking. Fc{gamma}RI was cross-linked using a specific Ab as described in Figure 1 . IgGFAB secondary Ab was applied for different periods of time (0.5–24 h) as indicated. A 20-fold excess of the unlabeled NF-{kappa}B was included as a cold competitor (comp). Bottom graphs show quantitation of DNA binding determined by using the NIH Image Analyzer program. (A) Nuclear extracts (5 µg/sample) were evaluated in EMSA. (B) MO were stimulated with SEB (1 µg/ml) for 2 h with or without previous Fc{gamma}RI cross-linking for 0.5 h (0.5) or 2.75 h (2.75).

 
NF-{kappa}B activation by Fc{gamma}RI cross-linking is linked to rapid I{kappa}B degradation in human MO
The common pathway for NF-{kappa}B activation is via phosphorylation and degradation of I{kappa}B{alpha} [14 , 15 ]. Cytoplasmic extracts obtained from MO at the indicated times after Fc{gamma}RI cross-linking were evaluated for degradation of I{kappa}B{alpha}. Decreasing cytoplasmic I{kappa}B{alpha} (Fig. 3A ) levels correlated with the activation of NF-{kappa}B at 0.5 and 2.75 h (Fig. 2A) , suggesting that NF-{kappa}B activation by Fc{gamma}RI is mediated via I{kappa}B{alpha} in MO. Cytoplasmic I{kappa}B{alpha} levels also decreased after Fc{gamma}RII but not Fc{gamma}RIII cross-linking, consistent with NF-{kappa}B activation by Fc{gamma}RII but not Fc{gamma}RIII stimulation (Fig. 3B) . In supershift assays using mAb against p50, p65, or c-Rel, we found that activation of NF-{kappa}B by Fc{gamma}RI was specific for the p65/p50 heterodimer, and the complex contained no c-Rel (Fig. 3C) .



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  		Figure 3. Fc{gamma}RI and -II but not -III cross-linking are associated with I{kappa}B degradation and p65/p50 nuclear binding. MO Fc{gamma}RI, -II, and -III were cross-linked using specific mAb followed by cross-linking with IgGFAB as described in Materials and Methods. (A) Equal amounts of cytoplasmic extracts (20 µg/group) from Fc{gamma}RI-cross-linked samples were subjected to immunoblotting using anti-I{kappa}B{alpha} Ab (Santa Cruz Biotechnology). Densitometric values for the individual bands are indicated below each lane. (B) Equal amounts of cytoplasmic extracts (20 µg/group) from Fc{gamma}RII- and -III-cross-linked samples were subjected to immunoblotting using anti-I{kappa}B{alpha} Ab (Santa Cruz Biotechnology). Densitometric values for the individual bands are indicated below each lane. (C) Supershift analysis of NF-{kappa}B in Fc{gamma}RI-cross-linked MO. Nuclear extracts from MO 2.75 h after Fc{gamma}RI cross-linking were admixed with rabbit anti-p50 (p50), anti-p65 (p65), or anti-cRel (cRel) Ab (2µg) after addition of [32P]NF-{kappa}B oligonucleotide and were incubated for 30 min prior to EMSA. Shifted bands were detected by their retarded mobility. A 20-fold excess of the unlabeled NF-{kappa}B was included as a cold competitor (comp).

 
Cross-linking any of the three Fc{gamma}R down-regulates MO IL-12 production
Fc{gamma}R cross-linking with anti-RhO(D) human Ig-treated RhO(D)+ erythrocytes, which cross-link all three Fc{gamma}R, resulted in substantially decreased IL-12 production (IFN-{gamma}+SEB: 79±5% inhibition, n=4; IFN-{gamma}+LPS: 53±12% inhibition, n=4; Fig. 4A ). Differences in IL-12 production were more evident when the Fc{gamma}R cross-linked, Fc{gamma}R-positive and the Fc{gamma}R noncross-linked, Fc{gamma}R-negative MO subpopulations were separated prior to stimulation with SEB, LPS, or IFN-{gamma} (Fig. 4B) . We found that the Fc{gamma}R-negative MO subpopulation produces higher levels of the p70 (bioactive) and p40 chains of IL-12 (Fig. 4C) .



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  		Figure 4. MO-IL-12 production is inhibited by Fc{gamma}R cross-linking. (A) Adherence isolated blood MO or MO stimulated with Fc{gamma}R cross-linking via IgG-coated human Rh+ erythrocytes (FcR crosslinked MO) were cultured in medium or in the presence of IFN-{gamma} (100 U/ml) plus LPS (1 µg/ml) or SEB (1 µg/ml) for 24 h. The levels of bioactive IL-12 (p70) were determined in the supernatants. Results from two different blood donors are shown out of four experiments with similar results. (B) IL-12 production capacity is different between Fc{gamma}R-positive and -negative MO subpopulations. Fc{gamma}R-cross-linked (Fc{gamma}R+) or non-Fc{gamma}R-cross-linked (Fc{gamma}R-) MO were separated and then stimulated with 1 µg/ml SEB, 1 µg/ml LPS, or a combination of 100 U/ml IFN-{gamma} plus 1 µg/ml SEB or 1 µg/ml LPS for 24 h. IL-12 (p70) levels were determined in the cell-free supernatants in a specific ELISA. Mean and standard error are shown from 11 experiments with SEB (left panel) and from five experiments with LPS stimulation (right panel). (C) The Fc{gamma}R-negative MO are the main source of p40 and p70 in blood MO. Fc{gamma}R-cross-linked (Fc{gamma}R+) and noncross-linked (Fc{gamma}R-) MO were stimulated as described in B with SEB or IFN-{gamma} plus SEB for 24 h. MO supernatants were tested for IL-12 in an ELISA that measures human p40 and p70. Mean values and standard deviation of seven different experiments/blood donors are shown.

 
Next, the role of the individual Fc{gamma}R, Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16) was investigated. We found that cross-linking Fc{gamma}RI, -II, or -III resulted in significantly reduced IL-12 production in response to IFN-{gamma} plus LPS or IFN-{gamma} plus SEB stimulation in blood MO (Fig. 5 ). MO treated only with the secondary Ab (Ab control) showed IL-12 production levels comparable with the control MO. Lower, absolute IL-12 levels in experiments with Fc{gamma}RIII cross-linking represent individual-to-individual variability in IL-12 production capacity between blood donors. The down-regulatory signal by Fc{gamma}R cross-linking lasted for at least 24 h, as Fc{gamma}R-crosslinked MO IL-12 production remained decreased even when a 24-h recovery period was provided after Fc{gamma}RI cross-linking prior to an IFN-{gamma} plus SEB challenge (data not shown).



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  		Figure 5. MO IL-12 production is inhibited by cross-linking Fc{gamma}RI, -II, or -III. Fc{gamma}R were cross-linked using specific Ab against Fc{gamma}RI, -II, or -III (0.5 h), followed by cross-linking with IgGFAB (0.5 h) as described in Figure 1 . Control Ab-treated MO, Fc{gamma}R-cross-linked (Fc crosslinked), and control MO (non-crosslinked M/) were stimulated with IFN-{gamma} (100 U/ml) plus SEB (1 µg/ml) or LPS (1 µg/ml) for 24 h. MO supernatants were tested for IL-12 (p70) by ELISA p70. Mean values and standard deviation are shown from two to nine experiments/blood donors as indicated.

 
Fc{gamma}R-cross-linked MO were shown to produce high levels of TNF-{alpha} after bacterial stimulation [7 , 8 ], whereas IL-12 levels were decreased in our current experiments. Thus, the potential of inhibition of IL-12 by TNF-{alpha} was explored in Fc{gamma}RI-cross-linked MO. Decreased IL-12 levels after Fc{gamma} cross-linking stimulation were not a result of an inhibitory effect of TNF-{alpha}, as IL-12 levels were not increased in the presence of neutralizing anti-TNF-{alpha} Ab during stimulation for 24 h (Fig. 6 ). IL-12 production is pivotal for T cell activation during antigen presentation [19 ]. We found that the antigen presentation capacity of the Fc{gamma}R-negative MO was significantly greater than that of the Fc{gamma}R-cross-linked MO subpopulation (Table 1 ). These data collectively imply that inhibition of IL-12 production by Fc{gamma}R cross-linking may contribute to reduced antigen presentation capacity of Fc{gamma}R-expressing MO.



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  		Figure 6. Decreased MO IL-12 production in Fc{gamma}RI-cross-linked cells is not a result of increased TNF-{alpha} production. Fc{gamma}RI were cross-linked using specific Ab (10 µg/ml) as primary Ab directed against Fc{gamma}RI (0.5 h), as described in Materials and Methods. IgGFAB (20 µg/ml) was used as a secondary, cross-linking Ab (0.5 h). Fc{gamma}RI cross-linked were stimulated with IFN-{gamma} (100 U/ml) plus SEB (1 µg/ml) for 24 h. One group was treated additionally with a TNF-{alpha}-neutralizing or a control Ab. MO supernatants were tested for IL-12 (p40/p70) by ELISA p40/p70 and TNF-{alpha} by ELISA hTNF-{alpha}.

 

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  		Table 1. Fc{gamma}R Cross-linking Inhibits Monocyte Antigen Presentation in Tetanus Toxoid-Induced T Cell Proliferation

 
Fc{gamma}RI cross-linking of MO does not affect their maturation to functionally active DC
Peripheral blood MO can mature to myeloid DC in vivo and in vitro upon antigen or cytokine stimulation [20 , 21 ]. Our results so far suggested that Fc{gamma}RI cross-linking induces acute inflammatory pathways (NF-{kappa}B and TNF-{alpha}) at the expense of the APC function in blood MO. Thus, we investigated whether Fc{gamma}RI cross-linking of MO would adversely affect their capacity to develop into MO-derived myeloid DC. Adherent blood MO were stimulated via Fc{gamma}RI cross-linking, then matured in the presence of IL-4 and GM-CSF for 7 days, as described before [24 ]. There was no difference in the morphology of the DC derived from Fc{gamma}RI-cross-linked MO compared with the control MO. We found no difference in a MLR in the accessory cell function between DC derived from control or Fc{gamma}RI-cross-linked MO (Fig. 7A ). The surface expression of the accessory molecules CD80 and CD86 and of the immature DC marker CD1a (Fig. 7B) or morphologic typical veiled cell appearance (data not shown) was not different between control and Fc{gamma}RI-MO-derived DC. These results suggest that Fc{gamma}RI cross-linking does not modulate blood MO maturation to functionally active DC.



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  		Figure 7. Fc{gamma}RI cross-linking does not affect myeloid DC generation from MO. (A) DC were generated in the presence of IL-4 plus GM-CSF as described in Materials and Methods from unstimulated MO (DC), MO after Fc{gamma}RI cross-linking (Fc-DC), or control Ab treatment (Fc-control DC). On day 7, DC were collected and cocultured with allogeneic T lymphocytes at the indicated DC/T cell ratios for a total of 5 days. T cell proliferation was assessed by incorporation of 3H-thymidine during the last 16 h of the proliferation assay. Data are shown as mean ± SD from four experiments. Proliferation of T lymphocytes or DC alone was less than 400 cpm. (B) DC were generated as described in A. MO were maintained in culture medium without additional stimulation. Surface expression of CD1a, CD80, and CD86 was analyzed on day 7 by FACS (Becton Dickinson). Data are shown as mean ± SD of four experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human blood MO represent a heterogeneous population in which subpopulations can be defined on the basis of surface expression of various receptors including Fc{gamma}R, CD14, and CD4 [2 , 6 7 8 9 , 29 , 30 ]. Differential expression of receptors provides a basis for selective activation of MO. Stimulation of human MO via Fc{gamma}R has been shown to induce production of inflammatory mediators, including TNF-{alpha} and MCP-1 [3 , 6 7 8 , 28 ]. We show for the first time that Fc{gamma}RI or Fc{gamma}RII but not Fc{gamma}RIII cross-linking increases NF-{kappa}B activation, a regulatory element in inflammatory gene activation, in human MO. The NF-{kappa}B activation was time-dependent after Fc{gamma}R cross-linking. Our data show that NF-{kappa}B activation in Fc{gamma}RI- and -II-cross-linked human MO involves degradation of I{kappa}B{alpha} and formation of the p50/p65 NF-{kappa}B complex. In addition, this is the first report in human MO to show that cross-linking any of the three Fc{gamma}R can down-regulate IL-12 production. Thus, Fc{gamma}R cross-linking induced induction of NF-{kappa}B in combination with decreased IL-12 production and antigen presentation capacity, which are major determinants of the functional capacity of the Fc{gamma}R-positive MO subpopulation. Our data also demonstrate for the first time that in contrast to inhibition of antigen presentation by MO, Fc{gamma}RI cross-linking does not inhibit development of functionally active myeloid DC.

Activation of NF-{kappa}B by Fc{gamma}RI and -II cross-linking in human MO correlates with the increased production of TNF-{alpha}, which was reported earlier by our laboratory [8 , 24 ]. MO stimulation by immune complexes (Fc{gamma}RI and -II stimulation) was also shown to induce MCP-1 and NF-{kappa}B activation [28 ]. NF-{kappa}B is common to the promoter region of many of the inflammatory genes induced by Fc{gamma}RI cross-linking, including TNF-{alpha}, MCP-1, and procoagulant activity [12 , 14 , 28 ]. Our results demonstrate that Fc{gamma}RI- and Fc{gamma}RII-mediated NF-{kappa}B activation involves I{kappa}B{alpha} degradation and is mediated by p65/p50 NF-{kappa}B/Rel heterodimer. Thus, NF-{kappa}B activation after Fc{gamma}RI or -II cross-linking mediates induction of inflammatory responses in MO. In addition to rapid induction of inflammatory responses after Fc{gamma}R cross-linking, MO phenotypic changes also include reduced antigen presentation capacity, reduced T cell stimulatory capacity, and IL-12 production [8 , 9 , 31 ]. Although the IL-12 promoter has a NF-{kappa}B repeat site, IL-12 production was inhibited by cross-linking Fc{gamma}RI, -II, or -III [32 ]. In addition to NF-{kappa}B, IL-12 gene activation is also regulated by PU.1, which was up-regulated in mouse macrophages after FcR stimulation [32 ]. Based on our experiments, the role of NF-{kappa}B in IL-12 regulation after Fc{gamma}R cross-linking cannot be ruled out, as the NF-{kappa}B sequence used in our experiments (consensus sequence) was different from that in the IL-12 promoter.

Our data demonstrate that cross-linking Fc{gamma}RI and -II but not -III induced NF-{kappa}B, and cross-linking any of the Fc{gamma}R down-regulated IL-12 production in MO, suggesting that the signaling mechanisms for NF-{kappa}B activation and IL-12 down-regulation are different. Fc{gamma}RI and Fc{gamma}RII consist of single polypeptide chains and possess high-sequence homology with each other in their extracellular domains, but their cytoplasmic tails are unrelated by amino acid sequence [33 ]. Fc{gamma}RI interacts with the FcR {gamma}-chain, which is essential for surface expression and signaling through the Fc{gamma}RI [34 35 36 ]. Fc{gamma}RI, like T cell and B cell receptors, do not contain intrinsic tyrosine kinase activity, but upon receptor cross-linking, Src-like and syk/ZAP70 family members of nonreceptor tyrosine kinases are activated [37 38 39 40 41 ]. Macrophage activation via Fc{gamma}RI increases intracellular calcium levels, a mechanism suggested for inhibition of IL-12 production in murine bone marrow-derived macrophages after Fc{gamma}RII/III cross-linking [17 , 42 ].

IL-12, as a result of its proinflammatory and immunoregulatory effects, plays an important role in innate and acquired immunity. In inflammatory responses, IL-12 has been shown to be rapidly induced in models of acute bacterial infection, and mice lacking IL-12 rapidly succumb to sepsis and have impaired bacterial clearance [43 , 44 ]. In the acquired immune response, IL-12 is critical for development of a Th1-type immune response and pivotal in host defense against intracellular pathogens including Mycobacterium tuberculosis, Listeria monocytogenes, and Toxoplasma gondii [45 46 47 ]. Regulation of IL-12 production by MO and DC involves increased IL-12 production capacity after priming with IFN-{gamma} or GM-CSF, whereas IL-4, IL-10, IL-13, and TGF-ß inhibit IL-12 production [48 49 50 ]. Unlike soluble bacterial derivatives such as LPS or SEB, phagocytosis of latex beads reportedly does not induce IL-12 [51 ].

Furthermore, cross-linking surface Fc{gamma}RII/III or Mac-1 resulted in suppression of LPS-mediated IL-12 p40 mRNA induction in murine bone marrow-derived macrophages [17 ]. In the same study, decreased p40 levels were consistent with suppressed IL-12 p70 levels in Fc{gamma}R-cross-linked macrophages in response to IFN-{gamma} plus LPS stimulation. Our data show that Fc{gamma}R cross-linking substantially down-regulates IL-12-producing capacity in human MO. Decreased IL-12 production in the Fc{gamma}R-cross-linked population was prolonged and could not be overcome by IFN-{gamma} treatment of Fc{gamma}R+ MO prior to a subsequent bacterial challenge. This was unlikely to be a result of a loss of IFN-{gamma} receptors in Fc{gamma}R-cross-linked MO, based on a recent study showing no change in IFN-{gamma}R expression in blood MO after Fc{gamma}RI/II-cross-linking stimulation [52 ]. Further, down-regulation of IL-12 in the Fc{gamma}R-cross-linked MO was selective, as TNF-{alpha} levels were greater in this MO subpopulation compared with the Fc{gamma}R-noncrosslinked MO. Neutralization of TNF-{alpha} could not restore decreased IL-12 in Fc{gamma}R-cross-linked MO, suggesting that Fc{gamma}R cross-linking inhibits IL-12 independent of TNF-{alpha}.

Reduced IL-12 production after MO Fc{gamma}R cross-linking correlated with decreased antigen presentation in our experiments. Although IL-12 is pivotal in support of Th1-type T cell activation, impaired T cell stimulatory capacity of MO after Fc{gamma}R cross-linking may also involve reduced costimulatory molecule (CD80) expression and increased PGE2 [8 , 31 ]. Further, IL-10 levels were increased in the Fc{gamma}R-cross-linked MO in our experiments (data not shown), similar to that previously reported in murine macrophages [53 ]. Thus, IL-10-mediated inhibition of T cell proliferation and/or IL-12 production is a possible component of the reduced MO accessory cell function after Fc{gamma}R cross-linking stimulation.

The Fc{gamma}R-mediated signal transduction pathway leading to the decreased MO IL-12 production is yet to be defined. Our data suggest that Fc{gamma}R cross-linking is likely to interfere with signal transduction pathways involved in IL-12. One of the potential signaling pathways involved in Fc{gamma}R cross-linking-induced inhibition of IL-12 is the tyrosine phosphorylation of p91 (Stat 1) transcription factor that is involved in IFN-{gamma}-induced signaling [52 ]. However, our experiments show that Fc{gamma}RI-cross-linked MO produce decreased amounts of IL-12 even when stimulation is different from IFN-{gamma}, suggesting that the modulating effect of Fc{gamma}RI cross-linking on IL-12 production is not limited to induction by IFN-{gamma}. Stimulation of p72syk protein tyrosine kinase and tyrosine phosphorylation of the {gamma} subunit of murine and human Fc{gamma}RI and Fc{gamma}RIII has been demonstrated during receptor-mediated phagocytosis [39 40 41 ]. Other reports demonstrated association of syk nonreceptor protein tyrosine kinase with the {gamma}-chain subunit of Fc{gamma}RI and with cross-linking Fc{gamma}RI or -II in human monocytic cell lines [54 ]. Thus, Fc{gamma}RI-associated tyrosine phosphorylation and additional downstream signaling elements may be involved in Fc{gamma}R cross-linking-induced modification of MO IL-12 production. In addition, activation of mitogen-activated protein kinase p42 has been shown to be necessary for Fc{gamma}RII and Fc{gamma}RII-cross-linking-induced TNF-{alpha} synthesis in murine bone marrow-derived macrophages [55 ]. The other potential mechanism affected by Fc{gamma}R is Ca2+ mobilization. A recent report suggested that Fc{gamma}RII-induced elevation in intracellular Ca2+ level down-regulated IL-12 production in murine bone marrow-derived macrophages [17 , 53 ]. In addition, IL-12 production can be down-regulated by an elevation in intracellular cyclic adenosine monophosphate (cAMP) levels. Nevertheless, in previous reports, elevation in intracellular cAMP was shown to augment antigen presentation capacity in the high, IL-12-producing, Fc{gamma}R-negative MO, indirectly suggesting that the cAMP pathway is unlikely to be involved [56 ].

Finally, our data show that although Fc{gamma}RI cross-linking inhibits accessory cell features (antigen presentation and IL-12 production) in MO, it does not prevent MO maturation into DC. In vitro DC maturation was induced by cytokine stimulation with IL-4 plus GM-CSF even in Fc{gamma}RI cross-linking-stimulated MO, suggesting that subsequent selective stimulation may overcome the inhibitory effects of Fc{gamma}RI cross-linking on APC functions.

In summary, these observations suggest that a population of peripheral blood MO with high-density Fc{gamma}R expression can be selectively induced via Fc{gamma}R cross-linking for distinct functional capacity including decreased IL-12 production and antigen presentation capacity. In contrast, non-Fc{gamma}R-cross-linked MO can become high IL-12-producing APC. These results imply that surface receptor expression is likely to determine the type of signals MO can receive from the microenvironment, and in turn, those signals are likely to contribute to functional disparity between blood MO subpopulations.


   ACKNOWLEDGEMENTS
 
This work was supported by NIAAA grants #AA08577 and #AA11576, and its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIAAA.

Received September 21, 2001; revised January 11, 2002; accepted June 3, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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