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RI on Macrophages and Mast Cell Lines and Demonstrate Heterogeneity Among Subcutaneous and Other Dendritic Cells 1
* Austin Research Institute, Austin and Repatriation Medical Center, Heidelberg, Victoria, Australia;
The Scripps Research Institute, La Jolla, CA 92037;
Department of Biological Sciences, University of California, Santa Barbara, CA 93106;
Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; and
¶ Institut Curie, Section de Recherche, Paris, France
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Abstract |
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RI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the FcRI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected FcRI encoded by the more common Fcgr1a and Fcgr1b alleles, and although they identified different epitopes, none inhibited the binding of IgG to FcRI. When bound to FcRI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of FcRI. Resting and IFN--induced macrophages expressed FcRI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed FcRI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of FcRI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8
, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, FcRI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI is a key receptor in the development of immune responses (1, 2, 3) and links innate and adaptive immunities via its dual roles as a low affinity receptor for the T-independent IgG3 and as a high affinity receptor for the T-dependent (Th1) IgG2a (1, 2).
In these dual roles there is a clear and elegant relationship among cytokine regulation of receptor behavior, IgG development, and immune responses (1, 2). IgG3 is the earliest IgG class produced, and its switch from IgM is driven by IFN- produced largely by NK cells (4, 5), which simultaneously also up-regulate FcRI expression. As the T-dependent response matures, the Th1 response produces affinity-matured IgG2a Abs, which are the high affinity ligand for FcRI and out-compete the low affinity, T-independent IgG3 Abs. A comparison of normal and FcRI-deficient mice has shown that the capacity of FcRI to capture, internalize, and deliver Ag in immune complexes to Ag presentation pathways is at least 10-fold greater than that of the low affinity receptors (2, 3). Moreover, the receptor is important in the regulation of Ab responses generally (2).
While the development of FcRI-deficient mice has proven to be a major advance in the analysis of this receptor, its characterization has been severely hampered by the complete lack of specific mAb. Such mAb have proven difficult to generate due to the lack of genetic polymorphism between mouse strains and the intrinsically high affinity of the receptor for mouse IgG2a or rat IgG, which confounded screening protocols. Since the alleles of more FcRI have now been identified in BALB/cJ and NOD/Lt mice (6), we have used polymorphic differences to generate alloantibodies specific for FcRI. These unique Abs were characterized to define their specificities, affinities, epitopes, and functions and the cellular distribution of the receptor, which showed it to be restricted largely to macrophages and dendritic cells (DC),3 especially a population of Langerhans cell-derived DC found uniquely in s.c. lymph nodes that are highly active in Ag processing (7).
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Materials and Methods |
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Anti-FcRI Abs were produced by immunizing NOD/Lt mice with a fusion protein consisting of the extracellular region of the FcRI from BALB/c (8) and the Fc fragment of human IgG1. Mice were immunized i.p. with 100 µg of the fusion protein on three occasions, 14 days apart, and then boosted. Three days later spleen cells were fused with NS-1 myeloma cells. Hybridomas were generated as described previously and were selected in hypoxanthine/aminopterin/thymine medium (9). Hybridoma supernatants were screened by ELISA for Abs binding to the immunizing FcRI:IgG fusion protein, but not human IgG. Selected hybridomas were cloned by limiting dilution, and ascites was produced in (BALB/c x NOD/Lt)F1 mice. Three mAb hybridomas were obtained: X54-5/7.1 (IgG1), X54-37/10.4 (IgG2b), and X54-3/4 (IgG2b). F(ab')2 of X54-5/7.1 were obtained by pepsin (Sigma-Aldrich, St. Louis, MO) digestion at an enzyme to substrate ratio of 1/500 for 3.5 h at 37°C, pH 3.5. Fab of X54-5/7.1 were obtained by papain (Sigma-Aldrich) digestion at an enzyme to substrate ratio of 1/100 for 8 h at 37°C, pH 5.5. Fab of the two IgG2b mAbs were produced using immobilized pepsin beads (Pierce, Rockford, IL) for 1.5 h at 37°C, pH 4.8. Fc fragments and undigested IgG were removed, and purified Fab and F(ab')2 were shown to be free of whole Ig and Fc by SDS-PAGE. Ab fragments were labeled with biotin using biotin-X-NHS (Calbiochem, Darmstadt, Germany) at room temperature for 1 h in the presence of 0.1 M NaHCO3 and 0.1 M NaCl. Biotinylated mAb fragments exhibit only a minimal loss of Ag-binding activity compared with whole mAb by flow cytometric analysis.
Mouse strains
The following mouse strains were used in these studies and maintained in our facilities: the in-bred mice BALB/cJ, NOD/Lt, DBA/2, C57Bl/6J, CBA/J, SJL/J, C3H/HEJ, 129/SvJ, BALB/c.scid, and NZW; FcRI-deficient (2); and Fc
RI chain deficient strains.
Maintenance of cell lines and stable transfectants
L-929 (L cells), murine myelomonocytic leukemia WEHI-3 cells, human monocytic cells (U937), mouse mastocytoma (P815), mouse embryo mast cells (10P2), and Chinese hamster ovary (CHO-K1) cells were cultured in RPMI 1640 (CSL, Melbourne, Australia) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (CSL), and 0.05 mM 2-ME (Koch-Light, Suffolk, U.K.) at 37°C in 10% CO2. CHO cells transfected with human FcRI (Tf2-13), BALB/c-FcRI (1N3-2), and NOD-FcRI (2B5-13C) DNA were maintained in the same medium, with the addition of 0.6 mg/ml G418 (Life Technologies, Paisley, Scotland). COS-7 cells used for the transient expression of different mutants, allelic variants, and chimeric forms of FcRI were grown in DMEM (CSL Ltd.) supplemented as described above. IIa1.6 cells (B cell line lacking surface FcRs) was stably transfected with FcRI and FcR-associated -chain cDNA, and cells containing the receptor and -chain or expression vector were cultured in the presence of 10 µg/ml puromycin (Sigma-Aldrich) and 0.7 mg/ml geneticin (Life Technologies). FcRI+ cells were sorted several times using MoFlo (Cytomation, Fort Collins, CO), and -chain expression was detected on Western blot using an anti--chain rabbit polyclonal Ab.
Transient expression of chimeric FcR
Chimeric receptors composed of different combinations of domains derived from mouse FcRI and FcRII had been previously described (10). The cDNA encoding these proteins was transiently transfected into COS-7 cells growing at 
50–70% confluence. Cells were incubated with a transfection mix containing 1 µg/ml cDNA, 0.2 mg/ml DEAE-dextran (Pharmacia Biotech, Uppsala, Sweden), and 0.1 mM chloroquine (Sigma-Aldrich) in DMEM containing 10% Nu-Serum (Flow Laboratories, Sydney, Australia) for 3.5 h at 37°C. The DNA mix was then removed, and the cells were exposed to 10% DMSO in PBS for 1 min and washed. Cells were cultured in fully supplemented DMEM for 48–72 h and then analyzed by flow cytometry or immunoprecipitation. Negative controls included mock-transfected cells.
Cell isolation and culture
Macrophages.
Bone marrow-derived macrophages (BMM) were obtained by culturing femoral bone marrow cells from various mouse strains for 4 days in RPMI supplemented with 30% L cell-conditioned medium. Nonadherent cells were cultured in the same medium for an additional 3 days, during which macrophages would adhere to the culture dish. Peritoneal exudate cells were obtained from mice injected i.p. with 0.5 ml of thioglycolate and were cultured overnight in the same medium as BMM. The identity of macrophages was confirmed by the expression of F4/80. For stimulation of macrophages, recombinant mouse IFN- (Genzyme, Cambridge, MA) was added at 300 U/ml for 24 or 48 h before flow cytometric analysis.
Neutrophils. Exudate neutrophils were isolated from mice injected i.p. with 1 ml of 0.4% sodium caseinate 20 and 3–4 h before sacrifice. Cells were obtained by flushing the peritoneal cavity with 10 ml of PBS. Bone marrow cells were obtained from the femur of C57BL/6J mice. Blood neutrophils were isolated from mice and were mixed with PBS/Percoll (10/1 ratio) before centrifugation. The buffy coat was harvested, and the cells were treated with 0.83% NH4Cl for 5 min at 37°C to remove RBC before flow cytometric analysis. Neutrophils were identified in flow cytometry by the expression of Ly6B.2.
Bone marrow-derived mast cells (BMMC).
BMMC were obtained as previously described (11). Briefly, 3 x 105 cell/ml femoral bone marrow cells from wild-type or FcRI-deficient mice were cultured in supplemented RPMI or StemPro-34 (Life Technologies) with 40% WEHI-3B cell-conditioned medium containing IL-3 (W3CM-IL3) in the absence or the presence of recombinant mouse stem cell factor (SCF). The medium was replaced every 5–7 days. After 3 wk, >95% of the cells grown in W3CM-IL3 alone were BMMC, and cells in W3CM-IL3 and SCF were connective tissue-type mast cells, as determined by Alcian Blue-Safranin staining (12). Growth factor cytokines were used at the following concentrations as recommended: 100 or 500 U/ml IFN- (Genzyme), 10 ng/ml IL-4, 10 ng/ml IL-6, and 20 ng/ml IL-10 (PeproTech, Rocky Hill, NJ). Cells were harvested 3 days later for flow cytometric analysis.
Bone marrow-derived DC (BM-DC).
BM-DC were prepared as follows. Bone marrow cells from C57BL/6, FcRI-deficient mice or FcRIII-deficient mice were cultured for 2 wk in IMDM (Sigma-Aldrich) containing 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 50 mM 2-ME, and 30% conditioned medium from GM-CSF-producing J558 cells. Cells were MHC class II+, CD86/B7-2+, CD40+, and Gr1- in flow cytometric analysis.
Lymphoid organ-derived DC.
DC from thymus, spleen, and lymph nodes were isolated from wild-type and FcRI-deficient mice as previously described (13). Briefly, organs were digested at room temperature with collagenase-DNase for 20 min and then treated with EDTA to disrupt T cell-DC complexes. All subsequent steps were performed at 0–4°C in a divalent-metal free medium. Light density cells were selected by centrifugation in Nycodenz medium (Nycomed Pharma, Oslo, Norway). Non-DC were then depleted by coating them with titrated levels of anti-CD3 (KT3-1), anti-CD4 (GK1.5), anti-Thy1 (T24/31.7), anti-B220 (RA36B2), anti-Gr-1 (RB68C5), and anti-erythrocyte (TER119) and then removing the Ab-coated cells with anti-rat Ig-coupled magnetic beads (Dynabeads; Dynal, Oslo, Norway). To identify DC, anti-class II MHC (N22) and anti-CD11c (N418) were used. Anti-CD8 (YTS169.4) was used to delineate DC subsets, and analysis was performed on a FACStar Plus instrument (BD Biosciences, San Jose, CA) using anti-DEC205 (NLDC145), anti-CD40 (FGK45.5), anti-CD86 (GL1), and anti-FcRI (X54-37/10.4, X54-5/7.1, or X54-3/4). Blocking of nonspecific staining was achieved by incubation with purified mouse Ig before staining. Mouse IgG1 (OKT6) and IgG2b (OKT4) isotype-matched controls were used to verify staining with the anti-FcRI mAb.
Emigrant DC. Ears were removed from mice and briefly washed in 70% ethanol. The ears were split, removing the dorsal skin from the cartilage. The dorsal skin was placed split side down in 1 ml of modified mouse osmolarity RPMI 1640 containing 10% FCS for 2–4 h at 37°C in a humidified 10% CO2-in-air incubator, to eliminate non-DC initially released. The skin was then transferred into another 1 ml of medium and cultured for 24 h. Cells migrating into the culture medium were harvested and kept in cold medium. The skin was transferred to fresh medium, and the incubation was repeated. The cells migrating from the skin over the first and second 24-h incubations were then pooled. Skin emigrant DC were identified using anti-class II MHC and were analyzed as described above for the DC isolated from thymus, spleen, and lymph node (7).
Flow cytometry
Cells (1 x 106 cells in 25 µl of PBS/0.5% BSA or Leibovitz’s L-15 (Life Technologies)/0.5% BSA) were incubated with saturating amounts of mAb (25 µl of hybridoma supernatant, 1 µg of purified whole Ig, or 5 µg of Fab/F(ab')2) for 30 min on ice and washed. Anti-mouse IgG F(ab')2-FITC (Silenus, Melbourne, Australia) or streptavidin-PE (BD PharMingen, San Diego, CA) was then added for an additional 30 min on ice. For experiments using intact IgG, cells were preincubated with 25 µl of 100 mg/ml human Ig (Sandoglobulin; Sandoz, Sydney, Australia) for 30 min on ice to block Fc binding. Viable cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Abs to cell surface markers such as F4/80 (macrophage cell marker; Serotec, Oxford, U.K.), CD45, either Ly 5.2 or B220, CD4 (BD PharMingen), CD8 (BD PharMingen), and Ly 6B.2 (PMN marker) were used. Cells obtained directly from mice were incubated for 5 min at 37°C with 0.83% NH4Cl to lyse RBC before the addition of mAb.
Immunoprecipitation and SDS-PAGE analysis
Iodination of 1–4 x 107 cells was performed using lactoperoxidase (Sigma-Aldrich) and hydrogen peroxide for 4 min, before lysis in 1 ml of Nonidet P-40 lysis buffer (0.01 M Tris-HCl, 0.15 M NaCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 1 mM PMSF, and 0.5% BSA) for 10 min on ice. Insoluble proteins and nuclear debris were removed, and cell lysates were precleared with 50 µl of packed Sepharose 4B beads coupled to protein A (Pharmacia Biotech) for 30 min at 4°C. Saturating amounts of mAb (200 µl of tissue culture supernatant or 1 µg of purified mAb) were added for 1 h at 4°C. Immunoprecipitations were conducted with 25 µl of 50% GammaBind G Sepharose (Pharmacia Biotech) for an additional 30 min at 4°C. Samples were washed in lysis buffer, placed in 20 µl of SDS sample buffer (0.1 M Tris-HCl (pH 6.8), 4% SDS, 0.2% bromophenol blue, and 20% glycerol), and analyzed by SDS-PAGE and autoradiography.
Erythrocyte Ab (EA) rosetting
Sheep erythrocytes were sensitized with rabbit anti-sheep erythrocyte IgG for 30 min at room temperature, followed by incubation for 5 min at 37°C. These EA complexes were added to monolayers of transfected COS-7 cells in six-well plates and incubated for 5 min at 37°C. The plates were then centrifuged briefly and placed on ice for 30 min. Unbound EA complexes were removed by washing with PBS containing 0.5% BSA (CSL). Rosette formation was detected by light microscopy.
Scatchard analysis
Scatchard analysis of mAb binding was performed as previously described (14). Briefly, 20 µg of whole mAb was radiolabeled with 125I (Amersham, Little Chalfont, U.K.) using 5 µg of Iodogen (Pierce). Cells (2 x106/ml of CHO cells) expressing BALB/c-FcRI were added to radiolabeled mAb in PBS/0.5% BSA and incubated for 2 h on ice. Bound mAb was separated from unbound mAb by centrifugation through 200 µl of phthalate oil (3/2, v/v, dibutyl-phthalate/diocyl phthalate; Fluka Chemika, Buchs, Switzerland), and samples were analyzed on a Wallac 1470 Wizard automatic gamma counter (Wallac, Turku, Finland). Data were normalized against an isotype control. The Kd values were determined by Scatchard plot analysis, using CurveFit (Kevin Raner software; CSIRO, Melbourne, Australia).
Calcium mobilization
FcRI+ IIa1.6 cells with or without FcR-associated -chain were used to determine the function of the mAb in triggering biochemical signal. Briefly, 1 x 107 cells were loaded with 4 µM Fura-2/AM (Calbiochem) and 0.05% pluronic acid (Molecular Probes, Eugene, OR) in the presence of 1 mM Ca2+, 138 mM NaCl, 6 mM KCl, 1 mM MgSO4·7H2O, 1 mM Na2HPO4·2H2O, 5 mM NaHCO3, 6.5 mM glucose, 20 mM HEPES, and 0.1% (w/v) BSA, pH 7.4, for 30 min at 37°C. Unlabeled Fura-2/AM was removed, and cells were resuspended in 250 µM sulfinylpyrazole (Sigma-Aldrich), 100 µM Ca2+, 138 mM NaCl, 6 mM KCl, 1 mM MgCl2·6H2O, 6.5 mM glucose, and 20 mM HEPES, pH 7.4. Detection of calcium released was performed using a Hitachi model F-2000 fluorescence spectrometer at excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm at 37°C for 5 min. Each reaction used 1 x 106 cells, and 25 µg/ml of F(ab')2 anti-mIgG, biotinylated F(ab')2 mAb X54-5/7.1, or streptavidin was added at different times. Baseline calcium release was established using cells stimulated with streptavidin alone or PBS.
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Results |
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RI
Fusion protein consisting of the three extracellular domains of BALB/c-FcRI, and the Fc portion of human IgG1 was injected to NOD/Lt mice for the generation of allele-specific FcRI mAb. Three hybridomas testing positive for the fusion protein but negative for human IgG1 were obtained, cloned, and designated X54-5/7.1 (IgG1), X54-37/10.4 (IgG2b), and X54-3/4 (IgG2b); their properties are listed in Table I.
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Several approaches were undertaken to establish the specificity of the mAb. First, the mAb were tested by flow cytometry using untransfected CHO-K1 cells or CHO-K1 cells expressing BALB/c-FcRI (Fig. 1). Specific binding of Fab or F(ab')2 was also observed for all three mAb only on BALB/c-FcRI+ CHO cells and not on untransfected CHO-K1 cells (data not shown). Equivalent binding to FcRI+ CHO cells was seen with the ligand IgG2a, but isotype control IgG1 and IgG2b did not bind to either cell type (data not shown). None of the mAb bound to NOD-FcRI, mouse FcRII, or human FcRI expressing CHO-K1 cells (data not shown), indicating that these mAb are specific for BALB/c-FcRI. Second, the binding to BMM was determined by flow cytometry (Fig. 1). All three mAb bound significantly to BMM isolated from wild-type mice, but not that from from FcRI-deficient mice. Third, mAb X54-5/7.1 and X54-3/4 immunoprecipitated a 72-kDa moiety from BALB/c-FcRI+ CHO cells (Fig. 2A) or wild-type BMM (Fig. 2B), but not from FcRI-deficient BMM. This 72-kDa protein moiety corresponds to the size of mouse FcRI, obtained using IgG2a, but not with PBS or isotype control IgG1 and IgG2b. It should be noted that mAb X54-3/4 could also detect unreduced FcRI in Western blots; reduced FcRI was not detectable (Table I).
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RI+CHO cells was determined by Scatchard analysis (Table I). X54-5/7.1 and X54-3/4 exhibit similar affinities for FcRI (Kd = 1.5 x 10-8 and 1.4 x 10-8 M, respectively), and X54-37/10.4 has a lower affinity (Kd = 5.6 x 10-8 M).
Binding of mAbs to mouse FcRI is not inhibited by human IgG
To determine whether the binding epitopes of the mAb generated overlapped with the ligand binding site, mAb binding to BALB/c-FcRI+ CHO cells was performed in the presence of up to 50 mg/ml IgG (Table I). All three mAb bound to the cells, indicating that the binding epitopes of these mAb are not on or in close proximity to the IgG binding site, in contrast to the binding of mIgG2a, which was inhibited in the presence of IgG as expected.
Epitope mapping using chimeric FcRs
The epitopes detected by the three mAb were defined using COS-7 cells expressing chimeric FcRs containing different domains of mouse FcRI and FcRII (10) (Fig. 3). The expression of these receptors was first confirmed by the binding of IgG-EA. Direct binding studies showed that all three mAb detected distinct epitopes. X54-5/7.1 bound only to receptors containing both domains 2 and 3 of FcRI, i.e., FcRI and FcRII-I-I, but not FcRII-II-I containing only domain 3 of FcRI nor FcRI-I containing both domains 1 and 2, but not domain 3 (Fig. 3). This indicates a requirement for both domains 2 and 3 to form the epitope. Abs X54-37/10.4 and X54-3/4 bound only to FcRI, but not to any other chimeric receptor (Fig. 3). Clearly the epitopes for these two mAb are dependent on the integrity of the entire extracellular region of FcRI. However, the epitopes can be distinguished, as X54-3/4 detects the nonreduced form of FcRI in Western blots, but X54-37/10.4 does not (Table I).
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The capacity of these anti-receptor mAb to induce biochemical signals was analyzed by calcium mobilization in cells transfected with the FcRI alone or in conjunction with the FcR -chain (Fig. 4). Cells cotransfected with FcRI and FcR -chain were treated with biotin-conjugated F(ab')2 of X54-5/7.1, and no calcium mobilization was seen. Subsequent cross-linking with streptavidin induced a rapid calcium mobilization as expected. There was no response of cells treated with streptavidin alone. Calcium mobilization was also shown when bound X54-37/10.4 and X54-3/4 were cross-linked (data not shown), suggesting an activation role of these mAb on receptor. The response was similar to that induced by aggregation of the Ag receptor on these cells. Moreover, the mAb was unable to induce calcium mobilization on cells expressing only FcRI in the absence of the -chain, which was expected (Fig. 4B). FACS analysis showed that the receptor expression, with or without -chain, was equivalent on these cells, indicating that the difference in calcium mobilization was due to the presence or absence of the -chain.
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The mouse strain distribution pattern of the mAb was determined. We have previously identified seven alleles of the mouse Fcgr1 gene (6). Flow cytometry using Fab or F(ab')2 of the mAb showed binding to peritoneal exudate cells from mice carrying the allele Fcgr1a (C57BL/6, BALB/c, DBA/2) or Fcgr1b allele (C3H/HeJ, CBA/J, NZW, SJL/J, 129/SvJ), but, as expected, not the Fcgr1d allele (NOD/Lt; data not shown), demonstrating that these mAbs recognized the more common FcRI alleles.
Expression of FcRI on lymphoid cells from spleen, thymus, lymph node, peritoneal exudate macrophages, and neutrophils
To further define the cellular distribution of FcRI, the binding of X54-5/7.1 to cells from spleen, thymus, and lymph nodes obtained from wild-type or FcRI-deficient mice was examined by flow cytometry. No reactivity was detected on cells from the wild-type lymphoid tissues or on isolated CD4+, CD8+, or B220+ cells (Fig. 5). The binding of X54-5/7.1 to peritoneal exudate macrophages from wild-type mice was comparable to that seen with BMM, confirming the high surface expression of FcRI on macrophages (Fig. 1). Neutrophils were also isolated from the peritoneal cavity, peripheral blood, and bone marrow of wild-type and FcRI-deficient mice, and the expression of FcRI was examined. Insignificant binding of X54-5/7.1 was detected in neutrophils from wild-type mice, suggesting that FcRI is either absent or at very low levels on these cells (Fig. 5).
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RI on mastocytoma cell lines, but not on primary mast cell cultures
Two mast cell lines, P815 and 10P2, were found to express significant levels of mouse FcRI (Fig. 6A). The receptor was functional, as demonstrated by the ability to bind mouse IgG2a ligand (data not shown). However, this mAb reactivity to mast cell lines was not found in BMMC (c-Kit+, FcRI+, Alcian-Blue+, Safranin-) cultured in IL-3 alone (Fig. 6B), IL-3 with SCF, and IL-3 with SCF, IL-4, and IL-10 (data not shown). Metcalfe et al. (15) has described the expression of a functional FcRI on IFN--treated human mast cells cultured in the presence of SCF, IL-3, and IL-6. However, the expression of FcRI was not detected when these conditions were applied to mouse mast cells (data not shown).
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RI expression on BM-DC
BM-DC obtained from normal, FcRI-deficient, and FcRIII-deficient bone marrow cells were grown for 2 wk in the presence of GM-CSF. These cells all expressed MHC class II, CD86/B7-2, and CD40, but not Gr1 (a granulocyte surface marker; data not shown). The expression of FcRI was detected on normal and FcRIII-deficient BM-DC with X54-5/7.1, but not in FcRI-deficient BM-DC (Fig. 7). All BM-DC, including those from FcRI-deficient mice, showed similar levels of the low affinity FcR, which were detected by the FcRII/FcRIII-specific mAb 2.4G2, indicating the specific reactivity of X54-5/7.1 for FcRI on BM-DC.
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RI expression on freshly isolated lymphoid DC
DC from thymus, spleen, and lymph nodes and those migrating from the skin were isolated from normal or FcRI-deficient mice. The expression of FcRI on these DC was analyzed by flow cytometry using mAb X54-37/10.4, X54-5/7.1, X54-3/4, or their Fab. No binding of the mAb was observed in DC from FcRI-deficient mice. DC from normal thymus (data not shown) showed relatively low levels of binding of the mAb. A higher level of binding was apparent in spleen, lymph node, and skin-emigrant DCs (Fig. 8A). The highest binding was seen in a population of lymph node DC, which upon closer investigation was found to be comparable to a previously described population of MHC class IIhigh, DEC205+, CD40+, CD86+ cells unique to the cutaneous draining lymph node and largely absent from the mesenteric lymph node (7). Gating on MHC class II and CD8 for the lymph node population revealed that the FcRIhigh cells also expressed very high levels of MHC class II, but low to very low levels of CD8 (Fig. 8B). The FcRIhigh population also expressed high levels of DEC205, CD40, and CD86 (data not shown), indicating that these were highly active, mature DC, correlating to the DC derived from Langerhans cells in the skin and with the greatest Ag processing potential (7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, and three such Abs were produced. Despite the existence of mAb technology for >25 years, the production of this particular Ab has proved difficult. Following a comprehensive analysis of genetic polymorphisms of FcRI in mouse strains, we exploited these differences (6) and immunized NOD/Lt mice, which expressed the Fcgr1d allele with a fusion protein of the extracellular domains of FcRI isolated from BALB/c mice, which expressed the Fcgr1a allele. As a consequence, three Abs (X54-5/7.1 (IgG1), X54-37/10.4 (IgG2b), and X54-3/4 (IgG2b)) were produced. Their specificity was confirmed by 1) binding to cells transfected with FcRI of BALB/c, but not NOD; 2) binding to macrophages from normal, but not FcRI-deficient, mice; and 3) immunoprecipitation of a 72-kDa protein, which is consistent with the expected molecular size of this receptor detected by ligand. When bound to FcRI, these mAb has the capacity to induce receptor-specific biological functions, which was shown in the release of calcium upon cross-linking. Such characteristics of the mAb allows the surrogate use of these mAb in eliciting cellular responses seen only when receptor was cross-linked by complexed ligand.
Epitope mapping and competitive binding studies show that the Abs detected three distinct conformation-dependent epitopes distinct from the IgG binding site. X54-5/7.1 recognized a conformational determinant formed between domains 2 and 3, while X54-37/10.4 and X54-3/4 required all three domains for detection. Interestingly, X54-3/4 was the only mAb able to detect FcRI in Western blots, but only in a nonreduced state, indicating that despite the dependence of the epitope on the presence of all three domains, this Ab was still able to recognize partially denatured receptor. Scatchard analysis showed that X54-5/7.1 and X54-3/4 have similar affinities for FcRI (108 M-1), while X54-37/10.4 had a slightly lower affinity (5 x 108 M-1).
The mouse strain distribution analysis showed that all three mAb detected the FcRI encoded by the Fcgr1a and Fcgr1b alleles, but, as expected, not the Fcgr1d allele of the NOD mouse (6). The Fcgr1a and Fcgr1b alleles differ from each other by five nucleotides, which encode three amino acid differences in the extracellular domains. These alleles, in turn, differ from the Fcgr1d allele, which encodes 20 amino acid changes present in the receptor protein, of which 17 occur in the extracellular domains (16). The production of Fcgr1a allele-specific mAb in NOD mice implies the fundamental differences in the structure of the two allelic receptors, whose phenotype in ligand binding affinity and specificity are distinct as well. The major difference is the insertion of four contiguous amino acids in the junction between domains 2 and 3 of the NOD receptor, which has profound effects on receptor specificity and affinity (17). The addition of these four NOD-derived amino acids to Fcgr1a-encoded receptor (BALB/c) abolished the epitope recognized by X54-5/7.1, and the removal of these sequences from NOD FcRI allowed the binding of X54-5/7.1 (data not shown). While this result suggests that the epitope may be located in the D2/D3 junction, this appears unlikely, as a 20-mer synthetic peptide from this region was not bound by the mAb (data not shown).
Recent findings in FcRI-deficient mice showed that FcRI has a major role in Ag presentation and presumably in the development of immune responses (2), but at what level is not known. We undertook an analysis of leukocyte classes to define the cell types that express FcRI, and, as expected, FcRI was absent from purified T and B lymphocytes. However, we then analyzed other leukocyte groups, and there were a number of surprising findings.
Of particular interest was the observation that FcRI expression in the mouse was largely restricted to cell types known to be involved in Ag presentation and in greatest quantity on cells with high processing capacity. Macrophages, either exudate or bone marrow-cultured cells, expressed the receptor, which could be up-regulated by IFN- (2). The expression of FcRI was also detected on DC in spleen, lymph nodes, and skin emigrant, with the highest high level found on Langerhans cell-related DC that are unique in lymph nodes draining the skin. This subpopulation is absent in mesenteric lymph nodes and thymus and has a high capacity to process Ag (7). These cells are larger in size and expressed low levels of CD8 as well as high levels of DEC205, CD40, and CD86, distinguishing them from the mesenteric and splenic DC. Importantly, the very high levels of MHC class II and langerin suggest a high capacity for Ag presentation and processing. It should be noted that additional subsets of DCs also expressed FcRI, but at a lower level, including the Langerhans-derived DCs in the skin, which have not yet reached full maturation, but have a high migratory potential, or fully matured DCs, which are typically characterized by a diminished capacity for Ag processing, but have become professional presenting cells and are found in lymphoid tissues for the priming of T cells. This is also consistent with the analysis of BM-DCs cultured in the presence of GM-CSF that also showed the expression of FcRI. Cells cultured under such conditions are again somewhat immature, but still have a high capacity to process Ag with a lower capacity to present than their matured counterparts. The correlation between high FcRI expression and a capacity to process and present Ag was interesting and consistent with its known roles in immunity. FcRI, as the high affinity receptor, binds IgG at lower concentrations than the low affinity receptors and has the capacity to capture immune complexes and the Ag therein earlier in immune responses. In this context, Langerhans-derived DCs or other less mature DCs would be able to take up Ag quickly and before migration to lymphoid organs. Reports of immune complex-induced DC maturation and MHC class I- and II-restricted presentation of peptides from IgG-complexed Ags also imply a role of the high affinity IgG receptor in Ag presentation and subsequent priming of T cells in the mouse (18, 19).
Besides APC, established mastocytoma P815 and mouse embryonic mast cell line 10P2 also expressed functional FcRI. However, primary cultured mast cells derived from bone marrow of the mucosal type (expanded with IL-3 alone) or the serosal type (IL-3 plus SCF) did not express FcRI. Moreover, addition of IFN-, IL-4, IL-6, or IL-10 did not stimulate the expression of FcRI on these cells, which contrasts with the recent report by Okayama et al. (15) demonstrating the expression of FcRI on a subset of human mast cells. These human CD34+ peripheral blood stem cells were cultured in the presence of SCF, IL-6, and IL-3 (15) and, after stimulation with IFN-, expressed functional FcRI. There are two possible explanations for the apparent contradiction between the expression of FcRI on established immortalized cell lines and the lack of expression on primary cultured mast cells. Firstly, human CD34+ peripheral blood stem cell-derived FcRI+ mast cells may have an equivalent in the mouse that has not yet been identified. Alternatively, these transformed cell lines may have acquired expression of the receptor as a consequence of their transformation. If the later is true, the absence of FcRI on mouse mast cells may simply reflect one of the fundamental differences in the roles of FcRI in human and mice, and indeed, we have already shown at least one difference between the cellular distributions of human and mouse FcRI. We were not able to show the expression of FcRI on mouse neutrophils, which coincides with another report demonstrating that both resting and G-CSF-stimulated mouse neutrophils do not express FcRI (3). The expression of FcRI on human neutrophils, on the other hand, can be induced by IFN- and has been shown to participate in Ab-dependent cellular cytotoxicity (20). The lack of FcRI on mouse mast cells is in contrast to the human FcRI on CD34+ stem cell-derived mast cells, which upon aggregation of the receptor caused degranulation and cytokine release. With the expression of mouse FcRI only limited to macrophages and DCs (APC), the main role of FcRI in mouse is suggested to be in Ag internalization and delivery to processing pathways, linking low quantities of immune complexes to the production of highly specific Abs (21) and subsequent T cell effector functions.
It is interesting that FcRI expression appears to be intimately tied into the development of immune responses. The evidence is accumulating that this is the case. First, the inactivation of FcRI in vivo profoundly effects the development of Ab responses and renders APC less able to capture Ag for subsequent representation. Second, frontline APC, i.e., skin-emigrant DC express high levels of FcRI in conjunction with a high capacity for Ag processing. Third, during the development of immune responses, T-independent Ab production of IgG3 in the mouse is IFN- driven as is the FcRI up-regulation. The subsequent development of the adaptive immune response involving more complex cell:cell interactions and cytokine production produces affinity-matured Abs of the IgG2a class, which is the high affinity ligand of FcRI. Such Abs would inevitably compete with and displace the earlier produced T-independent IgG3. These observations taken together with the detection of FcRI expression on APC indicate that the high affinity IgG FcRI has a key and central role in the development of immunity.
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2 Address correspondence and reprint requests to Prof. P. Mark Hogarth, Austin Research Institute, Austin and Repatriation Medical Center, Studley Road, Heidelberg, Victoria 3084, Australia. E-mail address: pm.hogarth{at}ari.unimelb.edu.au
3 Abbreviations used in this paper: DC, dendritic cell; BM-DC, bone marrow-derived DC; BMM, bone marrow-derived macrophage; BMMC, bone marrow-derived mast cell; EA, erythrocyte Ab; SCF, stem cell factor.
Received for publication October 1, 2002. Accepted for publication December 31, 2002.
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RI-deficient mice show multiple alterations to inflammatory and immune responses. Immunity 16:379.[Medline]
RI (CD64.) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection. Immunity 16:391.[Medline]
: a model for T cell-independent class switching in response to T cell-independent type 2 antigens. J. Exp. Med. 175:1367.RI: identification and functional characterization of five new alleles. Immunogenetics 51:206.[Medline]
RI, on human mast cells: up-regulation by IFN-. J. Immunol. 164:4332.RI of NOD mice: implications for the evolution of the Ig superfamily. EMBO J. 17:3850.[Medline]
receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371. receptors and cross-presentation in dendritic cells. J. Exp. Med. 195:F1.
RI/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. Invest. 97:331.[Medline]This article has been cited by other articles:
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|
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|
|
|
|
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) but not on isotype control IgG1 (
). B, BMMC from normal (solid line) or Fc
