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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by van Egmond, M. Articles by van de Winkel, J. G.J. Search for Related Content PubMed PubMed Citation Articles by van Egmond, M. Articles by van de Winkel, J. G.J. Related Collections Phagocytes Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 93 No. 12 (June 15), 1999: pp. 4387-4394 Human Immunoglobulin A Receptor (FcRI, CD89) Function in Transgenic Mice Requires Both FcR  Chain and CR3 (CD11b/CD18) By Marjolein van Egmond, A.J. Hanneke van Vuuren, H. Craig Morton, Annemiek B. van Spriel, Li Shen, Frans M.A. Hofhuis, Takashi Saito, Tanya N. Mayadas, J. Sjef Verbeek, and Jan G.J. van de Winkel From the Department of Immunology and Medarex Europe, University Medical Center Utrecht, Utrecht, The Netherlands; the Department of Immunohistochemistry and Immunopathology, University of Oslo, Oslo, Norway; the Department of Immunology and Microbiology, Dartmouth Medical School, Lebanon, NH; the Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba, Japan; and the Department of Pathology, Brigham and Women's Hospital, Boston, MA.     ABSTRACT TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR  chain or the 2 integrin CR3 (CD11b/CD18) in FcRI biological function, FcRI transgenic mice were crossed with either FcR  chain / or CR3 / mice. In contrast to in vitro data, FcR  chain was essential for surface expression of hFcRI in vivo. Functional studies in hFcRI/ /mice were, therefore, limited. In vitro studies showed FcR  chain to be necessary for phagocytosis. Neither hFcRI expression nor phagocytosis, triggered via hFcRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcRI transgenic/ CR3/ mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcRI-mediated antibody-dependent cellular cytotoxicity (ADCC). © 1999 by The American Society of Hematology.     INTRODUCTION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES RECEPTORS FOR THE Fc part of immunoglobulins (FcR) play a crucial coordinating role in host defense. Leukocyte receptors for immunoglobulin (Ig) G, E, and A classes have been characterized, and each bear unique ligand-binding -chains. Both receptors for IgG (FcR) and IgE (FcR) have been extensively studied in vitro and in vivo.1,2 In contrast, knowledge about IgA receptors (FcR) is limited and no information is available about their role in vivo. In the early nineties, a human myeloid receptor for IgA (FcRI, CD89) was isolated and biochemically and genetically characterized.3-5 Human FcRI (hFcRI) is constitutively expressed as a 55- to 75-kD protein on neutrophils and monocytes/macrophages or as a 70- to 100-kD glycoprotein on eosinophils due to increased glycosylation. It has a medium affinity (Ka 5 × 107 mol/L1) for IgA1, IgA2, and secretory IgA, and receptor expression is regulated by cytokines.6 In vitro studies documented the capacity of hFcRI to trigger release of inflammatory mediators and phagocytosis of IgA-coated particles.7,8 Moreover, tumor cells are effectively lysed using IgA antitumor antibodies or bispecific antibodies (BsAb) directed to hFcRI and tumor antigens.9 Recent work identified hFcRI as a promising target for immunotherapy of malignant and infectious diseases.10,11 At present, neither a FcRI equivalent is known in mice, nor is an appropriate experimental model available. We, therefore, generated a novel transgenic mouse model expressing human FcRI using its own promoter and regulatory elements. To study involvement of (co-) signaling molecules in hFcRI function, transgenic mice were crossed with mice lacking such units, eg, FcR  chain- or CR3-deficient mice. It has been documented that, like most other leukocyte Fc receptors, hFcRI complexes with the FcR  chain signaling molecule.12 Both proximal (eg, calcium release) and distal (eg, cytokine production) signaling events initiated via hFcRI were shown dependent on association with the FcR  chain.13 The integrity of the ITAM signaling motif within the FcR  chain was essential for hFcRI signaling ability.14 Work from several laboratories indicated CR3 (CD11b/CD18) to be involved in FcR function (see Brown15). Antibodies directed to CR3 were able to inhibit IgG-mediated phagocytosis by monocytes.16 In addition, CD11/CD18 devoid polymorphonuclear leukocytes (PMN) from patients with leukocyte adhesion deficiency failed to amplify phagocytosis of IgG-opsonized particles on inflammatory stimuli.17 In the present study, a FcRI transgenic mouse is described in which expression, regulation, and function of the receptor mimics the situation in man. Crosses of hFcRI transgenic mice with mice deficient for (co)-signaling molecules and in vitro studies in transfectant models showed that FcR  chain was important for expression and phagocytosis, whereas CR3 was selectively involved in FcRI-mediated antibody-dependent cellular cytotoxicity (ADCC).     MATERIALS AND METHODS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Antibodies and flow cytometry.   Surface expression of hFcRI or CR3 was determined using fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of antihuman FcRI monoclonal antibody (MoAb) (A77-FITC) (Medarex, Annandale, NY) or MoAb M1/70-FITC (Boehringer, Mannheim, Germany), respectively. Neutrophils were defined with Gr-1 (PharMingen, San Diego, CA) and monocytes/macrophages with F4/80 (Serotec, Oxford, UK) and on the basis of light scatter characteristics. CD45R/B220 and anti-T-cell receptor (TCR) served to distinguish lymphocytes. Biotin-conjugated MoAb were detected with phycoerythrin (PE)-labeled Streptavidin (Becton Dickinson, San Jose, CA). Whole blood of mice was incubated with MoAb (10 µg/mL) for 15 minutes at room temperature (RT) and then subjected to FACS Lysing Solution (Becton Dickinson). Peritoneal cells (2 × 105), either freshly isolated or cultured, were incubated with MoAb for 30 minutes at 4°C. To examine IgA binding, whole blood of mice was incubated with human serum IgA (Cappel, Aurora, OH; 250 µg/mL) for 1 hour at 4°C. After washing, cells were incubated with PE-labeled F(ab')2 fragments of GHIgA antibody (Southern Biotechnology, Birmingham, AL). Cells were analyzed on a FACScan (Becton Dickinson). Rabbit anti-Candida albicans (C. albicans) IgG was obtained from Biodesign (Kennebunk, MA). BsAb A77xCan, BsAb A77x520C9,10 and A77x ox erythrocyte (A77xOE) were prepared as described.18 The anti-HER-2/neu MoAb 520C9 (Medarex) and TA-1 (Calbiochem, La Jolla, CA) recognize different epitopes on HER-2/neu, a proto-oncogene product overexpressed on human carcinoma cells. Transgenic mice.   A cosmid clone (R31931, 41 kb) of chromosome 19 carrying the 12-kb human FcRI gene was used to generate transgenic FVB/N mice. The cosmid clone was kindly provided by Dr L.K. Ashworth (Human Genome Center, Livermore, CA).19 DNA was linearized, isolated by electroelution, and microinjected into fertilized oocytes. Two different transgenic founders were mated with FVB/N mice. Heterozygous transgenic offspring were identified by analyzing peripheral blood neutrophils for hFcRI expression using anti-hFcRI MoAb A77. Southern blots.   Southern blots were performed as described.20 Samples with different amounts of DNA were digested with EcoRI, electrophoresed through 0.8% agarose gels, and blotted onto Qiabrane nylon plus filters. Blot hybridization was performed using a random prime 32P-labeled 0.9 kb human FcRI coding region probe.4 Copy numbers of the transgenes were determined by quantitating intensity of bands using ImageQuant PhosphorImager software (Molecular Dynamics, Inc, Sunnyvale, CA). Human genomic DNA digested with EcoRI served as a reference. Cell culture.   The breast carcinoma cell line SK-BR-3, overexpressing HER-2/neu was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI 1640 medium (GIBCO BRL, Grand Island, NY), supplemented with 10% fetal calf serum (FCS) and antibiotics, and harvested using trypsin-EDTA (Life Technologies, Paisley, UK). The murine IIA1.6 cell line was transfected with the pCAV vector containing human FcRI cDNA (generously provided by Dr C. Maliszewski, Immunex, Seattle, WA) and pNUT vector containing either wild-type murine FcR  chain or mutated Y65F-Y76F cDNA.14 Cells were cultured in RPMI, 10% FCS supplemented with 5 mmol/L methotrexate to allow selection for hFcRI/ or hFcRI/Y65F-Y76F positive cells. Mouse bone marrow cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L glucose, 10% FCS, and antibiotics, with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL) or tumor necrosis factor- (TNF-) (50 ng/mL). After 24 hours, nonadherent cells were harvested and stained with A77-FITC and Gr-1-PE. Adherent cells were harvested after 8 days and stained with F4/80 and A77-FITC. Peritoneal macrophages were cultured for 24 hours with 50 ng/mL GM-CSF/TNF- to induce hFcRI before functional studies. To increase blood neutrophil counts, mice were injected subcutaneously with granulocyte colony-stimulating factor (G-CSF) (1.6 µg/mouse/day) for 4 days.21 Whole mouse blood was incubated for 1 minute in 0.2x phosphate-buffered saline (PBS) to lyse erythrocytes. Murine GM-CSF and murine G-CSF were generously provided by Dr J. Andresen (Amgen, Thousand Oaks, CA). Murine TNF- was a kind gift from Dr W. Buurman (University of Limburg, Limburg, The Netherlands). Crossing of transgenic mice.   Generation of FcR  chain-deficient mice22,23 and CR3-deficient mice24 has been described before. The hFcRI transgenic mouse was crossed either with FcR  chain-deficient or CR3-deficient mice. Heterozygous offspring was crossed back with FcR  chain-deficient or CR3-deficient mice, yielding four different genotypes: NTg,+/, NTg,/, Tg,+/, and Tg,/. FcR  chain genotype was detected by genomic polymerase chain reaction (PCR).22 CR3 phenotypes were determined with flow cytometry and genotypes were confirmed by genomic PCR using sense 5'-TGA GCT ATC CAG AGG TAG AC-3' and antisense 5'-CAT ACC TGT GAC CAG AAG AGC-3' primers to detect wild-type CR3 alleles or sense and antisense 5'-ATC GCC TTC TTG ACG AGT TC-3' primers to detect mutant CR3 alleles. Step program was 94°C, 1 minute; 56°C, 1 minute; 72°C, 2 minutes, 40 cycles. hFcRI PCR was performed using sense primer 5'-GAG CAC AGT CAG TAG ACT TC-3' and antisense primer 5'-GAT TCC GAG CGT GAG TCC A-3' (94°C, 1 minute; 60°C, 1 minute, 72°C, 2 minutes; 30 cycles). Phagocytosis.   Phagocytic capacity mediated via hFcRI was investigated using ox erythrocytes (OE) or C. albicans as targets. OE were washed three times with PBS and labeled by incubation with FITC (0.4 mg/mL; Sigma, St Louis, MO) in 0.1 mol/l NaH2PO4/Na2HPO4 buffer, pH 9.6 for 30 minutes at RT. OE were washed three times and coated with BsAb A77 × anti-OE. Peripheral blood neutrophils were incubated with OE (E:T = 1:20) for 25 minutes at 37°C. Nonphagocytosed OE were lysed in 0.2 × PBS for 1 minute before microscopical or flow cytometrical analysis. Phagocytosis of C. albicans,10 or phagocytosis of Staphylococcus Aureus Wood bacteria by hFcRI-transfected IIA1.6 cells was performed as described previously25 with minor modifications. Bacteria (1 × 108) were stained with the fluorochrome PKH26 (2 × 103 mmol/L, 15 minutes, RT; Sigma) and opsonized with human serum IgA (Cappel; 1 mg/mL, 30 minutes, 37°C). Cell-surface bound bacteria were detected using F(ab')2 fragments of GHIgA-FITC (Southern Biotechnology). Samples were analyzed by flow cytometry. C. albicans kill.   Killing of C. albicans by peritoneal PMN was analyzed using a colony-forming unit assay.26 Freshly grown yeast particles (1 × 105) were incubated with 1 × 105 PMN in RPMI 1640 medium alone or medium with 10 µg/mL BsAb A77xCan for 2 hours at 37°C. PMN were lysed by incubation for 30 minutes at -70°C, which did not affect C. albicans viability. Samples (quadruplicate) were spread over Sabouraud 4% glucose agar plates (Merck, Darmstadt, Germany), and colony-forming units were calculated after a 24-hour incubation at 37°C. ADCC.   A 51Chromium release assay21 was used to evaluate the capacity of mouse blood cells to trigger lysis of tumor cells. A total of 1 × 106 SK-BR-3 tumor cells overexpressing HER-2/neu were incubated with 150 µCi of 51Cr (Amersham, Little Chalfont, UK) for 2 hours at 37°C, washed three times, and plated (5 × 103/well) in 96-well round bottom microtiter plates. A total of 50 µL whole blood of G-CSF-treated mice and various concentrations of BsAb A77x520C9 were added. Cells were incubated at 37°C for 4 hours, after which 51Cr-release in supernatants was measured. Immunoadsorption and Western blots.   To verify physical interaction of hFcRI with murine FcR  chain, we performed immunoadsorption experiments.22 Peritoneal exudate neutrophils (1 × 107) were incubated with A77 hybridoma culture supernatant, washed, and lysed in 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) buffer containing several protease inhibitors. Lysates were centrifuged to remove insoluble material and incubated overnight at 4°C with Protein G-Sepharose Beads (Pharmacia, Uppsala, Sweden) in the presence of GMIgG1 (Southern Biotechnology). Adsorbed material was separated on 12.5% nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. Membranes were stained for FcR  chain using a rabbit anti-FcR  chain antiserum (kindly provided by Dr J.-P Kinet, Harvard, Boston, MA). Statistics.   Statistical analyses were performed with unpaired two-tailed Student's t-tests. P < .05 was considered significant.     RESULTS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Expression of human FcRI by myeloid cells of transgenic mice.   A 41-kb cosmid insert containing the hFcRI gene (Fig 1A) was injected into FVB/N oocytes, to generate transgenic mice. Two transgenic mouse lines, designated 2107 and 2126, were established, and copy numbers of transgenes were estimated to be between 1 to 2, and 2 to 3, respectively, by semiquantitative Southern blots (Fig 1B). Neutrophils showed hFcRI expression, whereas only a subpopulation of monocytes expressed hFcRI. Macrophages isolated from the peritoneal cavity and nonmyeloid cells, such as lymphocytes (Fig 1C), platelets, endothelial cells, and hepatocytes (data not shown) exhibited no hFcRI expression. The transgenic receptor was recognized by CD89 MoAb defining different epitopes on hFcRI (A77 and A59; Monteiro et al27) and bound human serum IgA (Fig 1D) and IgA-opsonized S. aureus bacteria (results not shown). Both transgenic lines exhibited identical cell expression patterns. However, due to higher expression level of hFcRI, only results obtained from experiments with line 2126 are presented in this report. View larger version (29K): [in this window] [in a new window]   Fig 1. Generation of transgenic mice expressing human FcRI. (A) Structure of the transgene consisting of a 41-kb cosmid insert carrying the gene encoding FcRI. Exons are represented by closed boxes. S1, S2, signal peptide; S3, putative additional signal exon39; EC1 and EC2, extracellular Ig-like domains; TM/C, transmembrane and cytoplasmic region; E, EcoRI; H, HindIII. (B) Southern blot analysis of hFcRI transgenic mice. Genomic DNA from transgenic (Tg) line 2107 (lane 1), line 2126 (lane 2), a nontransgenic (NTg) mouse (lane 3), and human DNA (lane 4) were digested with EcoRI and hybridized with hFcRI cDNA probe. Sizes of DNA fragments (kb) are indicated on the left. (C) Flow cytometric analysis of FcRI surface expression on mouse blood cells and peritoneal macrophages. Cells of nontransgenic (thin lines) and transgenic (bold lines) mice were stained with anti-FcRI MoAb A77-FITC. Cells were stained with Gr-1-PE or F4/80-biotin/streptavidin-PE to identify granulocytes and monocytes/macrophages, respectively. Anti-CD45/B220 and anti-TCR served to distinguish lymphocytes. This experiment was repeated at least five times, yielding essentially identical results. (D) Human IgA binding to transgenic neutrophils. Cells of NTg (thin line) and Tg (bold line) were incubated with human serum IgA and PE-labeled antihuman IgA antibody. Regulation of human FcRI expression by cytokines.   To test whether the proper regulatory elements were present in the transgenic construct, cytokine regulation of the transgene was assessed. Bone marrow cells of transgenic mice were cultured with mouse TNF- and mouse GM-CSF, as both cytokines were documented to upregulate hFcRI expression on myeloid cells.6,28,29 Both TNF- and GM-CSF upregulated hFcRI expression on bone marrow-derived neutrophils (Fig 2A). No additional upregulation was observed when neutrophils were cultured with GM-CSF and TNF-. Bone marrow-derived macrophages were grown for 8 days in medium alone or with cytokines. GM-CSF induced hFcRI expression on macrophages and greatly enhanced cell growth, whereas cells cultured without cytokines grew slowly and did not express surface hFcRI (Fig 2B). Growing cells with TNF- alone inhibited cell growth, and after 8 days, no cells could be detected. However, when macrophages were cultured with both GM-CSF and TNF-, an additional upregulation of hFcRI was found. Similar data were generated using peritoneal macrophages (results not shown). G-CSF, interferon (IFN)- or interleukin (IL)-10, which all increase hFcRI (CD64) expression on phagocytes,20,30 had no effect on hFcRI expression of either macrophages or neutrophils (n = 3). View larger version (11K): [in this window] [in a new window]   Fig 2. Cytokine regulation of hFcRI expression in transgenic mice. (A) Bone marrow-derived neutrophils were cultured overnight with TNF- and/or GM-CSF and stained for surface expression of FcRI. Cells cultured in the presence of cytokines (bold lines) were compared with cells cultured in medium alone (thin lines). Gr-1-PE was used to identify granulocytes. (B) Effect of GM-CSF and TNF- on hFcRI expression on bone marrow-derived macrophages. Cells of nontransgenic (thin lines) and transgenic (bold lines) mice were cultured for 8 days. Cells were stained with anti-hFcRI MoAb A77-FITC and counterstained with F4/80-biotin/streptavidin-PE to define macrophages. This experiment was repeated four times with similar results. Phagocytosis mediated by human FcRI.   Phagocytic capacity of peripheral blood PMN was assessed using OE as targets. Neutrophils were incubated with FITC-labeled and anti-hFcRI BsAb (A77xOE)-coated OE. Before analysis, nonphagocytosed OE were lysed. Only neutrophils of Tg mice, but not of NTg control mice, phagocytosed BsAb-coated OE (Fig 3A, upper panels), whereas nonopsonized OE were not ingested. Fluorescence of neutrophils after ingestion of FITC-OE was determined with flow cytometry (Fig 3A, lower panels). Phagocytosis experiments were also performed with C. albicans yeast particles as targets. Uptake of C. albicans by Tg PMN was effectively enhanced in the presence of BsAb A77xCan (Fig 3B, right panel), compared with phagocytosis without BsAb (results not shown). PMN from NTg litter mates were unable to phagocytose C. albicans (Fig 3B, left panel). After phagocytosis, enhanced C. albicans killing by Tg PMN was found in the presence of hFcRI-targeted BsAb (Fig 3C), but not by control NTg PMN. View larger version (33K): [in this window] [in a new window]   Fig 3. Biological functions triggered via FcRI on transgenic neutrophils. (A) Phagocytosis of BsAb-coated OE by granulocytes of nontransgenic (upper left panel) and transgenic (upper right panel) mice analyzed by microscopy. Flow cytometric analysis is shown in lower panels. White blood cells were incubated with nonopsonized OE-FITC (thin lines) or A77xOE BsAb opsonized OE-FITC (bold lines). Nonphagocytosed OE were lysed. FITC-fluorescence of granulocytes reflects phagocytosed OE. Gr-1-PE was used to identify granulocytes. (B) Microscopic analysis of phagocytosis of BsAb-coated C. albicans by granulocytes of nontransgenic (left panel) or transgenic (right panel) mice. (C) C. albicans killing by PMN from transgenic and nontransgenic mice in the presence of medium alone (white bars) or 10 µg/mL BsAb A77xCan (black bars). *P < .05 versus NTg control. (D) Capacity of FcRI to trigger whole blood ADCC. Whole blood of NTg () and Tg () mice was incubated with 51Cr-labeled SK-BR-3 tumor cells in the presence of hFc RI-directed BsAb. 51Cr release from duplicates was measured. *P < .05 versus values of NTg. The data shown (mean ± standard deviation [SD]) are representative of results obtained in four separate experiments. Antibody-dependent cell-mediated cytotoxicity triggered via hFcRI.   The ability of hFcRI to trigger ADCC in whole blood was tested using HER-2/neu-overexpressing SK-BR-3 tumor cells as targets. Cells of Tg mice were capable of lysing tumor cells even in the presence of low concentrations (0.08 µg/mL) of anti-hFcRI x anti-HER-2/neu BsAb (A77x520C9). No tumor cell lysis was observed using blood of NTg mice (Fig 3D). In parallel experiments, we verified the capacity of NTg cells to lyse tumor target cells via endogenous mouse FcR. In the presence of the parental mouse IgG1 anti-HER-2/neu MoAb 520C9 (2 µg/mL), 70.3% ± 12.1% of tumor cells were lysed (n = 3). Forty-five percent of the cells in whole blood of G-CSF-treated animals was hFcRI-expressing neutrophils, while only few other cells (monocytes) expressed hFcRI. It was therefore likely that neutrophils were responsible for the observed tumor cell lysis (Fig 3D). To test this, experiments were performed with isolated peritoneal neutrophils from mice injected with thioglycollate. Both neutrophils of PBS- or G-CSF-treated Tg mice induced lysis of tumor cells in the presence of anti-hFcRI × anti-HER-2/neu BsAb (65% lysis with 2 µg/mL A77x520C9; n = 2). As a control, we also tested a human FcRI (CD64)-directed BsAb, (MDX-H210), which is an anti-FcRI × anti-HER-2/neu BsAb.21 This BsAb did not exhibit any cytotoxic activity with FcRI Tg neutrophils as effector cells (n = 3; data not shown). hFcRI interaction with the FcR  chain signaling molecule.   To elucidate the necessity of FcR  chain for hFcRI function in vivo, hFcRI Tg mice were crossed with FcR  chain-deficient animals, resulting in hFcRI transgenic, FcR  chain-deficient mice (Tg,/). Genotypes were checked by genomic PCR (Fig 4A). hFcRI PCR showed a 900-bp band. PCR of wild-type or mutant FcR  chain yielded bands of 300 bp (closed arrowhead), or 950 bp (open arrowhead), respectively. Surface hFcRI expression was checked on mouse cells (Fig 4B). Remarkably, neutrophils of Tg,/ mice did not express hFcRI on their membrane. Also, monocytes or GM-CSF/TNF- cultured macrophages entirely lacked hFcRI expression (n = 4). Immunoadsorption of hFcRI from neutrophils showed mouse FcR  chain, indeed, to be physically associated with hFcRI in Tg,+/mice (Fig 4C). View larger version (47K): [in this window] [in a new window]   Fig 4. The FcR  chain is essential for both surface expression of and phagocytosis by hFcRI. (A) Genomic DNA from wild-type and FcR  chain-deficient mice was checked for the presence of hFcRI and FcR  chain by PCR. Wild-type FcR  chain PCR products are marked by the closed arrowhead and positions of mutant FcR  chains are marked by the open arrowhead. (B) Surface expression of hFcRI on granulocytes of Tg,+/ (bold line), Tg,/ (thin line), or NTg,+/ (filled area) analyzed by flow cytometry. Cells were stained with A77-FITC. (C) Physical interaction between FcR  chain and hFcRI was shown in Tg mice by FcR  chain immunoadsorption with MoAb directed to hFcRI. The gel was run under nonreducing conditions and the blot was stained with a rabbit anti-FcR  chain antiserum. Position of FcR  chain homodimers is marked by the arrowhead. (D) Phagocytosis of IgA-coated PKH26-labeled bacteria by hFcRI/ chain transfectants is dependent on a functional ITAM. Human secretory IgA-opsonized bacteria were incubated with hFcRI transfectant cells. FL-2 fluorescence represents bacterial binding to transfectants. After incubation at either 4°C or 37°C, remaining cell-surface bound bacteria was detected using GhIgA-FITC. A decrease in FITC-fluorescence (FL-1) reflects phagocytosis. One representative experiment of five is shown. The cytoplasmic tail of FcR  chain contains an ITAM signaling motif that proved crucial for calcium mobilization after hFcRI cross-linking.14 To test whether this ITAM was important for hFcRI phagocytosis, both tyrosines (present at amino acid positions 65, and 76 within the FcR  chain cytoplasmic tail) within this ITAM were changed into phenylalanines (:Y65F-Y76F). IIA.1.6 cells expressing this mutant FcR  chain were evaluated for their ability to bind and ingest human serum IgA-coated S. Aureus bacteria. Cells expressing hFcRI/ or hFcRI/:Y65F-Y76F showed comparable FL-2 fluorescence intensities (indicating comparable binding of IgA-opsonized bacteria). Only cells expressing hFcRI/, however, phagocytosed bacteria, represented by a drop in FITC-fluorescence, (shown in FL-1) on incubation at 37°C (Fig 4D, upper right panel). CR3 (CD11b/CD18) plays a role in hFcRI-triggered ADCC, but not phagocytosis.   To examine whether the integrin CR3 (CD11b/CD18) was involved in hFcRI function, CR3-deficient animals were crossed with hFcRI transgenic mice. Phenotypes of offspring were tested by flow cytometry (Fig 5B and C), and genotypes were confirmed by genomic PCR (Fig 5A). hFcRI PCR showed a 900-bp band, whereas wild-type CR3 and mutant CR3 yielded bands of 300 bp (closed arrowhead), and 600 bp (open arrowhead), respectively. No differences were observed in hFcRI expression levels between Tg,CR3+/ and Tg,CR3/ (Fig 5B). View larger version (33K): [in this window] [in a new window]   Fig 5. The 2 integrin CR3 (CD11b/CD18) does not affect surface expression of FcRI in transgenic mice. (A) Detection of FcRI and CR3 in genomic DNA from wild-type and CR3-deficient mice by PCR. Wild-type CR3 (lanes 1) is marked by the closed arrowhead and mutant CR3 (lane 2) by the open arrowhead. (B and C) Flow cytometric analyses of hFcRI and CR3 expression on peripheral blood granulocytes of Tg,CR3+/ (bold lines), Tg,CR3/ (thin lines), or NTg,CR3+/ (filled area) mice. Cells were stained with A77-FITC to detect hFcRI (B) or M1/70-FITC to assess CR3 expression (C). Experiments were repeated four times, yielding identical results. ADCC capacity of Tg,CR3/ blood cells was determined with a 51Cr-release assay. In the presence of 0.08 µg/mL anti-hFcRI BsAb (A77x520C9), 37.5% of tumor cells were lysed by Tg,CR3+/cells, reaching 65% lysis with 2 µg/mL BsAb (Fig 6A). Remarkably, cells of Tg,CR3/ mice were unable to lyse tumor cells. To test the possibility that neutrophils exhibit defective binding to tumor cells, we studied adherence of PMN to BsAb-coated tumor target cells (Fig 6B and C). No differences in binding capacity were found between Tg,CR3+/ and Tg,CR3/ (Fig 6C). To test phagocytic capacity in CR3/ cells, neutrophils were incubated with C. albicans. Both Tg,CR3+/ and Tg, CR3/ avidly phagocytosed C. albicans in the presence of A77xCan BsAb (Fig 6D), whereas cells were unable to ingest yeast particles without BsAb (results not shown). View larger version (49K): [in this window] [in a new window]   Fig 6. ADCC, but not phagocytosis triggered via hFcRI, depends on the presence of CR3 (CD11b/CD18). (A) hFcRI-mediated ADCC is absent in CR3-deficient mice. Whole blood of NTg,CR3+/(), Tg,CR3+/ (), or Tg,CR3/ () mice was incubated with 51Cr-labeled SK-BR-3 tumor cells. 51Cr release from duplicates was quantitated as reflection of tumor cell lysis. *P < .05, **P < .001 versus values of NTg. (B) Binding of neutrophils to A77x520C9 BsAb-opsonized tumor cells is unaffected in CR3-deficient mice. (C) Binding of neutrophils to tumor cells was quantitated on cytospin preparations (binding index = number of granulocytes/100 tumor cells). **P < .001 versus NTg controls. (D) Microscopical analysis of hFc RI phagocytosis. Peritoneal granulocytes of Tg,CR3+/ (left panel) or Tg,CR3/ (middle panel) were incubated with C. albicans in the presence or absence of BsAb A77xCan. Phagocytic index (phagocytosed C. albicans/100 cells) was determined on cytospins (right panel). **P < .001 versus NTg controls.     DISCUSSION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Although much progress has been made in our understanding of the role Fc receptors play in immunity,1,2 the function of receptors for IgA is poorly understood. Existing data on hFcRI are based on in vitro experiments, and because no hFcRI equivalent has been identified in mice, in vivo data are scarce. To overcome this difficulty, two lines of transgenic mice were generated in which hFcRI expression was restricted to the myeloid lineage. The receptor was recognized by MoAb defining different epitopes on hFcRI27 and could bind human IgA. Levels of hFcRI expression were solely regulated by cytokines known to influence FcRI expression on human cells, eg, GM-CSF28 and TNF-,29 and not by cytokines that regulate FcRI expression, eg, G-CSF, IFN-, and IL-10.20,30 Furthermore, hFcRI was capable of triggering phagocytosis and ADCC. Taken together, these results indicated hFcRI to be expressed in a functional way using its own promoter and regulatory elements, thus mimicking the situation in man.6 It has been documented that hFcRI complexes with the FcR  chain signaling molecule.12 To assess the role of FcR  chain in hFcRI signaling, Tg mice were crossed with FcR  chain/ animals. Although the hFcRI gene was clearly present in Tg,/ mice, no hFcRI expression was detected on the membrane of neutrophils. This is notably different from earlier in vitro data in IIA1.6 cells that indicated FcR  chain to be important for hFcRI function, but not for expression per se.13 Importantly, the present data document FcR  chain to be essential for hFcRI expression in vivo on PMN and monocytes/macrophages, which parallels the situations for FcRI,22 FcRIII,31 and FcRI.32 Due to the heterologous nature of our transgenic mouse model, however, we cannot formally exclude subtle differences to exist between transgenic and human phagocytes. Still, our data emphasize the importance of performing studies on phagocytic receptors naturally expressed on cells. Lack of receptor expression in Tg,/ mice, however, precluded studying FcR  chain involvement in hFcRI-function in in vivo derived cells. Phagocytic capacity was therefore tested in vitro, using IIA1.6 cells expressing hFcRI, and wild-type or mutant FcR  chains. Although both types of transfectant cells could bind IgA-coated bacteria, only FcRI/ cells exhibited phagocytosis. The integrity of the FcR  chain ITAM was, thus, shown critical for hFcRI phagocytic capacity. Previous work showed signaling via hFcRI to require a functional ITAM14 and to involve both Src family and Syk protein tyrosine kinases.33 Several laboratories have documented a requirement for CR3 in both FcR-mediated ADCC34 and phagocytosis.15,17 To study whether CR3 is involved in hFcRI function, Tg mice were crossed with CR3-deficient mice. No differences were observed in hFcRI expression or phagocytic capacity between Tg,CR3+/ and Tg,CR3/cells. However, the ability of neutrophils of Tg,CR3/ mice to lyse tumor cells via hFcRI was absent, indicating CR3 to be essential for ADCC, but not for phagocytosis. Importantly, F(ab')2 fragments of anti-CR3 MoAb 44a abrogate the capacity of human neutrophils to lyse tumor cells via FcRI (A.B. van Spriel, unpublished data), as well. These data suggest qualitative differences in the requirement for select signaling molecules between hFcRI-mediated phagocytosis and ADCC. The molecular basis underlying the involvement of CR3 in Fc receptor function is incompletely understood. Because immune complexes can block the binding of anti-CR3 Fab fragments to monocytes16 and FcR were shown to cocap with CR3,35 a close physical contact between these classes of receptors has been suggested. CR3 possibly provides an essential costimulatory signal, which is absent in Tg,CR3/ cells. FcRIII and CR3 were reported to cooperate in generation of a neutrophil respiratory burst in normal PMN.36 Work on FcR-mediated phagocytosis supports a more direct interaction between IgG receptors and CR3: (1) FcRIIIb-expressing NIH3T3 cells were able to bind, but unable to ingest IgG-coated targets. Cotransfection of CR3 enabled phagocytosis of these particles.37 (2) CR3 could, furthermore, restore phagocytic capacity of a phagocytosis-defective FcRIIa tail-minus mutant in 3T3 transfectant models.38 In conclusion, the present data uncover a novel level of complexity in IgA receptor function in phagocytes: while FcR  chain was shown to be crucial for hFcRI expression and the capacity of the receptor to trigger phagocytosis, CR3 proved indispensable for hFcRI-triggered ADCC. These results highlight the cooperative nature of different classes of receptors in phagocyte function. This transgenic model provides a valuable tool to further dissect the role of hFcRI function in vivo. Because hFcRI was recently identified as a candidate therapeutic target,9-11 these transgenics may provide a suitable model for preclinical evaluation of hFcRI-directed therapies.     ACKNOWLEDGMENT We thank Toon Hesp, Els Dorresteijn, and Herma Boere for excellent animal care.     FOOTNOTES Submitted October 12, 1998; accepted February 4, 1999. Supported by Grant No. 901-12-214 from Netherlands Organization for Scientific Research (NWO) and Grants No. AI 22816 and DK 51643 from the National Institutes of Health (NIH). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. Address reprint requests to Jan G.J. van de Winkel, PhD, Department of Immunology, Immunotherapy Laboratory, KC.02-085.2, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands; e-mail: J.vandewinkel{at}lab.azu.nl.     REFERENCES TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES 1. van de Winkel JGJ, Capel PJA (eds): Human IgG Fc Receptors. Austin, TX, Landes, 1996. 2. Ravetch JV: Fc receptors. Curr Opin Immunol 9:121, 1997[Medline] [Order article via Infotrieve] 3. Monteiro RC, Kubagawa H, Cooper MD: Cellular distribution, regulation, and biochemical nature of an Fc receptor in humans. J Exp Med 171:597, 1990[Abstract/Free Full Text] 4. Maliszewski CR, March CJ, Schoenborn MA, Gimpel S, Shen L: Expression cloning of a human Fc receptor for IgA. J Exp Med 172:1665, 1990[Abstract/Free Full Text] 5. de Wit TPM, Morton HC, Capel PJA, van de Winkel JGJ: Structure of the gene for the human myeloid IgA Fc receptor (CD89). 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Worth RG, Mayo Bond L, van de Winkel JGJ, Todd RF III, Petty HR: CR3 (M2; CD11b/CD18) restores IgG-dependent phagocytosis in transfectants expressing a phagocytosis-defective FcRIIA (CD32) tail-minus mutant. J Immunol 157:5660, 1996[Abstract] 39. Toyabe S, Kuwano Y, Takeda K, Uchiyama M, Abo T: IgA nephropathy-specific expression of the IgA Fc receptor (CD89) on blood phagocytic cells. Clin Exp Immunol 110:226, 1997[Medline] [Order article via Infotrieve] © 1999 by The American Society of Hematology.   0006-4971/99/9312-0008$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J. M. Beekman, C. E. van der Poel, J. A. van der Linden, D. L. C. van den Berg, P. V. E. van den Berghe, J. G. J. van de Winkel, and J. H. W. Leusen Filamin A Stabilizes Fc{gamma}RI Surface Expression and Prevents Its Lysosomal Routing J. Immunol., March 15, 2008; 180(6): 3938 - 3945. [Abstract] [Full Text] [PDF] M. A. 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