Alternative Endocytic Pathway for Immunoglobulin A Fc Receptors (CD89) Depends on the Lack of FcRgamma Association and Protects against Degradation of Bound Ligand*

Pierre LaunayDagger , Claire Patry§, Agnès LehuenDagger , Benoit PasquierDagger , Ulrich Blankparallel , and Renato C. MonteiroDagger **

From Dagger  INSERM, Unité 25, Hôpital Necker, 75743 Paris, § Unité Mixte de Recherche, Institut Curie/CNRS 144, 75005 Paris, and the  Unité d'Immunoallergie, Institut Pasteur, 75015 Paris, France

    ABSTRACT
Top
Abstract
Introduction
References

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (Fcalpha R) are expressed with or without the gamma  chain on monocytes and neutrophils. gamma -less Fcalpha R represent a significant fraction of surface Fcalpha R molecules even on cells overexpressing the gamma  chain. The Fcalpha R-gamma 2 association is up-regulated by phorbol esters and interferon-gamma . To characterize gamma -less Fcalpha R functionally, we generated mast cell transfectants expressing wild-type human Fcalpha R or a receptor with a point mutation (Arg right-arrow Leu at position 209) which was unable to associate with the gamma  chain. Mutant gamma -less Fcalpha R bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of Fcalpha R-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant Fcalpha R were localized mainly in early endosomes, those containing Fcalpha R-gamma 2 were found in endo-lysosomal compartments. Mutant gamma -less Fcalpha R recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of Fcalpha R, associated or unassociated with gamma  chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.

    INTRODUCTION
Top
Abstract
Introduction
References

In humans, IgA is found in the systemic and mucosal compartments; it is the second most common antibody class in blood and the major immunoglobulin at mucosal surfaces (1, 2). More IgA is produced daily than all of the other immunoglobulin classes together (3). In serum, IgA is mainly monomeric and has a half-life around five times shorter than that of IgG because of its fast catabolism (2, 4). Although the implications of secretory IgA in host defenses are well established (2), much less is known about the antibody-mediated functions of serum IgA in human blood. Serum IgA has been considered an anti-inflammatory isotype capable of inhibiting several functions mediated by other isotypes including inhibition of IgG phagocytosis, bactericidal activity, oxidative burst, and cytokine release (5-10). The molecular basis of these inhibitory functions is poorly understood; however, IgA-immune complexes can trigger effector cells after aggregation of IgA Fc receptor(s) (Fcalpha R,1 CD89), resulting in various immune effector functions such as phagocytosis, oxidative burst, and cytokine release (11-13).

Fcalpha R are expressed on myeloid cells as heterogeneously glycosylated type I transmembrane proteins that can bind both IgA1 and IgA2 isotypes at the boundary between the Calpha 2 and Calpha 3 domains (14-18). Polymeric IgA binds more efficiently to Fcalpha R than does monomeric IgA (19, 20). Fcalpha R exist as at least two isoforms (a.1 and a.2) differing by a deletion in their extracellular domains and expressed alternatively on monocytes and alveolar macrophages (21). Several other splice variants, the corresponding native proteins of which have not been identified, have also been reported (21-25). Fcalpha R are associated with the disulfide-linked FcR gamma  chain homodimer (26-28). This interaction is resistant to treatment with Nonidet P-40 detergent, which contrasts with the dissociation of gamma  chains from Fcepsilon RI or Fcgamma RI in certain detergents (26, 29, 30). This strong interaction can be explained by the presence of two oppositely charged residues (Arg+/Asp-) in the transmembrane domain of the Fcalpha R and gamma  chain, respectively (28). The gamma  chain contains a common immunoreceptor tyrosine-based activation motif in its cytoplasmic tail. Recently, it has been shown that signaling through Fcalpha R-gamma 2 involves several tyrosine kinases including lyn, syk, and Btk (31, 32). Recruitment and phosphorylation of syk and Btk were modulated by stimulation with interferon-gamma (IFN-gamma ) and/or phorbol esters, indicating that activation of tyrosine kinases through Fcalpha R depends on the priming state of the cell (32).

FcR without signaling motifs in their cytoplasmic tails are associated with specialized subunits, such as gamma  or beta  chains, and depend on their specific retention motifs to be fully expressed on the cell surface (33). In the absence of the gamma  chain they are degraded rapidly in the endoplasmic reticulum as in the case of Fcgamma RIII (34). One remarkable feature of Fcalpha R is that these receptors can be expressed fully at the surface of COS cells after transfection, without the signaling gamma  subunits (16). Despite the role of the gamma  chain in downstream Fcalpha R signaling (28), we wondered whether the Fcalpha R could exist and function as receptors when unassociated with the gamma  chain (gamma -less Fcalpha R) on myeloid cells. We have identified significant amounts of gamma -less Fcalpha R in several cell types, including monocytes, neutrophils, and transfected cells overexpressing the gamma  chain. gamma -less Fcalpha R and Fcalpha R-gamma 2 are expressed on the same cells, and this constitutes the basis for differential endocytosis pathways of IgA, in which gamma -less receptors recycle IgA toward the cell surface whereas Fcalpha R-gamma 2 undergo endo-lysosomal sorting for IgA degradation.

    EXPERIMENTAL PROCEDURES

Antibodies-- The following mouse mAb were used: A59 (IgG1kappa ), A77 (IgG1kappa ) mAb specific for Fcalpha R (35), an irrelevant IgG1kappa control mAb (clone 7.1 anti-glutathione S-transferase protein). My43 anti-Fcalpha R mAb (IgM) was a gift from Dr. L. Shen, Dartmouth Medical School, Lebanon, NH (36). Phycoerythrin-labeled anti-Fcalpha R A59 (A59-PE) was purchased from PharMingen (San Diego). 4D8 anti-FcR-gamma chain mAb (IgG2bkappa ) was a gift from Drs. D. Presky and J. Kochan, Hoffman-la-Roche, Nutley, NJ (37). Complete digestion and F(ab')2 purity were verified by SDS-PAGE. Rabbit anti-mouse Ig (RAM) antibodies were obtained from rabbits immunized with an IgG1 (clone A59). F(ab')2 fragments of A59, A77, and IgG1kappa and RAM IgG fractions were prepared by pepsin digestion (Sigma) as described previously (38) and purified on DEAE columns. Rabbit antiserum specific for the FcR-gamma chain was a gift from Dr. J. P. Kinet, Harvard Medical School, Boston (39). Fluorescein isothiocyanate (FITC)-conjugated goat Ab specific for mouse (GAM) and rabbit Ig (GAR) and horseradish peroxidase-conjugated goat anti-rabbit IgG were purchased from Southern Biotechnology Associates (Birmingham, AL). IgA myeloma proteins were purified as described previously (19). Monomeric and polymeric IgA preparations (>98% pure) were biotinylated.

Cells-- The human monocytic cell line U937 was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Polymorphonuclear cells and mononuclear cells were isolated from whole blood by Ficoll-Hypaque (Amersham Pharmacia Biotech) gradient centrifugation. Granulocytes were purified from red cell pellets by dextran sedimentation as described previously (15). Enriched monocyte populations (60-80% pure) were obtained by subjecting mononuclear cells to rosette formation with 2-aminoethylisothiouronium bromide-treated SRBC, and nonrosetting cells were submitted to plastic adherence as described in Ref. 19. In some experiments, cells were cultured for 18 h with 10-7 M PMA (Sigma) (15), 50 units/ml human recombinant IFN-gamma (Genzyme, Cambridge, MA), 5 µM ionomycin (Calbiochem, San Diego), 100 pM human recombinant GM-CSF (Sandoz AG, Basel, Switzerland), or 50 units/ml interleukin (IL)-1beta (Rhône-Poulenc Santé, Vitry, France). Rat basophilic leukemia cells (RBL-2H3) (40) were transfected with human Fcalpha R and/or human gamma  chain and were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 2 mM glutamine, 1.5 µg/ml puromycin (Sigma) and/or 1.0 mg/ml of G418 (Life Technologies, Inc.).

Constructs, Expression Vectors, and Transfection-- Human Fcalpha R containing the R209L mutation was constructed by amplifying base pairs 591-891 of a previously described Fcalpha R a.1 cDNA (21). The sense primer included the BanII restriction site at position 601 and introduced the R209L mutation (at base pairs 688-690, with CTG replacing CGC). The amplified fragment was ligated to the remainder of the NH2-terminal cDNA via the BanII restriction site. The construct was checked by sequencing as described in Ref. 21. The Fcalpha R a.1, Fcalpha R (R209L), and human gamma  chain (41) were subcloned into pSRalpha Neo (kindly provided by Dr. J. Di Santo, INSERM U429) a modified version of the pcDL-SRalpha promoter-based expression vector (42). RBL-2H3 cells were first transfected with 30 µg of DNA by electroporation at 250 V and 1,050 microfarads using an Easyjet+ apparatus (Eurogenetec, Seraing, Belgium), then grown under 1 mg/ml G418 selection; resistant clones were selected for Fcalpha R expression by means of flow cytometry. One Fcalpha R-expressing clone was chosen and cotransfected with pSRalpha Neo-human gamma -chain (30 µg) and pSRalpha -Puro (4 µg) (43).

Cell Iodination, Immunoprecipitation, and Immunoblotting-- Cell surface iodination with Na125I (1 mCi; Amersham Pharmacia Biotech) was carried out by the lactoperoxidase method (44). For immunoprecipitation of Fcalpha R, cells (107/ml) were lysed for 30 min at 4 °C in PBS containing 1% digitonin (Aldrich), 0.02% sodium azide, 1% aprotinin, 1 mM diisopropylfluorophosphate, 5 mM iodoacetamide, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 14,000 × g for 30 min to remove insoluble materials, cleared lysates were immunodepleted of Fcgamma R by using human IgG, 32.2 and IV.3 mAb, and precipitated with test mAb as described previously (35). Bound materials were treated or not treated with N-glycanase (Genzyme), and samples were subsequently prepared for SDS-PAGE (45). For immunoblotting, immunoprecipitated proteins were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose Hybond-C (Amersham Pharmacia Biotech) filter for 18-20 h (46). The blots were incubated in blocking buffer composed of 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl (TBS) containing 3% bovine serum albumin and 0.1% Tween 20 and then incubated with anti-gamma (1:500) for 2 h at room temperature. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used a secondary Ab. Filters were developed using the Enhanced Chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).

Coimmunoprecipitation of Receptor-bound 125I-Anti-Fcalpha R mAb-- This was carried out as described in Ref. 47. Briefly, F(ab')2 fragments of A77 mAb or mouse IgE were labeled with Na125I using the IODO-GEN method (48). Cells (5 × 106) were incubated with 125I-labeled test mAb (25-35 µg/ml) for 1 h, washed in PBS and 0.1% NaN3, and then lysed in 0.5 ml of 1% digitonin buffer containing protease inhibitors as described above. After centrifugation, lysates were divided into two aliquots for 2-h incubations with either 20 µg of 4D8 anti-gamma chain mAb or 50 µg of RAM Ig Ab coupled to Sepharose 4B. These amounts of antibodies had been identified as saturating concentrations for precipitation of labeled Ab complexes. The percentage of specifically precipitated counts was calculated for each Ab after subtraction of nonspecific counts obtained using either irrelevant IgG1-coupled beads or lysates that had been preincubated with a 100-fold excess of unlabeled anti-Fcalpha R mAb (nonspecific counts were always <3%). For RBL transfectants, 3 µl of polyclonal rabbit anti-gamma chain antiserum plus protein A-coupled beads were used to coprecipitate both rat and human gamma  chains (49). Normal rabbit serum was used to determine background precipitation.

Immunofluorescence and Flow Cytometry-- RBL transfectants (1 × 106) were stained with 10 µl of biotinylated A77 F(ab')2 or irrelevant IgG1 F(ab')2 fragments (at 0.1 mg/ml) for 30 min at 4 °C followed by 10 µl of 1/50 diluted streptavidin PE (Southern Biotechnology Associates) as developing reagent. For IgA binding, cells preincubated with human IgG (10 mg/ml) to block Fcgamma R were incubated with 10 µl of biotinylated purified IgA (0.5 mg/ml) for 1 h at 4 °C followed by streptavidin PE. For two-color immunofluorescence analysis, viable U937 cells (2 × 106), preincubated with an excess of human IgG (10 mg/ml) to block Fcgamma R, were stained directly with 10 µl of PE-labeled A59 anti-Fcalpha R mAb (0.1 mg/ml) or with an irrelevant PE-labeled IgG1 control for 30 min at 4 °C. After washing, cells were fixed with PBS containing 1% paraformaldehyde, permeabilized with PBS containing digitonin (10 µg/ml) for 5 min at 4 °C, and stained with anti-gamma chain rabbit antiserum (10 µl at 1:100 dilution) or a control rabbit serum for 30 min at 4 °C in PBS containing 0.05% Tween 20. After washes, cells were incubated with 10 µl of FITC-labeled goat anti-rabbit antibodies (25 µg/ml; human- and mouse-adsorbed, purchased from Southern Biotechnology Associates) for 30 min at 4 °C and analyzed by flow cytometry using a FACScalibur apparatus (Becton Dickinson). In some experiments, cytoplasmic molecules were evaluated on cells cytospun onto glass slides, fixed, and permeabilized for 20 min at -20 °C with 95% ethanol and 5% acetic acid solution, washed, and incubated for 20 min with 4D8, A59, or control IgG1 mAb (0.05 mg/ml). FITC-labeled anti-mouse Ig Ab (0.05 mg/ml) was added as a developing reagent and mounted on coverslips.

beta -Hexosaminidase Assay-- This was based on a method described previously (43). Briefly, transfectants were plated at 5 × 104 cells in 100 µl of complete DMEM in the absence of G418 and sensitized with anti-dinitrophenyl IgE Abs (1/200) or F(ab')2 fragments of A77 mAb (0.01 mg/ml) for 4 h at 37 °C. Cells were washed in Hanks' balanced saline solution containing 1% fetal calf serum and resuspended in the same buffer containing 100 ng/ml dinitrophenyl-human serum albumin (Sigma) or F(ab')2 fragment RAM (40 µg/ml), respectively. To determine spontaneous release, cells were incubated in the absence of Ag (for Fcepsilon RI stimulation) or with irrelevant IgG1 F(ab')2 fragments (for Fcalpha R stimulation). Maximal release was determined with 100 nM PMA plus 1 µM ionomycin as stimulant. After incubation for 1.5 h, hexosaminidase secretion was analyzed in test supernatants by adding p-nitrophenyl N-acetyl-beta -D-glucosamine (1.3 mg/ml Sigma). The total cellular content of beta -hexosaminidase was determined by lysis of adherent cells in 0.5% Triton X-100. Absorbance was determined at 410 nm in a microplate reader.

Internalization and Recycling Assays-- This was performed as described elsewhere (50). Briefly, 1 × 106 cells were incubated with 1 µg of 125I-F(ab')2 fragments of A77 anti-Fcalpha R mAb for 1 h at 4 °C. After extensive washing, 10 µl of F(ab')2 fragments of rabbit anti-mouse antibodies (1 mg/ml) was added for 30 min. Excess antibody was removed, and endocytosis was induced by incubating cells at 37 °C in RPMI 1640, 25 mM HEPES, 5% fetal calf serum for the times indicated. The reaction was stopped by placing the cells on ice. Any residual antibodies on the surface were removed by acid stripping (PBS, pH 2.5, at 4 °C for 5 min). This acid treatment routinely removes 85-90% of surface-bound anti-Fcalpha R F(ab')2. After pelleting, cell-associated counts were detected in a gamma counter. In recycling experiments the cells were incubated with 125I-polymeric IgA1kappa or 125I-Fab fragments of A77 alone for 1 h on ice, washed, and then either treated or not treated for 20 min with 0.6 mM primaquine (Sigma) before incubation at 37 °C. Nonspecific counts were obtained by preincubating cells with a 100-fold excess of nonlabeled mAb or IgA. Data are expressed as percentages of total initial cell-associated counts and presented as the means ± S.D. of at least three separate experiments.

Measurement of IgA Proteolysis after Internalization-- RBL transfectants were plated in the absence of G418 at 0.5 × 106 cells/ml in 24-well Costar tissue culture plates. 24 h later, the cells were incubated with biotinylated, 125I-labeled dimeric IgA1kappa (1 µg/well) in 0.2 ml of 0.1% bovine serum albumin, DMEM at 4 °C for 1 h. The medium was removed after 1 h, and the cells were washed three times at 4 °C. Cells were then incubated with streptavidin-labeled PE (10 µg/ml) at 4 °C for 15 min to induce receptor aggregation. After washings, cells were cultured in DMEM containing 0.1% bovine serum albumin and 100 µg/ml unlabeled IgA1kappa for the times indicated. After incubation, the medium was removed, proteins were precipitated in 10% trichloroacetic acid, and acid-soluble and acid-insoluble radioactivities were counted in a gamma counter as described in Ref. 51.

Endocytosis Procedure by Confocal Microscopy-- Adherent cells on glass slides were incubated at 4 °C for 30 min with 100 µl of 0.1 mg/ml A77 anti-Fcalpha R F(ab')2 fragments in PBS and 0.2% bovine serum albumin. After washings, cells were incubated with 100 µl of 0.04 mg/ml RAM or FITC-coupled GAM for 30 min on ice. When indicated, cells carrying unlabeled antibodies were incubated further with 0.005 mg/ml GAR coupled to FITC to amplify aggregation. To visualize transferrin receptor-recycling vesicles, cells were cultured in serum-free DMEM for 30 min to deplete endogenous transferrin and incubated on ice with 100 nM human transferrin coupled to Cy3 (kindly provided by Dr. A. Benmerah, CJF-97-10, Necker Institute) together with anti-Fcalpha R mAb as above. The slides were either warmed to 37 °C for various times or kept at 4 °C. Cells were washed, fixed in 3% paraformaldehyde for 10 min, and quenched twice in PBS containing 1 M glycine. For intracellular gamma  chain staining, cells were permeabilized with 0.05% saponin (Sigma) and stained with 3.5 µg/ml purified anti-gamma chain polyclonal Ab plus 3.5 µg/ml GAR coupled to Texas Red. To visualize the plasma membranes, cells were stained after endocytosis for 5 min at 4 °C with 10 µg/ml of wheat germ agglutinin coupled to Texas Red (38). After washing, the slides were mounted in 10% Moviol, 25% glycerol, Tris-HCl (100 mM, pH 8.5). Confocal laser scanning microscopy was carried out with a TCS4D confocal microscope based on a DM microscope interfaced with an argon/krypton laser. Simultaneous double fluorescence acquisitions were made with the 488 nm and 568 nm laser lines to excite FITC and Texas Red dyes using a 100 × oil-immersion Plan Apo objective (numerical aperture 1.4). The fluorescence was selected using appropriate double-fluorescence dichroic mirror and band-pass filters (52).

    RESULTS

Identification of gamma -less Fcalpha R on Human Myeloid Cells-- Because Fcalpha R can be fully expressed at the surface of COS cells after transfection without the signaling gamma  subunits (16), we investigated whether all Fcalpha R expressed on PMA-treated U937 cells were associated with the gamma  chain homodimer by means of confocal microscopy. Because gamma -less and gamma -associated Fcalpha R could not initially be distinguished on the cell surface, we performed short term endocytosis of Fcalpha R·anti-Fcalpha R mAb complexes (FITC-labeled) to examine whether all internalized receptors colocalized with the gamma  chain (Texas Red-labeled) in the vesicles. As shown in Fig. 1, two types of intracellular vesicles were detected in single cells, in which Fcalpha R was either colocalized (yellow) or not colocalized (green) with the gamma  chain. These results strongly suggest the existence of gamma -less and gamma -associated Fcalpha R in the same cells.


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 1.   Lack of colocalization between gamma  chain and some Fcalpha R within intracellular vesicles after short term endocytosis. PMA-treated U937 cells preincubated with human IgG (10 mg/ml) to block Fcgamma R were incubated with anti-Fcalpha R A77 F(ab')2 fragments plus GAM coupled to FITC on ice as described under "Experimental Procedures." After Fcalpha R staining, cells were incubated at 37 °C for 3 min, fixed, permeabilized, and stained with anti-gamma chain polyclonal Ab plus GAR coupled to Texas Red. Cells were observed under a confocal microscope and optically sectioned at 1.5-µm intervals. A representative medial section of the horizontal slices is shown. No staining was observed when FITC-labeled secondary Ab or irrelevant IgG1 was used.

To investigate whether the gamma -less receptor population could also be detected in detergent extracts, we performed immunodepletion experiments using digitonin-solubilized cells because the Fcalpha R-gamma 2 interaction is resistant to digitonin treatment (26). As shown in Fig. 2, immunoprecipitation of surface-iodinated Fcalpha R from blood neutrophils and PMA-treated U937 cells resulted in the appearance of the expected broad band of 55-75 kDa. Precipitation with an anti-gamma chain antibody gave rise to similarly sized species. Treatment of anti-gamma chain mAb-associated glycoproteins with N-glycanase resulted in 32- and 36-kDa bands that comigrated with those observed with the anti-Fcalpha R mAb (Fig. 2A). After extensive immunodepletion with anti-gamma chain mAb, anti-Fcalpha R mAb-reactive molecules could still be precipitated (Fig. 2A). Similar results were obtained with PMA-treated U937 cells using a polyclonal anti-gamma chain Ab (Fig. 2B), in which the same treatment eliminated most of the gamma  chain molecules (Fig. 2B, bottom). Conversely, immunoadsorptions with an anti-Fcalpha R mAb (A59) completely eliminated 55-75 kDa-4D8 mAb reactive proteins (not shown). These results reveal two forms of Fcalpha R, gamma -less and gamma -associated that are expressed on the surface of U937 cells and blood neutrophils. Because of the extensive immunoadsorption it was, however, impossible to quantify the two forms of Fcalpha R.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   Identification of gamma -less Fcalpha R on human myeloid cells. 2 × 107 blood neutrophils (panel A) and PMA-activated U937 cells (panel B) were surface labeled with Na125I, and the membrane proteins were solubilized using a 1% digitonin lysis buffer as described under "Experimental Procedures." Lysates were divided into three aliquots and incubated with irrelevant IgG1kappa (lanes 1 and 4), anti-Fcalpha R mAbs (lanes 2 and 5; A, A59 and B, A77 F(ab')2), or anti-gamma Abs (lanes 3 and 6; A, 4D8 and B, rabbit antiserum) plus RAM Ig Ab (panel A) or protein G (panel B) coupled to Sepharose 4B beads. Eight immunoadsorptions were performed with an excess of monoclonal (panel A) or polyclonal anti-gamma chain (panel B) and followed by immunoprecipitations with test Abs. In panel A, immunoprecipitates were digested or not digested by N-glycanase, as indicated (N-gly) and analyzed by 10% SDS-PAGE (2ME+) and autoradiography. In panel B, immunoprecipitated 125I-surface proteins were separated by 12.5% SDS-PAGE (2ME-), transferred onto a nitrocellulose membrane, and analyzed by autoradiography (top) and immunoblotting (bottom) using anti-gamma chain polyclonal Ab and horseradish peroxidase-conjugated anti-rabbit Ig Ab plus ECL.

To estimate the amounts of gamma -less Fcalpha R we used a coimmunoprecipitation assay validated previously for Fcepsilon RI (47). Cells were loaded with 125I-labeled anti-Fcalpha R mAb F(ab')2 fragments. Labeled receptor-Ab complexes were solubilized in the presence of 1% digitonin and precipitated with either anti-gamma chain (4D8)- or anti-mouse Ig Ab. As shown in Fig. 3A, whereas total precipitable amounts of 125I-Ab·Fcalpha R complexes using anti-mouse Ig Abs exceeded 70% (total Fcalpha R), those precipitated with the anti-FcRgamma mAb were significantly lower on U937 cells, monocytes, and neutrophils (16 ± 5%; 21 ± 3%, and 29 ± 5%, respectively). The results were almost similar when 125I-labeled Fab fragments of anti-Fcalpha R were used instead of F(ab')2 fragments in two comparative experiments (17 ± 0.5% versus 23.5 ± 1.5% in monocytes and 26 ± 3% versus 32 ± 1% in neutrophils, respectively). Similar results were also obtained using a rabbit anti-gamma chain antiserum (data not shown). We then analyzed the time-dependent dissociation of the gamma  chain from the Fcalpha R in digitonin lysates. Fcalpha R-gamma complexes were very stable over a 24-h period, ruling out the possibility that partial association of Fcalpha R with the gamma  chain was the result of their instability in detergents (Fig. 3B).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   A. gamma -less Fcalpha R represents the major fraction of Fcalpha R molecules on the cell surface. 5 × 106 of U937 cells, monocytes, and neutrophils (empty, hatched, and filled bars, respectively) were incubated with 125I-labeled A77 anti-Fcalpha R F(ab')2 fragments (25-35 µg/ml) for 1 h at 4 °C, washed, and solubilized in 1% digitonin lysis buffer. The lysates were divided into two aliquots and incubated for 2 h at 4 °C with either RAM Ig Ab or 4D8 mAb anti-gamma chain coupled to Sepharose 4B. After washes, precipitated counts versus total counts were determined. Nonspecific precipitated counts were obtained using either irrelevant IgG1-coupled beads or lysates that had been preincubated with a 100-fold excess of unlabeled anti-Fcalpha R. Bars (mean ± S.D. of at least three experiments performed in triplicate) show the calculated percentage of specifically precipitated 125I-labeled A77 bound to cell surface receptors. Panel B, time-dependent stability of Fcalpha R-gamma association in the mild detergent, digitonin. PMA-activated U937 cells were incubated with 125I-labeled A77 anti-Fcalpha R F(ab')2 fragments for 1 h at 4 °C, washed, lysed (time 0) for 15 min on ice in 1% digitonin lysis buffer, and centrifuged for 15 min at 14,000 × g. Lysates were then incubated at 4 °C for different time periods and immunoprecipitated by 4D8 anti-gamma chain mAb coupled to beads for 2 h at 4 °C and analyzed as described in panel A.

Partial Association of Fcalpha R with the gamma  Chain Is Not Dependent on the Amounts of Expressed gamma  Chain-- To examine the effect of the amounts of expressed gamma  chain, we transfected a previously established Fcalpha R+ RBL transfectant (32) with the human gamma  chain. One clone (alpha gamma ) expressing large amounts of human gamma  chain was selected (Fig. 4, A and B). Coimmunoprecipitation experiments, using a polyclonal Ab that recognizes both rat and human gamma  chains, showed that in these transfectants the fraction of gamma -associated receptors did not increase compared with cells transfected with Fcalpha R only (Fig. 4C). As a control we coprecipitated the Fcepsilon RI using the same anti-gamma Ab. In agreement with previous observations (47), a major fraction of Fcepsilon RI (>50%) was coprecipitated in these transfectants (data not shown). This demonstrated that the amount of anti-gamma Ab used in the assay was not limiting. These results also indicated that association of the gamma  chain with Fcalpha R did not depend on the amount of expressed gamma  chain.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Reconstitution of the partial association of gamma  chains with human Fcalpha R on the rat mast cell line, RBL-2H3. Panel A, overexpression of human gamma  chain. Transfectants expressing human Fcalpha R a.1 (alpha ) (32) were cotransfected with gamma  chain and selected on the basis of the reactivity with anti-human gamma  chain 4D8 mAb. 107 cells were then solubilized in 0.5% Nonidet P-40 lysis buffer and immunoprecipitated using anti-human (hu) gamma  chain mAb (4D8)-coupled Sepharose 4B beads. Precipitated proteins were separated by 12.5% SDS-PAGE under nonreducing conditions and analyzed by immunoblotting using rabbit anti-gamma chain polyclonal Ab (poly) and horseradish peroxidase-conjugated anti-rabbit Ig Ab. One out of five clones was chosen (alpha gamma ). PMA-treated U937 cells were used as positive control. Panel B, Fcalpha R expression. 5 × 105 nontransfected (NT), human Fcalpha R-transfected (alpha ) and human Fcalpha R/gamma transfected (alpha gamma ) cells were stained with biotinylated A77 anti-Fcalpha R mAb (solid lines) or biotinylated irrelevant IgG1kappa (dotted line, for alpha gamma ) and with streptavidin PE followed by FACS analysis. Panel C, partial association of Fcalpha R with the gamma  chain on transfectants. Because human Fcalpha R molecules were previously found to be associated with rat gamma  chain on transfectants (32), experiments were performed using a polyclonal Ab that recognizes both rat and human gamma  chains. 5 × 106 cells were incubated with 125I-labeled A77 anti-Fcalpha R F(ab')2 fragments and digitonin solubilized. The lysates were divided into two aliquots and incubated for 2 h at 4 °C with either RAM Ig or polyclonal anti-gamma chain plus protein A-coupled beads, and the percentage of precipitated surface receptors was determined as described in Fig. 3A. Maximum percentages of precipitable 125I-Ab-receptor complexes using anti-mouse Ig Abs exceeded 80%.

Fcalpha R and gamma  Chain Are Coexpressed in a Single Cell Population-- The experiments shown in Fig. 1 suggested that both gamma -less and gamma -associated Fcalpha R are expressed on the same cells. To further exclude the possibility that partial association was caused by heterogeneity in gamma  chain expression on a given cell population, we carried out two-color FACS analysis of cell surface Fcalpha R and intracellular gamma  chain. Fig. 5 shows the expression of both Fcalpha R and gamma  chain on U937 cells, before and after IFN-gamma or PMA treatment. The majority of cells were Fcalpha R+/gamma +. Even though the expression level of both molecules was heterogeneous, the absence of two independent contour plots ruled out the existence of a subpopulation expressing only one of these proteins. The coexpression of Fcalpha R and gamma  chain was also found in neutrophils as determined by cytoplasmic stainings (not shown).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Fcalpha R and gamma  chain are mostly expressed in the same cells. Viable U937 cells preincubated with an excess of human IgG to block Fcgamma R were stained directly with PE-labeled A59 anti-Fcalpha R mAb or with an irrelevant PE-labeled IgG1 control. After washes, digitonin-permeabilized cells were stained with anti-gamma chain rabbit antiserum or a control rabbit serum and with FITC-labeled goat anti-rabbit antibodies as a developing reagent, as described under "Experimental Procedures." Two-color immunofluorescence analysis was then carried out by flow cytometry. The values inside the boxes represent the percentage of cells.

Modulation of the gamma  Chain Association with Fcalpha R on U937 Cells by Phorbol Esters and IFN-gamma -- Because a variety of agents have been described to modulate surface expression of Fcalpha R (15, 17, 38, 53, 54), we investigated whether they also affected expression of Fcalpha R-gamma 2 complexes as determined by coimmunoprecipitation. U937 cells were cultured with IL-1beta , IFN-gamma , GM-CSF, PMA, or ionomycin for 18 h. Table I shows that Fcalpha R expression on the cell surface was enhanced significantly by PMA or GM-CSF, whereas ionomycin diminished receptor expression by about half. Interestingly, despite the lack of effect on Fcalpha R surface expression, IFN-gamma promoted a significant increase (about 1.5-fold) in gamma  chain association with Fcalpha R. PMA also significantly favored gamma  chain association with Fcalpha R (about 1.8-fold). Treatment with IL-1beta , GM-CSF, or ionomycin had no significant effect on gamma  chain association with Fcalpha R.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Modulation of the gamma  chain association with Fcalpha R on U937 cells

gamma -less Fcalpha R Binds IgA but Does Not Induce Exocytosis-- We first established stable transfectants expressing either a wild-type or a mutant (R209L) Fcalpha R by using the Fcalpha R-negative rat mast cell line RBL-2H3. As shown in Fig. 6A, the selected transfectants expressed similar levels of Fcalpha R. In contrast to the wild-type, the mutant receptor did not associate with endogenous gamma  chains of RBL cells (Fig. 6B). Both types of receptor specifically bound monomeric and polymeric IgA1 or IgA2 molecules, as this binding was inhibited by My43 anti-Fcalpha R mAb (Table II). However, gamma -less Fcalpha R bound more IgA than wild-type Fcalpha R, despite their similar levels of Fcalpha R expression (evaluated using mAb A77). The capacity of wild-type and mutant (R209L) receptors to mediate downstream events was examined by measuring the capacity of cells to degranulate in response to receptor stimulation. As a control the releasing capability of each individual transfectant was tested by stimulating cells through Fcepsilon RI. Maximal release was obtained with PMA and Ca2+ ionophore. As shown in Fig. 7A, activation through mutant receptors did not lead to significant release of the granular enzyme, beta -hexosaminidase, whereas the response to stimulation through wild-type Fcalpha R was comparable to that induced by Fcepsilon RI.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Characterization of Fcalpha R transfectants. Panel A, expression of wild-type (WT) or mutant (R209L) Fcalpha R on RBL transfectants. 5 × 105 cells were stained with biotinylated A77 anti-Fcalpha R mAb (solid lines) or with biotinylated irrelevant IgG1kappa (dotted lines) and with streptavidin PE as described in Fig. 4. Panel B, absence of gamma  chain association with R209L Fcalpha R.Nontransfected (NT) RBL cells and transfectants expressing wild-type or R209L Fcalpha R (107) were solubilized in 1% digitonin lysis buffer and immunoprecipitated using A77 anti-Fcalpha R F(ab')2 fragments coupled to Sepharose 4B beads. Precipitated proteins were separated by 12.5% SDS-PAGE in nonreducing conditions and analyzed by immunoblotting using rabbit anti-gamma chain polyclonal Ab, horseradish peroxidase-conjugated anti-rabbit Ig Ab, and ECL as described in the legend of Fig. 2. Similar results were obtained with two other clones.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Binding of human IgA to RBL-2H3 transfectants expressing either wild-type or R209L mutant (gamma -less) human Fcalpha R


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Functions of gamma -associated and gamma -less Fcalpha R. Panel A, release of beta -hexosaminidase through Fcalpha R depends on their ability to associate with gamma  chains. RBL transfectants expressing either wild-type (open bars) or R209L (solid bars) Fcalpha R were incubated either with A77 anti-Fcalpha R mAb F(ab')2 followed by RAM F(ab')2 or mouse IgE plus dinitrophenyl-human serum albumin, as described under "Experimental Procedures." Secreted beta -hexosaminidase was analyzed in the supernatants. Maximal release was obtained after incubation of cells with PMA plus ionomycin. Similar results were obtained with two other clones. Panel B, kinetics of Fcalpha R-mediated endocytosis are independent of their association with gamma  chains. RBL transfectants expressing either wild-type (closed circles) or R209L (open circles) Fcalpha R were loaded with 125I-A77 anti-Fcalpha R mAb F(ab')2 at 4 °C for 1 h, washed, and incubated for 30 min with RAM F(ab')2 fragments. Cells were warmed rapidly to 37 °C for the indicated time periods followed by acid treatment at 4 °C to remove cell surface-bound mAbs. Non-acid-releasable counts were determined and expressed as percentage of total initial cell-associated counts and presented as the mean ± S.D. from at least three separate experiments.

gamma -less Fcalpha R·IgA Complexes Are Rapidly Endocytosed and Recycled to the Cell Surface-- We next examined endocytosis as a second function for both types of Fcalpha R. As shown in Fig. 7B, wild-type and mutant receptors internalized immune complexes at similar rates and amounts, indicating a potential endocytic function of gamma -less Fcalpha R molecules. Analysis of endocytosis by means of confocal microscopy revealed numerous intracellular vesicles containing Fcalpha R in transfectants expressing gamma -less or gamma -associated Fcalpha R (Fig. 8). However, close inspection revealed a marked difference in the localization of intracellular endocytic vesicles between the mutant and wild-type Fcalpha R transfectants. The internalized mutant (R209L) Fcalpha R was localized very close to the periphery, whereas wild-type receptors were also found in vesicles deeper inside the cell. Colocalization experiments revealed that Fcalpha R was partially found within recycling vesicles that stained positively for transferrin receptors in both types of transfectants (Fig. 9). Recycling was also suggested by flow cytometry experiments in which high amounts of receptor complexes were still detectable on the cell surface even after endocytosis for 90 min (Fig. 10A). R209L-Fcalpha R mutant transfectants had significantly more anti-Fcalpha R mAb-receptor complexes on the cell surface than wild-type transfectants (exceeding 50% of initial fluorescence intensity values), suggesting a preferential role of gamma -less receptors in recycling. To demonstrate receptor recycling, we took advantage of the recycling inhibitor primaquine. This drug blocks endocytic recycling vesicles from reaching the cell surface, thus accumulating the internalized ligand inside the cell, as described for recycling of internalized monomeric IgG by Fcgamma RI (50). 125I-Polymeric IgA was bound to transfected Fcalpha R molecules and allowed to internalize for various periods in the presence or absence of primaquine. As shown in Fig. 10B, primaquine-induced accumulation of internalized 125I-polymeric IgA was significantly higher in R209L-Fcalpha R+ mutant transfectants than in cells expressing wild-type receptors, indicating that gamma -less Fcalpha R recycles IgA toward the cell surface.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8.   Intracellular localization of internalized gamma -associated and gamma -less Fcalpha R after 60-min endocytosis. Adherent RBL cells (wild-type (WT) and R209L mutant) were incubated successively for 30 min on ice with F(ab')2 of the anti-Fcalpha R A77 mAb, with F(ab')2 fragments of RAM and with GAR coupled to FITC before incubation of the cells at 37 °C for 60 min as described under "Experimental Procedures." Cells were finally incubated or not with wheat germ agglutinin coupled to Texas Red (WGA) to delimit the plasma membrane. Cells were observed under a confocal microscope and optically sectioned at 1.5-µm intervals. A representative medial section of the horizontal slices is shown. No staining was observed when FITC-labeled secondary Ab or irrelevant IgG1 F(ab')2 was used.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 9.   Colocalization of Fcalpha R and transferrin receptors (TfR) in intracellular vesicles after 15-min endocytosis. Adherent RBL cells (wild-type (WT) and R209L mutant) were first cultured in serum-free DMEM for 30 min to deplete endogenous transferrin and then successively incubated with anti-Fcalpha R A77 mAb, RAM, and GAR coupled to FITC on ice as described in Fig. 8. After Fcalpha R staining, cells were incubated with 100 nM human transferrin coupled to Cy3 before incubation of the cells at 37 °C for 15 min. Cells were observed under a confocal microscope and optically sectioned at 1.5-µm intervals. A representative medial section of the horizontal slices is shown. No staining was observed when FITC-labeled secondary Ab or irrelevant IgG1 F(ab')2 was used.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 10.   gamma -less Fcalpha R recycles IgA and protects against degradation. Panel A, detection of Fcalpha R·anti-Fcalpha R mAb complexes on the cell surface after endocytosis. Wild type (open circles) and R209L mutant (closed circles) Fcalpha R transfectants were stained with A77 anti-Fcalpha R F(ab')2 fragments and then with RAM F(ab')2 fragments before incubation of cells at 37 °C for the times indicated. Cells were then surface stained with PE-labeled F(ab')2 fragments of goat anti-rabbit IgG Ab and analyzed by FACS. Results were calculated as follows 100 - [(100 × (x of A77 mAb incubated at 37 °C - x of negative control incubated at 37 °C)/(x of A77 mAb incubated at 0 °C - x of negative control incubated at 0 °C) in which x is the computer mean fluorescence intensity value of each FACS profile. *p < 0.05 in Student's t test. Panel B, increased intracellular accumulation of gamma -less Fcalpha R·IgA complexes in the presence of primaquine, a recycling inhibitor. Wild-type (open bars) and R209L mutated (closed bars) Fcalpha R transfectants were loaded with 125I-polymeric IgA1kappa at 4 °C for 1 h and then warmed rapidly to 37 °C in the presence or absence of 0.6 mM primaquine. Non-acid-releasable counts were determined at the time points indicated, calculated as a percentage of total initial cell-associated counts, and presented as the ratio of primaquine-treated to untreated cells. Results are expressed as the mean ± S.D. of three separate experiments. *p < 0.03 in Student's t test. Panel C, degradation of IgA bound to Fcalpha R. Cells expressing wild-type (open bars) or R209L mutant (closed bars) plated in triplicate for each time point were preincubated in serum-free medium for 30 min at 37 °C followed by incubation with 125I-labeled IgA for 1 h at 4 °C with streptavidin PE. Internalization of aggregated receptors was induced by incubating the cells at 37 °C. After the indicated times, trichloroacetic acid (TCA)-soluble radioactivity released into the medium was determined as described under "Experimental Procedures." Each experimental point is expressed as a percentage of the total radioactivity recovered. Results of three experiments are presented as the mean ± S.D.

gamma -less Fcalpha R Protects IgA from Degradation-- As it has recently been shown that gamma  chains mediate endocytic trafficking to lysosomes and are important for ligand degradation and antigen presentation (55), we examined the ability of wild-type and gamma -less Fcalpha R to sort for IgA degradation. Biotinylated and iodinated dimeric IgA was bound to Fcalpha R on cells, followed by cross-linking using streptavidin PE to induce internalization. IgA proteolysis was monitored by determining the fraction of trichloroacetic acid-soluble radioactive counts in the supernatant between 30 and 120 min after endocytosis induction. As shown in Fig. 10C, no degradation of dimeric IgA1kappa was observed in R209L transfectants, whereas time-dependent IgA1 degradation was measured in wild-type Fcalpha R containing gamma -associated receptors. No IgA degradation was detected in the absence of cross-linking in both types of transfectants (data not shown).

    DISCUSSION

In this study, we report the existence of both gamma -associated and gamma -less surface Fcalpha R on blood monocytes and neutrophils as well as on U937 cells. This is demonstrated by three different technical approaches, which included confocal microscopy after Fcalpha R endocytosis, SDS-PAGE analysis of Fcalpha R immunodepleted in gamma  chains, and finally by coimmunoprecipitation assay of Fcalpha R using anti-gamma Ab. Results of this last assay suggest that gamma -less Fcalpha R represent a significant fraction of surface Fcalpha R molecules. The majority of cells express the two types of Fcalpha R.

Although the positively charged arginine residue at position 209 of the Fcalpha R transmembrane domain is critical for the interaction with the gamma  chain (Ref. 28 and our results), the mechanism underlying and regulating the partial association of Fcalpha R with the gamma  chain is unknown. Genomic cloning has revealed a single gene encoding Fcalpha R (56),2 suggesting that the partial association of Fcalpha R and the gamma  chain cannot be explained by the presence of a structurally different Fcalpha R protein. This is further supported by experiments showing partial gamma  association in RBL cells transfected with the Fcalpha R cDNA. It is also unlikely that intracellular amounts of gamma  chains are limiting for Fcalpha R-gamma 2 expression because the amount of coprecipitated receptors with anti-gamma was unchanged even when human gamma  chains were overexpressed. Rather, our data suggest that gamma  chain association with Fcalpha R may be a regulated process susceptible to modulation by a variety of agents. In this context, it is interesting to note that IFN-gamma favored gamma  chain association with Fcalpha R independently of surface expression of the corresponding alpha  chains, whereas phorbol esters increased both the amount of total Fcalpha R and the percentage of Fcalpha R-gamma 2. GM-CSF enhanced total Fcalpha R but not Fcalpha R-gamma 2. Furthermore, recruitment of the tyrosine kinases syk and Btk after Fcalpha R activation is modulated by these agents (32) and may be a consequence of amounts of Fcalpha R-gamma 2 complexes. Our results do not rule out the presence of another uncharacterized chain that would compete with the gamma  chain for association with the arginine residue in the Fcalpha R transmembrane domain. Taken together these results indicate that the formation of multimeric Fcalpha R is independent of the amounts of gamma  chains expressed and can be regulated by environmental factors that could be of physiologic relevance at inflammatory sites.

To examine the functional role of gamma -less Fcalpha R we established transfectants expressing gamma -less Fcalpha R (R209L mutants) or both types of receptor using the mast cell line RBL-2H3. We found that mutant gamma -less Fcalpha R bound IgA as efficiently, or even better, than wild-type receptors that contained Fcalpha R-gamma 2. This seems to be different from Fcgamma RI and III where coexpression with gamma  chain enhances ligand affinity (57). Our results confirm that gamma  chain association with Fcalpha R is not essential for IgA binding (16, 20). After cross-linking, cells expressing gamma -less Fcalpha R failed to release the granular marker beta -hexosaminidase after Fcalpha R aggregation, suggesting that gamma  chains play a key role in Fcalpha R-mediated signaling pathways leading to exocytosis. A role for IgA in eosinophil degranulation has been demonstrated previously (58). Our study also corroborates previous observations on B cell transfectants expressing R209L Fcalpha R in which downstream signals such as Ca2+ mobilization and IL-2 release were absent (28).

Although gamma -less Fcalpha R were unable to mediate downstream signaling, we found that both wild-type and mutant gamma -less Fcalpha R were able to endocytose after receptor clustering. Mutant gamma -less Fcalpha R was as efficient as wild-type receptors for internalization. Thus, endocytosis mediated by mutant gamma -less Fcalpha R does not depend on the presence of tyrosine-based motifs in the cytoplasmic tail. Indeed, this has also been shown for other FcR lacking the gamma  chain as is the case of Fcgamma RI and Fcgamma RIIb2 that mediate endocytosis of immune complexes (59-61). Our results point to major differences in endocytic pathways between these two forms of Fcalpha R. In particular, internalized gamma -less Fcalpha R were only localized close to the periphery, whereas internalized wild-type Fcalpha R (containing gamma -less and gamma -associated receptors) underwent deeper compartmentalization, suggesting gamma  chain sorting for the endo-lysosomal pathway. Fcalpha R endo-lysosomal compartmentalization has been demonstrated previously on blood monocytes by their colocalization with cathepsin D (38). Furthermore, a role for gamma  chains in mediating endocytic sorting to lysosomes that leads to antigen presentation has been demonstrated recently for Fcgamma R (55). Therefore, we focused on the characterization of biological functions mediated by gamma -less Fcalpha R. Intracellular vesicles containing gamma -less Fcalpha R colocalized with those containing transferrin receptors, suggesting that they were involved in recycling of Fcalpha R and its bound ligand. Further evidence for the recycling of IgA by mutant gamma -less Fcalpha R was provided by the effects of primaquine, an inhibitor of receptor recycling. The significant increase in internalized polymeric IgA by transfectants expressing mutant gamma -less Fcalpha R treated by primaquine is strongly indicative of Fcalpha R-ligand recycling. Reflux of IgA toward the cell surface has been observed in blood monocytes from patients with alcoholic liver cirrhosis who have increased levels of serum IgA (38). Finally, our results indicate that mutant gamma -less Fcalpha R are unable to sort for IgA degradation even when cells are cultured for 2 h with cross-linked dimeric IgA, whereas cells expressing gamma -associated Fcalpha R degraded bound IgA.

Taken together, these findings point to the existence of myeloid cells expressing two types of Fcalpha R with or without gamma  chains. They differ in the type of endocytic pathway used for the internalized ligand, which led us to propose the existence of an alternative mechanism that protects IgA from degradation. Because IgA bound to wild-type Fcalpha R is not degraded without cross-linking, the use of each pathway may depend on the degree of aggregation of Fcalpha R molecules. Cross-linking by IgA-immune complexes could thus increase numbers of Fcalpha R-gamma 2 complexes delivering signals for downstream events such as degradation of IgA-immune complexes, processing, and antigen presentation. In agreement with this proposal, a previous study has shown that only large sized macromolecular IgA are efficiently and rapidly cleared from the circulation in humans, whereas clearance of smaller sized IgA polymers is considerably slower (62).

The protective role of gamma -less Fcalpha R may be important in view of maintaining serum IgA concentrations that would certainly counterbalance the rapid catabolism of IgA through other receptors such as the hepatocyte asialoglycoprotein receptor, which interacts with IgA through their carbohydrates (63, 64). Simultaneous expression of gamma -associated and gamma -less Fcalpha R might thus increase cellular flexibility in carrying out alternative functions, independently, which either mediate IgA-antigen processing and presentation to major histocompatibility complex molecules or recycle the IgA monomer continuously to achieve serum IgA homeostasis.

    ACKNOWLEDGEMENTS

We thank Drs. D. Presky, J. Kochan, J. P. Kinet, and L. Shen for providing the antibodies; G. Gautier for help; R. Rousseau and B. Iannascoli for technical assistance; Dr. J. Salamero for advice in confocal microscopy; Drs. M. Benhamou and M. Throsby for critical reading; and M. Lillie-Kadouche and M. Netter for preparing prints for this manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Supported by the Association pour la Recherche sur le Cancer Grant 1110.

** Supported by the Association pour la Recherche sur le Cancer Grant 6490 and by the FRM-Sidaction. To whom correspondence should be addressed: INSERM U25, Hôpital Necker, 161, rue de Sèvres, 75743 Paris Cedex 15, France. Tel.: 33-1-4449-5366; Fax: 33-1-4306-2388; E-mail: monteiro{at}necker.fr.

2 R. C. Monteiro and U. Blank, unpublished results.

    ABBREVIATIONS

The abbreviations used are: FcR, Fc receptor(s); Ab, antibody(ies); mAb, monoclonal antibody(ies); PE, phycoerythrin; PAGE, polyacrylamide gel electrophoresis; RAM, rabbit anti-mouse Ig; GAM, goat antibody specific for mouse Ig; GAR, goat anti-rabbit Ig; FITC, fluorescein isothiocyanate; PMA, phorbol 12-myristate 13-acetate; IFN-gamma , interferon-gamma ; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; RBL, rat basophilic leukemia; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter.

    REFERENCES
Top
Abstract
Introduction
References
  1. Conley, M. E., and Delacroix, D. L. (1987) Ann. Int. Med. 106, 892-899[Medline] [Order article via Infotrieve]
  2. Mestecky, J., and Russell, M. W. (1997) Biochem. Soc. Trans. 25, 457-462[Medline] [Order article via Infotrieve]
  3. Solomon, A. (1980) Cancer Markers, p. 57, Humana Press, Clifton, NJ
  4. Heremans, J. F. (1974) The Antigens, p. 365, Academic Press, New York
  5. Van Epps, D. E., and Williams, J. R. (1976) J. Exp. Med. 144, 1227-1242[Abstract]
  6. Wilton, J. M. A. (1978) Clin. Exp. Immunol. 34, 423-428[Medline] [Order article via Infotrieve]
  7. Wolf, H. M., Fischer, M. B., Puhringer, H., Samstag, A., Vogel, E., and Eibl, M. M. (1994) Blood 83, 1278-1288[Abstract/Free Full Text]
  8. Nikolova, E. B., and Russell, M. W. (1995) J. Leukocyte Biol. 57, 875-882[Abstract]
  9. Wolf, H. M., Hauber, I., Gulle, H., Samstag, A., Fischer, M. B., Ahmad, R. U., and Eibl, M. M. (1996) Clin. Exp. Immunol. 105, 537-543[Medline] [Order article via Infotrieve]
  10. Russell, M. W., Sibley, D. A., Nikolova, E. B., Tomana, M., and Mestecky, J. (1997) Biochem. Soc. Trans. 25, 466-470[Medline] [Order article via Infotrieve]
  11. Morton, H. C., van Egmond, M., and van de Winkel, J. G. J. (1996) Crit. Rev. Immunol. 16, 423-440[Medline] [Order article via Infotrieve]
  12. Levy Polat, G., Laufer, J., Fabian, I., and Passwell, J. H. (1993) Immunology 80, 287-292[Medline] [Order article via Infotrieve]
  13. Patry, C., Herbelin, A., Lehuen, A., Bach, J. F., and Monteiro, R. C. (1995) Immunology 86, 1-5[Medline] [Order article via Infotrieve]
  14. Albrechtsen, M., Yeaman, G. R., and Kerr, M. A. (1988) Immunology 64, 201-205[Medline] [Order article via Infotrieve]
  15. Monteiro, R. C., Kubagawa, H., and Cooper, M. D. (1990) J. Exp. Med. 171, 597-613[Abstract]
  16. Maliszewski, C. R., March, C. J., Schoenborn, M. A., Gimpel, S., and Shen, L. (1990) J. Exp. Med. 172, 1665-1672[Abstract]
  17. Monteiro, R. C., Hostoffer, R. W., Cooper, M. D., Bonner, J. R., Gartland, G. L., and Kubagawa, H. (1993) J. Clin. Invest. 92, 1681-1685[Medline] [Order article via Infotrieve]
  18. Carayannopoulos, L., Hexham, J. M., and Capra, J. D. (1996) J. Exp. Med. 183, 1579-1586[Abstract]
  19. Chevailler, A., Monteiro, R. C., Kubagawa, H., and Cooper, M. D. (1989) J. Immunol. 142, 2244-2249[Abstract/Free Full Text]
  20. Reterink, T. J. F., Vanzandbergen, G., Vanegmond, M., Klarmohamad, N., Morton, C. H., van de Winkel, J. G. J., and Daha, M. R. (1997) Eur. J. Immunol. 27, 2219-2224[Medline] [Order article via Infotrieve]
  21. Patry, C., Sibille, Y., Lehuen, A., and Monteiro, R. C. (1996) J. Immunol. 156, 4442-4448[Abstract]
  22. Morton, H. C., Schiel, A. E., Janssen, S. W. J., and van de Winkel, J. G. J. (1996) Immunogenetics 43, 246-247[CrossRef][Medline] [Order article via Infotrieve]
  23. Pleass, R. J., Andrews, P. D., Kerr, M. A., and Woof, J. M. (1996) Biochem. J. 318, 771-777[Medline] [Order article via Infotrieve]
  24. Reterink, T. J. F., Verweij, C. L., Vanes, L. A., and Daha, M. R. (1996) Gene (Amst.) 175, 279-280[CrossRef][Medline] [Order article via Infotrieve]
  25. Vandijk, T. B., Bracke, M., Caldenhoven, E., Raaijmakers, J. A. M., Lammers, J. W. J., Koenderman, L., and Degroot, R. P. (1996) Blood 88, 4229-4238[Abstract/Free Full Text]
  26. Pfefferkorn, L. C., and Yeaman, G. R. (1994) J. Immunol. 153, 3228-3236[Abstract/Free Full Text]
  27. Saito, K., Suzuki, K., Matsuda, H., Okumura, K., and Ra, C. (1995) J. Allergy Clin. Immunol. 96, 1152-1160[Medline] [Order article via Infotrieve]
  28. Morton, H. C., van de Herik-Oudijk, I. E., Vossebeld, P., Snijders, A., Verhoeven, A. J., Capel, P. J. A., and van de Winkel, J. G. J. (1995) J. Biol. Chem. 270, 29781-29787[Abstract/Free Full Text]
  29. Kinet, J. P., Alcaraz, G., Leonard, A., Wank, S., and Metzger, H. (1985) Biochemistry 24, 4117-4124[Medline] [Order article via Infotrieve]
  30. Scholl, P. R., and Geha, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8847-8850[Abstract]
  31. Gulle, H., Samstag, A., Eibl, M. M., and Wolf, H. M. (1998) Blood 91, 383-391[Abstract/Free Full Text]
  32. Launay, P., Lehuen, A., Kawakami, T., Blank, U., and Monteiro, R. C. (1998) J. Leukocyte Biol. 63, 636-642[Abstract]
  33. Daeron, M. (1997) Annu. Rev. Immunol. 15, 203-234[CrossRef][Medline] [Order article via Infotrieve]
  34. Lobell, R. B., Arm, J. P., Raizman, M. B., Austen, K. F., and Katz, H. R. (1993) J. Biol. Chem. 268, 1207-1212[Abstract/Free Full Text]
  35. Monteiro, R. C., Cooper, M. D., and Kubagawa, H. (1992) J. Immunol. 148, 1764-1770[Abstract/Free Full Text]
  36. Shen, L., Lasser, R., and Fanger, M. W. (1989) J. Immunol. 143, 4117-4122[Abstract/Free Full Text]
  37. Schöneich, J. T., Wilkinson, V. L., Kado-Fong, H., Presky, D. H., and Kochan, J. P. (1992) J. Immunol. 148, 2181-2185[Abstract/Free Full Text]
  38. Silvain, C., Patry, C., Launay, P., Lehuen, A., and Monteiro, R. C. (1995) J. Immunol. 155, 1606-1618[Abstract]
  39. Letourneur, O., Kennedy, I. C., Brini, A. T., Ortaldo, J. R., O'Shea, J. J., and Kinet, J. P. (1991) J. Immunol. 147, 2652-2656[Abstract/Free Full Text]
  40. Barsumian, E. L., Isersky, C., Petrino, M. G., and Siraganian, R. P. (1981) Eur. J. Immunol. 11, 317-323[Medline] [Order article via Infotrieve]
  41. Kuster, H., Thompson, H., and Kinet, J. P. (1990) J. Biol. Chem. 265, 6448-6452[Abstract/Free Full Text]
  42. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol. 8, 466-472[Medline] [Order article via Infotrieve]
  43. Roa, M., Paumet, F., Lemao, J., David, B., and Blank, U. (1997) J. Immunol. 159, 2815-2823[Abstract]
  44. Goding, J. W. (1980) J. Immunol. 124, 2082-2088[Free Full Text]
  45. Laemmli, U. K. (1970) Nature 277, 680-684
  46. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
  47. Miller, L., Blank, U., Metzger, H., and Kinet, J. P. (1989) Science 244, 334-337[Medline] [Order article via Infotrieve]
  48. Salisbury, J. G., and Graham, J. M. (1981) Biochem. J. 194, 351-355[Medline] [Order article via Infotrieve]
  49. Tadou, G., Varin-Blank, N., Jouin, H., Marchand, F., Weyer, A., and Blank, U. (1993) Int. Arch. Allergy Immunol. 100, 344-350[Medline] [Order article via Infotrieve]
  50. Harrison, P. T., Davis, W., Norman, J. C., Hockaday, A. R., and Allen, J. M. (1994) J. Biol. Chem. 269, 24396-24402[Abstract/Free Full Text]
  51. White, S., Hatton, S. R., Siddiqui, M. A., Parker, C. D., Trowbridge, I. S., and Collawn, J. F. (1998) J. Biol. Chem. 273, 14355-14362[Abstract/Free Full Text]
  52. Saudrais, C., Spehner, D., de La Salle, H., Bohbot, A., Cazenave, J. P., Goud, B., Hanau, D., and Salamero, J. (1998) J. Immunol. 160, 2597-2607[Abstract/Free Full Text]
  53. Weisbart, R. H., Kacena, A., Schuh, A., and Golde, D. W. (1988) Nature 332, 647-648[CrossRef][Medline] [Order article via Infotrieve]
  54. Shen, L., Collins, J. E., Schoenborn, M. A., and Maliszewski, C. R. (1994) J. Immunol. 152, 4080-4086[Abstract/Free Full Text]
  55. Amigorena, S., Lankar, D., Briken, V., Gapin, L., Viguier, M., and Bonnerot, C. (1998) J. Exp. Med. 187, 505-515[Abstract/Free Full Text]
  56. Dewit, T. P. M., Morton, H. C., Capel, P. J. A., and van de Winkel, J. G. J. (1995) J. Immunol. 155, 1203-1209[Abstract]
  57. Miller, K. L., Duchemin, A. M., and Anderson, C. L. (1996) J. Exp. Med. 183, 2227-2234[Abstract]
  58. Abu-Ghazaleh, R. I., Fujisawa, T., Mestecky, J., Kyle, R. A., and Gleich, G. J. (1989) J. Immunol. 142, 2393-2400[Abstract/Free Full Text]
  59. Davis, W., Harrison, P. T., Hutchinson, M. J., and Allen, J. M. (1995) EMBO J. 14, 432-441[Abstract]
  60. Miettinen, H. M., Rose, J. K., and Mellman, I. (1989) Cell 58, 317-327[Medline] [Order article via Infotrieve]
  61. Amigorena, S., Bonnerot, C., Drake, J. R., Choquet, D., Hunziker, W., Guillet, J. G., Webster, P., Sautes, C., Mellman, I., and Fridman, W. H. (1992) Science 256, 1808-1812[Medline] [Order article via Infotrieve]
  62. Rifai, A., Schena, P., Montinaro, V., Mele, M., D'Addabbo, A., Nitti, L., and Pezzullo, J. C. (1989) Lab. Invest. 61, 381-388[Medline] [Order article via Infotrieve]
  63. Brown, T. A., Russell, M. W., and Mestecky, J. (1982) J. Immunol. 128, 2183-2186[Abstract/Free Full Text]
  64. Stockert, R. J., Kressner, M. S., Collins, J. C., Sternlieb, I., and Morell, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6229-6231[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.