Alternative Endocytic Pathway for Immunoglobulin A Fc Receptors
(CD89) Depends on the Lack of FcR
Association and Protects against
Degradation of Bound Ligand*
Pierre
Launay
,
Claire
Patry§,
Agnès
Lehuen
,
Benoit
Pasquier
,
Ulrich
Blank¶
, and
Renato C.
Monteiro
**
From
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 |
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) (Fc
R) are
expressed with or without the
chain on monocytes and neutrophils.
-less Fc
R represent a significant fraction of surface Fc
R
molecules even on cells overexpressing the
chain. The Fc
R-
2
association is up-regulated by phorbol esters and interferon-
. To
characterize
-less Fc
R functionally, we generated mast cell
transfectants expressing wild-type human Fc
R or a receptor with a
point mutation (Arg
Leu at position 209) which was unable to
associate with the
chain. Mutant
-less Fc
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 Fc
R-mediated endocytosis; however, the endocytosis
pathways of the two types of receptor differed. Whereas mutant Fc
R
were localized mainly in early endosomes, those containing Fc
R-
2 were found in endo-lysosomal compartments. Mutant
-less Fc
R recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of Fc
R, associated or unassociated with
chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.
 |
INTRODUCTION |
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) (Fc
R,1
CD89), resulting in various immune effector functions such as phagocytosis, oxidative burst, and cytokine release (11-13).
Fc
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 C
2 and C
3 domains (14-18). Polymeric
IgA binds more efficiently to Fc
R than does monomeric IgA (19, 20).
Fc
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). Fc
R are associated with the
disulfide-linked FcR
chain homodimer (26-28). This interaction is
resistant to treatment with Nonidet P-40 detergent, which contrasts
with the dissociation of
chains from Fc
RI or Fc
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
Fc
R and
chain, respectively (28). The
chain contains a
common immunoreceptor tyrosine-based activation motif in its
cytoplasmic tail. Recently, it has been shown that signaling through
Fc
R-
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-
(IFN-
) and/or
phorbol esters, indicating that activation of tyrosine kinases through
Fc
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
or
chains, and depend on
their specific retention motifs to be fully expressed on the cell
surface (33). In the absence of the
chain they are degraded rapidly
in the endoplasmic reticulum as in the case of Fc
RIII (34). One
remarkable feature of Fc
R is that these receptors can be expressed
fully at the surface of COS cells after transfection, without the
signaling
subunits (16). Despite the role of the
chain in
downstream Fc
R signaling (28), we wondered whether the Fc
R could
exist and function as receptors when unassociated with the
chain
(
-less Fc
R) on myeloid cells. We have identified significant
amounts of
-less Fc
R in several cell types, including monocytes,
neutrophils, and transfected cells overexpressing the
chain.
-less Fc
R and Fc
R-
2 are expressed on the same cells, and
this constitutes the basis for differential endocytosis pathways of
IgA, in which
-less receptors recycle IgA toward the cell surface
whereas Fc
R-
2 undergo endo-lysosomal sorting for IgA degradation.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
The following mouse mAb were used: A59
(IgG1
), A77 (IgG1
) mAb specific for Fc
R (35), an irrelevant
IgG1
control mAb (clone 7.1 anti-glutathione
S-transferase protein). My43 anti-Fc
R mAb (IgM) was a
gift from Dr. L. Shen, Dartmouth Medical School, Lebanon, NH (36).
Phycoerythrin-labeled anti-Fc
R A59 (A59-PE) was purchased from
PharMingen (San Diego). 4D8 anti-FcR-
chain mAb (IgG2b
) 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 IgG1
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-
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-
(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)-1
(Rhône-Poulenc
Santé, Vitry, France). Rat basophilic leukemia cells (RBL-2H3)
(40) were transfected with human Fc
R and/or human
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
Fc
R containing the R209L mutation was constructed by amplifying base
pairs 591-891 of a previously described Fc
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 Fc
R a.1, Fc
R (R209L), and human
chain (41) were subcloned into pSR
Neo (kindly provided by Dr. J. Di Santo, INSERM U429) a modified version of the pcDL-SR
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
Fc
R expression by means of flow cytometry. One Fc
R-expressing
clone was chosen and cotransfected with pSR
Neo-human
-chain (30 µg) and pSR
-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 Fc
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
Fc
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-
(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-Fc
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-
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-Fc
R mAb (nonspecific counts were always <3%). For RBL transfectants, 3 µl
of polyclonal rabbit anti-
chain antiserum plus protein A-coupled beads were used to coprecipitate both rat and human
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 Fc
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 Fc
R, were stained directly with 10 µl of
PE-labeled A59 anti-Fc
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-
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.
-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 Fc
RI stimulation) or with irrelevant IgG1 F(ab')2 fragments (for Fc
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-
-D-glucosamine (1.3 mg/ml Sigma).
The total cellular content of
-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-Fc
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-Fc
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 IgA1
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 IgA1
(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 IgA1
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-Fc
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-Fc
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
chain
staining, cells were permeabilized with 0.05% saponin (Sigma) and
stained with 3.5 µg/ml purified anti-
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
-less Fc
R on Human Myeloid
Cells--
Because Fc
R can be fully expressed at the surface of COS
cells after transfection without the signaling
subunits (16), we
investigated whether all Fc
R expressed on PMA-treated U937 cells
were associated with the
chain homodimer by means of confocal microscopy. Because
-less and
-associated Fc
R could not
initially be distinguished on the cell surface, we performed short term endocytosis of Fc
R·anti-Fc
R mAb complexes (FITC-labeled) to examine whether all internalized receptors colocalized with the
chain (Texas Red-labeled) in the vesicles. As shown in Fig. 1, two types of intracellular vesicles
were detected in single cells, in which Fc
R was either colocalized
(yellow) or not colocalized (green) with the
chain. These results strongly suggest the existence of
-less and
-associated Fc
R in the same cells.

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Fig. 1.
Lack of colocalization between
chain and some Fc R within
intracellular vesicles after short term endocytosis. PMA-treated
U937 cells preincubated with human IgG (10 mg/ml) to block Fc R were
incubated with anti-Fc R A77 F(ab')2 fragments plus GAM
coupled to FITC on ice as described under "Experimental
Procedures." After Fc R staining, cells were incubated at 37 °C
for 3 min, fixed, permeabilized, and stained with anti- 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
-less receptor population could also be
detected in detergent extracts, we performed immunodepletion experiments using digitonin-solubilized cells because the Fc
R-
2 interaction is resistant to digitonin treatment (26). As shown in Fig.
2, immunoprecipitation of
surface-iodinated Fc
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-
chain antibody gave rise to
similarly sized species. Treatment of anti-
chain mAb-associated
glycoproteins with N-glycanase resulted in 32- and 36-kDa
bands that comigrated with those observed with the anti-Fc
R mAb
(Fig. 2A). After extensive immunodepletion with anti-
chain mAb, anti-Fc
R mAb-reactive molecules could still be
precipitated (Fig. 2A). Similar results were obtained with
PMA-treated U937 cells using a polyclonal anti-
chain Ab (Fig.
2B), in which the same treatment eliminated most of the
chain molecules (Fig. 2B, bottom). Conversely,
immunoadsorptions with an anti-Fc
R mAb (A59) completely eliminated
55-75 kDa-4D8 mAb reactive proteins (not shown). These results reveal
two forms of Fc
R,
-less and
-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 Fc
R.

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Fig. 2.
Identification of
-less Fc 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 IgG1 (lanes 1 and
4), anti-Fc R mAbs (lanes 2 and 5;
A, A59 and B, A77 F(ab')2), or
anti- 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- 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- chain polyclonal Ab and horseradish
peroxidase-conjugated anti-rabbit Ig Ab plus ECL.
|
|
To estimate the amounts of
-less Fc
R we used a
coimmunoprecipitation assay validated previously for Fc
RI (47).
Cells were loaded with 125I-labeled anti-Fc
R mAb
F(ab')2 fragments. Labeled receptor-Ab complexes were
solubilized in the presence of 1% digitonin and precipitated with
either anti-
chain (4D8)- or anti-mouse Ig Ab. As shown in Fig.
3A, whereas total precipitable
amounts of 125I-Ab·Fc
R complexes using anti-mouse Ig
Abs exceeded 70% (total Fc
R), those precipitated with the
anti-FcR
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-Fc
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-
chain antiserum (data not shown). We then analyzed the time-dependent dissociation of the
chain from the
Fc
R in digitonin lysates. Fc
R-
complexes were very stable over
a 24-h period, ruling out the possibility that partial association of
Fc
R with the
chain was the result of their instability in
detergents (Fig. 3B).

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Fig. 3.
A. -less
Fc R represents the major fraction of
Fc 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-Fc 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- 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-Fc 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
Fc R- association in the mild detergent, digitonin. PMA-activated
U937 cells were incubated with 125I-labeled A77 anti-Fc 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- chain mAb coupled to beads for
2 h at 4 °C and analyzed as described in panel
A.
|
|
Partial Association of Fc
R with the
Chain Is Not Dependent
on the Amounts of Expressed
Chain--
To examine the effect of
the amounts of expressed
chain, we transfected a previously
established Fc
R+ RBL transfectant (32) with the human
chain. One clone (
) expressing large amounts of human
chain was selected (Fig. 4, A
and B). Coimmunoprecipitation experiments, using a
polyclonal Ab that recognizes both rat and human
chains, showed
that in these transfectants the fraction of
-associated receptors
did not increase compared with cells transfected with Fc
R only (Fig. 4C). As a control we coprecipitated the Fc
RI using the
same anti-
Ab. In agreement with previous observations (47), a major
fraction of Fc
RI (>50%) was coprecipitated in these transfectants
(data not shown). This demonstrated that the amount of anti-
Ab used in the assay was not limiting. These results also indicated that association of the
chain with Fc
R did not depend on the amount of expressed
chain.

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Fig. 4.
Reconstitution of the partial association
of chains with human
Fc R on the rat mast cell line, RBL-2H3.
Panel A, overexpression of human chain. Transfectants
expressing human Fc R a.1 ( ) (32) were cotransfected with chain and selected on the basis of the reactivity with anti-human chain 4D8 mAb. 107 cells were then solubilized in 0.5%
Nonidet P-40 lysis buffer and immunoprecipitated using anti-human
(hu) 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- chain polyclonal Ab (poly) and horseradish
peroxidase-conjugated anti-rabbit Ig Ab. One out of five clones was
chosen ( ). PMA-treated U937 cells were used as positive control.
Panel B, Fc R expression. 5 × 105
nontransfected (NT), human Fc R-transfected ( ) and
human Fc R/ transfected ( ) cells were stained with
biotinylated A77 anti-Fc R mAb (solid lines) or
biotinylated irrelevant IgG1 (dotted line, for  )
and with streptavidin PE followed by FACS analysis. Panel C,
partial association of Fc R with the chain on transfectants.
Because human Fc R molecules were previously found to be associated
with rat chain on transfectants (32), experiments were performed
using a polyclonal Ab that recognizes both rat and human chains.
5 × 106 cells were incubated with
125I-labeled A77 anti-Fc 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- 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%.
|
|
Fc
R and
Chain Are Coexpressed in a Single Cell
Population--
The experiments shown in Fig. 1 suggested that both
-less and
-associated Fc
R are expressed on the same cells. To
further exclude the possibility that partial association was caused by heterogeneity in
chain expression on a given cell population, we
carried out two-color FACS analysis of cell surface Fc
R and intracellular
chain. Fig. 5 shows the
expression of both Fc
R and
chain on U937 cells, before and after
IFN-
or PMA treatment. The majority of cells were
Fc
R+/
+. 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 Fc
R and
chain
was also found in neutrophils as determined by cytoplasmic stainings
(not shown).

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Fig. 5.
Fc R and
chain are mostly expressed in the same cells.
Viable U937 cells preincubated with an excess of human IgG to block
Fc R were stained directly with PE-labeled A59 anti-Fc R mAb or
with an irrelevant PE-labeled IgG1 control. After washes,
digitonin-permeabilized cells were stained with anti- 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
Chain Association with Fc
R on U937
Cells by Phorbol Esters and IFN-
--
Because a variety of
agents have been described to modulate surface expression of Fc
R
(15, 17, 38, 53, 54), we investigated whether they also affected
expression of Fc
R-
2 complexes as determined by
coimmunoprecipitation. U937 cells were cultured with IL-1
, IFN-
,
GM-CSF, PMA, or ionomycin for 18 h. Table
I shows that Fc
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 Fc
R surface expression, IFN-
promoted a significant increase (about 1.5-fold) in
chain
association with Fc
R. PMA also significantly favored
chain
association with Fc
R (about 1.8-fold). Treatment with IL-1
,
GM-CSF, or ionomycin had no significant effect on
chain association
with Fc
R.
-less Fc
R Binds IgA but Does Not Induce Exocytosis--
We
first established stable transfectants expressing either a wild-type or
a mutant (R209L) Fc
R by using the Fc
R-negative rat mast cell line
RBL-2H3. As shown in Fig. 6A,
the selected transfectants expressed similar levels of Fc
R. In
contrast to the wild-type, the mutant receptor did not associate with
endogenous
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-Fc
R mAb (Table
II). However,
-less Fc
R bound more
IgA than wild-type Fc
R, despite their similar levels of Fc
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 Fc
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,
-hexosaminidase, whereas the response to stimulation through
wild-type Fc
R was comparable to that induced by Fc
RI.

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Fig. 6.
Characterization of Fc R transfectants.
Panel A, expression of wild-type (WT) or mutant
(R209L) Fc R on RBL transfectants. 5 × 105 cells
were stained with biotinylated A77 anti-Fc R mAb (solid
lines) or with biotinylated irrelevant IgG1 (dotted
lines) and with streptavidin PE as described in Fig. 4.
Panel B, absence of chain association with R209L
Fc R.Nontransfected (NT) RBL cells and transfectants
expressing wild-type or R209L Fc R (107) were solubilized
in 1% digitonin lysis buffer and immunoprecipitated using A77
anti-Fc 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- 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.
|
|
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Table II
Binding of human IgA to RBL-2H3 transfectants expressing either
wild-type or R209L mutant ( -less) human Fc R
|
|

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Fig. 7.
Functions of -associated and -less
Fc R. Panel A, release of -hexosaminidase through
Fc R depends on their ability to associate with chains. RBL
transfectants expressing either wild-type (open bars) or
R209L (solid bars) Fc R were incubated either with A77
anti-Fc 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 -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 Fc R-mediated
endocytosis are independent of their association with chains. RBL
transfectants expressing either wild-type (closed circles)
or R209L (open circles) Fc R were loaded with
125I-A77 anti-Fc 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.
|
|
-less Fc
R·IgA Complexes Are Rapidly Endocytosed and
Recycled to the Cell Surface--
We next examined endocytosis as a
second function for both types of Fc
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
-less Fc
R molecules. Analysis of
endocytosis by means of confocal microscopy revealed numerous intracellular vesicles containing Fc
R in transfectants expressing
-less or
-associated Fc
R (Fig.
8). However, close inspection revealed a
marked difference in the localization of intracellular endocytic
vesicles between the mutant and wild-type Fc
R transfectants. The
internalized mutant (R209L) Fc
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 Fc
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-Fc
R
mutant transfectants had significantly more anti-Fc
R mAb-receptor
complexes on the cell surface than wild-type transfectants (exceeding
50% of initial fluorescence intensity values), suggesting a
preferential role of
-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
Fc
RI (50). 125I-Polymeric IgA was bound to transfected
Fc
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-Fc
R+ mutant transfectants than in cells expressing
wild-type receptors, indicating that
-less Fc
R recycles IgA
toward the cell surface.

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Fig. 8.
Intracellular localization of
internalized -associated and
-less Fc 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-Fc 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.
|
|

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Fig. 9.
Colocalization of
Fc 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-Fc R A77 mAb, RAM, and GAR coupled to FITC on ice as described
in Fig. 8. After Fc 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.
|
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Fig. 10.
-less Fc R recycles IgA and protects
against degradation. Panel A, detection of
Fc R·anti-Fc R mAb complexes on the cell surface after
endocytosis. Wild type (open circles) and R209L mutant
(closed circles) Fc R transfectants were stained with A77
anti-Fc 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 -less
Fc R·IgA complexes in the presence of primaquine, a recycling
inhibitor. Wild-type (open bars) and R209L mutated
(closed bars) Fc R transfectants were loaded with
125I-polymeric IgA1 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 Fc 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.
|
|
-less Fc
R Protects IgA from Degradation--
As it has
recently been shown that
chains mediate endocytic trafficking to
lysosomes and are important for ligand degradation and antigen
presentation (55), we examined the ability of wild-type and
-less
Fc
R to sort for IgA degradation. Biotinylated and iodinated dimeric
IgA was bound to Fc
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 IgA1
was observed in R209L transfectants, whereas time-dependent IgA1 degradation was measured in
wild-type Fc
R containing
-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
-associated and
-less surface Fc
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 Fc
R
endocytosis, SDS-PAGE analysis of Fc
R immunodepleted in
chains,
and finally by coimmunoprecipitation assay of Fc
R using anti-
Ab.
Results of this last assay suggest that
-less Fc
R represent a
significant fraction of surface Fc
R molecules. The majority of cells
express the two types of Fc
R.
Although the positively charged arginine residue at position 209 of the
Fc
R transmembrane domain is critical for the interaction with the
chain (Ref. 28 and our results), the mechanism underlying and
regulating the partial association of Fc
R with the
chain is
unknown. Genomic cloning has revealed a single gene encoding Fc
R
(56),2 suggesting that the
partial association of Fc
R and the
chain cannot be explained by
the presence of a structurally different Fc
R protein. This is
further supported by experiments showing partial
association in RBL
cells transfected with the Fc
R cDNA. It is also unlikely that
intracellular amounts of
chains are limiting for Fc
R-
2
expression because the amount of coprecipitated receptors with anti-
was unchanged even when human
chains were overexpressed. Rather,
our data suggest that
chain association with Fc
R may be a
regulated process susceptible to modulation by a variety of agents. In
this context, it is interesting to note that IFN-
favored
chain
association with Fc
R independently of surface expression of the
corresponding
chains, whereas phorbol esters increased both the
amount of total Fc
R and the percentage of Fc
R-
2. GM-CSF
enhanced total Fc
R but not Fc
R-
2. Furthermore, recruitment of
the tyrosine kinases syk and Btk after Fc
R activation is modulated
by these agents (32) and may be a consequence of amounts of Fc
R-
2
complexes. Our results do not rule out the presence of another
uncharacterized chain that would compete with the
chain for
association with the arginine residue in the Fc
R transmembrane
domain. Taken together these results indicate that the formation of
multimeric Fc
R is independent of the amounts of
chains expressed
and can be regulated by environmental factors that could be of
physiologic relevance at inflammatory sites.
To examine the functional role of
-less Fc
R we established
transfectants expressing
-less Fc
R (R209L mutants) or both types
of receptor using the mast cell line RBL-2H3. We found that mutant
-less Fc
R bound IgA as efficiently, or even better, than wild-type receptors that contained Fc
R-
2. This seems to be
different from Fc
RI and III where coexpression with
chain
enhances ligand affinity (57). Our results confirm that
chain
association with Fc
R is not essential for IgA binding (16, 20).
After cross-linking, cells expressing
-less Fc
R failed to release the granular marker
-hexosaminidase after Fc
R aggregation,
suggesting that
chains play a key role in Fc
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 Fc
R in which downstream signals such as Ca2+
mobilization and IL-2 release were absent (28).
Although
-less Fc
R were unable to mediate downstream signaling,
we found that both wild-type and mutant
-less Fc
R were able to
endocytose after receptor clustering. Mutant
-less Fc
R was as
efficient as wild-type receptors for internalization. Thus, endocytosis
mediated by mutant
-less Fc
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
chain as is the case of
Fc
RI and Fc
RIIb2 that mediate endocytosis of immune complexes
(59-61). Our results point to major differences in endocytic pathways
between these two forms of Fc
R. In particular, internalized
-less
Fc
R were only localized close to the periphery, whereas internalized
wild-type Fc
R (containing
-less and
-associated receptors)
underwent deeper compartmentalization, suggesting
chain sorting for
the endo-lysosomal pathway. Fc
R endo-lysosomal compartmentalization
has been demonstrated previously on blood monocytes by their
colocalization with cathepsin D (38). Furthermore, a role for
chains in mediating endocytic sorting to lysosomes that leads to
antigen presentation has been demonstrated recently for Fc
R (55).
Therefore, we focused on the characterization of biological functions
mediated by
-less Fc
R. Intracellular vesicles containing
-less
Fc
R colocalized with those containing transferrin receptors,
suggesting that they were involved in recycling of Fc
R and its bound
ligand. Further evidence for the recycling of IgA by mutant
-less
Fc
R was provided by the effects of primaquine, an inhibitor of
receptor recycling. The significant increase in internalized polymeric
IgA by transfectants expressing mutant
-less Fc
R treated by
primaquine is strongly indicative of Fc
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
-less
Fc
R are unable to sort for IgA degradation even when cells are
cultured for 2 h with cross-linked dimeric IgA, whereas cells
expressing
-associated Fc
R degraded bound IgA.
Taken together, these findings point to the existence of myeloid cells
expressing two types of Fc
R with or without
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 Fc
R is
not degraded without cross-linking, the use of each pathway may depend
on the degree of aggregation of Fc
R molecules. Cross-linking by
IgA-immune complexes could thus increase numbers of Fc
R-
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
-less Fc
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
-associated and
-less Fc
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.
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-
, interferon-
;
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.
 |
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