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Blood, 1 December 2001, Vol. 98, No. 12, pp. 3429-3434
PHAGOCYTES
The cytoplasmic domain of Fc RIIA
(CD32) participates in phagolysosome
formation
Randall G. Worth,
Laura Mayo-Bond,
Moo-Kyung Kim,
Jan G. J. van de
Winkel,
Robert F. Todd III,
Howard R. Petty, and
Alan D. Schreiber
From the Department of Biological Sciences, Wayne State
University, Detroit, MI; Division of Hematology and Oncology,
University of Michigan School of Medicine, Ann Arbor, MI; Department of
Immunotherapy, Medarex Europe, University Medical Center, Utrecht, The
Netherlands; and Division of Hematology and Oncology, University of
Pennsylvania School of Medicine, Philadelphia, PA.
 |
Abstract |
Signaling motifs located within the cytoplasmic domain of certain
receptors contribute to lysosome fusion. Most studies have described
lysosome fusion with respect to endocytic receptors. Phagolysosome
fusion has not been extensively studied. To test the hypothesis that
the tail of Fc RIIA participates in phagolysosomal fusion, a
"reverse" genetic complementation system was used. It was
previously shown that complement receptor type 3 (CR3) can rescue the
phagocytic activity of a mutant FcRIIA lacking its cytoplasmic
domain (tail-minus form). This system has allowed us to study Fc
receptor-dependent phagocytosis and phagolysosome fusion in the
presence and absence of the cytoplasmic domain of FcRIIA.
Fluorescent dextran was used to label lysosomes. After target
internalization, wild-type FcRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of
FcRIIA containing a full-length cytoplasmic tail with the 2 ITAM
tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2)
CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that
observed with the wild-type receptor. In contrast, in the presence of
CR3 and the tail-minus form of FcRIIA, internalized particles did
not colocalize with dextran. Electron microscopy revealed that the
lysosomal enzyme acid phosphatase colocalized with immunoglobulin
G-coated targets internalized by wild-type FcRIIA but not by
tail-minus FcRIIA and CR3. Thus, the tail of FcRIIA contributes
to phagolysosome fusion by a mechanism that does not require a
functional ITAM sequence.
(Blood. 2001;98:3429-3434)
© 2001 by The American Society of Hematology.
| |
Introduction |
Phagocytosis is a crucial step toward the
eventual destruction of foreign particles by the immune system. After
separation from the plasma membrane, a phagosome must traffic to and
fuse with lysosomes. Lysosomes contain a battery of hydrolytic enzymes within a low pH environment.1 Endosome-to-lysosome
recognition is mediated by signaling motifs located within the
cytoplasmic domains of certain receptors.2,3 Some
investigators have suggested that the tyrosine-containing ITAM motif
found within the cytoplasmic domain of receptors such as the chain
of Fc receptor I (FcRI) and FcRIII and the cytoplasmic domain
of FcRIIA is responsible for phagocytic signaling in
antibody-dependent phagocytosis.4,5 It is possible that
signal sequences located in the cytoplasmic tails of these receptors
are responsible for mediating trafficking to lysosomes and the eventual
fusion of the 2 organelles. However, when the cytoplasmic domain of
FcRIIA or its crucial tyrosine residues are removed, phagocytosis is abolished.4,5 After the phagocytic activity of the
receptor is lost, there is no mechanism to study downstream properties of the receptors such as lysosome fusion.
FcRs are known to cooperate with complement receptors during
immunoglobulin G (IgG)-dependent phagocytosis and oxidant
production.6-9 One mechanism of Fc-to-complement
receptor cooperation involves physical association of these
receptors.10-13 These receptor interactions and
cooperation phenomena have been extended to urokinase-type plasminogen
activator receptors and CD14.8,14 It has previously been
observed that complement receptor type 3 (CR3) ( M  2, CD11b/CD18) rescues the phagocytic activity of the disabled tail-minus form of
FcRIIA.11 The ability of CR3 to complement the
phagocytic function of tail-minus FcRIIA has allowed us to study the
role of the cytoplasmic domain of FcRIIA in phagolysosomal fusion.
In the current study we observed that CR3 reconstitutes
FcR-dependent phagocytosis in a FcRIIA mutant where tyrosine
residues are mutated to phenylalanine (FcRIIA-ITAM mutant). By
employing a reverse genetic complementation strategy and 2 mutant forms of FcRIIA, we have studied the cytoplasmic domain's role in
phagolysosome formation.
We examined the hypothesis that the cytoplasmic domain of FcRIIA
contributes to phagolysosome formation and observed that the
cytoplasmic domain of FcRIIA participates in phagolysosome fusion.
This was shown by colocalization of IgG-coated cells with either
fluorescent dextran or acid phosphatase, 2 independent experimental
strategies to label lysosomes. Although wild-type FcRIIA supported
phagolysosome formation, the tail-minus form of FcRIIA did not.
However, FcRIIA (ITAM mutant), complemented with CR3 to restore
phagocytosis, retained their intrinsic lysosome signaling capacity.
Thus, the cytoplasmic tail of FcRIIA contributes to fusion of
phagosomes with lysosomes.
| |
Materials and methods |
Cell culture and transfections
Chinese hamster ovary (CHO) cells were transfected by
electroporation with a mixture of 1.5 µg pSVneo, 5 µg pBACD11b
(generated by replacing the CD11a complementary DNA in
pBACD11a15 with the CD11b complementary
DNA,16 a gift from D. Hickstein (University of Washington,
Seattle, WA), 5 µg pCMVBACD18, and 5 µg of either pRcCMVCD32 or a
variant of this CD32 plasmid containing a tail-minus mutation, as
described.4 Expansion and selection were performed as
previously described.11 Seven different clones were
generated: 161-24, which was not transfected but exposed to the
transfection protocol; 161-84, which expressed only CR3; 131-3, which
expressed wild-type FcRIIA; 135-12, expressing FcRIIA tailless
alone; 169-8 and 169-24, which both express the FcRIIA tailless and CR3; and 173-46, expressing both the wild-type FcRIIA and CR3.
In addition, one crucial experiment is to compare wild-type FcRIIA
with both FcRIIA tailless and an ITAM mutant of FcRIIA that
expresses a full-length FcRIIA cytoplasmic domain with Tyr Phe mutations in both of the ITAM motifs (FcRIIA ITAM mutant).
Therefore, we transiently transfected wild-type FcRIIA, tailless
FcRIIA, and the ITAM mutant FcRIIA into an untransfected CHO cell
line or a CR3-expressing CHO cell line using FuGene6 transfection
reagent. For experiments, cells were seeded onto 25-mm2
coverslips and allowed to adhere overnight at 37°C in 5%
CO2. Cells were tested for expression using both indirect
immunofluorescence flow cytometry and fluorescence microscopy as
previously described.11 Transient expression of FcRIIA
and FcRIIA (ITAM mutant) was equivalent as detected by
fluorescence-activated cell sorting. Mean fluorescence intensities of
the transiently transfected receptors are shown in the figure legends
(Figure 3).
Lysosome labeling
Transfectants were grown on glass coverslips (Corning, Corning,
NY) overnight at 37°C. A total of 5 µg rhodamine-conjugated dextran (10 000 molecular weight; Molecular Probes, Eugene,
OR) was added to each coverslip for 90 minutes at 37°C. Cells were washed with phosphate-buffered saline followed by addition of fresh
media to the coverslips as described by Oh and Swanson.17 Imaging of lysosomes was performed using an Axiovert
135 fluorescence microscope (Carl Zeiss, Thornwood, NY) using
mercury illumination. Optical filters for rhodamine excitation and
emission were 530DF22 and 590DF30, respectively (Omega, Brattlesboro,
VT). Images were observed using an intensified charge-coupled device
(ICCD) (Hamamatsu, Hamamatsu City, Japan) coupled to a
Scion LG-3 (Scion, Frederick, MD) image capture board on a Dell
Precision 410 Workstation (Round Rock, TX). Images were processed using
Scion Image software.
Phagocytosis of erythrocytes
Sheep red blood cells (SRBCs) (Alsever; Rockland
Scientific, Gilbertsville, PA) were opsonized with the highest
subagglutinating concentration of IgG rabbit antisheep erythrocyte
antibody (ICN, Costa Mesa, CA). Subsequently, antibody-coated cells
(EAs) were added at a target-to-effector ratio of 10:1
(EA/transfectant). The EAs were incubated with transfectants for 45 minutes at 37°C in culture media. Coverslips were then placed on ice
to stop phagocytosis. Bound external EAs were either removed by
hypotonic lysis in 0.25 × phosphate-buffered saline or labeled with a
secondary fluorescent anti-IgG. Therefore, external EAs become
fluorescent, and internal EAs are not susceptible to the
secondary labeling.
Fluorescence microscopy
Goat antirabbit IgG F(ab')2 fragments conjugated
with fluorescein isothiocyanate (ICN) were added to the coverslips for
30 minutes on ice to detect the external EAs. The coverslips were observed using bright-field microscopy or by fluorescence microscopy using the system described above. Narrow band-pass discriminating filters were used with excitation at 482 nm and emission at 530 nm for
fluorescein isothiocyanate fluorescence (not
shown).11
Electron microscopy
Transfectants expressing either wild-type FcRIIA (131-3) or
tailless FcRIIA with CR3 (169-123) were incubated with opsonized sheep erythrocytes for 45 minutes at 37°C in culture media. The cells
were washed and then fixed with glutaraldehyde overnight at 4°C. To
detect the lysosomal compartment, we stained for acid phosphatase using
modified Gormori media consisting of 13.9 mM -glycerophosphate, 1 mM
Pb(NO3)2, 0.05 M acetate buffer, 0.08% CaCl2, and 5% sucrose. Cells were treated with the acid
phosphatase stain for 1 hour at 37°C with gentle agitation. The cells
were washed extensively with cacodylate buffer and then postfixed with osmium tetroxide for 1 hour at room temperature. The cells were dehydrated and embedded in Spurr resin as described
previously.18 Thin sections were viewed with a Joel 35e
(Tokyo, Japan) electron microscope. Micrographs were taken using an
in-column digital camera system coupled to a Macintosh G3 computer and
processed with Adobe Photoshop 5.0. Quantitative data are
combined with fluorescence data. Individual quantitative data are shown
in the figure legends (Figures 2 and 4).
| |
Results |
Receptor expression and phagocytosis
Transfected CHO cells were studied for expression of FcRIIA and
CR3 using flow cytometry (Figure 1).
Several cell lines were produced. Clone 131-3 expressed wild-type
FcRIIA; 135-12 expressed the tail-minus mutant of FcRIIA; and
161-24 expressed neither of the receptors but was exposed to the
transfection protocol. Clones 169-8 and 169-23 both expressed the
tailless mutant FcRIIA in combination with CR3. We also constructed
a wild-type FcRIIA and CR3 clone (173-46). As shown in Figure 1,
indirect immunofluorescence analysis confirmed the phenotypes of the
cell lines. In addition, we used a phagocytosis-defective FcRIIA
that had a full-length cytoplasmic domain with only the tyrosine
residues in each of the ITAM motifs mutated to phenylalanine (FcRIIA
ITAM mutant). This mutation has previously been shown to abolish
IgG-dependent phagocytosis via FcRIIA.5 We also
transfected wild-type FcRIIA, FcRIIA tailless, and FcRIIA
(ITAM mutant) transiently into untransfected CHO cells or a
CR3-expressing cell line. Expression was determined via indirect
immunofluorescence quantitated by flow cytometry. Expression of
wild-type FcRIIA (MFI 97/89), tailless FcRIIA (MFI 87/96), and
this FcRIIA (ITAM mutant) (MFI 93/91) were equivalent in
CHO/CR3-transfected cells.
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| Figure 1.
Indirect immunofluorescence flow cytometric analysis of
cell lines expressing FcRIIA (CD32) or CR3 (CD11b/CD18).
The indicated 5 clones were subjected to indirect immunofluorescence
using primary murine monoclonal antibodies specific for FcRIIA
(CD32), CR3 (CD11b/CD18), or a negative control reagent. In each panel,
the solid line represents cells stained with negative control reagent,
whereas the dotted line indicates staining with the appropriate
anti-CD antibody.
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To confirm that the receptors were functional, we examined phagocytosis
using IgG-coated sheep erythrocytes (EAs). After incubation of EAs with
the transfectants for 30 minutes at 37°C, we observed that the
wild-type FcRIIA (clone 131-3) was capable of internalizing IgG-coated erythrocytes. However, the FcRIIA tailless (clone 135-12)
and the FcRIIA (ITAM mutant) were not able to phagocytose EAs, as
previously reported.4,5,11 We observed that the coexpression of CR3 with either of the mutant FcRIIAs restored FcR-dependent phagocytosis (Table 1).
Fluorescence detection of phagosome-lysosome fusion
We next studied whether the cytoplasmic tail of FcRIIA
participates in phagolysosomal fusion. Fluorescently labeled dextran was used to label lysosomes.17 Fluorescent dextran is
taken up by pinocytosis and then delivered to lysosomes. This allows the fluorescent dextran to spill from the preloaded lysosomes into the
phagosome. After incubation with dextran, the transfectants exhibited
dextran located in small punctate vesicles when viewed with
fluorescence microscopy (data not shown).
Previous work has shown that coexpression of CR3 and a
phagocytosis-defective tailless FcRIIA restored IgG-dependent
phagocytosis.11 Similarily, studies have suggested that
CR3 does not mediate phagolysosome fusion by itself (R.G.W., L.M.-B.,
R.F.T., H.R.P., unpublished observations, September
1999).19 Therefore, we used this approach, cotransfection of FcRIIA and CR3, to examine postphagocytic events in the presence and absence of the cytoplasmic tail of FcRIIA or in
an ITAM mutant of FcRIIA. As shown in Figure
2, wild-type FcRIIA (clone 131-3)
transfectants exhibited colocalization of fluorescent dextran with the
internalized IgG-coated particle. This effect was seen as soon as 15 minutes after addition of targets and did not change significantly up
to 60 minutes after phagocytosis. In addition, more than 95% (101 of
104 dextran studies) of the internalized targets were positive for
lysosome fusion as determined by rhodamine dextran colocalization
(Figure 3 and Table 1). However, when the
cell lines containing the mutant tailless form of FcRIIA in the
presence of CR3 were studied (clones 169-8 and 169-23), very little
colocalization of IgG-coated cells with the dextran was observed
(Figures 2, 3, and Table 1). Little or no colocalization of dextran
with EAs was observed from 15 minutes to 60 minutes after phagocytosis.
Internalized targets displayed fusion with lysosomes in 6.4% and 8.7%
of the cells for clones 169-8 and 169-23, respectively. Table 1 shows
pooled data from both fluorescent dextran and acid phosphatase studies.
Individually, clone 169-8 showed colocalization in 2 of 35 targets
studied. Similarily, clone 169-23 contained 3 of 41 targets colocalized
with dextran. In addition, tailless FcRIIA was transiently
transfected into a CR3-expressing stable cell line (Figure 3, column
d). Similar phagolysosomal fusion data were observed as with cells
stably expressing both CR3 and tailless FcRIIA. The FcRIIA ITAM
mutant without CR3 is unable to induce phagocytosis of IgG-coated cells and, therefore, no lysosomal fusion can occur (Figure 3, column a).
However, in the presence of CR3 and FcRIIA ITAM mutant, phagocytosis was restored, and near wild-type levels of lysosome fusion was detected
(Figure 3, column b). We also studied clone 173-46, which expressed
wild-type FcRIIA and CR3 to determine if CR3 might affect
phagolysosome formation. As seen in Figure 3, expression of CR3 did not
affect the ability of wild-type FcRIIA to participate in
phagolysosome fusion (173-46 and Figure 3, column c).
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| Figure 2.
Differential interference contrast and
fluorescence micrographs displaying colocalization of internalized
targets and fluorescent dextran.
(A,C) Differential interference contrast (DIC) images of
transfectants. (B,D) Fluorescent micrographs indicating the location of
the preloaded fluorescent dextran. Panels A and B show clone 131-3 (n = 3) expressing wild-type FcRIIA. Panels C and D show clone
169-23 (n = 5), which expresses mutant tail-minus FcRIIA and CR3.
Colocalization of the fluorescent dextran (arrowheads) can be observed
with the wild-type FcRIIA but not in the clone expressing mutant
tailless FcRIIA and CR3 (169-23). Of 104 internal targets, 101 were
colocalized with fluorescent dextran in wild-type FcRIIA-transfected
cells. However, tailless FcRIIA only showed target
colocalization with dextran in 3 of 41 cases (original
magnification × 1100). TRITC indicates tetramethylrhodamine
isothiocyanate.
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| Figure 3.
Percent lysosome marker colocalization with internalized
target.
Cells were preloaded with rhodamine dextran and then allowed to
internalize opsonized erythrocytes or were stained for acid phosphatase
after phagocytosis. Lysosome fusion is determined by the colocalization
of the internalized target and the lysosomal marker. As shown, clones
131-3 and 173-46 expressing wild-type FcRIIA show more than 97% of
the internalized targets colocalized with one of the markers. However,
internalization via tailless FcRIIA, using CR3 to mediate
phagocytosis (clones 169-8 and 169-23, n = 5 for both lines),
exhibited very little colocalization of the targets with either
fluorescent dextran or acid phosphatase (P < .001
comparing wild-type FcRIIA with tailless FcRIIA). Columns a-d
represent experiments with transient transfections of the FcRIIA
constructs. FcRIIA immunoreceptor tyrosine-based activation motif
(ITAM) mutants (MFI 93) displayed no colocalization of targets
and marker due to the absence of phagocytosis (column a). However, in
the presence of CR3 to restore phagocytosis, FcRIIA ITAM mutants
(column b) (MFI 91) displayed near wild-type FcRIIA (column c) (MFI
89) levels of target/marker colocalization. Tailless FcRIIA (column
d) transiently transfected (MFI 96) displayed very little
colocalization of targets with dextran.
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Electron microscopy of phagosome-lysosome fusion
As a second independent means of detecting phagosome-lysosome
fusion following phagocytosis, we employed electron microscopy using a
specific lysosomal stain. Acid phosphatase is an enzyme specific for
lysosomes and has been used extensively to stain CHO
cells.20 Therefore, we used this enzyme to detect the
localization of lysosomal enzymes inside cells. After incubation of
transfectants expressing either wild-type FcRIIA or tailless
FcRIIA in the presence of CR3 with IgG-coated sheep erythrocytes,
the cells were fixed and stained for acid phosphatase. After embedding, thin sections were viewed with an electron microscope. Acid phosphatase appeared as dark electron dense patches, revealing the location of
lysosomal enzyme activity. Figure 4 shows
representative micrographs of experiments repeated on 4 independent
occasions. As shown, in the presence of the wild-type FcRIIA (clone
131-3) we observed acid phosphatase staining near the internalized
target, indicating phagolysosomal fusion in 61 of 63 cases studied with
acid phasphatase (Figure 4A). However, cells expressing the tail-minus
form of FcRIIA (clone 169-23) did not support phagolysosome
formation (only 9 of 97 targets colocalized with acid phosphatase).
Thus, the acid phosphatase staining was found throughout the entire cytoplasm as punctate granules and was not localized near internalized targets (Figure 4B). These results suggest that the cytoplasmic domain
of FcRIIA targets the internalized particle for fusion with
lysosomes.
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| Figure 4.
Electron micrographs showing location of acid
phosphatase, a lysosomal enzyme.
Micrographs are representative examples of experiments repeated 4 times. Transfectant CHO cells were allowed to internalize IgG-coated
EAs and then fixed and stained for acid phosphatase. (A)
Internalization via wild-type FcRIIA (clone 131-3) exhibits strong
acid phosphatase activity near the internalized target in 61 of 63 internal targets counted (n = 4 for both lines). (B) However,
internalization via tail-minus FcRIIA (clone 169-23), using CR3 to
mediate the phagocytic signal, does not show colocalization of the
target with acid phosphatase activity (arrows). When counted, only 9 of
97 internal targets show colocalization with acid phasphatase (original
magnification × 6000).
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| |
Discussion |
The goal of this study was to determine if the cytoplasmic tail of
FcRIIA participates in phagolysosomal fusion. Previous studies have
shown that the cytoplasmic tail of FcRIIA is necessary for
phagocytosis.4,5 Specifically, the tail's ITAM
(YXXL) sequence of the FcRIIA cytoplasmic domain is
required for phagocytosis.5 Although it is known that a
dileucine motif located in the cytoplasmic domain of FcRIIB mediates
endocytosis and basolateral sorting in MDCK cells,2 the
potential role of FcRIIA's cytoplasmic domain in lysosomal delivery
is unknown. Because the FcRIIA tail does not possess a known
phagolysosomal delivery sequence such as a dileucine motif, the tail's
potential ability to support phagolysosome fusion is uncertain.
However, because phagolysosome formation requires phagocytosis,
phagocytic signaling must remain intact. To dissect the mechanisms
involved in phagolysosome formation from phagocytosis (internalization)
per se, we have genetically complemented, with CR3, the phagocytic
function of a FcRIIA tail-minus mutant and ITAM mutants of
FcRIIA. Thus, in this study we use CR3 as a mechanism to allow
particles to be internalized in the absence of the normal FcRIIA
phagocytic machinery. We now show for the first time that the
cytoplasmic tail of FcRIIA participates in phagolysosomal fusion and
that this signal is distinct from a functional ITAM.
As mentioned above, one consensus sequence for endolysosome formation
is the dileucine sequence. This motif has been described in various
receptors such as FcRIIB, the cation-dependent mannose 6-phosphate
receptor, and the LDL receptor.2,21,22 These motifs may be
involved in the direct interaction of the receptors with lysosomes, or
they may act to recruit other cytoplasmic helper proteins such as Rho,
which could then direct the receptors toward lysosomes. Alternatively,
the endolysosome fusion signal may not be a linear sequence of amino
acids but, rather, a 3-dimensional motif such as that found on nascent
lysosomal enzymes in the endoplasmic reticulum.21,22
Several motifs have been shown to be important in mediating
phagocytosis, delivery to intracellular compartments, and cytoskeletal manipulation. Various ITAMs or ITAM-like motifs and their likely 3-dimensional structures have been implicated in the recruitment and
binding of signaling molecules.23-25 One such heavily
studied molecule is Syk kinase. Syk has been suggested to be a crucial mediator of Fc receptor-mediated phagocytosis and transport to lysosomes.23,24 In addition, one study has suggested that
kinase activity is required for directing FcRI to the lysosomal
compartment.26 It is likely that the ability to recruit
Syk leads to the further recruitment of other downstream signaling molecules.
Phagolysosome delivery motifs likely contribute to the recruitment of
additional signaling components such as GTP-binding proteins (eg, Rho).
Rho has also been shown to be a crucial partner in mediating Fc
receptor phagocytosis27 and in altering the actin
cytoskeleton in response to extracellular signals.28-32
Other molecules such as phosphatidylinositol-3 kinase are also
recruited to the sites of engulfment to mediate phagocytosis in other
cell types, possibly participating in receptor-mediated
phosphorylation.33,34 Additional studies have shown the
importance of Rho in the regulation of endosome
dynamics.35 These data show that the effects we observed
in the transfectant studies, specifically where the wild-type receptor
aggregated many internalized targets into one or more large phagosomes
while the tailless FcRIIA with CR3 did not mediate this aggregation
of endosomes, may be due to Rho recruitment. FcRIIA may contain a
signal motif in the cytoplasmic domain that is responsible for the
eventual recruitment of Rho.
In this study, using a transfectant CHO model system, we have shown
that the cytoplasmic tail of FcRIIA participates in phagolysosome fusion. The data presented in this study are relevant to studies involving phagocytosis and phagolysosome fusion of various microbes such as Mycobacterium tuberculosis and Toxoplasma
gondii.36,37 The suggestion that M
tuberculosis does not allow phagolysosome fusion unless opsonized
with IgG supports our studies that the cytoplasmic tail of FcRs (in
this study FcRIIA) participates in phagolysosome formation. Overall,
lysosomal targeting/fusion may be a more complex phenomenon than
initially hypothesized. The approach used in this study may be useful
in future studies involving other receptor interactions and
intracellular signaling pathways in addition to elucidating the
mechanism(s) by which particles are internalized and trafficked
throughout the cytoplasm.
| |
Acknowledgments |
The authors thank Dr Linda Hazlett and Ron Barrett of the electron
microscope core facility of Wayne State University School of Medicine
for their assistance with the electron microscopy.
| |
Footnotes |
Submitted September 26, 2000; accepted July 31, 2001.
Supported by grants AI-27409 (H.R.P.), CA-39064 (R.F.T.),
HL-40387 and HL-28207 (A.D.S.), and training grant 5T32-AR07442 (R.G.W.), all from the National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Alan D. Schreiber, Div of Hematology and Oncology,
University of Pennsylvania School of Medicine, 705 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104; e-mail:
schreibr{at}mail.med.upenn.edu.
| |
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Abstract
Introduction