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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
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 Fc␥RIIA 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 Fc␥RIIA. Fluorescent dextran was used
to label lysosomes. After target internalization, wild-type Fc␥RIIA–mediated phagolysosome formation was observed as indicated by colocalization of fluorescent
dextran and the phagosome. In addition,
when studying mutants of Fc␥RIIA 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
Fc␥RIIA, 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 Fc␥RIIA but not by tail-minus
Fc␥RIIA and CR3. Thus, the tail of Fc␥RIIA
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 (Fc␥RI) and Fc␥RIII and the cytoplasmic domain of
Fc␥RIIA is responsible for phagocytic signaling in antibodydependent 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 Fc␥RIIA 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.
Fc␥Rs 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 Fc␥RIIA.11
The ability of CR3 to complement the phagocytic function of
tail-minus Fc␥RIIA has allowed us to study the role of the
cytoplasmic domain of Fc␥RIIA in phagolysosomal fusion.
In the current study we observed that CR3 reconstitutes
Fc␥R-dependent phagocytosis in a Fc␥RIIA mutant where
tyrosine residues are mutated to phenylalanine (Fc␥RIIA-ITAM
mutant). By employing a reverse genetic complementation
strategy and 2 mutant forms of Fc␥RIIA, we have studied the
cytoplasmic domain’s role in phagolysosome formation.
We examined the hypothesis that the cytoplasmic domain of
Fc␥RIIA contributes to phagolysosome formation and observed
that the cytoplasmic domain of Fc␥RIIA 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
Fc␥RIIA supported phagolysosome formation, the tail-minus form
of Fc␥RIIA did not. However, Fc␥RIIA (ITAM mutant), complemented with CR3 to restore phagocytosis, retained their intrinsic
lysosome signaling capacity. Thus, the cytoplasmic tail of Fc␥RIIA
contributes to fusion of phagosomes with lysosomes.
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.
Institutes of Health.
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
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
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: [email protected].
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.
© 2001 by The American Society of Hematology
3429
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3430
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
WORTH et al
Materials and methods
Narrow band-pass discriminating filters were used with excitation at 482
nm and emission at 530 nm for fluorescein isothiocyanate fluorescence
(not shown).11
Cell culture and transfections
Electron microscopy
Chinese hamster ovary (CHO) cells were transfected by electroporation
with a mixture of 1.5 ␮g pSVneo, 5 ␮g 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 ␮g pCMVBACD18, and 5 ␮g 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 Fc␥RIIA; 135-12,
expressing Fc␥RIIA tailless alone; 169-8 and 169-24, which both express
the Fc␥RIIA tailless and CR3; and 173-46, expressing both the wild-type
Fc␥RIIA and CR3.
In addition, one crucial experiment is to compare wild-type Fc␥RIIA
with both Fc␥RIIA tailless and an ITAM mutant of Fc␥RIIA that expresses
a full-length Fc␥RIIA cytoplasmic domain with Tyr3Phe mutations in both
of the ITAM motifs (Fc␥RIIA ITAM mutant). Therefore, we transiently
transfected wild-type Fc␥RIIA, tailless Fc␥RIIA, and the ITAM mutant
Fc␥RIIA 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 Fc␥RIIA and Fc␥RIIA (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 ␮g 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 phosphatebuffered 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.
Transfectants expressing either wild-type Fc␥RIIA (131-3) or tailless
Fc␥RIIA 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 Fc␥RIIA and
CR3 using flow cytometry (Figure 1). Several cell lines were
produced. Clone 131-3 expressed wild-type Fc␥RIIA; 135-12
expressed the tail-minus mutant of Fc␥RIIA; 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 Fc␥RIIA in combination with CR3. We also constructed a
wild-type Fc␥RIIA 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
Fc␥RIIA that had a full-length cytoplasmic domain with only the
tyrosine residues in each of the ITAM motifs mutated to phenylalanine (Fc␥RIIA ITAM mutant). This mutation has previously been
shown to abolish IgG-dependent phagocytosis via Fc␥RIIA.5 We
also transfected wild-type Fc␥RIIA, Fc␥RIIA tailless, and Fc␥RIIA
(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 Fc␥RIIA (MFI 97/89), tailless Fc␥RIIA (MFI 87/96),
and this Fc␥RIIA (ITAM mutant) (MFI 93/91) were equivalent in
CHO/CR3-transfected cells.
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 Fc␥RIIA (clone 131-3) was capable of
internalizing IgG-coated erythrocytes. However, the Fc␥RIIA
tailless (clone 135-12) and the Fc␥RIIA (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 Fc␥RIIAs
restored Fc␥R-dependent phagocytosis (Table 1).
Fluorescence detection of phagosome-lysosome fusion
We next studied whether the cytoplasmic tail of Fc␥RIIA 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
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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
CD32 IN PHAGOLYSOSOME FORMATION
Figure 1. Indirect immunofluorescence flow cytometric analysis of cell lines
expressing Fc␥RIIA (CD32) or CR3 (CD11b/CD18). The indicated 5 clones were
subjected to indirect immunofluorescence using primary murine monoclonal antibodies specific for Fc␥RIIA (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.
3431
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 Fc␥RIIA 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 Fc␥RIIA and CR3, to
examine postphagocytic events in the presence and absence of the
cytoplasmic tail of Fc␥RIIA or in an ITAM mutant of Fc␥RIIA. As
shown in Figure 2, wild-type Fc␥RIIA (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 Fc␥RIIA 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 Fc␥RIIA 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
Fc␥RIIA. The Fc␥RIIA 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 Fc␥RIIA ITAM mutant, phagocytosis was
restored, and near wild-type levels of lysosome fusion was detected
Table 1. Phagocytosis of IgG-coated SRBCs by transfected CHO cells
Fc␥RIIA
wt
Fc␥RIIA
tailless
Fc␥RIIA
mutant
CR3
No.
CN
161 -24
⫺
⫺
⫺
⫺
3
352
4
0
0
161 -84
⫺
⫺
⫺
⫹
3
349
2
0
0
135 -12
⫺
⫹
⫺
⫺
3
348
395
0
0
131 -3*
⫹
⫺
⫺
⫺
3
123
238
167
162
169 -23*
⫺
⫹
⫺
⫹
5
584
672
54
4
169 -8
⫺
⫹
⫺
⫹
5
592
635
35
2
173 -46
⫹
⫺
⫺
⫹
3
230
440
210
204
163
Clone No.
SRBC
bound
SRBC
internal
Fused
lysosomes
Receptors expressed
Receptors expressed transiently
⫹†
⫺
⫺
⫹
2
193
253
171
⫺
⫹‡
⫺
⫹
2
248
293
38
⫺
⫺
⫹§
⫺
4
936
693
⫺
⫺
⫹¶
⫹
3
649
734
4㛳
127
3
0㛳
114
No. indicates number of experiments performed; CN, total number of transfected CHO cells counted; SRBC bound, total number of sheep erythrocytes bound; SRBC
internal, total number of internalized erythrocytes; Fused lysosomes, number of internalized SRBCs colocalized with dextran.
*Data presented by combining experiments with fluorescent dextran and acid phosphatase.
†Mean fluorescence intensity (MFI) of 89 for Fc␥RIIA wild-type transfected into CR3 permanent line.
‡MFI of 96 for Fc␥RIIA tailless transfected into CR3 permanent line.
§MFI of 93 for Fc␥RIIA (ITAM mutant) transfected into CHO cells.
㛳Too few internal targets to quantitate phagolysosome fusion.
¶MFI of 91 for Fc␥RIIA mutant transfected into CR3 permanent line.
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3432
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
WORTH et al
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 Fc␥RIIA. Panels C and D
show clone 169-23 (n ⫽ 5), which expresses mutant tail-minus Fc␥RIIA and CR3.
Colocalization of the fluorescent dextran (arrowheads) can be observed with the
wild-type Fc␥RIIA but not in the clone expressing mutant tailless Fc␥RIIA and CR3
(169-23). Of 104 internal targets, 101 were colocalized with fluorescent dextran in
wild-type Fc␥RIIA-transfected cells. However, tailless Fc␥RIIA only showed target
colocalization with dextran in 3 of 41 cases (original magnification ⫻ 1100). TRITC
indicates tetramethylrhodamine isothiocyanate.
cells.20 Therefore, we used this enzyme to detect the localization of
lysosomal enzymes inside cells. After incubation of transfectants
expressing either wild-type Fc␥RIIA or tailless Fc␥RIIA 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 Fc␥RIIA (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 Fc␥RIIA (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 Fc␥RIIA targets the
internalized particle for fusion with lysosomes.
Discussion
(Figure 3, column b). We also studied clone 173-46, which
expressed wild-type Fc␥RIIA 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 Fc␥RIIA to participate in
phagolysosome fusion (173-46 and Figure 3, column c).
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
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 Fc␥RIIA show
more than 97% of the internalized targets colocalized with one of the markers.
However, internalization via tailless Fc␥RIIA, 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 Fc␥RIIA with tailless Fc␥RIIA). Columns a-d represent experiments with
transient transfections of the Fc␥RIIA constructs. Fc␥RIIA immunoreceptor tyrosinebased 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, Fc␥RIIA ITAM mutants (column b) (MFI 91) displayed
near wild-type Fc␥RIIA (column c) (MFI 89) levels of target/marker colocalization.
Tailless Fc␥RIIA (column d) transiently transfected (MFI 96) displayed very little
colocalization of targets with dextran.
The goal of this study was to determine if the cytoplasmic tail of
Fc␥RIIA participates in phagolysosomal fusion. Previous studies
have shown that the cytoplasmic tail of Fc␥RIIA is necessary for
phagocytosis.4,5 Specifically, the tail’s ITAM (YXXL) sequence of
the Fc␥RIIA cytoplasmic domain is required for phagocytosis.5
Although it is known that a dileucine motif located in the
cytoplasmic domain of Fc␥RIIB mediates endocytosis and basolateral sorting in MDCK cells,2 the potential role of Fc␥RIIA’s
cytoplasmic domain in lysosomal delivery is unknown. Because
the Fc␥RIIA 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 Fc␥RIIA tail-minus mutant and ITAM
mutants of Fc␥RIIA. Thus, in this study we use CR3 as a
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
Fc␥RIIA (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 Fc␥RIIA (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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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
CD32 IN PHAGOLYSOSOME FORMATION
mechanism to allow particles to be internalized in the absence of
the normal Fc␥RIIA phagocytic machinery. We now show for the
first time that the cytoplasmic tail of Fc␥RIIA 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 Fc␥RIIB, the cationdependent 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 Fc␥RI 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
3433
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 Fc␥RIIA with CR3
did not mediate this aggregation of endosomes, may be due to Rho
recruitment. Fc␥RIIA 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 Fc␥RIIA 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 Fc␥Rs (in this study
Fc␥RIIA) 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.
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2001 98: 3429-3434
doi:10.1182/blood.V98.12.3429
The cytoplasmic domain of FcγRIIA (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
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