(Received for publication, July 17, 1995)
From the
CD16, the low affinity Fc receptor III for IgG
(Fc
RIII), exists as a polypeptide-anchored form (Fc
RIIIA or
CD16A) in human natural killer cells and macrophages and as a
glycosylphosphatidylinositol-anchored form (Fc
RIIIB or CD16B) in
neutrophils. CD16A requires association of the
subunit of
Fc
RI or the
subunit of the TCR-CD3 complex for cell surface
expression. The CD16B is polymorphic and the two alleles are termed NA1
and NA2. In this study, CD16A and the two alleles of CD16B have been
expressed in Chinese hamster ovary (CHO) cells and their ligand binding
and phagocytic properties analyzed. The two allelic forms of CD16B
showed a similar affinity toward human IgG1. However, the NA1 allele
showed approximately 2-fold higher affinity for the IgG3 than the NA2
allele. Although all three forms of CD16 efficiently bound rabbit
IgG-coated erythrocytes (EA), only CD16A coexpressed with the
subunit phagocytosed EA. The phagocytosis mediated by CD16A expressed
on CHO cells was independent of divalent cations but dependent on
intact microfilaments. CHO cells expressing CD16A-
and CD16A-
chimeras also phagocytosed EA. The phagocytosis was specifically
inhibited by tyrphostin-23, a tyrosine kinase inhibitor. In summary,
our results demonstrate that glycosylphosphatidylinositol-anchored
CD16B alleles differ from CD16A in their ability to mediate
phagocytosis. Furthermore, since studies with other Fc
Rs have
shown that CHO cells lack the phagocytic pathway mediated by the
cytoplasmic domain of Fc
Rs, the phagocytosis of EA by CHO cells
stably transfected with CD16A and CD16A-subunit chimera provides an
ideal system to dissect the phagocytic signaling pathways mediated by
these Fc
R-associated subunits.
The binding of the Fc region of IgG to specific receptors
(FcRs) (
)expressed on hematopoietic cells results in a
wide array of cellular responses that include release of inflammatory
mediators, lymphokine production, cytotoxic triggering, cell
activation, regulation of antibody production, and phagocytosis of
antibody coated
particles(1, 2, 3, 4, 5, 6) .
Thus Fc
R interaction with IgG bridges the cellular and humoral
immune responses. Three major distinct types of Fc
Rs have been
described. They are Fc
RI (CD64), Fc
RII (CD32), and
Fc
RIII (CD16). CD64 is a high affinity receptor for monomeric IgG
expressed on monocytes and activated neutrophils. CD32 is a low
affinity receptor expressed on B cells, monocytes, neutrophils, and
nonhematopoietic cells such as epithelial cells. CD16 is also a low
affinity receptor for monomeric IgG and is expressed on neutrophils,
macrophages, NK cells, activated endothelial cells(7) , and
placental trophoblasts(8) . Thus many cells coexpress more than
one type of Fc
Rs. Moreover, each type of Fc
Rs is polymorphic
and expressed in different structural forms.
In humans, CD16 is
expressed as two distinct (CD16A and CD16B) forms (9, 10, 11, 12, 13, 14, 15) which
are products of two different highly homologous genes. CD16B is
expressed on neutrophils in a glycosylphosphatidylinositol
(GPI)-anchored form, whereas CD16A is expressed on NK cells,
macrophages, and placental trophoblasts as a polypeptide-anchored
transmembrane
protein(8, 16, 17, 18) . The
GPI-anchored CD16B exists as two allelic forms termed NA1
(CD16B) and NA2 (CD16B
)(19) . The
polypeptide-anchored CD16A expressed on NK cells and macrophages is
associated with subunits such as the
chain of the TCR-CD3 complex (20, 21) or the
chain of
Fc
RI(22, 23, 24) . The NA1 and NA2
alleles of CD16B are 95% homologous to each other and are 95-97%
homologous to CD16A in their extracellular domain(12) . The
functional significance of the existence of membrane isoforms and the
polymorphism of CD16 are not clear. However, some studies have shown
that CD16A differs from CD16B by triggering killing of tumor targets
and signaling for IL-2 production (16, 25, 26) .
We established CHO cell
lines expressing isoforms of CD16 and determined their ligand binding
and phagocytic properties. The results show that the
polypeptide-anchored CD16A is able to mediate phagocytosis of
antibody-coated target cells, whereas under similar conditions the NA1
and NA2 alleles of GPI-anchored CD16B are not. Moreover, chimeric
molecules created by replacing the cytoplasmic domain of CD16A with
that of or
chains also delivered signal for phagocytosis in
CHO cells. These results show that the membrane anchor and associated
subunits have a profound influence on the biological properties of cell
surface receptors.
cDNAs encoding NA1 and NA2 alleles of CD16B in a CDM8 vector
were provided by Dr. B. Seed (Massachusetts General Hospital, Boston,
MA). cDNAs encoding CD16A in a pSVL vector and the subunit of rat
Fc
RI also in a pSVL vector were kind gifts from Drs. J. Ravetch
(Sloan-Kettering Institute for Cancer Research, New York) and J. P.
Kinet (National Institutes of Health, Bethesda, MD), respectively.
Hygromycin resistance gene in a plasmid pSV2 vector was kindly provided
by Dr. C. L. Saxe III (Emory University, Atlanta, GA).
CD16 cells were selected for their ability to form
strong rosettes with rabbit anti-DNP IgG-opsonized TNP-coated SE.
Briefly, SE (2% packed cell volume) were washed in PBS, 5 mM EDTA, resuspended in 10 ml of trinitrobenzene sulfonic acid (6
mg/ml) in 50 mM cacodylate buffer, pH 6.9, and incubated for
30 min at room temperature with constant slow rotation. TNP-E was
washed once in HBSS, incubated for 5 min at room temperature in HBSS
with 40 mM glycine, and washed three times in HBSS. Finally,
the TNP-E were resuspended in 10 ml of RPMI 1640, 10% FBS. TNP-E (1.5
ml) were incubated with a subagglutinating concentration of rabbit
anti-DNP IgG for 1 h at room temperature. After washing the cells in
RPMI 1640, 10% FBS twice, the TNP-E treated with rabbit anti-DNP IgG
was used on the same day.
The opsonized EA were incubated with CD16
transfected CHO cells in 1 ml of HBSS, 5 mM EDTA, 1% FBS
(100:1 ratio) at 25 °C for 30 min. Rosetted CHO cells were
separated from unrosetted CHO cells by Histopaque-1077 density gradient
centrifugation. The heavily rosetted cells pelleted with free E were
washed in RPMI, 10% FBS, and the surface-bound E were removed by
hypotonic lysis in distilled HO for 20 s. CD16+ cells
were expanded, and this selection procedure was repeated four more
times to get cells expressing higher level of CD16. CHO cell
transfectants were maintained in CHO-S-SFM II serum-free medium (Life
Technologies, Inc.) supplemented with 0.1% heatinactivated IgG low FBS
(Life Technologies, Inc.) and 200 µg/ml hygromycin B.
CHO cell
lines expressing CD16A- and CD16A-
chimeric molecules
described previously (28) were maintained in CHO-S-SFM II, 0.1%
IgG free FBS with 0.4 mg/ml neomycin analog, Geneticin (Life
Technologies, Inc.). For the purpose of clarity, the CD16A expressed in
CHO cells after cotransfection of the CD16A gene with the
subunit
gene will be referred as CD16A, and the CD16A chimeric molecules made
with either the
subunit or the
subunit will be referred as
CD16A-
and CD16A-
, respectively.
CHO cell transfectants (50 µl of 5
10
cells/ml in RPMI 1640, 5% heat-inactivated IgG
low FBS, Hepes, pH 7.4) were incubated with 50 µl of IC (5
µg/ml final concentration) in the presence and absence of 50 µl
of IgG1 (
) or IgG3 (
) for 1 h at 4 °C with constant
gentle shaking in a microtiter plate. After washing, the cells were
resuspended in peroxidase assay buffer (0.1 M citrate buffer,
pH 4.0, with 0.1% Triton X-100). Peroxidase activity was developed
using ABTS as substrate with H
O
(0.03%) in 0.1 M citrate buffer, pH 4.0. The color produced was read in an
ELISA reader (Bio-Rad) at 405 nm.
Figure 6: Sequential photomicrographs of phagocytosis of EA by a CHO cell expressing CD16A. The human erythrocytes were opsonized with CLBFcgran1, an anti-CD16 mAb, and allowed to adhere to CHO cells in a micromanipulation chamber. A CHO cell with a bound EA (indicated by arrow) was captured by a micropipette (A) and the entire phagocytic process (B and C) was viewed with brightfield microscopy, and the images were recorded using a closed circuit video system.
Figure 7: Brightfield (A, C, and E) and fluorescent (B, D, and F) images of a human EA (A and B), a CD16A-positive CHO cell (C and D), and a CD16A-positive CHO cell that has engulfed an EA (E and F). The fluorescent images were obtained using an ultrasensitive liquid nitrogen-cooled CCD camera. No fluorescent dyes were used; therefore, the images seen were from autofluorescence of the cells. The fluorescent images shown in B, D, and F were the same cells as those brightfield images shown in A, C, and E. It can be seen that the originally nonfluorescent CHO cell (D) became fluorescent after engulfing the autofluorescent EA (F). For comparative purposes Fig. 6C is reproduced as E.
Figure 1:
Immunofluorescent flow cytometry
analysis of CHO cell transfectants expressing CD16 isoforms. CHO cell
stable transfectants expressing CD16B,
CD16B
, and CD16A isoforms were stained with the indicated
mAbs followed by goat (Fab`)
anti-mouse IgG. CLBFcgran1, an
anti-CD16 mAb, binds to all isoforms, whereas CLBgran11 (GRAN11) and
GRM1 are specific for CD16B
and CD16B
,
respectively. X63 is a nonbinding mouse myeloma IgG1. Dashed lines represent the staining after PIPLC
treatment.
The cell surface expression of
CD16 isoforms on stable cell lines was further confirmed by cell
surface labeling with I and immunoprecipitation. The CD16
isoforms moved as a broad band on SDS-PAGE with a molecular mass of
40-60 kDa (Fig. 2). The mobility of CD16B
,
CD16B
, and CD16A from CHO stable cell lines was slightly
faster than that of CD16 from NA1/NA1, NA2/NA2 homozygous neutrophils,
and NK cells, respectively. However after N-glycanase
digestion, the mobility of CD16 isoforms expressed on CHO cells was
identical to that of CD16 isoforms expressed on neutrophils and NK
cells, suggesting that the difference in mobility may be due to
differences in cell-specific glycosylation. Since the most of the
subunit resides on the cytoplasmic side of the membrane surface
radiolabeling was not possible. Therefore, the expression of the
subunit in CD16A transfectants was confirmed by analyzing mRNA specific
for the
subunit. Oligonucleotide primers specific for the 5` end
(23-40 base pairs) and 3` end (263-280 base pairs) of
subunit (34) were synthesized, and reverse transcription
polymerase chain reaction was performed using Life Technologies, Inc.
superscript kit as per the manufacturer's instructions. The
results showed that a single product of 260 base pairs could be
obtained from CD16A CHO cell transfectants that comigrated with the
polymerase chain reaction product obtained using pSVL vector containing
subunit cDNA (data not shown). Under identical conditions no
reverse transcription polymerase chain reaction product was obtained
using mRNA from CHO cells expressing CD16B
. This suggests
that the
subunit was expressed in CD16A but not in CD16B CHO cell
transfectants.
Figure 2:
SDS-PAGE analysis of CD16 isoforms. CD16
was immunoprecipitated from lysate of I-surface-labeled
granulocytes from a CD16B
homozygous donor (lane
1), CD16B
homozygous donor (lane 2), NK
cells (lane 3) from peripheral blood and CHO cell stable
transfectants expressing CD16B
(lane 4),
CD16B
(lane 5), and CD16A (lane 6)
isoforms. CD16 was immunoprecipitated from the lysates using
CLBFcgran1-Sepharose 4B and treated with or without N-glycanase and run on SDS-PAGE under nonreducing conditions.
Molecular mass markers are indicated. Lane 1 was obtained from
a different gel run under identical
conditions.
The stable cell lines expressing CD16 isoforms were
analyzed for their functional ability to bind to IC. Radiolabeled,
soluble transferrin/rabbit anti-transferrin IgG IC was used for binding
studies. As shown in Fig. 3, I-IC bound to CHO
cells expressing CD16 isoforms, but not to untransfected CHO cells.
This binding was completely inhibited in the presence of anti-CD16
mAbs, 3G8 and CLBFcgran1, but not by the nonbinding mouse myeloma IgG1,
X63, and IV.3, a Fc
RII mAb.
Figure 3:
Binding of I-labeled immune
complex by CHO cell transfectants expressing CD16 isoforms. The
untransfected CHO cells (A) or CHO cell stable transfectants
expressing CD16B
(B), CD16B
(C), or CD16A (D) were used for IC binding.
I-transferrin rabbit anti-transferrin IgG IC was formed
as described under ``Materials and Methods.'' Cells (5
10
) were incubated in medium with
I-IC in the absence or presence of anti-CD16 mAbs,
CLBFcgran1 (CLB), 3G8, or anti-Fc
RII mAb IV.3. X63, a nonbinding
mouse myeloma IgG1, served as control. Data are represented as mean
± S.D. from triplicates.
The binding of human IgG1 and IgG3 to CD16B allelic forms was
further investigated to determine the consequences of CD16B
polymorphism on the relative binding affinity of the human IgG
subclasses. IgG1() or IgG3(
) was complexed with mAb HP 6156,
a peroxidase-conjugated mouse anti-human Ig
chain mAb, as
described under ``Materials and Methods.'' Either human IgG1
(
) or IgG3 (
) was used for competition studies since neither
cross-reacts with the anti-human IgG
chain-specific mAb used for
preparing IC.
IgG1 or IgG3 complexes bound to stable cell lines
expressing CD16B allelic forms and were specifically inhibited with
anti-CD16 mAb. Competitive inhibition of IgG1() IC binding by
various concentration of monomeric IgG1(
) showed that
CD16B
and CD16B
follow a similar inhibition
pattern, suggesting that CD16B
and CD16B
have a similar affinity toward IgG1 (Fig. 4A). At
100 µg/ml of monomeric IgG1, nearly 84 and 72% of IgG1 IC
inhibition was observed for CD16B
and
CD16B
, respectively. In contrast, the inhibition studies
with IgG3 showed a striking difference, with 100 µg/ml of monomeric
IgG3(
) inhibiting IgG3(
) IC nearly 98% for CD16B
and 50% for CD16B
(Fig. 4B). The
IC
for monomeric IgG3 inhibition of IgG3(
) complex
binding was 300 nM (45 µg/ml) for CD16B
and
667 nM (100 µg/ml) for CD16B
, suggesting
that CD16B
has at least 2-fold higher affinity for IgG3
than CD16B
. However, the results from rosetting studies
described above showed that CD16 isoforms rosetted less with E-IgG3
than E-IgG1. This could be due to a higher susceptibility of IgG3 than
IgG1 for chemical coupling procedures.
Figure 4:
Binding of CD16 isoforms to IgG1 or IgG3
complexes. Human IgG1() or IgG3(
) were incubated with
peroxidase-conjugated mouse anti-human Ig
light chain mAb HP6156
to obtain a soluble complex as described under ``Materials and
Methods.'' CHO cell transfectants expressing CD16B
(
) or CD16B
(
) were allowed to bind
the IgG1 complex in the presence of monomeric IgG1 (A).
Similarly, binding of the IgG3 complex to CD16 isoforms was done in the
presence of monomeric IgG3 (B). The binding of IgG1 or IgG3
complexes was quantitated by assaying the peroxidase activity
associated with the bound complex. The values are mean ± S.D. of
triplicates.
Figure 5:
Phagocytosis of rabbit IgG-opsonized SE by
CD16 isoforms. CHO cell stable transfectants expressing
CD16B, CD16B
, or CD16A were assayed for
phagocytosis of EA. CHO cells (1
10
) in RPMI 1640,
2% IgG low FBS, 10 mM Hepes, pH 7.3, were incubated at 4
°C for 45 min with rabbit anti-DNP IgG-opsonized DNP-coated E (80
10
in 50 µl) in the absence or presence of
CLBFcgran1 or cytochalasin D (2 µg/ml). The phagocytosis assay was
performed as described under ``Materials and Methods.'' The
pseudoperoxidase activity of hemoglobin from ingested E was assayed and
read in an ELISA reader.
To confirm that the methodology we used to measure the phagocytosis
by CHO cells truly reflects the internalization of antibody-coated
particles, we have analyzed the phagocytosis of fluorescein
isothiocyanate-labeled Candida albicans coated with rabbit
antibody using fluorescent microscopy. The fluorescence from
uninternalized C. albicans was quenched by trypan blue. CHO
cells expressing CD16A phagocytosed the C. albicans, whereas
no phagocytosis was observed by CHO cells expressing either
CD16B or CD16B
No phagocytosis was also
observed by CD16A-expressing CHO cells kept at 4 °C or treated with
cytochalasin D. Microscopic observations further showed that the number
of C. albicans phagocytosed by CHO cells varied; most of the
cells phagocytosed more than one C. albicans.
We have also
directly visualized the phagocytosis of EA by CHO cells at the single
cell level using micropipette manipulation. This technique has been
successfully used to observe and manipulate phagocytosis of yeast by
granulocytes (35) and macrophages. ()CHO cells were
added to the test chamber in PBS containing 1% bovine serum albumin and
allowed to spontaneously rosette with EA. As shown in Fig. 6A, a single conjugated pair of cells, CHO cell
and EA, was captured and held by a micropipette to enable close
observation of the phagocytic process in detail. After 5 min the CHO
cell began to engulf the attached EA (Fig. 6B). In an
additional 6 min the phagocytic process was completed, and ruffling of
CHO cell membrane was observed (Fig. 6C). To further
confirm the ingestion of the EA by the CHO cell, an ultrasensitive
liquid nitrogen-cooled CCD camera was used to image the cells under
epi-fluorescence illumination. When examined separately, the EA was
autofluorescent (Fig. 7B), whereas the CHO cell was not (Fig. 7D). After engulfing the EA, however, the CHO
cell became fluorescent, as can be seen in Fig. 7F.
These results demonstrate that CD16A-expressing CHO cells can
phagocytose EA and confirm the phagocytosis data obtained using
colorimetric and fluorescence methods described above.
Integrins are
known to cooperate with FcRs in neutrophil and macrophage
phagocytosis(36, 37, 38, 39) .
Integrins require divalent cations such as Ca
and
Mg
for ligand binding. To determine the requirement
of extracellular divalent cations for CHO cell phagocytosis, the
phagocytosis assay was carried out in a Ca2
- and
Mg2
-free medium, in the presence of Ca
or Mg
or both. As shown in Fig. 8the
phagocytosis mediated by CD16A on CHO cells is independent of
extracellular Ca
and Mg
concentration, suggesting that integrins are not involved in CHO
cell phagocytosis.
Figure 8:
Effect of divalent cations on phagocytosis
of EA by CHO cells expressing CD16A. Phagocytosis of rabbit anti-DNP
IgG-opsonized DNP-E by CHO cells expressing CD16A was performed in one
of the following buffers: HBSS with
Ca/Mg
, HBSS without
Ca
/Mg
(buffer A), buffer A with 5
mM EDTA, or buffer A with 5 mM EGTA. Phagocytosis was
determined by assaying the pseudoperoxidase activity of hemoglobin from
ingested E as described under ``Materials and
Methods.''
Figure 9:
Phagocytosis of rabbit IgG-sensitized
DNP-E by CD16A- and CD16A-
chimeras. CHO cell transfectants
expressing CD16B
, CD16B
, CD16A
cotransfected with the
subunit of Fc
RI, and CD16A-
and
CD16A-
chimeras were used in the phagocytosis assay. CD16.3
represents a control CD16A CHO cell transfectant with no significant
surface expression of CD16A.
Existence of more than one FcR on neutrophils has
complicated the analysis of specificity and affinity of CD16B
and CD16B
for human IgG subtypes. Expression of
cloned genes in Fc
R negative cell lines offers the advantage of
analyzing the properties of individual isoforms of Fc
R. Our study
provides a comparison of ligand binding by CD16B alleles in an isolated
system. The results presented in this paper also demonstrate that the
CD16A isoform differs from CD16B
and CD16B
in its phagocytic properties and that both the
and
subunits of CD16A can deliver signals for phagocytosis of IgG-coated
particles in nonhematopoietic cells such as CHO cells.
The CD16 isoforms expressed on CHO cells bind IC complexes and IgG-coated cells. As expected, the NA1 and NA2 alleles of CD16B were susceptible to PIPLC treatment, whereas CD16A was not. Biochemical characterization of CD16 expressed on CHO cells showed that the mobility of CD16-expressed CHO cells is slightly faster than corresponding forms expressed on NK cells and neutrophils. However, the apparent size of the polypeptide was similar after N-glycanase treatment. This suggests that the difference in mobility is due to difference in cell-specific glycosylation of CD16A. A slight shift in electrophoretic mobility due to heterogeneity in glycosylation has also been observed for CD16A expressed on NK cells and macrophages(16, 17) .
It
is well established that polymorphism leads to functional differences
in various receptors, including FcRII(41) . However, only
very limited comparative studies are available on the allelic forms of
CD16B. We have compared the ligand binding and phagocytic properties of
CD16 isoforms expressed on transfected CHO cell lines. Erythrocytes
coated with human IgG1 and IgG3 subtypes efficiently formed rosettes,
whereas IgG2-E mediated weak rosetting with CD16 isoforms. IgG4-E did
not form rosettes with CD16B and consistently showed very few rosettes
with CD16A. The relative affinity of CD16B allelic forms to IgG1 and
IgG3 was further compared by analyzing the competitive inhibition of
soluble IC with the respective monomeric IgG. These competition binding
studies are an indirect measure of the relative affinity of CD16 for
the IgG subtypes. The inhibition of IgG1 complex binding by monomeric
IgG1 to CD16B
and CD16B
followed a similar
pattern, suggesting that CD16B
and CD16B
have a similar affinity for IgG1. The inhibition pattern with IgG3
reveals that the NA1 allele has a 2-fold higher affinity for IgG3 than
the NA2 allelic form of CD16B. Monomeric IgG3 inhibited IC more
efficiently than monomeric IgG1, suggesting that CD16B alleles have a
higher affinity for IgG3 than IgG1. Our results of the IgG subtype
binding pattern of CD16B alleles expressed in CHO cells are similar to
those reported specificities for CD16 on NK cells and neutrophils (42, 43, 44) .
The ability of the two
allelic forms of GPI-anchored CD16B and CD16A expressed on CHO cell
transfectants to mediate phagocytosis of EA was tested. CHO cells
expressing CD16B and CD16B
did not
phagocytose EA, whereas under the same conditions CHO cells expressing
CD16A ingested EA. The phagocytosis was specifically blocked by
anti-CD16 mAb and was independent of extracellular divalent cations.
Treatment of CHO cells with cytochalasin D also blocked CD16A-mediated
phagocytosis, suggesting that intact microfilaments are necessary for
the phagocytic process. These results demonstrate that GPI-anchored
CD16B does not deliver a phagocytic signal, whereas the
polypeptide-anchored CD16A that is coexpressed with the
subunit
can deliver a signal for phagocytosis of EA.
The signaling and
phagocytosis by GPI-anchored CD16B are controversial. Salmon et
el. (45, 46) have shown that neutrophil CD16B can
mediate phagocytosis of E coated with concanavalin A. Moreover it has
been observed (46) that neutrophils from NA2 homozygous
individuals show depressed phagocytosis of E coated with IgG or
concanavalin A when compared with NA1 homozygous individuals. On the
other hand, studies using bispecific antibodies by Anderson et al.(47) have shown that CD16B expressed on neutrophils cannot
mediate phagocytosis. These contradictory observations on the
phagocytic ability of CD16B could be due to the difference in the type
of ligand used or cell type in which CD16B is expressed. It is possible
that neutrophils, unlike CHO cells, may have other proteins that under
certain conditions can associate with GPI-anchored CD16B and provide
phagocytic signals. The GPI-anchored CD16B expressed on neutrophils can
deliver signals for lysosomal enzyme release, Ca mobilization, actin assembly, and antibody-dependent cellular
cytotoxicity of chicken
E(48, 49, 50, 51, 52) ,
whereas CD16B transfected into T cell lines does not signal for
Ca
mobilization (26) . Thus the cell-specific
differences in signaling by CD16B further suggests that CD16B by itself
cannot transduce signals from outside to inside the cell but may do so
by associating with cell-specific components. Precedents for
association of signaling molecules with GPI-anchored receptors such as
CD59 and CD55 have been reported (53, 54, 55) .
In contrast to our
observations on CD16A, the human FcRIIA expressed on CHO cell
transfectants with intact transmembrane and cytoplasmic domains did not
mediate phagocytosis(56) . However, under similar conditions
the Fc
RIIA transfected into a macrophage cell line-mediated
phagocytosis, suggesting that CHO cells lack the machinery for Fc
R
mediated phagocytosis. The phagocytic signal delivered by Fc
RIIA
was mapped to a distinct region in the cytoplasmic domain(56) .
It should be noted that Fc
RIIA does not require any subunits for
cell surface expression, whereas CD16A cannot be expressed on the cell
surface without association of a
or a
subunit. Therefore,
it is possible that the phagocytosis observed in CHO cell transfectants
may be due to the CD16A-associated
subunit. To address this we
have analyzed the phagocytic ability of chimeric CD16A-
and
CD16A-
molecules expressed on CHO cells. These chimeras were made
by joining the extracellular domain of CD16A and transmembrane and
cytoplasmic domains of a
or
chain(28) . Both
chimeras mediated phagocytosis of EA as efficiently as CD16A expressed
in association with the
chain, demonstrating that both
and
chains can deliver a phagocytic signal in CHO cells. This
suggests that CHO cells are capable of mediating phagocytosis if the
phagocytic signal is delivered by
and
chains. Therefore, it
may be possible that other types of Fc
Rs can also mediate
phagocytosis of IgG-coated particles in CHO cells if they are
coexpressed with the
or
chain. Indeed, a recent study shows
that Fc
RI expressed on COS cells requires coexpression of the
subunit to mediate phagocytosis(57) .
The phagocytosis
by CD16A-expressing CHO cell transfectants was blocked by
tyrphostin-23, a protein tyrosine kinase inhibitor, suggesting that
protein tyrosine phosphorylation plays an important role in the
phagocytosis events initiated by CD16A-associated subunits. Studies by
others have also shown that the phagocytosis mediated by FcRs
associated with the
or
subunit can be blocked by
tyrphostin-23 (57, 58) . Protein tyrosine
phosphorylation, including the phosphorylation of the associated
subunit, is an essential signaling pathway in Fc
R-dependent
phagocytosis mediated by mouse macrophages(59, 60) .
Triggering of cells via TCR or Fc receptors results in phosphorylation
of associated
or
chain at tyrosine
residues(26, 61, 62, 63, 64) .
However, it was shown that CD16A cross-linking does not induce
phosphorylation of
or
subunits in CHO cells(28) .
This suggests that tyrphostin-23 may interfere with phosphorylation of
proteins other than
or
subunits that are essential for the
phagocytic process. However, although tyrphostin-23 is a more specific
inhibitor of tyrosine kinases than genistein (40) and has been
widely used in many signal transduction studies, our results did not
rule out nonspecific inhibition of phagocytosis by tyrphostin-23 in CHO
cells. Recently, Park et al.(58, 65) have
shown that COS cells transiently cotransfected with CD16A and the
or
subunit can mediate phagocytosis of EA. They have further
shown that conserved tyrosine residues on the
or
chain are
necessary for both the phagocytosis and Ca
mobilization in COS cells. Unlike CD16A expressed on COS
cells(65) , CD16A expressed on CHO cells does not induce
intracellular Ca
mobilization(26, 28) , suggesting that the
phagocytic events initiated by
or
subunits in CHO cells do
not require intracellular Ca
mobilization.
Ca
-dependent and Ca
-independent
phagocytosis has been described in human neutrophils and
macrophages(66, 67, 68) . Therefore, it is
possible that the phagocytosis mediated by CD16A and its chimeras in
CHO cells may represent the Ca
-independent phagocytic
pathway observed in macrophages and neutrophils.
The results shown
here with CD16A chimeras, in conjunction with the observations on
FcRIIA(56) , suggest that at least two distinct components
can deliver signals for Fc
R-mediated phagocytosis, i.e. one is the cytoplasmic domain of Fc
Rs as in the case of
Fc
RIIA, and the second is the Fc
R-associated
and
subunits. Depending upon the type of cells and Fc
Rs, either one or
both pathways may be involved in phagocytosis. At present it is not
clear whether the Fc
R-associated subunits are obligatory
components of the phagocytic machinery of all Fc
R-dependent
phagocytosis mediated by cells such as macrophages and neutrophils.
Recently, it has been shown that Fc
RI and Fc
RII associate
with the
chain, although they do not require subunit association
for expression(69, 70) . Phagocytic cells such as
neutrophils and macrophages constitutively express the
chain that
may associate with Fc
Rs and thus provide a phagocytic
signal(22, 71) . Very recently, Takai et al.(72) have shown that macrophages from
chain-deficient mice are defective in Fc
-R mediated phagocytosis,
suggesting that the
chain expression is crucial for
Fc
R-mediated phagocytosis in macrophages. The phagocytosis
mediated by the CD16A-subunit chimeras expressed on CHO cells described
in this report provides an ideal model system to study the
Ca
-independent phagocytic signaling pathway initiated
by Fc
R-associated subunits. Furthermore, the CHO cell system could
also be used to determine whether other types of Fc
Rs also use the
or
subunit-initiated signaling pathway to mediate
phagocytosis, even though they do not require the subunits for surface
expression.