(Received for publication, June 20, 1995; and in revised form, July 24, 1995)
From the
Differing roles for
transients in
Fc
R-mediated phagocytosis have been suggested based on the
observations that antibody-opsonized erythrocyte phagocytosis by human
neutrophils shows a [Ca
]
dependence, while that by murine macrophages appears
[Ca
]
-independent. To
explore whether this difference might reflect different receptor
isoforms or different cell types, we studied the
[Ca
]
dependence of
receptor-initiated phagocytosis by human Fc
RIIa and a panel of
Fc
RIIa cytoplasmic domain mutants expressed in murine P388D1 cells
and by human Fc
R endogenously expressed on human neutrophils and
monocytes. Wild-type and point mutants of huFc
RIIa stably
transfected into murine P388D1 cells have different capacities to
initiate a [Ca
]
transient, which are closely correlated with quantitative
phagocytosis (r = 0.94, p < 0.0001).
Phagocytosis both by huFc
RIIa in P388D1 cells and by huFc
RIIa
endogenously expressed on neutrophils and blood monocytes shows
[Ca
]
dependence.
Phagocytosis of antibody-opsonized erythrocytes by neutrophils
demonstrated greater susceptibility to
[Ca
]
quenching
compared with Fc
RIIa-specific internalization with E-IV.3,
suggesting that the phagocytosis activating property of Fc
RIIIb in
neutrophils also engages a
[Ca
]
-dependent
element. In contrast, phagocytosis by human Fc
RIa, endogenously
expressed on blood monocytes, is
[Ca
]
-independent.
Despite the importance of a consensus tyrosine activation motif for
both receptors, Fc
RIa and Fc
RIIa engage at least some
distinct signaling elements to initiate phagocytosis. The recognition
that both of the phagocytic receptors on murine macrophages and human
Fc
RIa associate with the Fc
RI
-chain, which contains a
tyrosine activation motif distinct from that in the Fc
RIIa
cytoplasmic domain, suggests that
[Ca
]
-independent
phagocytosis is a property associated with the utilization of
-chains by Fc
R.
Receptors for the Fc region of IgG (FcR) (
)provide a link between antibody-antigen complexes and
cellular-based effector functions and are critical in the regulation of
the inflammatory response(1, 2) . Significant
structural diversity between the three gene families encoding Fc
R
is
observed(1, 2, 3, 4, 5) .
Nonetheless, Fc
R share certain intracellular signaling pathways.
The common themes in Fc
R signaling pathways involve the activation
of protein tyrosine kinases followed by a transient rise in
intracellular Ca
levels. The
[Ca
]
increase is
essential for many cellular functions and is required for the phagocyte
Fc
R-induced oxidative burst(6, 7) .
Many lines
of evidence in both human and murine systems indicate that tyrosine
phosphorylation events are critical for phagocyte FcR functions,
including phagocytosis(8, 9, 10) . In
addition, in many systems examined, tyrosine kinase activity is
required for the receptor-induced rise in
[Ca
]
(presumably
through tyrosine phosphorylation of phospholipase C
1 and
generation of inositol 1,4,5-trisphosphate). However, the role of
[Ca
]
in Fc
receptor-mediated phagocytosis has been controversial. For example,
work in murine macrophage cell lines suggests that transients in
[Ca
]
are not essential
for phagocytosis of antibody-opsonized erythrocytes (EA) (11, 12, 13) . In contrast, phagocytosis of
EA by human neutrophils is significantly impaired by chelation of
intracellular calcium and abrogation of
[Ca
]
transients(14, 15) . The ability of
[Ca
]
-depleted
neutrophils to mediate phagocytosis initiated by other cell surface
receptors suggests that the
[Ca
]
-dependent EA
phagocytosis by human neutrophils may reflect a particular property of
the Fc
receptors on these cells(16) .
Indeed, each of
the studies of the
[Ca]
-dependence of
Fc
receptor mediated phagocytosis has used EA probes that engage
all available Fc
receptor types and has not systematically
distinguished between different cell types or cells derived from
different species. Recent data indicate that important species
differences do exist for Fc
receptors. For example, human
Fc
RIIA and Fc
RIIIB, expressed on neutrophils, do not have
murine homologues(1, 2, 3) . Human
Fc
RIIA stably transfected into the murine macrophage cell line
P388D1 mediates receptor-specific phagocytosis but in a
[Ca
]
-dependent
fashion(17) . In contrast, murine Fc
RII (the IIb isoform)
does not have a tyrosine activation motif nor does it trigger a
[Ca
] transient or protein tyrosine
phosphorylation(18) . Murine Fc
RII is also unable to
mediate phagocytosis in macrophages in either a
[Ca
]
-dependent or
independent fashion(19) . These observations, coupled with the
recent data of Stendahl and co-workers (20) that
[Ca
]
storage
organelles accumulate at contact sites during phagocytosis in human
neutrophils prompted us to reexamine the question of the
[Ca
]
dependence of
Fc
receptor-mediated phagocytosis by human Fc
receptors,
endogenously expressed by human cells and stably transfected into the
P388D1 murine macrophage cell line.
Our data indicate that human
FcRIIa uses
[Ca
]
-dependent
elements to mediate receptor-specific phagocytosis and that
huFc
RIIa point mutants with a varying ability to initiate a
[Ca
]
transient show a
closely corresponding variation in quantitative phagocytosis. Human
Fc
RIIa, endogenously expressed on neutrophils and blood monocytes,
also shows partial [Ca
]
dependence for phagocytosis. In contrast, phagocytosis by
human Fc
RIa, endogenously expressed on blood monocytes, is
[Ca
]
-independent.
Despite the importance of tyrosine phosphorylation for phagocytosis and
the use of a consensus tyrosine activation motif by both
receptors(1, 2, 3, 4, 5, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) ,
they engage at least some distinct signaling elements to initiate
phagocytosis. The recognition that the human Fc
RIa associates with
the Fc
RI
-chain, which contains the tyrosine activation motif (21, 22) as do both of the phagocytic receptors on
murine macrophages(23) , suggests that
[Ca
]
-independent
phagocytosis is a property associated with Fc
R utilizing
-chains. Human Fc
RIIa, while engaging many elements in common
with human Fc
RIa such as p72
and
phospholipase
C
1(25, 26, 27, 28, 29, 30) ,
must also engage
[Ca
]
-dependent
elements.
Fresh anti-coagulated human peripheral blood was separated by
centrifugation through a discontinuous two-step Ficoll-Hypaque gradient (33) . Mixed mononuclear cells were isolated from the upper
interface and washed with Hanks' balanced salt solution.
Neutrophils were isolated from the lower interface and washed with
Hanks' balanced salt solution. Contaminating erythrocytes in the
neutrophil harvest were lysed with hypotonic saline (0.2% NaCl) for 20
s followed by 1.6% NaCl and a final wash with Hanks' balanced
salt solution. After final washes, cells were resuspended to 5
10
cells/ml in PBS prior to immunofluorescent staining or
in RPMI containing 10% fetal calf serum prior to phagocytosis.
Treatment of cells with BAPTA (to quench intracellular
Ca levels) or genistein (to block protein tyrosine
kinase activity) was performed as described
previously(10, 17, 34) . Briefly, cells were
incubated with BAPTA-AM (1-100 µM) in buffer without
free Ca
for 30 min at room temperature (neutrophils
and monocytes) or 37 °C (P388D1 transfectants) followed by one
wash. Buffer containing Ca
was then added and handled
as described below. For genistein treatment, cells were preincubated
with 100 µg/ml genistein for 30 min, and then the genistein was
maintained at the same concentration through the phagocytic assay.
Controls included loading cells with the BAPTA-AM and genistein solvent
(dimethyl sulfoxide) at appropriate concentrations for the same period
of time. As an alternative to BAPTA-AM treatment for quenching
[Ca
]
, cells were allowed to
drain their intracellular Ca
stores as described by
Rosales et al.(35) . Levels of intracellular
Ca
were directly determined in all cases as described
below.
mAb-conjugated
erythrocytes were prepared by incubating E with dilutions
of biotinylated anti-Fc
R mAb(37) . mAb-coated E
were resuspended in RPMI 1640-fetal calf serum, an aliquot was
removed for analysis by indirect immunofluorescence, and the remaining
cells were used immediately for the phagocytosis or attachment assays.
EA were prepared by incubating bovine erythrocytes with a 1:4 dilution
of the maximal subagglutinating titer of rabbit anti-bovine erythrocyte
IgG as described previously(38) .
For quantitation of
mAb-coated E or EA phagocytosis by fresh human cells,
erythrocytes were mixed with 100 µl of fresh neutrophils (5
10
cells/ml) or fresh mononuclear cells (5
10
monocytes/ml, determined by myeloperoxidase staining) at a ratio
of 25:1 (mAb-coated E
) or 50:1 (EA) (37) . The
cell mixture was pelleted for 5 min at room temperature at 44
g and then incubated at 37 °C for 20 min (neutrophils) or
1 h (mononuclear cells). After the nonphagocytosed erythrocytes were
lysed with hypotonic saline (0.2% NaCl for 20 s followed by the
addition of an equal volume of 1.6% NaCl), phagocytosis was quantitated
by light microscopy. The data are expressed as phagocytic index (PI,
the number of ingested particles/100 neutrophils or monocytes).
Phagocytosis by transfected P388D1 cells was determined in an
adherent assay system. P388D1 cells (5 10
cells/ml)
were allowed to adhere to round glass coverslips in culture dishes
overnight at 37 °C. Coverslips were then transferred to clean
culture dishes and EA- or mAb-coated E
were added (50
µl at 5
10
erythrocytes/ml) were added and
incubated for 1 h at 37 °C. Noninternalized erythrocytes were lysed
by brief immersion of the coverslip in dH
O followed by
immersion in buffer. Phagocytosis was quantitated by light microscopy
and expressed as phagocytic index as described above.
Heat-treated
and serum-treated zymosan were prepared as described
previously(38) . Briefly, heat-treated zymosan were prepared by
boiling 10 mg of zymosan for 10 min. Serum-treated zymosan were
prepared by incubating 2 mg of zymosan with 2 ml of normal human serum
for 30 min at 37 °C. Following washing, both heat-treated zymosan
and serum-treated zymosan were resuspended to 2.5 108/ml. For
phagocytosis, heat-treated zymosan or serum-treated zymosan were mixed
with neutrophils (5:1, zymosan/neutrophil ratio), pelleted, and
incubated for 20 min at 37 °C. Phagocytosis was assessed by light
microscopy.
Figure 4:
A, receptor-specific fluxes in
[Ca]
were elicited in
Indo-1-loaded neutrophils by cross-linking Fc
RIIa with mAb IV.3
Fab and GAM F(ab`)
. Pretreatment of cells with increasing
concentrations of BAPTA-AM led to a progressive decline in the
[Ca
]
response. Each
sample was calibrated as described by Grynkiewicz et
al.(35) . A representative experiment is shown (n = 3). B, in parallel experiments, neutrophils were
assayed for phagocytosis of E-IV.3 by Fc
RII (
), of rabbit
antibody opsonized erythrocytes (EA), which engage both
Fc
RIIa and Fc
RIIIb (
), and of heat-treated and
serum-treated zymosan (
,
, respectively). Maximal
inhibition of both E-IV.3 and EA phagocytosis was reached by 50
µM BAPTA, while phagocytosis of the zymosan particles was
unaffected by the same dose of BAPTA. The untreated control PI for EA
of 33 was not different from the untreated control PI for E-IV.3 of 29 (p = not significant). The means and standard
deviations are shown, and each point represents 5-15 independent
determinations.
Several recent observations including information on species
differences in Fc receptor isoforms and function (1, 2, 17, 19) have prompted a
reconsideration of the studies supporting
[Ca
]
-dependent and
[Ca
]
-independent Fc
receptor-mediated phagocytosis. For example, studies by Stendahl et
al.(20) have shown that there is an accumulation of
[Ca
]
storage organelles during
phagocytosis in human neutrophils. These observations suggest the
possibility that the Fc
receptors expressed in human neutrophils
are functionally distinct from murine Fc
receptors in engaging
[Ca
]
-dependent elements for
phagocytosis. Indeed, initial studies of human Fc
RIIA truncation
mutants stably transfected into P388D1 cells have shown that all
truncations unable to initiate a [Ca
]
transient are unable to mediate receptor-specific
phagocytosis(17) . The evidence for an essential role for
[Ca
]
in Fc
RIIa
phagocytosis is strengthened by the ability of BAPTA, a chelator of
[Ca
]
, to block phagocytosis by
Fc
RIIa wild-type receptor in P388D1 cells(17) .
Accordingly, we have examined these relationships in a series of
Fc
RIIa transfectants with point mutations in the region of the
cytoplasmic domain containing the YXXL tyrosine activation
motif. Mutations in this region (Fig. 1) can lead to altered
binding and activation of p72
, which in turn
phosphorylates phospholipase C
-1 leading to the generation of
inositol 1,4,5-trisphosphate and [Ca
]
transients(41) . Studies of transfected Fc
R mutants
indicate that receptor-mediated phagocytosis is also altered (42, 43
Figure 1:
Comparison
of the tyrosine activation consensus motif (46) with that for
human FcRIIA and human
-chain of Fc
RI shows that
Fc
RIIA has a 12-residue, rather than a 7-residue, sequence between
the two YXXL motifs (A). Point mutations were
designed to alter both the YXXL motifs and adjacent residues (B).
Fifteen mutants were constructed (Fig. 1), and stable transfectants expressing between 1.1 and
2.6 10
receptors/cell were selected. The
[Ca
] transient observed after cross-linking
each mutant receptor was measured and ranged from no response for
several mutants of tyrosine residues within the tyrosine activation
motif to a flux of approximately 500 nM (Fig. 2). The
measured [Ca
]
transients were
abrogated by pretreatment of cells with BAPTA, were unaffected by 10
mM EGTA extracellularly, and therefore were due to
mobilization of [Ca
]
from
intracellular stores. Among the 15 cell lines expressing different
mutant Fc
RIIa, there was no significant relationship between
quantitative receptor expression measured by flow cytometry and peak
[Ca
]
flux (p >
0.10; not significant).
Figure 2:
A , receptor-specific fluxes in
[Ca]
were initiated in
Indo-1-loaded transfected P388D1 cells by cross-linking human
Fc
RIIa with mAb IV.3 Fab and GAM F(ab`)
as described
previously in selected mutant Fc
RIIa expressing cells. Each sample
was calibrated as described by Grynkiewicz et al.(36) . B, receptor-specific phagocytosis in
selected mutated Fc
RIIa transfected P388D1 cells was determined
using mAb IV.3 Fab coated erythrocytes as described
previously(37) . The mean and the standard deviation are shown (n = 6 for each mutant).
The same mutants were probed for
FcRIIa-specific phagocytosis using the receptor-specific mAb IV.3
Fab conjugated to erythrocytes via a streptavidin bridge (E-IV.3). The
density of mAb IV.3 conjugation to erythrocytes was monitored by flow
cytometry. Nonbiotinylated erythrocytes and biotinylated but
unconjugated erythrocytes neither bound nor were internalized by
transfected or nontransfected P388D1 cells. Furthermore, erythrocytes
coupled to an IgM anti-H2-D
via the streptavidin bridge
bound to P388D1 as expected but were not internalized (mean attachment
index = 133; phagocytic index = 0; n =
3). Quantitative phagocytosis of E-IV.3 ranged from no internalization
for the same tyrosine mutants that failed to elicit a
[Ca
]
transient to a maximum
phagocytic index of 126.5 ± 16 E-IV.3 ingested per 100 cells (Fig. 2B). Among the 15 different mutants, there was no
significant relationship between quantitative receptor expression and
E-IV.3 ingestion (p > 0.10; not significant). As with
wild-type Fc
RIIa(17) , the protein tyrosine kinase
inhibitor genistein inhibited by >95% phagocytosis of E-IV.3 by the
phagocytic mutant forms of Fc
RIIa and of EA by the native murine
Fc
R in parental P388D1.
There was, however, a striking
relationship between peak [Ca]
and Fc
RIIa-specific phagocytosis (Fig. 3) with a
correlation coefficient of 0.94 (p < 0.0001). Importantly,
even the most phagocytic of the mutant receptors was sensitive to
chelation of [Ca
]
by 50
µM BAPTA (Fig. 3, inset).
Figure 3:
Peak
[Ca]
flux, as demonstrated in Fig. 2, was strongly correlated with quantitative,
receptor-specific phagocytosis measured with E-IV.3 (correlation
coefficient = 0.94; p < 0.0001). Of note, the
EE239-240QQ mutant, which is located in a region outside of the
YXXL dyad and has a high
[Ca
]
flux occurs
within the 7 residues defined as essential for
[Ca
]
by Kolanus et al.(43) . Inset, pretreatment of
phagocytic cells with 50 µM BAPTA-AM-abrogated
phagocytosis. A representative experiment is shown (n =
3 for each determination).
The strong
correlation between [Ca]
and
phagocytosis for human Fc
RIIa even in the environment of a murine
macrophage cell line and the dependence of Fc
RIIa-mediated
phagocytosis on [Ca
]
suggested
that this property might reflect the characteristics of human
Fc
RIIa per se. Since previous studies in human and murine
cells had not probed Fc
receptor function in a receptor-specific
fashion(11, 12, 13, 14, 15) ,
we sought to explore the properties of Fc
RIIa expressed
endogenously on neutrophils. Our previous studies with neutrophils
indicated that Fc
RIIa alone can mediate phagocytosis(10) .
Therefore, using receptor-specific engagement of Fc
RIIa with mAb
IV.3 Fab and cross-linking with GAM F(ab`)
, we defined the
ability of BAPTA pretreatment of neutrophils to blunt the
[Ca
]
response (Fig. 4A). Correspondingly, we assessed quantitative
receptor-specific phagocytosis (Fig. 4B). A BAPTA
dose-dependent inhibition of the Fc
RIIa-mediated rise in
[Ca
]
was observed with complete
inhibition achieved by 50 µM BAPTA. BAPTA also reduced
phagocytosis in a dose-dependent fashion with
50% reduction at a
loading concentration of 50 µM (p < 0.002).
Neither phagocytosis of heat-treated zymosan nor of serum-treated
zymosan was altered by 50 µM BAPTA pretreatment (both
99-100% of untreated control cells; p > 0.5, not
significant) (Fig. 4B). Higher loading concentrations
of BAPTA did not lead to further decrement of E-IV.3 internalization
(PI = 50.6% of control at 100 µM). 50 µM BAPTA was also sufficient to abrogate detectable
[Ca
]
transients initiated by
10
FMLP (
[Ca
]
indistinguishable from base line, n = 7).
As
an alternative technique to deplete intracellular free Ca levels, we allowed neutrophils to incubate in
Ca
/Mg
-free media to exhaust
intracellular Ca
stores (Fig. 5) as described
by Rosales and Brown(35) . Fc
RIIa-specific phagocytosis by
neutrophils treated in this manner was markedly blunted relative to
control cells in the presence of physiologic levels of
Ca
/Mg
(PI = 48.0 ±
13.1% of control, n = 3). The extent of inhibition was
comparable with that achieved by BAPTA pretreatment.
Figure 5:
Incubation of neutrophils in
Ca/Mg
-free buffer empties
intracellular Ca
stores. Addition of FMLP
(10
M) results in a rapid rise in
[Ca
] in control neutrophils (neutrophils
that were Ca
depleted and then repleted), and this
response is completely blunted in Ca
-depleted cells.
A representative experiment is shown (n =
4).
To compare our
results in neutrophils with those of Lew and co-workers (14) ,
we also probed neutrophils with EA for the effects of
[Ca]
chelation on phagocytosis
engaging both Fc
RIIa and Fc
RIIIb. Although the GPI-anchored
Fc
RIIIb does not mediate phagocytosis itself, it does elicit a
[Ca
]
transient and functions
synergistically with Fc
RIIa for an enhanced phagocytic
response(10) . As reported by Lew(14) , EA phagocytosis
was profoundly reduced by [Ca
]
chelation (Fig. 4B) and to a significantly
greater extent than E-IV.3 (EA = 23 ± 15% (n = 10) of control compared 47 ± 20% (n = 12) of control for E-IV.3 at 50 µM BAPTA, p < 0.005; over all doses of BAPTA, EA versus E-IV3, two-way analysis of variance: F = 13.2, p < 0.0002). These observations suggested that the
Fc
RIIIb-initiated [Ca
]
transients might play an important quantitative role in
Fc
RIIa internalization.
To test this hypothesis as the basis
for the known difference in EA phagocytosis by individuals homozygous
for the two different alleles of
FcRIIIb(38, 40) , we examined the relative
ability of Fc
RIIIb in NA1 and NA2 homozygotes to elicit
[Ca
]
fluxes in neutrophils.
Engagement of Fc
RIIIb by anti-receptor mAb 3G8 and cross-linking
with either GAM or with streptavidin leads to a
[Ca
]
flux derived from
intracellular stores (34) . When the anti-Fc
RIII mAb 3G8
IgG, a murine IgG1, was used to initiate the
[Ca
]
transient, consistent
differences between donors were noted that were attributable to the
His-131/Arg-131 polymorphism of Fc
RIIa and presumably the ability
of Fc
RIIa to engage the Fc region of 3G8 IgG and form heterotypic
Fc
RIIa-Fc
RIIIb receptor clusters (Fig. 6A).
However, no consistent difference in the magnitude of the
[Ca
]
flux could be attributed
to the phenotype of Fc
RIIIb engaged by 3G8 F(ab`)
with
subsequent cross-linking in five matched pairs of NA homozygous donors (Fig. 6B). Taken together, these observations suggest
that a [Ca
]
-sensitive signaling
element is engaged by Fc
RIIa during EA phagocytosis but that the
magnitude of the Fc
RIIIb-induced
[Ca
]
flux per se does
not explain the difference in quantitative phagocytosis between NA1 and
NA2 homozygous donors.
Figure 6:
A, the ability of intact mAb 3G8
IgG to elicit a [Ca]
flux in Indo-1-loaded neutrophils was assessed in a series of
donor pairs. A consistent difference in quantitative
[Ca
]
was noted
between donors homozygous for the Fc
RIIa His-131 (LR) and
Arg-131 (HR) alleles. Homozygous Arg-131 (HR) donors
are able to bind murine IgG1 more effectively to form a receptor
cluster of Fc
RIIIb via Fab binding and Fc
RIIa via Fc binding
thereby triggering a more vigorous
[Ca
]
response (n = 6 pairs of donors). B, 3G8 F(ab`)
,
which engages only Fc
RIIIb, elicits a
[Ca
]
when cross-linked
with goat anti-mouse F(ab`)
, but no difference between NA1
and NA2 homozygotes could be defined (n = 5 pairs of
donors examined in at least two independent
experiments).
Demonstration of the
[Ca]
dependence of E-IV.3
phagocytosis in neutrophils and in murine P388D1 cells resolves the
apparent controversy between DiVirgilio and others about the role of
[Ca
]
in
phagocytosis(11, 12, 13, 14, 15) ,
but it does not address the issue of whether a human homologue of a
``[Ca
]
-independent''
murine receptor would also be [Ca
]
independent for phagocytosis. Accordingly, we examined the
effects of BAPTA on Fc
RIIa-specific (E-IV.3) and
Fc
RIa-specific (E-22) phagocytosis by human peripheral blood
monocytes. Both receptors mediate
[Ca
]
transients after receptor
cross-linking that are blocked by 50 µM BAPTA (Fig. 7A). As anticipated, E-IV.3 showed more than a
50% decrement in phagocytosis after pretreatment with 50
µM BAPTA (Fig. 7B; PI = 40.5
± 13.5% of control, p < 0.002). In contrast E-22,
the specific probe for Fc
RIa, showed only a minimal change that
was significantly less than E-IV.3 (p < 0.002) and not
significantly different from control (Fig. 7B, p >
0.5). These data for human Fc
RIa are similar to those for
phagocytic murine Fc
receptors on elicited peritoneal
macrophages(12, 44) .
Figure 7:
A, both FcRIIa and
Fc
RIa can initiate a [Ca
]
flux in human blood monocytes with cross-linking of
receptors, which is completely blocked with 50 µM BAPTA (n = 3). B, but only Fc
RIIa-specific
phagocytosis is significantly affected by pretreatment of cells with 50
µM BAPTA-AM (mean and standard deviation is shown, n = 4). *, p < 0.002, 50 µM BAPTA
treated versus control.
Recent observations have prompted a re-evaluation of the role
of [Ca]
in Fc
receptor-mediated phagocytosis. In previous work, both murine
macrophage cell lines and human neutrophils were used, which may have
confused the issues since the cells express structurally distinct
Fc
receptors and may have intrinsically different signaling
capacities and cell programs. Indeed, the two Fc
receptors
constitutively expressed on human neutrophils do not have corresponding
murine homologues. While human Fc
RIIa is phagocytic, murine
Fc
RII (the Fc
RIIb isoform) on macrophages is unable to
mediate [Ca
] transients, induction of
tyrosine phosphorylation, or phagocytosis(18, 19) .
These observations, coupled with the
[Ca
]
dependence of
huFc
RIIa-mediated phagocytosis in stably transfected cells,
suggest that the apparent controversy might simply reflect structurally
different receptors each engaging at least some distinct signaling
elements.
Our data indicate that human FcRIIa, both in a murine
macrophage environment and endogenously expressed in human neutrophils,
shows significant [Ca
]
dependence for phagocytosis. This is in contrast to human
Fc
RIa, which like its murine homologue, shows minimal or no
[Ca
] dependence. These observations
emphasize that Fc
RIIa, which has a cytoplasmic tyrosine activation
motif distinct from that in the
-chain associated with Fc
RIa
(due to 12 versus 7 amino acids separating the YXXL
sequences, respectively), engages some signaling elements distinct from
Fc
RIa. The fact that Fc
RIIa phagocytosis in native cells was
not completely [Ca
]
-dependent
suggests, however, that this receptor may engage several signaling
pathways, a property now recognized for other Fc
receptors(45) .
The mechanism underlying the variation in
[Ca]
transients and
phagocytosis for different Fc
RIIa mutants may relate to variable
efficiency in engaging the SH2 domain of the protein tyrosine kinase
p72
. Fc
RIIa does bind
p72
(25, 26, 28, 29) ,
and the binding of the ZAP70 homologue to a similar YXXL
tyrosine activation motif shows a high degree of sensitivity to
mutations in the flanking sequences (41, 46, 47, 48) . In the Fc
RI
model system, which incorporates the YXXL tyrosine activation
motif in
-chain, p72
binding and phosphorylation
leads to tyrosine phosphorylation of phospholipase C
1 (either
directly by p72
or through an intermediary kinase(s)),
inositol lipid breakdown with generation of inositol
1,4,5-trisphosphate, and a [Ca
]
flux(49, 50, 51, 52, 53) .
Disruption of this sequence by inhibition of p72
with
piceatannol or specific inhibitory peptide abrogates the
[Ca
]
flux(53, 54) . The essential role for
[Ca
]
in Fc
RIIa-mediated
phagocytosis is strongly supported by the correlation of
[Ca
]
with phagocytosis between
the 15 different Fc
RIIa mutants, by the ability of BAPTA to
abrogate phagocytosis by both wild-type and mutant receptors (Fig. 3) and by the ability of BAPTA to abrogate phagocytosis by
Fc
RIIa in neutrophils without affecting ingestion of zymosan and
in monocytes without affecting internalization by Fc
RIa. However,
the critical [Ca
]
-dependent
element(s) for Fc
RIIa signaling remain unidentified at present.
Preliminary experiments with cyclosporin A, an inhibitor of calcineurin
that is a [Ca
]
-dependent
phosphatase, show little or no effect on human Fc
RIIa phagocytosis
in transfected P388D1 cells, although calcineurin activity is essential
for neutrophil motility in some circumstances(55) . Recent data
suggest that L-plastin, a
[Ca
]
-regulated actin-bundling
protein, may be a candidate for a
[Ca
]
-dependent element
essential for Fc receptor-mediated phagocytosis in neutrophils (56) .
The greater susceptibility of EA phagocytosis to
Ca buffering compared with Fc
RIIa-specific
phagocytosis with E-IV.3 suggests that the activation of phagocytosis
by Fc
RIIIb in human neutrophils has Ca
-dependent
elements. Since Fc
RIIIb elicits a
[Ca
]
flux and functions
synergistically with Fc
RIIa for phagocytosis, an allele-specific
variation in Fc
RIIIb-initiated
[Ca
]
would provide a
straightforward mechanism for the quantitative difference in
phagocytosis shown by donors homozygous for different Fc
RIIIb
alleles. Although we could define consistent differences in
[Ca
]
transients between
individuals, with those elicited by intact anti-receptor IgG relating
to the His-131/Arg-131 polymorphism of
Fc
RIIa(57, 58, 59) , donors homozygous
for the NA1/NA2 Fc
RIIIb alleles were not different in their
ability to generate Fc
RIIIb-specific fluxes in
[Ca
]
. Thus, some other
mechanism, perhaps relating to the differences in glycosylation of the
NA1 and NA2 alleles and potential carbohydrate-mediated interactions
with other cell surface molecules such as CD11b/CD18 (60, 61) must be involved. While we cannot explain the
NA1/NA2 difference in phagocytosis on the basis of quantitative
differences in [Ca
]
interacting
with the partially
[Ca
]
-dependent phagocytosis of
Fc
RIIa, the difference in [Ca
]
dependence between Fc
RIIa and Fc
RIa underscores the
fact that different Fc
receptors can engage distinct signaling
elements. Whether these distinct elements reflect primary sequence
differences in the tyrosine activation motifs used by each of the
receptors or some modifying contribution of the cytoplasmic domain of
the ligand binding Fc
RIa
chain (compare (52) )
remains to be determined. These distinct elements may converge on some
cell programs, but they also provide the foundation for differences in
elicited cell programs and for selective therapeutic intervention.