(Received for publication, December 8, 1994)
From the
Human type 1 Fc receptors (Fc
RI) bind with high
affinity (K
=
10
M) Fc regions of monomeric IgG1 and
IgG3. As demonstrated in this report, interaction of IgG-Fc with the
ligand binding site on Fc
RI alters its capacity for
aggregation-dependent signaling. This Fc-dependence was demonstrated in
normal monocytes and U937-10.6 cells exposed to monomeric IgG and
then to anti-Fc
RI F(ab`)
that cross-link the receptor.
Using O
production to measure cell
signaling, we found that binding by high affinity IgGs of various
species, as well as by murine hybrid IgGs containing only one high
affinity heavy chain, resulted in a marked increase in
Fc
RI-mediated signaling. Preaggregated Fc
RI/IgG had a ratio
of one. IgG binding after aggregation of unligated Fc
RI did not
restore signaling. Dose responses indicated that concentrations of IgG
that saturated Fc
RI optimized transductional activity. The
inclusion of unligated with ligated Fc
RI in aggregates depressed
activity, indicating a lack of trans-activation of unligated Fc
RI.
Significantly, IgG-binding markedly increased aggregation-dependent
tyrosine phosphorylation of Fc
RI
-chains and the association
of tyrosine phosphorylated Syk. Thus, the consequences of IgG-Fc
binding were increases in aggregation-dependent phosphorylation of
Fc
RI
-chains, recruitment of pp72Syk to Fc
RI, and
signaling of the NADPH oxidase pathway.
Fc receptors (Fc
R) (
)bind the Fc region of
immunoglobulin G (IgG) and are expressed on the surface of a variety of
cells. The human high affinity Fc
R (Fc
RI) is a multichain
receptor comprised of a 70-kDa glycoprotein
-chain and homodimeric
largely intracellular
-chain subunits(1, 2) .
Surface expression of the receptor on monocytes and U937 cells is
induced by IFN-
(3, 4, 5) . Fc
RI
binds with high affinity (K
=
3 nM) human IgG subclasses IgG1 and IgG3. It has one log
less affinity for IgG4 and no apparent affinity for
IgG2(6, 7) . The receptor binds with high affinity
certain IgGs from other species that possess sequence homologies in the
C
2 Fc regions(6) . Fc
RI engagement of complexed IgG
or anti-receptor antibodies(8, 9) aggregates the
receptor(10, 11) , triggering several very early
responses including fluxes of intracellular
Ca
(12, 13) , tyrosine
phosphorylations of several proteins (14) and activation of the
NADPH oxidase pathway(12) .
Myelocytic signaling functions
of FcRI, Fc
RIIIA, Fc
RI, and Fc
R (15) are
mediated by tyrosine activation of
-chains (10, 11, 13) in motifs recognized by members
of src family(16) . Src kinases Hck and Lyn associate
with Fc
RI
-chains to constitute a preaggregation multichain
transduction unit(17) . Aggregation of Fc
RI induces the
tyrosine phosphorylation of its
-chains(15, 18) and the tyrosine phosphorylation of Syk
kinase(18, 19, 20) . Syk, a 72-kDa structural
homologue of the T-cell signal-transducing ZAP70, is recruited to
-chains on activated Fc
RI(21) .
We have previously
reported that an equimolar mixture of anti-FcRI monoclonal
antibodies 32 and 22 is a potent combination for triggering
Fc
RI-mediated NADPH oxidase activity(8, 9) .
Subsequently, we found that the F(ab`)
fragments of these
anti-Fc
RI trigger poorly, and that this situation is reversed by
binding high affinity IgG to the receptors(22) . For this
report, we analyzed the consequences of IgG-binding on signaling
through Fc
RI. By engaging the ligand binding site on Fc
RI
with monomer ligand and then cross-linking the receptors through
anti-Fc
RI F(ab`)
, we show evidence of a novel
dependence on IgG-Fc for Fc
RI transductional activity. We show
that IgG-Fc binding increased aggregation-dependent
O
production, tyrosine phosphorylation of
Fc
RI
-chains, and the association of pp72Syk kinase to
activated Fc
RI.
Figure 2:
Effects of Igs of various species, class,
and subclass on FcRI signaling. Cells were preincubated in various
Igs and, after a brief wash, were analyzed for the percent occupancy of
Fc
RI-ligand binding sites and for Fc
RI-mediated
O
production. A, preincubations
contained hIgG1 (H), goat IgG (G), rabbit IgG (R) (10 µg/ml), or medium (none). B,
preincubation media were hybridoma supernatants (200 µl) containing
murine (m) or rat (r) mAbs of IgG subclasses 2a (2a), 1 (1), and 2b (2b) or of the IgM (M), A (A), or E (E) classes. Identities of
mAbs are listed under ``Materials and Methods''; those with
reactivity toward monocyte surface antigens are indicated (*). Assays
of mAb-preincubated cells included control cells preincubated with
hIgG1 or medium. In B, the O
production by cells receiving 32F(ab`)
and 22F(ab`)
but no IgG was subtracted. Therefore, data in B represent the enhancement of O
production due to ligand. (Negative values are slight depressions
in response by unligated receptors.)
Figure 1:
The effect of hIgG on
FcRI-mediated O
production. A, U937 10.6 cells, differentiated by IFN-
for 6 days,
were preincubated for 10 min in O
assay
medium with and without hIgG1 (G1) (10 µg/ml). IV.3Fab (25
µg/ml) was added to block Fc
RII. The preincubated cells were
added to luminol containing 32F(ab`)
and 22F(ab`)
(F) (5 µg/ml each), or 22F(ab`)
alone as
control. O
production was measured by
luminometry and is expressed in mV ± S.D. Controls were
subtracted. B, human monocytes cultured with IFN-
for 2
days were preincubated with or without hIgG1 for 5 min and stimulated
by the addition of cells to luminol containing 5 µg/ml intact mAbs
32 and 22 (W) or 32F(ab`)
and 22F(ab`)
(F). C, U937 10.6 cells were preincubated with
human IgG subclasses 1, 2, 3, and 4, or no IgG (none) for 10 min and then washed.
Duplicate cell samples were stimulated by the addition of
32F(ab`)
and 22F(ab`)
for an
O
assay. Another set of duplicates were
incubated with FITC-hIgG1 and analyzed by FACS to measure hIgG subclass
binding. O
production and IgG-binding to
Fc
R are expressed as a percent of the hIgG1 control. There was no
detectable binding by hIgG2. Data represent the mean of three
experiments.
A similar assay was performed with monocytes that were
treated with IFN- to induce the surface expression of newly
synthesized Fc
RI. Treatment was done in culture medium lacking IgG
so that induced receptors would be occupied by high affinity ligands.
When anti-Fc
RI F(ab`)
triggered receptors in the
absence of IgG, there was almost no activity (Fig. 1B),
but monocytes that had been preincubated with IgG produced a
respiratory burst that was comparable with that triggered by intact
anti-Fc
RI. We conclude that IgG binding profoundly affected
Fc
RI signaling in normal human receptors.
To determine whether
other human subclasses exert a similar effect, we preincubated 10.6
cells with hIgG subclasses 1, 2, 3, and 4; washed the cells briefly;
and then cross-linked their FcRI. As shown in Fig. 1C, hIgG1- and hIgG3-preincubated cells had
enhanced Fc
RI signaling, whereas hIgG4 preincubation produced an
intermediate response. FACS analysis showed that bound IgG-binding to
the cells (Fig. 1C) correlated with enhanced
O
production (Fig. 1C).
HIgG2 did not bind and had no effect on signaling over the IgG-free
control.
Certain other nonhuman IgGs bind human FcRI. To
analyze their effect specifically on Fc
RI signaling, we tested
cells after exposure to several murine and rat mAbs of various Ig
classes and subclasses as well as rabbit and goat polyclonal IgGs. As
shown in Fig. 2A, rabbit IgG, which binds with high
affinity to Fc
RI, supported signaling to the same extent as did
hIgG1, whereas goat IgG neither bound nor affected response above the
control. As shown in Fig. 2B, murine IgG2a and rat
IgG2b mAbs are the subclasses that bind to Fc
RI, whereas murine
mAbs of the IgA, IgE, and IgM classes, murine IgG1 and IgG2b mAbs, and
rat IgG2a mAbs bind poorly or not at all. As shown, only the mAbs that
bound enhanced O
production through
Fc
RI. Some mAbs had antigen specificities for non-Fc
RI
monocyte epitopes, but these had no apparent affect on
O
production attributable to antigen (Fig. 2A, asterisks). Some exceptions were
mIgG1 mAbs that recognize leukocyte function-associated antigens and an
IgE mAb. These mAbs reduced Fc
RI-binding sites without affecting
O
production. Overall, the results
suggest that ligand occupancy by high affinity IgG enhanced
Fc
RI-signaling for the oxidative burst.
Figure 3:
Hybrid Abs enhance FcRI-mediated
signaling. Cells were preincubated for 10 min with hIgG1 or hybrid
murine mAbs comprising two IgG2a chains (2a/2a), one IgG2a and
one IgG2b chain (2a/2b), or one IgG2a and one IgG1 chain (2a/1) (7 µg/ml each). Anti-Fc
RI F(ab`)
were added as in Fig. 2, and Fc
RI-mediated
O
production was measured. The
preincubations and assay were repeated in the presence of 25 µg/ml
IV.3Fab. Data are expressed as a percent of the hIgG1
control.
The effect of hybrids suggested a ligated structure involving one
receptor/IgG as opposed to a trimer composed of two receptors each
binding to one heavy chain on IgG. For a more direct approach in
establishing this ratio, we measured cell-associated 32, 32Fab, and
32F(ab`), to establish the number of Fc
RI on cells; we
measured cell-associated IgG2a, to establish the number of ligand
binding sites/cell. The results of a FACS analysis (Table 1) show
that IgG-binding sites equal the number of Fc
RI, indicating that
the ligated structure must have a ratio of one Fc
RI/bound IgG.
Figure 4:
Effect of IgG on the fMLP-triggered
respiratory burst. Cells were preincubated with medium (med),
hIgG1, 32F(ab`), or hIgG1 plus 32F(ab`)
. They
received 22F(ab`)
to trigger Fc
RI or fMLP (5
10
M) to stimulate chemotactic receptors.
O
production is expressed in
mV.
Measurements by FACS analysis of the
number of surface FcRI following a typical preincubation of cells
with IgG revealed no significant increase in Fc
RI numbers (Table 2). Thus, neither cell-priming nor increased numbers of
surface Fc
RI account for the increase in signaling.
Second, the
possibility that ligation might increase signaling through an increased
rate of anti-FcRI binding was tested. Rates of binding depend on
concentrations of the anti-Fc
RI
antibodies(8, 9) . Since there is a linear
relationship between the rates of binding of cross-linking antibody and
O
generation(8, 9) ,
times of peak rates of response correspond to peak rates of binding.
Therefore, peak times could be used as an index for the rate of
binding. As shown in Table 4, peak times were changed by antibody
concentration but not by IgG-ligation.
Third, FcRI were
aggregated through anti-Fc
RI F(ab`)
plus sheep or goat
anti-murine antibodies, or through 22Fab covalently conjugated
multimers that cross-link Fc
RI and would have a different gross
physical structure compared with 22F(ab`)
. In each case,
ligand-increased signaling was found (Table 5). These data show
that enhancement was not due to a unique mode of bridging by
22F(ab`)
. Collectively, the results indicate that
enhancement was not due to a specific mode of anti-Fc
RI binding.
Figure 5:
IgG-binding prior to
FcR-cross-linking produces the critical change in transductional
activity. A, cells were preincubated with hIgG1 and
22F(ab`)
, and individual samples of these cells were given
32F(ab`)
at 0, 3, 6, 9, or 12 min to stimulate
O
production. One sample received no
32F(ab`)
(none). B, cells were
preincubated in 22F(ab`)
for 10 min, and stimulated by the
addition of 32F(ab`)
. HIgG1 was added to individual samples
of these cells at 0, 3, 6, 9, or 12 min, or no hIgG1 was added (none). Results are presented as rate of
O
production, measured in mV/s and
expressed as a percent of the optimum.
Figure 6:
Effect of IgG concentration on
aggregation-dependent FcRI signaling. In sequential experiments,
cells were preincubated in O
assay medium
containing 0, 0.06, 0.125, 0.25, 0.5, 1, 2, and 4 µg/ml hIgG1 (opencircles) or 0.7, 7, 23, 70, 230, and 7000
µg/ml hIgG1 (filledcircles). Samples in each
were assayed for Fc
R-mediated respiratory bursts (A) and
hIgG1-binding (B). B was measured by FACS analysis
following an incubation with FITC-conjugated anti-hIgG antibody; data
is expressed as the mean fluorescence intensity of cells. C,
data were calculated from results in A and B to
determine O
production as a function of
hIgG1-binding. On a log plot, data falls on a straight line indicating
that O
production/ligated receptor
decreases by a simple exponential function (not shown). The saturation (SAT'N) point for IgG1-binding is indicated by an arrow. Data represent the mean ± the half-range of
seven assays.
Figure 7:
Effect of IgG on -chain
phosphorylation and the co-precipitation of pp72Syk. A, cells
preincubated with or without hIgG1 were incubated in
O
assay medium containing 22F(ab`)
or 22F(ab`)
and 32F(ab`)
. After 5 min at
37 °C, the cells were centrifuged through cold PBS/100 uM Na
VO
(1 ml) and solubilized in digitonin
lysis buffer. The receptors were precipitated by GAMk-AB1 beads,
separated by nonreducing SDS-polyacrylamide gel electrophoresis,
transferred, and analyzed by immunoblot using anti-phosphotyrosine
(anti-PY). The membrane was stripped and reblotted with
anti-
-chain (anti-
) antibody. Blots were developed using
horseradish peroxidase-conjugated second antibody and ECL.
Phospho-
2 is indicated by brackets. The anti-
-reactive
18-22-kDa proteins beneath phospho-
are unphosphorylated
-chain dimers. Arrow indicates pp72. B, anti-PY
and anti-Syk immunoblots of 72-kDa proteins that co-precipitated with
cross-linked Fc
RI.
In this report, we demonstrate that signaling resulting in
activation of the NADPH oxidase pathway and involving the tyrosine
phosphorylation of -chains on Fc
RI is markedly increased by
IgG-Fc interaction with the receptor ligand binding site. To show the
effect on Fc
RI-mediated NADPH oxidase activity, we triggered
through Fc
RI on monocytic cells exposed to a variety of IgGs and
found that all IgGs that bound to IFN-
-treated U937-10.6
monocytic cells, including rabbit, human subclasses 1 and 3, murine
subclass 2a, and rat subclass 2b, increased the Fc
RI-mediated
production of O
. Low affinity IgGs and
IgA, IgM, and IgE had no effect. Binding to some leukocyte integrins
caused slight Fc
RI blocking but did not increase Fc
RI
signaling.
Significantly, binding by IgG had a direct effect on the
receptor in that it increased the aggregation-dependent tyrosine
phosphorylation of FcRI
-chains(15, 18) .
This was accompanied by the binding to activated Fc
RI of a 72-kDa
tyrosine phosphoprotein, identified as pp72Syk. This result is
consistent with recent studies showing that Syk binds to tyrosine
phosphorylated
-chains in cells stimulated through
Fc
RI(18) , to
-chains in activated Fc
RI
complexes(21, 32) , and to homologous
tyrosine-phosphorylated motifs in activated B-cell receptor
complexes(33) . We propose that IgG-binding creates a change in
availability of Fc
RI
-chains that permits
aggregation-dependent tyrosine phosphorylation of
-chain motifs
and recruitment of pp72Syk. Another possibility is that ligand binding
influences the affinity of the
-subunits for associated Src
kinases (17) , perhaps modifying the kinetics of reactions in
aggregates.
This study also shows a correlation between
-phosphorylation, pp72Syk association, and
O
production, suggesting a link between
Fc
RI-associated tyrosine kinases and the activation of the NADPH
oxidase pathway.
Experiments to elucide possible effects of IgG
other than on signaling through FcRI demonstrated that enhancement
was not due to global cell-priming, because triggering through fMLP was
unaffected. It was not due to an increase in the numbers of surface
Fc
RI nor to specific modes of cross-linking by anti-Fc
RI
antibodies. It was also not due to a lack of aggregability of unligated
Fc
RI. These negative results help to support the proposed
hypothesis that aggregation without ligand is transductionally
insufficient.
The IFN--induced receptors on U937-10.6
cells appeared somewhat less IgG-Fc-dependent than those on monocytes.
However, induced receptors on premonocytic U937 cells were not tested
after a comparable expression time because several days of additional
culture are required to develop an oxidative capacity in the tumor
cells(23) . Furthermore, greater numbers of Fc
RI are
expressed through induction on the U937-10.6 subclone. Thus, age
or number (or some other factor) could have converted a minority of
receptors to what may be an easily achieved signal-efficient
configuration. Regardless of the reason, characterizing newly expressed
normal monocyte Fc
RI as IgG-Fc-dependent strengthens our
conclusions on the importance of Fc to Fc
RI signaling.
The
nature of the improved signaling is not yet understood. However,
several observations were made that partially characterize the
ligand/receptor relationship. (i) IgG that was bound to cells was not
itself cross-linked, making it unlikely that a mechanical strain
altered the receptor and more likely that the interaction with IgG-Fc perse brought about relevant changes. (ii)
Concentrations of IgG optimizing transductional activity were identical
to those saturating FcRI, supporting the biochemical linkage and
the specificity of the reaction. (iii) This interaction in
unaggregated, signal-quiescent Fc
RI did not increase
phosphorylation of
. Therefore, potentiation was not accompanied
by an obvious increase in activity of protein-tyrosine kinases
associating with
-chains(17) . However, binding could have
increased protein-tyrosine kinase affinity, a point presently under
investigation. (iv) When ligand was bound after receptor cross-linking,
there was no return to oxidase activation. This outcome is consistant
with the view that, with or without ligand, aggregation provides only a
transient opportunity (interval/receptor) for transductional activity (8, 9) and that the ligand is effecting a change
within this window. (v) At various subsaturating IgG concentrations,
anti-Fc
RI cross-linking must have produced mixed aggregates of
ligated and unligated Fc
RI, and in these cases,
O
production decreased exponentially with
the decrease in ligand-occupied receptors (Fig. 6C).
Evidently, the unligated receptors were not only refractory as triggers
but seemed obstructive to efficient signaling. The surprising
implication is that the alteration in ligated Fc
RI seems not to
have been transmitted to proximate unligated Fc
RI. If the
alteration were a matter of affinity or availability of Src kinases on
-chains, it is possible that the kinase concentration was diluted
below a critical level for a trans-activation of the unligated
Fc
RI. In any case, this evidence indicates that ligated and
unligated Fc
RI are markedly different in ways that are critical
and specific to transductional activity.
A further characterization
using hybrid antibodies indicated that signal-enhancement was achieved
through the binding of IgG containing only one high affinity heavy
chain capable of binding FcRI. This evidence alone would not have
excluded the possibility of hybrid low affinity chain engagement of a
second Fc
RI or an Fc
RII. However, we made direct measurements
that show that the potentiated Fc
RI structure has a ratio of one
receptor/hIgG1 ligand. This indicated that the other heavy chain on
hIgG1 does not engage with high affinity the ligand-binding site on a
second Fc
RI. Additionally, measured with IV.3Fab as a block, the
second heavy chain does not occupy the ligand binding site on
Fc
RII. Therefore, as predicted by the hybrids, only one of the two
heavy chains of IgG binds in a manner sufficient for potentiation.
Unlike the ligand-induced dimerization characteristic of several
cytokine receptor family members(34) , monomer ligand does not
dimerize Fc
RI.
The results of this study do not alter the
general view that aggregation, not a ligand-induced physical change in
FcR, is the key event in transmembrane signaling(35) . But in
addition to that, IgG-Fc binding must alter FcRI such that those
events normally involved in aggregation-dependent signal transduction
can occur. It is our speculation on this point that Fc binding
stabilizes an activatable configuration of the receptor, one that may
be present to varying extents in the nonphysiologic conditions of
ligand-free cell culture. Fc region involvement other than in passive
mediation of myelocytic antigen recognition has been suggested by Brown
and Koshland(36, 37) , and some distinctions in
FcR-mediated cytotoxicity due to hIgG-Fc in subclasses 1 and 3 have
also been described(38, 39) . Physicochemical studies
have shown that coupling of IgG to Fc
RI and IgE to Fc
RI
induces conformational alterations in the ligands (40) and
possibly in Fc
RI during an affinity shift(41) . Physical
distances between FcR in aggregates (42) and interactions with
cytoskeletal proteins may also be important(43) . Whether
ligation changes the configuration of Fc
RI and how binding is
communicated to cytoplasmic components will require further study.
In summary, the results in this report provide evidence that
FcRI is altered by IgG-Fc interaction with the Fc
RI
ligand-binding site and that this alteration permits efficient
signaling of the oxidative inflammatory response. Phosphorylation of
associated
-chains and association by pp72Syk kinase were also
increased by ligation of the receptors.