From the Division of Clinical Immunology and Rheumatology, Departments of Medicine and Microbiology, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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Human neutrophils express two structurally
distinct receptors for the Fc region of IgG, FcRIIa and Fc
RIIIb.
Although earlier studies have suggested that the functional properties
of these receptors are similar, mounting evidence suggests that these
receptors are capable of inducing distinct functional responses.
Accordingly, we have examined the regulation of intracellular
Ca2+ transients induced by each of these receptors
alone (homotypic receptor cross-linking) and together (heterotypic
receptor cross-linking). The glycosylphosphatidylinositol-anchored
Fc
RIIIb induces a rise in [Ca2+] after homotypic
cross-linking that is independent of ligand-mediated engagement of the
transmembrane Fc
RIIa. Both receptors were sensitive to the
protein-tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate, but
Fc
RIIa-induced signaling was uniquely sensitive to the
protein-tyrosine kinase inhibitor genistein. Fc
RIIa but not
Fc
RIIIb engages a cAMP-sensitive and inositol
1,4,5-trisphosphate-dependent pathway(s) that results in
the Ca2+-transient. When these receptors are cross-linked
into heterotypic clusters, a synergistic rise in [Ca2+]
is observed that is accompanied by synergistic increases in the
phospholipase C
-breakdown products inositol 1,4,5-trisphosphate and
diglyceride. These data provide a mechanism for the functional differences between these two receptors and suggest the possibility that they can be differentially modulated.
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INTRODUCTION |
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Receptors for the Fc region of IgG
(FcR)1 are critical
participants in inflammation and in the immune response by providing an
important link between the humoral and cellular immune systems. The
cluster of eight genes for human Fc
R on chromosome 1q encode a
diverse group of receptors that display similar extracellular domains
yet remarkably diverse transmembrane and cytoplasmic domains (1-3).
Human neutrophils constitutively express two distinct Fc
R:
Fc
RIIa and Fc
RIIIb. Fc
RIIa is a transmembrane receptor that
can initiate many neutrophil inflammatory responses including degranulation and the generation of reactive oxygen intermediates. Fc
RIIIb is a glycosylphosphatidylinositol (GPI)-linked protein that
can also initiate a number of neutrophil inflammatory responses.
Tyrosine phosphorylation events are essential for the early
intracellular signals initiated by FcR. Fc
RIIa has an
immunoreceptor tyrosine activation motif in the cytoplasmic domain (4),
and mutational analysis has shown the importance of the tyrosine
residues in this motif for the functional capacity of this receptor
(5-7). Cross-linking of Fc
RIIa results in the association of the
receptor with src-family tyrosine kinases (fgr in PMN,
lyn and hck in THP-1 cells) and Syk
(p72syk) (8-10). In myeloid cell lines, Fc
RIIa-induced
activation of PLC
by Syk results in a rapid IP3-mediated
[Ca2+] transient (11, 12). The mechanisms for early
tyrosine phosphorylation events triggered after cross-linking of
Fc
RIIIb are less clear. Most likely the result of preferential
partitioning of GPI-anchored proteins and palmitylated src-family
kinases in lipid domains in the plasma membrane (13), Fc
RIIIb, like
many GPI-anchored proteins (14), is associated with an src-family
kinase hck in certain detergent-insoluble complexes
(15).
An early view that FcRIIIb is simply a binding molecule without
signaling capacity (16, 17) has been revised by ample evidence that
Fc
RIIIb activates protein-tyrosine kinases and initiates
intracellular [Ca2+]i transients, degranulation,
and the respiratory burst (15, 18-20). Although some Fc
RIIIb
functions may overlap with Fc
RIIa, Fc
RIIIb does have a distinct
repertoire of cell programs that it initiates. Unlike Fc
RIIa,
Fc
RIIIb does induce a unique proinflammatory phenotype in
neutrophils (21). Although it is not a phagocytic receptor (17, 22),
Fc
RIIIb enhances Fc
RIIa-mediated internalization and functions
cooperatively with CD11b/CD18 in promoting phagocytosis and the
respiratory burst (22-24). Therefore, using changes in the
intracellular [Ca2+]i levels, which are induced
by Fc
RIIa and Fc
RIIIb and which are required for many receptor
functions (5, 25, 26), we have explored the possibility of differential
regulation of signaling by Fc
RIIa and Fc
RIIIb. Both receptors
elicit a brisk increase in [Ca2+]i derived
primarily from intracellular stores. Unlike Fc
RIIa, which engages a
cAMP-sensitive IP3-dependent pathway for
generation of [Ca2+]i transients, Fc
RIIIb
engages a cAMP-insensitive pathway that is also resistant to the
protein-tyrosine kinase inhibitor genistein. These two distinct
pathways can interact synergistically at the level of
phosphatidylinositol 4, 5-bisphosphate breakdown to lead to enhanced
transients in [Ca2+]i, reflecting both
intracellular and extracellular stores. Engagement of these two
distinct pathways may provide the basis for different cell programs
initiated by these receptors.
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EXPERIMENTAL PROCEDURES |
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Reagents and Buffers-- All buffers and solutions were made with ultra-purified endotoxin-free water (Millipore). Glassware was rendered endotoxin-free by either washing in chromic acid/nitric acid or by baking at 190 °C for 4 h. A modified PBS solution was prepared with 5 mM KCl and 5 mM glucose. Modified PBS plus Ca2+ and Mg2+ included 1.0 mM CaCl2 and 1.65 mM MgCl2. Solutions were confirmed to have <0.05 endotoxin units/ml by the limulus lysate assay (Associates of Cape Cod). Indo-1 acetoxymethyl ester (Molecular Probes, Eugene, OR), a cell permeant fluorogenic Ca2+ indicator, was prepared as a 0.5 mM stock in absolute ethanol.
mAb IV.3, a murine IgG2b recognizing human FcPreparation of PMN-- Fresh heparinized blood from healthy donors was diluted with an equal volume of modified PBS at 25 °C, and PMN were separated from the diluted blood by a two-step discontinuous density gradient consisting of ficoll-hypaque (density = 1.075 and 1.125 g/ml) (28). After two washes with modified PBS, the cells were treated with distilled water for 15 s to lyse contaminating erythrocytes, followed by an equal volume of 1.8% saline solution to restore isotonicity. The remaining PMN were resuspended in modified PBS at 1 × 107 cells/ml. By microscopic examination >95% of the cells were PMN. Separations were completed within 2 h, and all experimental procedures were completed within 5-6 h of phlebotomy.
Donors were typed for the FcAnalysis of Intracellular Ca2+
Concentrations--
Indo-1, a fluorescent dye with spectral properties
that change with the binding of free Ca2+, was used to
measure changes in intracellular calcium concentrations as we have
described (5, 18). PMN were incubated at 37 °C for 15 min with 5 µM indo-1 AM. After loading, the cells were washed once
with modified PBS and maintained at 25 °C in the dark. In most
experiments, an aliquot of cells (at a concentration of 1 × 107 cells/ml) was opsonized with anti-FcR mAb for 5 min
at 37 °C followed by one wash at room temperature. The cells were
then resuspended to 5 × 106 cells/ml in modified PBS,
and an aliquot was removed to quantitate mAb opsonization levels by
indirect immunofluorescence (see below). The cells were then warmed to
37 °C for 5 min in modified PBS plus 1.1 mM
Ca2+ and 1.6 mM Mg2+ before
analysis. Cells were loaded in an identical manner with fura-2 AM (2 µM) for single-cell Ca2+ analysis (see
below).
Quantitation of IP3 and Diglyceride
Formation--
To quantitate stimulus-induced changes in
[IP3], isolated PMN (pre-equilibrated with
Ca2+/Mg2+ as described above) were mixed with
mAb 3G8 IgG, fMLP, or opsonized E (see above) for various periods of
time followed by rapid addition of ice-cold 15% trichloroacetic acid
(TCA). Alternatively, cells were pre-opsonized with anti-FcR mAb Fab
or F(ab')2 fragments for 5 min at 37 °C. After one wash,
cells were re-equilibrated with Ca2+/Mg2+ and
then stimulated with F(ab')2 GAM and incubated for various periods of time followed by the rapid addition of ice-cold 15% trichloroacetic acid. In both cases, the precipitates were pelleted in
a microfuge for 15 min at 4 °C. The supernatants were extracted three times with 10 volumes of water-saturated diethyl ether and neutralized to pH 7.5 with NaHCO3. IP3 levels
were quantitated by competitive receptor binding assay with
[3H]IP3 and IP3-binding protein
(30) exactly according to the manufacturer's instructions (Amersham
Pharmacia Biotech).
Preparation of mAb Opsonized E-- Biotinylated mAb IV.3 Fab, mAb 3G8 F(ab')2, and bovine erythrocytes (E) were prepared as we have previously described (33). Biotinylated E were saturated with streptavidin and washed. The resulting E were coated with biotinylated mAb, and the level of mAb binding was verified by flow cytometry. For E-IV.3 Fab or E-3G8 F(ab')2-induced stimulation of IP3 and diglyceride production, E were added to PMN suspensions at a ratio of 25:1 (E:PMN) and gently pelleted for 15 s followed by incubation at 37 °C for various periods of time.
Immunofluorescent Flow Cytometry-- Aliquots of PMN at 5 × 106 cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4 °C. After two washes, the cells were incubated with saturating concentrations of phycoerythrin-conjugated goat anti-mouse IgG F(ab')2 at 4 °C for another 30 min. In addition, cells obtained from each [Ca2+]i measurement test cuvette were directly stained with saturating amounts of phycoerythrin-conjugated goat anti-mouse IgG F(ab')2 at 4 °C for 30 min. After washing, the cells were analyzed immediately for immunofluorescence using a Cytofluorograf IIS flow cytometer and a 2151 computer (Becton Dickinson Immunocytometry Systems, Westwood, MA).
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RESULTS |
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Homotypic FcRIIa and Fc
RIIIb Ca2+
Transients--
Fc
R-mediated neutrophil stimulation activates the
respiratory burst, which is dependent on receptor-induced elevations in the intracellular [Ca2+]. Since Fc
RIIa and Fc
RIIIb
associate with different tyrosine kinases (15), we hypothesized that
the rise in intracellular Ca2+ induced by these
structurally distinct receptors might be differentially regulated.
Accordingly, using anti-receptor mAb Fab and F(ab')2 fragments, we developed an experimental system to cross-link these receptors in a receptor-specific manner involving one type (homotypic) or both types (heterotypic) of receptors. When either neutrophil Fc
RIIa or Fc
RIIIb are cross-linked with anti-receptor mAb Fab or
F(ab')2 fragments (homotypic cross-linking), a brisk rise
in [Ca2+] is observed (Fig.
1A). Indeed, the rise in
[Ca2+] is similar in magnitude to the flux observed in
response to the potent neutrophil-activating peptide fMLP (Fig.
1A). This rise in [Ca2+] is due to release of
Ca2+ from intracellular stores. When either EDTA or EGTA is
added to the extracellular media, the Fc
R and fMLP-mediated
[Ca2+] fluxes are intact (results not shown) (18, 34).
The quantitative level of the Fc
R-induced Ca2+ flux is
dependent on the concentration of the stimulating anti-receptor mAb.
Over a subsaturating range of mAb concentrations, a dose response in
the quantitative rise in [Ca2+] was observed (Fig.
1B), and at saturating concentrations of anti-receptor mAb,
the rise in [Ca2+] induced by cross-linking Fc
RIIIb is
consistently higher in peak magnitude than the rise induced by
cross-linking Fc
RIIa (1417 ± 183 versus 846 ± 82 nM peak rise in [Ca2+], Fc
RIIa
versus Fc
RIIIb respectively, p < 0.005, n = 20).
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Differential Regulation of the FcRIIa and Fc
RIIIb
Ca2+ Transients--
Cross-linking of neutrophil Fc
R
results in tyrosine kinase activity (3, 15, 22). The dependence of the
Fc
RIIIb-induced Ca2+ transient on protein-tyrosine
kinase activity was shown with the tyrosine kinase inhibitor methyl
2,5-dihydroxycinnamate (100 µM), a stable erbstatin
analog; the Fc
RIIa- and Fc
RIIIb-mediated rise in
[Ca2+] was completely blocked by pretreatment with this
protein-tyrosine kinase inhibitor (Fig.
3). Cell viability was unaltered by the brief exposure (5 min) to this tyrosine kinase inhibitor as determined by exclusion of trypan blue (control, 90% cells viable; treated cells,
85% cells viable). Comparable results were obtained with the tyrosine
kinase inhibitors tyrophostin (40 µg/ml, n = 3), lavendustin A (50 µg/ml, n = 2),
2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid (1 µg/ml,
n = 3), and staurosporine, which inhibits both tyrosine
and ser/thr kinases (0.5 µg/ml, n = 3). However,
differential sensitivity to the tyrosine kinase inhibitor genistein was
observed for the Fc
RII- and Fc
RIII-mediated [Ca2+]
transients. When neutrophils were incubated with 100 µM
genistein for 5 min, the rise in [Ca2+] induced by
cross-linking Fc
RIIa (Fig. 3) and by fMLP (results not shown) was
completely abolished. Surprisingly, the Fc
RIIIb-induced rise in
[Ca2+] was not abrogated by 100 µM
genistein (Fig. 3). In four independent paired experiments, the ability
of Fc
RIIa but not Fc
RIIIb to initiate a rise in
[Ca2+] was abrogated by 100 µM genistein
(Fc
RIIa/Fc
RIIIb % control, 5 ± 4%/52 ± 6%;
n = 4, p < 0.001). The differential
sensitivity to genistein was also observed at 50 µM
genistein (Fc
RIIa/Fc
RIIIb % control, 26 ± 3%/63 ± 2%, n = 3, p < 0.001). These
concentrations of genistein have been shown to completely inhibit
neutrophil Fc
RIIa-induced tyrosine phosphorylation and phagocytosis
(37, 38) and to block neutrophil degranulation and superoxide
production in response to cross-linking of Fc
R and L-selectin (35,
36). Little or no sensitivity of the Fc
RIIa- or Fc
RIIIb-induced
Ca2+ transient to the tyrosine kinase inhibitor reduced
carboxamidomethylated and maleylated-lysozyme (100 µg/ml,
n = 2) or the ser/thr kinase inhibitors calphostin C,
H-7, H-8, and H-1004 was observed. These results demonstrate that both
Fc
RIIa and Fc
RIIIb initiate Ca2+ transients in a
tyrosine kinase-dependent manner. Despite this similarity,
the differential sensitivity to the tyrosine kinase inhibitor genistein
suggests that these receptors are engaging distinct intracellular
activation pathways.
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IP3-dependent and -independent
Ca2+ Transients--
Elevations in [cAMP] can induce
activation of the cAMP-dependent protein kinase, which in
turn can down-modulate PLC1 activity (46). The sensitivity of the
Fc
RIIa-induced, but not the Fc
RIIIb-induced, rise in
[Ca2+] to cAMP suggests that Fc
RIIa may engage an
IP3-dependent mechanism. Upon maximal homotypic
Fc
RIIIb cross-linking with saturating levels of mAb 3G8
F(ab')2 and F(ab')2 GAM, there was no
detectable increase in IP3 levels, which is in marked
distinction to the time-dependent increase in
IP3 observed after homotypic cross-linking of Fc
RIIa
with mAb Fab and F(ab')2 GAM (Table
I). As a positive control, the
fMLP-induced increase in IP3 levels is shown (Table I).
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Biochemical Characterization of the Heterotypic
FcRII+Fc
RIII Ca2+ Transient--
Our results
with receptor-specific IP3 data do not provide an
explanation for the vigorous IP3 response that has been
reported during antibody opsonized erythrocyte (EA) phagocytosis (47). EA can engage both Fc
RIIa and Fc
RIIIb, resulting in heterotypic cross-linking of these receptors. Since heterotypic cross-linking of
Fc
RIIa and Fc
RIIIb results in a synergistic phagocytic response (22) and since neutrophil Fc
R phagocytosis is dependent on a
receptor-induced rise in [Ca2+], we determined if
heterotypic cross-linking of Fc
RIIa and Fc
RIIIb results in a
synergistic [Ca2+] response. Neutrophils opsonized with
equivalent densities of either anti-Fc
RII mAb IV.3 Fab,
anti-Fc
RIII mAb 3G8 F(ab')2, or both IV.3 Fab and 3G8
F(ab')2 (verified by flow cytometric analysis) displayed
markedly different quantitative [Ca2+] responses with
heterotypic cross-linking, showing a synergistic rise in
[Ca2+] (Fig. 6).
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DISCUSSION |
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Neutrophil FcRIIIb elicits a rise in
[Ca2+]i that is independent of ligand engagement
of Fc
RIIa. Like the Fc
RIIa-induced change in
[Ca2+]i, the Fc
RIIIb-induced
[Ca2+]i transient is dependent on tyrosine
phosphorylation events. However, unlike Fc
RIIa, the
Fc
RIIIb-induced [Ca2+]i transient is resistant
to the protein-tyrosine kinase inhibitor genistein, resistant to
elevations in [cAMP], and independent of demonstrable changes in
[IP3]. These data demonstrate that the biochemical
regulation of Fc
RIIIb function is distinct from Fc
RIIa, and they
provide the initial basis for understanding the distinct repertoire of
cell programs initiated by Fc
RIIIb.
FcRIIa interacts with the src-family tyrosine kinase fgr
and with Syk through interactions between the phosphorylated
immunoreceptor tyrosine activation motif in the cytoplasmic domain of
Fc
RIIa and SH2 domains in the kinases (3, 12). One characteristic of
Syk activation is the tyrosine phosphorylation and activation of
PLC
, resulting in the breakdown of phosphatidylinositol
4,5-bisphosphate into IP3 and diacylglyceride (49, 50).
Indeed, in myelomonocytic cell lines, cross-linking of Fc
RIIa is
associated with a rapid rise in [IP3] (11, 12). Data
suggesting that stimulation of Fc
R in human neutrophils does not
lead to any change in the intracellular [IP3] (34, 51), a
finding at variance with our results (Table I, Fig. 7), may reflect
technical differences in the threshold for detection. We chose to use
an indirect receptor binding assay to quantitate IP3
levels, which avoids the biosynthetic labeling of cells with
myo-[3H]inositol, a process that is inherently
inefficient and difficult to perform in neutrophils due to the
necessarily short labeling periods.
Heterotypic neutrophil FcR cross-linking during Fc
R-mediated
phagocytosis or immune complex-induced Fc
R-stimulation, elicits an
IP3 burst (47, 52). Our data indicate that the nature of the Fc
R stimulus is critical; there is no detectable increase in
IP3 levels after homotypic cross-linking of Fc
RIIIb, a
small but detectable increase in [IP3] after
cross-linking of Fc
RIIa, and a substantial generation of
IP3 induced by heterotypic cross-linking of neutrophil
Fc
R with the IP3 response, comparable in magnitude to
the fMLP-induced IP3 response. In parallel with the
increased [IP3], we also observed significant increases
in the concentration of diacylglyceride, the other breakdown product of
phosphatidylinositol 4,5-bisphosphate. These data provide a mechanism
for the synergism between Fc
RIIa and Fc
RIIIb in the generation of
the early Ca2+ transient (Fig. 6) (53) and perhaps for
phagocytosis (22) and the oxidative burst (23). Since cross-linking of
Fc
RIIIb results in tyrosine phosphorylation of Fc
RIIa (22),
enhanced tyrosine phosphorylation of Fc
RIIa might enhance the
ability of this receptor to activate downstream effector molecules such as PLC
, leading to the IP3 and diacylglyceride responses
observed after heterotypic receptor cross-linking.
The ability of GPI-anchored proteins to generate intracellular signals
is now well established (14). Although the mechanisms may not be
completely understood, current data suggest that these proteins are
capable of activating src-family kinases. Some evidence suggests that
GPI-anchored proteins and myristylated src-family kinases are both
found in specialized lipid domains in the plasma membrane. Indeed,
neutrophil FcRIIIb co-precipitates in detergent-insoluble domains
with hck, which is in contrast to the association of
neutrophil Fc
RIIa with fgr (15). Differential association
and activation of src-kinases by neutrophil Fc
RIIa and Fc
RIIIb
may provide an explanation for the differences in sensitivity to the
protein-tyrosine kinase inhibitor genistein. These data also
demonstrate that it may be possible to differentially manipulate the
functional capacity of these receptors, a property that may be useful
in altering the response of neutrophils to circulating IgG
autoantibodies such an anti-neutrophil cytoplasmic antibodies.
Selective inactivation of Fc
RIIIb, which plays an important role in
anti-neutrophil cytoplasmic antibodies-positive Wegener's
granulomatosis (21, 54), might allow targeted down-modulation of
neutrophil-mediated injury mechanisms in that disease.
Although our results clearly show that FcRIIa does induce
IP3 production, Fc
RIIIb does not elicit an
IP3 response after homotypic cross-linking (Table I). This
lack of detectable IP3 cannot be due to a lack of
sensitivity, since homotypic engagement of Fc
RIIIb at receptor
saturation consistently results in a Ca2+ transient that is
higher in magnitude than the response elicited by homotypic
cross-linking of Fc
RIIa. Nonetheless, the Fc
RIIIb-induced Ca2+ is released from intracellular stores (18). The nature
of the intracellular Ca2+-mobilizing signal has yet to be
elucidated. Among the IP3-independent mechanisms, cyclic
ADP-ribose and sphingosine-1-phosphate are candidates for
intracellular Ca2+-mobilizing signals (55-57). Of course,
it is also possible that Fc
RIIa engages both
IP3-dependent and IP3-independent
Ca2+-releasing mechanisms. Future studies will be needed to
resolve the role of IP3, cyclic ADP-ribose, and sphingosine
kinase in neutrophil Fc
R-mediated Ca2+ transients.
There are significant implications in the finding that FcRIIIb does
not engage an IP3-mediated signaling pathway. Direct interactions between GPI-anchored proteins and src-family kinases provide one possible mechanism for the transmission of intracellular signal generation. Alternatively, GPI-anchored proteins may interact with transmembrane proteins to form multimolecular complexes. The
formation of multimolecular complexes in the membrane is a common theme
among plasma membrane receptors, including Fc
RIa, Fc
RIIIa,
Fc
RI, and Fc
RI (1, 3). The nature and identity of possible
Fc
RIIIb-associating structures is currently unclear. Elegant
co-capping and fluorescence resonance energy transfer studies have
shown that CD11b/CD18 in neutrophil membranes can associate with a wide
range of other cell surface receptors including Fc
RIIIb, leukocyte
function antigen-1 (LFA-1), and the urokinase receptor (58-60).
However, the lack of an Fc
RIIIb-induced increase in IP3
contrasts to the ability of CD11b/CD18 to induce increases in
[IP3] after cross-linking (47). Furthermore, the ability of Fc
RIIIb to activate CD11b/CD18 for phagocytosis, a function that
CD11b/CD18 cannot do alone in resting neutrophils, indicates that all
Fc
RIIIb signaling cannot be mediated through CD11b/CD18 (22).
Our results also provide the basis for understanding that the results
of FcR engagement on neutrophils will depend on which receptor
type(s) are engaged. An IgG2 ligand will selectively and homotypically
engage Fc
RIIa of the H131 genotype (61, 62). Anti-neutrophil
cytoplasmic antibodies may favor engagement of the more highly
expressed Fc
RIIIb. In contrast, multivalent immune complexes would
favor heterotypic cross-linking of Fc
RIIa, Fc
RIIIb, and perhaps
complement receptors as well. Each of these might result in the
engagement of different biochemical signal-transducing pathways and in
qualitatively and quantitatively different effector functions.
Delineation of receptor-specific pathways is essential in the
identification of kinases and kinase substrates that are important in
regulation of Fc
R-mediated signal transduction. Ultimately, an
understanding of these pathways will enable specific modulation of
Fc
R-initiated inflammatory processes in autoimmune diseases without
complete blockade of all Fc
R-mediated functions, which may be
essential in normal host defense.
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ACKNOWLEDGEMENTS |
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We thank Dr. Barbara J. Struthers and G. D. Searle for providing the prostaglandin E1 analog misoprostol and Dr. Bruce Rapuano (The Hospital for Special Surgery, New York, NY) for performing the diglyceride determinations. We are also grateful for the advice and discussions with our colleagues at the Hospital for Special Surgery where this work was performed.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1-AR42476 and RO1-AR33062). The flow cytometry core at the Hospital for Special Surgery was supported by National Institutes of Health Grant P60-AR38320.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Clinical
Immunology and Rheumatology, 1900 University Blvd., University of
Alabama, Birmingham, AL 35294. Tel.: 205-934-0894; Fax: 205-934-1564; E-mail: jedberg{at}uab.edu.
§ Present address: Dept. of Immunology, University of Washington, Seattle, WA 98195.
¶ Present address: Div. of Rheumatology, Dept. of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
1
The abbreviations used are: FcR, receptor for
the Fc region of IgG; GPI, glycosylphosphatidylinositol; PMN,
polymorphonuclear leukocyte; PLC, phospholipase C; IP3,
inositol 1,4,5-trisphosphate; PBS, phosphate-buffered saline; E,
erythrocyte; GAM, IgG F(ab')2 fragments of polyclonal goat
anti-mouse IgG; MP, misoprostol; fMLP,
formylmethionylleucylphenylalanine; NECA,
5'-(n-ethylcarboxamido)adenosine; Bt2cAMP,
dibutyryl cyclic AMP.
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REFERENCES |
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