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RIIa on HL-60 Granulocytes1 ,2
Departments of
*
Microbiology and
Internal Medicine, Ohio State University, Columbus, OH 43210; and
Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa, Syk kinase, and
phospholipase C2 in granulocytic HL-60 cells. CRP also
stimulated translocation to the membrane of both phospholipase C2
and phosphatidylinositol-3-kinase. The signaling response triggered by
CRP was a rapid, early event with kinetics similar to the response
elicited by human IgG. Both soluble-aggregated CRP and monomeric CRP
cross-linked FcRII to generate a signal of the same intensity. The
results are consistent with signaling through the intrinsic
immunoreceptor tyrosine-based activation motif of the cytoplasmic
domain of FcRIIa, the major CRP-receptor on monocytes and
neutrophils that is responsible for CRP-mediated phagocytosis. The
signaling events driven by CRP have the potential to regulate
infiltrating neutrophil activities. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-strands
in a single polypeptide chain of 206 aa arranged into two -sheets
with two bound Ca2+ ions per subunit (4, 5). The two Ca2+ ions coordinate the
binding of a single PC (5). The lectin-like PC-binding
sites are all on the same "recognition-face" of the pentamer
(3, 5). The opposite face of the pentamer is termed the
"effector-face," which contains the critical residues regulating
the binding and activation of C1q (6) and the amino acids
that interact with specific leukocyte CRP receptors (CRP-R) (7, 8). The rapid, greatly amplified CRP gene expression by
hepatocytes is driven by the synergistic interaction between IL-1
and IL-6 that together synergistically induce transcription factors
that also mediate specific immune responses (9, 10). The
well-documented biological activities ascribed to CRP led
to its appreciation as a link between innate and
specific immunity (3, 8, 11). This link is best supported
by the observations that CRP complexes activate the classical C pathway
and inhibit alternative C pathway activation by binding factor H
(12), as well as the conservation of its structure from
invertebrates through the vertebrates (13). CRP also
contributes directly to innate host defense by efficiently mediating
phagocytosis in a manner analagous to specific IgG Ab
(14). Expression of CRP transgenes, as well as passive
transfer of human CRP, protects mice against certain microbial
pathogens and endotoxin shock (8).
Specific leukocyte CRP-R until recently were thought to be distinct
from FcR, yet shared many of their molecular properties (8, 14). Recent evidence clearly shows that the major CRP-R on human
monocytes and neutrophils (polymorphonuclear neutrophil; PMN) is the
40-kDa low-affinity FcRII, which has a high affinity for CRP
(15, 16). The structurally related pentraxin, serum
amyloid P-component (SAP), has also recently been shown to use several
of the FcR classes, including FcRII, to mediate phagocytosis
(17). CRP-mediated signal transduction through FcRII
has not yet been fully explored. Because PMN are the most abundant
FcRII-bearing infiltrating cells to enter inflammatory sites where
CRP or CRP complexes can localize (18), we examined the
signaling response to CRP in differentiated HL-60 granulocytes (G),
which possess abundant FcRII and share many PMN functions (19, 20). In the experiments reported here, we show that acute phase
concentrations of CRP trigger tyrosine-phosphorylation (Tyr-P) of
the immunoreceptor-tyrosine based activation motif (ITAM) of human
FcRIIa and Syk kinase, as well as inducing both phosphorylation and
membrane localization of phospholipase C (PLC)2, translocation to
the membrane of phosphatidylinositol 3-kinase (PI-3K), and mobilization
of intracellular Ca2+ stores. These findings are
consistent with the selective activation of phagocytic leukocytes by
CRP, and provide a molecular basis for the role of CRP in host
protection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human promyelocytic cell line HL-60 was grown in RPMI 1640
supplemented with 4% defined FBS and 6% supplemented bovine calf
serum (HyClone Laboratories, Logan, UT). HL-60 cells were
differentiated into G cells by incubating 4 x 105
cells/ml with 1.25% DMSO for 5–7 days until >90% of the cells
reduced nitroblue tetrazolium dye and were capable of rapid reduction
of ferricytochrome c in response to PMA (19, 21). DMSO, Nonidet P-40, Na3VO4,
ionomycin, human IgG1 myeloma, and the proteinase inhibitors (PMSF,
leupeptin, pepstatin, chymostatin, and aprotinin) were purchased from
Sigma Aldrich (St. Louis, MO). The
p-aminophenyl-PC-Sepharose for purification of CRP was
from Pierce Chemical (Rockford, IL). Recombinant protein G-Sepharose
and mouse anti-Syk mAb were obtained from Zymed Laboratories
(Burlingame, CA). Affinity-purified rabbit IgG anti-human PLC2
(no. 407) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Biotin-labeled goat-anti-mouse IgG (-chain specific) and
HRP-labeled streptavidin were from Kirkegaard & Perry Laboratories
(Gaithersburg, MD). The mouse mAb specific for FcRIIa (IV.3) was
obtained from Medarex (Hanover, NH). A mixture of mAb to Tyr-P
consisting of Py20, Py72.10.5, and 4G10 in a ratio of 30:30:1 was used
to detect Tyr-P proteins. Rabbit anti-p85 (PI-3K) specific for the
human protein was generated to a purified GST-fusion protein as
described previously (22). The rabbit Ab to part of the
cytoplasmic tail of the 
-chain of FcRIIa (CT-GST) was
obtained from Dr. J.-L. Teillaud (Institut Curie, Paris, France)
(23). ECL reagents and HRP-conjugated
anti-rabbit-IgG were obtained from Kirkegaard & Perry Laboratories.
Cell activation and protein preparation
HL-60(G) cells were washed twice and resuspended in Earle’s balanced salt solution containing 10 mM HEPES (pH 7.4) at 10 x 106 cells/100 µl and kept on ice, and warmed to 37°C for 10 min just before the addition of 5–200 µg/ml of purified human CRP, purified human SAP, or human IgG1 myeloma. The cell activation reactions were stopped by sonication on ice for 1 min and centrifuging at 800 x g at 4°C for 10 min to remove nuclei and large debris. For immunoprecipitation the cells were lysed in Nonidet P-40 lysis buffer consisting of: 1% Nonidet P-40, 20 mM Tris at pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 3 mM Na3VO4.
Membrane preparation
The supernatant from the cell sonication was mixed with an extraction buffer of Tris (50 mM) at pH 7.5, EGTA (2 mM), Na3VO4 (10 mM), leupeptin (1 µg/ml), aprotinin (0.2 µg/ml), and fresh PMSF (2 mM). The suspension was centrifuged at 60,000 rpm at 4°C (TLA-100 rotor; Beckman Coulter, Fullerton, CA). The pellet was washed twice by resonicating in the extraction buffer to remove cytosolic proteins. The protein concentration was measured by the bicinchoninic acid method and the proteins solubilized in 1% Nonidet P-40 lysis buffer. Equal amounts of protein per lane (60–80 µg) were separated along with Rainbow protein m.w. markers by SDS-PAGE and immediately transferred to nitrocellulose membranes (Micron Separations, Westborough, MA) at 60 V for 1–2 h at 4°C. The membrane was saturated with 5% powdered milk in TBS plus 0.1% Tween 20, incubated for 2 h with one of the Ab probes, washed, and incubated with an HRP-labeled second reagent and the signal detected by chemoluminscence. Autoradiograms were developed on film (Kodak BioMax, Rochester, NY) and scanned for relative intensity.
Immunoprecipitation
Insoluble material from HL-60 (G) cells in the Nonidet P-40
lysis buffer was removed by centrifugation at 16,000 x
g for 10 min, and the supernant was immunoadsorbed
overnight at 4°C with 2 µg of specific IgG Ab mixed with 10 µl of
recombinant protein G-Sepharose. Following immunoadsorption, unbound
proteins were removed with four or five washes in the Nonidet P-40
lysis buffer containing 1 mM Na3VO4. Detection
of Tyr-P proteins was accomplished by immunoprecipitation of the
FcRIIa, Syk, or PLC2 from whole cell lysates using limiting amounts
of each of the following: mAb anti-FcRIIa (IV.3), mouse
anti-Syk mAb, rabbit IgG anti-human PLC2, and protein
G-Sepharose beads (10 µl). Tyr-P proteins were detected using the
anti-Tyr-P mixture and biotin-labeled goat-anti-mouse IgG and
HRP-streptavidin.
Purification of CRP and SAP
Human CRP and SAP were purified exactly as described elsewhere
(24, 25). Briefly, SAP was removed from CRP-containing
ascitic fluids by passage through a column of agarose beads (A-15m;
Bio-Rad, Hercules, CA). After extensive washing, the bound SAP was
eluted in 10 mM EDTA and sized on a Sepharose column (25).
The unbound protein from the agarose beads was passed through a 10 ml
(20 mm diameter) column of p-aminophenyl-PC-Sepharose.
The PC-Sepharose with bound protein was washed extensively with TBS
plus 2 mM Ca2+, and the bound protein eluted in TBS + 10 mM
EDTA. A second round of affinity purification on the PC-bearing matrix
was used to remove trace amounts of other proteins. The concentration
of CRP was determined by ELISA and radial immunodiffusion vs
sheep anti-human CRP. The protein was >99% CRP based on reactivity in
a competitive ELISA. The CRP preparations had 
0.01 µg/ml Ab (IgG +
IgA), equivalent to <0.01 µg IgG/mg of CRP. The concentration of
endotoxin in the purified CRP was 0.1–0.2 endotoxin U/mg CRP using the
chromogenic Limulus assay (BioWhittaker, Walkersville,
MD; LPS of <0.1 ng/mg of CRP).
Cytosolic Ca2+ measurement
Intracellular Ca2+ concentration ([Ca2+]i) was assessed by loading HL-60(G) cells with the noncharged ester form of indo-1 (indo-1-AM; Molecular Probes, Eugene, OR). Cells were washed, resuspended in medium at 5 x 106/ml, incubated with 1.0 µM indo-1 AM for 30 min at 37°C, washed, and resuspended to 2 x 106/ml in a buffer of 125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.1% glucose, 0.1% BSA, 0.5 mM MgCl2, 1 mM CaCl2, and 25 mM HEPES (pH 7.4). Aliquots (500 µl) of cells were added to matched quartz cuvettes with stirring at 37°C in a recording spectrofluorometer (LS-5, PerkinElmer, Wellesley, MA). The excitation wavelength was 331 nM with a 5 nm slit width; the emission was continuously recorded at 410 nm with a 10 nm slit width. A baseline level of [Ca2+]i was recorded for 3 min before the agonist was added, and the emission monitored for 10 min or until the emitting light returned to baseline levels. The maximum emission (Fmax) was determined by permeablizing the cells with 50 µM digitonin; the minimum emission (Fmin) was determined by adding 4 mM EGTA. The Fmin and Fmax, respectively, were determined after the response to the agonist was obtained. Ionomycin at 100 ng/ml was used as a control. The [Ca2+]i was calculated as described using the equation: [Ca2+]i = F - Fmin/(Fmax - F) (26). The F value is the maximum emission recorded in the presence or absence of an agonist, and was obtained directly from the recording trace. Values for the transient change in [Ca2+]i were obtained from independent, triplicate measurements for each treatment per experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa
Because the FcRII class of IgG receptors appears to account for
most of the high affinity, receptor-specific CRP binding to human
monocytes and PMN (15, 16), we examined whether CRP could
trigger signaling events via this receptor by detecting its
phosphorylation as Tyr-P. FcRIIa is the only human FcR class with
an endogenous ITAM (20, 27). Therefore, the FcRII
specific mAb IV.3 was used for immunoprecipitation to determine whether
CRP signals via this receptor in HL-60 (G) cells. When CRP binds to
HL-60(G) cells at 37°C in an aggregated, soluble form, Tyr-P of
FcRIIa occurred in a dose-dependent manner (Fig. 1
A). Maximum levels of
signaling were reproducibly recorded at acute phase levels of CRP of
100–200 µg/ml. Purified CRP that had not been deliberately
aggregated induced a similar level of phosphorylation of the receptor
(Fig. 1A, lane 4 vs 5).
Heat-aggregated, soluble human IgG1 at the same concentration triggered
more extensive Tyr-P of FcRIIa than CRP as judged by signal
intensity (Fig. 1A). Thus, the range of effective molar
concentrations for CRP (120 kDa) for FcRII signaling is similar to
that for human IgG1. CRP always induced an additional prominent Tyr-P
band of 
72 kDa that coprecipitated with FcRIIa. The kinetics of
FcRIIa Tyr-P was followed over an interval of 0.5–12 min with
aggregated CRP (100 µg/ml). The most pronounced signal occurred at
1.5 min (Fig. 2A). The
pattern of the time-course of the Tyr-P of FcRII was reproducible
and transient with a return to baseline levels by 10–12 min. A similar
quantity of FcRIIa protein was present in each lane (Fig. 2B). When either aggregated or monomeric CRP was allowed
to bind to HL-60(G) cells before the addition of aggregated IgG, the
signal intensity was not altered (data not shown). The 40 kDa signal
always appeared as a diffuse band because this FcR is heavily
glycosylated (20). These results indicate that FcRIIa
Tyr-P is a rapid and early event triggered by clustering of the
receptors by CRP.
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Srk gene family kinases are activated during the
earliest stages of FcR-signaling and phagocytosis, although the
activation mechanism for these tyrosine kinases by FcRIIa is not yet
understood (28). The phosphorylation of the ITAM in
FcRIIa permits the recruitment of the Src homology
(SH)2-domain containing 72 kDa Syk kinase, which is present in the
cytoplasm of resting leukocytes (28). Because Syk is
required for ITAM-dependent activation of actin assembly and subsequent
FcRIIa-mediated phagocytosis (29, 30), we assayed for
its activation in response to CRP. Tyr-P of Syk in HL-60(G) cells was
triggered by incubation with either aggregated or unaggregated CRP
(Fig. 3A). The Tyr-P
signal observed with acute phase concentrations of CRP (
100 µg/ml)
was similar to that induced by aggregated human IgG (Fig. 3A, lane 2 vs 5). Similar
amounts of Syk protein were compared (Fig. 3B). In a
separate time course experiment, the Tyr-P of Syk was detected as early
as 1 min after stimulation with CRP, with a maximum signal observed by
2–3 min, which decayed by 12–20 min. (Fig. 4). This result correlates with the
kinetics of phosphorylation of FcRIIa (Fig. 2
A),
suggesting that the Tyr-P of both FcRIIa and Syk are early, rapid
events induced by CRP.
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RIIa and Syk
Syk contains N-terminal tandem SH2-domains that bind to either of
the two Tyr-P within the ITAM of FcRIIa, and this binding is thought
to induce a conformational change in Syk that increases its kinase
activity (31). Therefore, we tested the effects of CRP on
the binding interaction between FcRIIa and Syk. HL-60 (G) cells were
stimulated with aggregated CRP, and FcRIIa was immunoprecipitated
and then probed with either anti-Tyr-P or anti-Syk. The
coprecipitating Syk became Tyr-P in response to CRP as well as
aggregated IgG (Fig. 5). In addition,
purified human SAP also induced the association of Syk with FcRIIa
(Fig. 5, lane 5).
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2 phosphorylation and membrane localization
One downstream signaling event following FcRIIa ligation and
Syk activation in human neutrophils is the phosphorylation and
recruitment to the membrane of PLC (32), which
generates inositol trisphosphate and diacylglycerol (33).
The major PLC isoform in PMN and HL-60(G) cells is PLC2 with a
molecular mass of 145 kDa (33). Therefore, HL-60(G)
cells incubated with increasing concentrations of aggregated CRP were
used to test for membrane bound PLC2. When CRP was used at an acute
phase level of 100 µg/ml, the phosphorylation of PLC2 increased
in a manner similar to the response to IgG (Fig. 6A), when equal amounts of
PLC2 protein were present in each lane (Fig. 6B).
Translocation of PLC2 from the cytosol to the membrane was also
readily detected in response to aggregated CRP (Fig. 7A). These results are
consistent with activation of PLC in response to CRP, and suggests
that CRP may induce changes in intracellular Ca2+ levels
(32).
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PI-3K plays a pivotal role in FcRIIa signaling because it
generates lipid secondary messengers such as phosphatidylinositol
3,4,5-trisphosphate that are required for phagocytosis
(34). Incubation of HL-60 (G) cells with acute phase
concentrations of CRP induced translocation to the membrane of PI-3K
when isolated membrane proteins were probed with an Ab to the p85
regulatory subunit of PI-3K (Fig. 7B). The results
indicate that CRP mobilizes PI-3K via FcR in a manner analogous to
human IgG.
Intracellular Ca2+ mobilization in response to CRP
The rapid mobilization of intracellular stores of Ca2+
in response to inositol trisphosphate is characteristic of signaling by
FcRs in human neutrophils (32, 35). Therefore, we
tested the ability of CRP to elevate [Ca2+]i
in indo-1 loaded HL-60 (G) cells. Both purified monomeric CRP and
aggregated CRP at concentrations as low as 5 µg/ml induced a rapid,
transient increase in [Ca2+]i that returned
to baseline levels by 5–6 min. (Table I). The maximum response correlated with
the dose of CRP with up to a 4-fold increase with acute phase levels of
CRP (Table I). The response to the heat aggregated, but soluble
complexes of CRP was significantly greater than the response to
nonaggregated CRP, but only at the suboptimal level of 5 µg/ml
(p < 0.05) (Table I). The transient
Ca2+ response to cross-linking of FcRIIa with the
receptor specific IV.3 mAb, with or without a second reagent, was of
the same magnitude as the response to CRP (Table I). The transient
change in [Ca2+]i in response to CRP was
attenuated by >50% when 5 mM EDTA was added to the cell suspension
just before the addition of CRP, suggesting that store-operated
Ca2+ entry into the cells was also elicited. The
intracellular Ca2+ mobilization response to CRP is entirely
consistent with a cell activation signal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa. The
distribution of this human FcR class on almost all
hematopoietically-derived cells (20) corresponds exactly
with the distribution for CRP-R (8, 14). In addition, the
FcRII class has been shown by DuClos and colleagues
(15, 16, 17) to serve as the major functional leukocyte CRP-R.
The unique property of FcRIIa is that the ITAM required for
propagating the initial steps of signaling is intrinsic to the
cytoplasmic domain of the receptor itself and not on an associated
protein (27, 36). The new findings revealed in this study
are that the CRP ligand, when presented as a monomer or as a multimer,
not only triggers the Tyr-P of the FcRII ITAM, but also propagates
the signal as indicated by the Tyr-P of the receptor-associated Syk
kinase (30, 31) and the downstream effector PLC2
(33). CRP-induced FcRIIa clustering also induced the
translocation to the membrane of both PLC2 and PI-3K, events
required for phagocytosis via this receptor, at least by human PMN
(32, 34, 35). These events triggered by CRP occurred with
the same kinetics as IgG immune complexes, albeit lower levels of
phosphorylation were observed with CRP indicating that perhaps an
inhibitory or regulatory signal may accompany the activating signal
(36). Overall, the findings are consistent with the
proposed link between CRP and its receptor in augmenting innate host
resistance (11, 18).
One issue raised by these findings is whether the signaling pathway
initiated by CRP via FcRIIa is identical with that initiated by IgG.
The proximal signal transduction events appear to be qualitatively the
same in terms of the critical components of the signaling pathway that
are activated (37). Furthermore, the relative efficiency
of activation as judged by the kinetics is very similar for CRP and
IgG. This result might have been anticipated if both agonists activate
the initial kinase for phosphorylating the two Tyr within the tandem
Y-X-X-I/L motifs of the ITAM of the receptor (27, 28). The
initiating phosphotyrosine kinase for the FcRs has so far only been
identified as a Src family kinase (28, 38).
Recent work from one of our laboratories suggests that both Syk and
PI-3K bind directly to the ITAMs through their SH2-domains
(38). Because PI-3K is essential for signal transduction
and for propagating downstream events leading to membrane movement and
phagocytosis (34, 39), the documentation of its
recruitment to the membrane in response to CRP is evidence that CRP is
capable of generating the intracellular mediators needed for
phagocytosis. In earlier experiments using the same system, we
demonstrated an increase in the kinase activity of PI-3K in membrane
fractions in response to CRP at levels >50 µg/ml (40).
The activated PI-3K generates phosphatidylinositol-3,4,5-trisphosphate
which promotes distal signaling events by binding to Pleckstrin
homology domains of many different enzymes, including PLC2 in
neutrophils (33, 34, 41). The localization of PLC2 to
the membrane and mobilization of intracellular
Ca2+ stores in response to CRP also represent
crucial downstream events required for leukocyte movement and
phagocytosis (32, 42).
Indirect evidence that CRP signaled via FcRIIa on PMN was first
gathered by Zeller et al. (43), who found that CRP
potentiated the aggregated IgG-induced activation of the respiratory
burst of human PMNs, but that mAb IV.3 could not block the potentiation
induced by CRP (44). Earlier attempts to define the CRP-R
with specific mAb reagents, such as the IV.3 mAb, for FcRs suggested
that the recognition sites on FcRIIa for IgG and CRP are distinct
(45). Because 93% of activated PMNs bind aggregated CRP
(43, 44) and virtually all HL-60(G) cells
(24), CRP may be able to "prime" PMNs for IgG-complex
activation through FcRIIa when both are present. The structural
basis for the functional similarity between CRP and the Fc of IgG has
yet to be resolved. However, DuClos and colleagues (46)
suggested that the shared, accessible sequence of
175YLGGP of CRP is involved in binding to
FcRI. Only recently, the Tyr175 residue was
shown to be critical for the binding of C1q to CRP, and is part of the
unusual extended deep cleft of the central pore of the pentraxin
(6). Because the pentagonal arrangement of the CRP
subunits with the effector face on the plane of the pentraxin opposite,
the PC-binding face is compatible with multipoint FcR clustering with
each subunit binding to a single FcR (47). This may
explain our observation that unaggregated monomers of CRP are
sufficient for clustering. The specific findings of signaling of CRP
via FcRIIa in this report does not preclude CRP-induced signaling
via FcRI (46), or even FcRIII, which bind CRP and/or
SAP (17). However, the HL-60 cell line, like PMN, does not
express FcRI unless treated with IFN- (36). The
evolution of specific IgG Ab-mediated phagocytosis may have depended on
the earlier existence of CRP-R. The regulation of CRP-mediated
activation of PMN may involve the use of negative signaling receptors
such as FcRIIb to recruit phosphatases (36). The
inflammatory milieu where CRP accumulates may provide the critical
density needed to eventually modulate infiltrating leukocyte
activities.
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Acknowledgments |
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Footnotes |
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2 This work was presented at the Society for Leukocyte Biology meeting, October 6, 2000, in Cambridge, MA.
3 Address correspondence and reprint requests to: Dr. Richard F. Mortensen, Department of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. E-mail address: mortensen.3{at}osu.edu
4 Abbreviations used in this paper: CRP, C-reactive protein; CRP-R, CRP receptor; G, granulocyte; PMN, polymorphonuclear neutrophil; SAP, serum amyloid P-component; ITAM, immunoreceptor tyrosine-based activation motif; PI-3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; Tyr-P, tyrosine phosphorylation or phosphotyrosine; [Ca2+]i, intracellular or cytosolic free Ca2+ concentration; PC, phosphocholine; SH, Src homology.
Received for publication August 20, 2001. Accepted for publication November 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(C/EBP) and Rel p50. J. Immunol. 166:2378.RIIa-transfected COS cells. J. Exp. Med. 190:585.RIIa on human monocytes and neutrophils is allele-specific. J. Clin. Invest. 105:369.[Medline]
receptors and opsonizes particles for phagocytosis. J. Immunol. 166:6735. receptor on human platelets through an ITAM: reconstitution with synthetic phosphopeptides. J. Biol. Chem. 271:10775.RIIa2 by activated platelets and inhibition of anti-CD9-mediated platelet aggregation by recombinant FcRIIa2. Blood 85:698. receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209. receptor signalling. J. Biol. Chem. 269:22732.R clusters evoke a synergistic Ca2+ response in human neutrophils. J. Biol. Chem. 270:10671.RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis. J. Immunol. 167:844.RI in transfected COS cells. J. Immunol. 155:2185.[Abstract]
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. The blot is representative of four similar
experiments.
