From the
Both Fc
The human polymorphonuclear neutrophil expresses two different
types of receptors that can bind the Fc domain of IgG antibodies in
immune complexes. These Fc
The results of some studies suggest an inability of Fc
The ability of immune complexes to
bind both to Fc
F(ab`)
Fab fragments were made by digestion with 4% (w/w) papain for 1.5 h
at 37 °C in PBS containing 10 mM cysteine and 5
mM EDTA. The reaction was terminated by addition of 20
mM iodoacetamide. Protein A affinity chromatography was used
to remove Fc fragments and intact antibodies. When F(ab`)
Antibodies were biotinylated with
biotin- N-hydroxysuccinimide ester (2 mg/mg IgG) for 4 h at
room temperature. Free biotin was removed by dialysis against PBS.
Cross-linking of antibodies was performed with polyclonal goat
anti-mouse immunoglobulin (GAM) F(ab`)
MAbs 3G8
and IV.3 were conjugated to fluorescein isothiocyanate (FITC) by
incubation for 2 h at room temperature with FITC (4 times molar excess
of FITC) at pH 9.5. Free FITC was removed by dialysis against PBS. All
mAb were stored at 4 °C in PBS with 0.01% azide. Heat-aggregated
IgG was freshly prepared for each experiment by incubating human IgG
(Central Laboratory of the Netherlands Red Cross Blood Transfusion
Service, Amsterdam, The Netherlands) at 30 mg/ml for 30 min at 63
°C. Insoluble aggregates were removed by centrifugation at 10,000
For intracellular Ca
In some experiments, neutrophils
were loaded with 10 µM BAPTA-AM
(1,2-bis-( O-aminophenoxyl)ethane- N,N,N`,N`-tetraacetic
acid) (Molecular Probes) as follows. Prewarmed neutrophils (2
Assessment of Ca
To
further explore the effect of cross-linking of Fc
Direct heterotypic cross-linking of one Fc
Recent studies have established that both Fc
Our results show that ligation of one receptor did not
induce a modulation of the Ca
It
has been suggested that cross-linking of both Fc
Our results show that a
synergistic effect by dual receptor activation is only observed when
cross-linking of Fc
Taken together, the interaction of Fc
During phagocytosis, accumulation of
Ca
The heterotypic
cross-linking of Fc
In conclusion,
our study shows that formation of heterotypic clusters of Fc
Peak increases above
resting values under the various conditions of stimulation were
calculated. Experimental details are given in the legend to Fig. 1.
Results are the mean ± S.E. of four independent experiments.
The results of different experiments on the combined Fc
Fixed neutrophils, Fc
The results of different experiments are shown in
which the BsAb response and the combined Fc
The simultaneous homotypic Fc
The
simultaneous homotypic Fc
We thank Lily Kannegieter for purification of the
bispecific antibody, Martin de Boer for technical assistance with
photography, and Masja de Haas for critically reading the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptors on human neutrophils (Fc
RIIa and
Fc
RIIIb) are capable of initiating signal transduction after
multivalent cross-linking. However, immune complexes most likely
activate neutrophils by a combined homotypic and heterotypic
cross-linking of Fc
Rs. We have investigated the effect of
homotypic and heterotypic Fc
R cluster formation on changes in the
intracellular free Ca
concentration. Combined
heterotypic and homotypic cluster formation resulted in a
Ca
response that was strongly enhanced as compared to
the sum of both individual Fc
R responses. This synergistic
response was caused by the formation of heterotypic clusters of
Fc
Rs and not by the simultaneous formation of homotypic clusters.
This conclusion was supported by experiments with a bispecific antibody
binding to both Fc
RIIa and Fc
RIIIb. The heterotypic Fc
R
cross-linking results in efficient activation of Ca
influx, probably caused by a more pronounced depletion of
intracellular Ca
stores. Stimulation with immune
complexes also induced Ca
influx in normal
neutrophils, but not in Fc
RIIIb-deficient neutrophils. The
synergism between both Fc
Rs was also apparent in other responses
of neutrophils, such as the activation of the respiratory burst. This
study shows that the two different Fc
Rs on neutrophils complement
each other in mediating an important cellular response.
receptors (Fc
RIIa and
Fc
RIIIb)
(
)
play a key role in host defense
mechanisms by linking the humoral immune response to the cell-mediated
effector system. The Fc
IIa receptor is a 40-kDa transmembrane
molecule with an expression of 10,000 to 20,000 molecules per
neutrophil. The Fc
IIIb receptor is a heavily glycosylated protein
with an apparent molecular mass of 50 to 80 kDa, linked via a
glycosylphosphatidylinositol-anchor to the membrane; per neutrophil
100,000 to 200,000 molecules are expressed
(1, 2) . The
individual quantitative and qualitative role of each receptor in
neutrophil activation has not yet been unraveled in detail. Multivalent
cross-linking of Fc
RIIa clearly induces signal transduction in the
neutrophil: a rise in
[Ca
]
, phagocytosis,
degranulation, and the respiratory burst can be initiated via
Fc
RIIa
(3, 4, 5, 6, 7, 8, 9) .
RIIIb to
transduce signals independently of Fc
RIIa
(3, 5, 8, 10, 11) . These
results, together with the lack of transmembrane and cytosolic protein
domains and the high expression level of this receptor on the cell
surface, have led to the belief that Fc
RIIIb is principally a
binding molecule that presents ligands to Fc
RIIa
(3, 11) . However, several lines of evidence have
emerged that point to a more extended role for Fc
RIIIb.
Multivalent cross-linking of this receptor alone initiates, by still
unknown mechanisms, signal transducing events such as membrane
potential changes and an increase in
[Ca
]
(4, 12) , and can lead to actin filament assembly
(13) . Moreover, several effector functions, such as killing of
chicken erythrocytes coated with anti-Fc
RIII-Fab
(14) ,
degranulation
(15) , phagocytosis of ConA-opsonized erythrocytes
(16) , and activation of the respiratory burst
(4, 17, 18) , have been observed to be induced
via Fc
RIIIb in neutrophils.
RIIa and Fc
RIIIb raises the possibility of
interactions between the two receptors or the signal transduction
elements connected to these receptors. Indirect evidence for such a
cross-talk between Fc
Rs on neutrophils has recently been obtained
(3, 4, 19, 20, 21) . In the
present study, we have investigated the effect of homotypic and
heterotypic Fc
R cluster formation on changes in the intracellular
free Ca
concentration
([Ca
]
). To achieve
controlled conditions of Fc
R cluster formation, monoclonal
antibodies against Fc
RII and Fc
RIII were cross-linked under
different conditions. We present evidence that heterotypic
cross-linking of Fc
RIIa and Fc
RIIIb produces a synergistic
Ca
response in human neutrophils. Induction of
Ca
influx from the extracellular medium is important
for this synergistic increase in
[Ca
]
. Furthermore, we
observed also synergism in other functional responses of neutrophils,
such as the activation of the respiratory burst.
Isolation of Neutrophils
Peripheral blood was
obtained from healthy individuals and from a healthy
FcRIIIb-negative donor, as described by Huizinga et al.(22) . Neutrophils were purified from buffy coats of 500 ml
of blood anticoagulated in 0.4% (w/v) trisodium citrate and centrifuged
through a Percoll layer with a specific gravity of 1.076 g/ml (1000
g, 18 min, 20 °C). Contaminating erythrocytes in
the pellet fraction were removed by lysis in ice-cold buffer containing
155 mM NH
Cl, 10 mM KHCO
, and
0.1 mM EDTA (pH 7.4). The neutrophils were washed twice in
phosphate-buffered saline (PBS) and resuspended in incubation medium
containing 132 mM NaCl, 6 mM KCl, 1 mM
CaCl
, 1 mM MgSO
, 1.2 mM
NaHPO
, 20 mM Hepes, 5.5 mM glucose, and
0.5% (w/v) human serum albumin (pH 7.4). The purity of the neutrophils
was more than 95%, the remaining cells were eosinophils.
Antibodies
The anti-human FcRIII mAb 3G8
(mIgG1)
(23) and the anti-human Fc
RII mAb IV.3 (mIgG2b)
(24) were purified from hybridoma culture supernatant by
precipitation with 50% saturated ammonium sulfate and subsequent
protein A affinity chromatography. The anti-major histocompatibility
complex (MHC) class I mAb W6/32 (mIgG1) was purified from ascites fluid
by protein A affinity chromatography.
fragments
were prepared by digestion with 2% (w/w) pepsin at pH 3.7 for 3G8 and
pH 4.0 for W6/32 for 16 h at 37 °C, followed by protein A affinity
chromatography to remove free Fc fragments and intact antibodies.
and Fab fragments were checked on SDS-PAGE, intact antibodies or
Fc fragments were not detectable.
fragments against
intact antibodies or against Fc domains of antibodies (Jackson
Immunoresearch, West Grove, PA). Streptavidin (10 µg/ml) was used
to cross-link biotinylated antibodies bound to neutrophils.
g for 10 min.
Synthesis of Bispecific F(ab`)
Fab fragments
of IV.3 (3 mg/ml in PBS), prepared by digestion with papain as
described above, were incubated for 5 h with regular shaking at room
temperature with a 10-fold molar excess of the heterobifunctional
cross-linker N-succinimidyl-3-(2-pyridyldithiol)propionate
(SPDP) (Pierce) added from a stock solution in ethanol. To remove
unbound SPDP, the reaction mixture was passed through a G-25 Sephadex
column equilibrated with PBS. Fab`-SH fragments of 3G8 were prepared by
reducing F(ab`) Antibody
(BsAb) Recognizing Fc
RII and Fc
RIII
fragments (2.8 mg/ml in PBS), obtained by
pepsin digestion as described above, with 15 mM
2-mercaptoethanol for 30 min at 30 °C. The reduced product was
passed through a G-25 Sephadex column in PBS to remove the
2-mercaptoethanol and was immediately added to the IV.3 Fab-SPDP
fragments. After the column step, a sample of 3G8 Fab`-SH was taken and
incubated for 14 h to control for spontaneous reoxidation of the
Fab`-SH fragments. Analysis on SDS-PAGE showed that this was not the
case. The mixture of Fab`-SH and Fab-SPDP fragments was concentrated to
one-third of the original volume in C30 Amicon microconcentrators
(Amicon, Beverly, MA). After 14 h of incubation at room temperature,
the mixture was passed through a fast protein liquid chromatograph
Superose 12 column equilibrated with PBS. Appropriate fractions were
pooled and analyzed on SDS-PAGE. The fraction containing dimers was
taken for further characterization as bispecific antibody against
Fc
RII and Fc
RIII (bsAb Fc
RIIxFc
RIII).
Characterization of BsAb
Fc
The fraction containing the bsAb
FcRIIxFc
RIII
RIIxFc
RIII was first tested for its ability to inhibit the
binding of the parent antibodies IV.3 and 3G8 to human neutrophils. For
this purpose, purified neutrophils were fixed in PBS containing 1%
(w/v) paraformaldehyde for 10 min at 4 °C. After washing in PBS
containing 1% (w/v) bovine serum albumin, 2
10
cells were incubated with bsAb (3 µg/ml) or with a control
mIgG1 antibody in the same concentration for 45 min at 4 °C. After
washing, the cells were incubated with GAM-FITC (62.5 µg/ml)
(Central Laboratory of the Netherlands Red Cross Blood Transfusion
Service, Amsterdam, The Netherlands), IV.3-FITC (2.5 µg/ml), or 3G8
F(ab`)
-FITC (2.5 µg/ml) for 30 min at 4 °C. After
another washing step, cell-associated fluorescence was measured in a
flow cytometer (Becton Dickinson FACScan, Palo Alto, CA). Further
characterization of the bsAb was performed with a Chinese hamster ovary
(CHO) cell line transfected with human Fc
RIIIb cDNA
(CHO
cells)
(25) . These cells did not
express human Fc
RII. Wild type CHO cells (CHO
cells)
were used in these experiments as a negative control.
Measurements of the Cytosolic Free Ca
Determination of
[CaConcentration
]
was performed as
described before
(26) . In short, neutrophils (2
10
/ml in incubation medium) were loaded with indo-1 by
incubation with 1 µM indo-1/AM (Molecular Probes, Eugene,
OR) for 40 min at 37 °C. The neutrophils were washed and
resuspended in incubation medium to 2
10
/ml and
kept in the dark at room temperature. Unless indicated otherwise, the
cells loaded with indo-1 were diluted to 2
10
/ml in
incubation medium with 1 mM Ca
and incubated
with the appropriate antibodies for 5 min at 37 °C followed by
washing and transfer to a cuvette. Fluorescence changes of the
neutrophil suspension, magnetically stirred and kept at 37 °C, were
monitored with a spectrofluorometer (model RF-540, Shimadzu
Corporation, Kyoto, Japan), with 340 and 390 nm as excitation and
emission wavelengths, respectively. To calibrate the indo-1
fluorescence
(27) as a function of
[Ca
]
, all trapped
indo-1 was saturated with Ca
by addition of digitonin
(10 µM), after which the indo-1 fluorescence was quenched
with MnCl
(0.5 mM). A dissociation constant of 250
nM for the indo-1
Ca
complex was used
to calculate [Ca
]
(28) .
measurements in the presence of EGTA, indo-1-loaded neutrophils
were diluted to 2
10
/ml just prior to stimulation
in incubation medium (without CaCl
) containing 1
mM EGTA. Before the calibration with digitonin, CaCl
(2 mM) was added.
10
/ml in incubation medium) were first incubated with 1
µM indo-1/AM for 10 min at 37 °C, and then 10
µM BAPTA-AM was added. After 30 min, the cells were washed
and resuspended in incubation medium without Ca
(2
10
/ml).
influx was carried out with the Mn
quenching
technique
(29) . For this purpose, an emission wavelength of 446
nm instead of 390 nm was chosen, resulting in the complete absence of
fluorescence changes upon addition of a Ca
mobilizing
stimulus (data not shown). For these experiments, the indo-1-loaded
neutrophils were incubated in incubation medium with Ca
(2
10
/ml) for 5 min at 37 °C with the
appropriate antibodies, washed, and resuspended (2
10
/ml) in medium containing 0.2 mM CaCl
(to favor Mn
entry)
(30) . Two min prior
to addition of the stimulus, 0.5 mM MnCl
was added
to the indo-1-loaded neutrophils (2
10
/ml) and
fluorescence changes were recorded in time as described above. Except
for the traces shown in Figs. 6 and 7, scanning of the fluorometer
traces was performed followed by smoothing in the computer program
Correldraw
.
Measurement of the Respiratory Burst
Activation of
the respiratory burst was measured with 1,2,3-dihydrorhodamine-loaded
cells, as described
(31, 32) . In short, neutrophils (2
10
/ml in incubation medium) were prewarmed at 37
°C for 10 min. Subsequently, the cells were incubated with 0.25
µM 1,2,3-dihydrorhodamine (Molecular Probes) and 2
mM NaN
for 5 min. The cells were incubated with
the appropriate antibodies, washed, and resuspended in warm incubation
medium, and were incubated with cross-linking agents for 30 min. The
reactions were stopped by addition of a 30-fold excess of ice-cold PBS
containing 1% (v/v) bovine serum albumin and the samples were kept on
ice. Cell-associated fluorescence was measured by flow cytometry
(FACScan, Becton Dickinson).
Statistical Analysis
For statistical analysis
paired Student t tests were performed. p values
exceeding 0.05 were not considered significant.
The Simultaneous Homotypic Response via Fc
We first investigated the CaRIIa and
Fc
RIIIb Shows an Incomplete Summation of the Individual
Responses
response of human neutrophils after simultaneous, but specific,
homotypic cross-linking of both Fc
R receptors (further referred to
as the ``simultaneous homotypic Fc
R response'') and
compared this response to the responses after cross-linking of each
receptor alone. For this purpose, neutrophils were incubated with mAb
IV.3 (anti-Fc
RII) and biotinylated 3G8 F(ab`)
(anti-Fc
RIII). Neither mAb IV.3 nor 3G8
F(ab`)
-biotin alone elicited an increase in
[Ca
]
(data not shown).
Subsequently, GAM F(ab`)
anti-Fc domains (to specifically
cross-link Fc
RIIa) and streptavidin (to specifically cross-link
Fc
RIIIb) were added simultaneously, which resulted in a
significant rise in [Ca
]
(Fig. 1 C and ). Neither GAM
F(ab`)
nor streptavidin alone induced a Ca
response. Reference responses were determined by multivalent
homotypic cross-linking of both receptors alone with the same agents
(Fig. 1, A and B, and ). The
simultaneous homotypic Fc
R response was significantly lower than
the sum of the separate Fc
RIIa and Fc
RIIIb responses: the
peak increase in [Ca
]
was only 79 ± 5% (mean ± S.E., n =
4) of this latter value ( p < 0.025).
Figure 1:
Changes in intracellular free
Ca after homotypic cross-linking of Fc
RIIa and
Fc
RIIIb on human neutrophils. Indo-1-loaded neutrophils were
preincubated for 5 min at 37 °C with: A, intact IV.3 mAb
(10 µg/ml); B, biotinylated anti-3G8 F(ab`)
(10 µg/ml); or C, with both mAb. The cells were then
washed and transferred to a cuvette. Cross-linking ( arrow) was
performed with: A, GAM F(ab`)
anti-Fc domains (15
µg/ml); B, streptavidin (10 µg/ml); or C,
with both cross-linkers together. Each curve is representative for four
independent experiments.
For this inhibition
to occur, ligation of one FcR, without multivalent cross-linking
was not sufficient. Neither the response after Fc
RIIa
cross-linking (the mean of the Ca
increase ±
S.E.: 288 nM ± 61, n = 3), nor the
response after Fc
RIIIb cross-linking (237 nM ± 70,
n = 7) was significantly changed by ligation of
Fc
RIIIb with 3G8 Fab (293 nM ± 70, n = 3) or Fc
RIIa with IV.3 Fab (272 nM ±
72 n = 7), respectively. The results depicted in
Fig. 1
and might suggest some competition between the
signal transduction pathways used in activation via homotypic
cross-linking of both Fc
R.
Synergistic Response Evoked by Heterotypic Cross-linking
of Fc
To mimic cross-linking of
FcRIIa and Fc
RIIIb
Rs by immune complexes, neutrophils were incubated with
anti-Fc
RII Fab fragments and anti-Fc
RIII Fab fragments, and
then GAM F(ab`)
was added. After cross-linking with GAM
F(ab`)
, which binds to both anti-Fc
RII Fab and
anti-Fc
RIII Fab, a pronounced increase in
[Ca
]
was observed (
Fig. 2
and ). Neither anti-Fc
RII Fab nor
anti-Fc
RIII Fab alone, nor both Fabs in combination, nor GAM
F(ab`)
alone induced a response (data not shown). The GAM
F(ab`)
concentration chosen induced a level of activation
of the separate Fc
receptors responses that was comparable or even
lower than these responses seen with the other reagents used for
cross-linking ( cf. Tables I and II).
Figure 2:
Changes in intracellular free
Ca after combined homotypic and heterotypic
cross-linking of Fc
RIIa and Fc
RIIIb on human neutrophils.
Indo-1-loaded neutrophils were preincubated for 5 min at 37 °C
with: A, IV.3 Fab (10 µg/ml); B, 3G8 Fab (10
µg/ml); or C, with both mAb. The cells were then washed
and transferred to a stirred cuvette. Cross-linking ( arrow)
was performed with GAM F(ab`)
(15 µg/ml). Curves are representative for five independent
experiments.
In the experiments
depicted in Fig. 2(further referred to as the ``combined
FcR response''), a combination of homotypic Fc
R
cross-linking with a substantial proportion of heterotypic
cross-linking of Fc
RIIa and Fc
RIIIb may be expected.
Immunofluorescence microscopy showed that, indeed, upon cross-linking
of anti-Fc
RII Fab (FITC-labeled) and anti-Fc
RIII Fab
(tetramethylrhodamine isothiocyanate-labeled) with GAM
F(ab`)
, localization of Fc
RIIa and Fc
RIIIb in the
same clusters on the cell surface was induced (data not shown). The
magnitude of the contribution of homotypic cross-linking to the
combined Fc
R response was determined by means of separate
cross-linking of both Fc
R with the same agents ( Fig. 2and
). The increase in
[Ca
]
of the combined
Fc
R response reached a much higher level than the sum of the
Ca
responses initiated by Fc
RIIa cross-linking
alone and Fc
RIIIb cross-linking alone. The ratio of the
``combined'' response to the sum of the separate responses
was 3.03 ± 0.71 (mean ± S.E., n = 5) at
antibody concentrations of 10 µg/ml and 1.62 ± 0.14 (mean
± S.E., n = 3) at antibody concentrations of 2.5
µg/ml. Thus, in the combined Fc
R response, in which besides
homotypic cross-linking also heterotypic cross-linking can occur, a
significant synergistic increase in
[Ca
]
is observed
( p < 0.05 for both antibody concentrations). Comparison of
the results depicted in Figs. 1 and 2 shows that the kinetics of the
increase in [Ca
]
are
dependent on the agent used for cross-linking the Fc
Rs.
Nevertheless, these results suggest that a synergistic increase in
[Ca
]
is evoked after
heterotypic Fc
RIIa
Fc
RIIIb cross-linking.
RIIa and
Fc
RIIIb together, the effect of stimulation with bsAb, directed
against both Fc
RIIa and Fc
RIIIb, was investigated. The bsAb
Fc
RIIxFc
RIII was able to inhibit binding of both IV.3-FITC
and, to a lesser extent, 3G8 F(ab`)
-FITC to human
neutrophils (I). To ensure the presence of the Fab
recognizing CD16, binding of the bsAb to CHO cells expressing human
Fc
RIIIb was studied (I). Although these cells did not
express Fc
RII, a clear binding of the bsAb was observed. Hence,
the bsAb consisted of IV.3 Fab and 3G8 Fab with intact antigen-binding
sites.
RIIa to one
Fc
RIIIb by addition of the bsAb to neutrophils did not initiate an
increase in [Ca
]
(). Additional cross-linking of the bsAb with GAM
F(ab`)
was required to elicit a
[Ca
]
response (further
referred to as the ``bsAb response'') ( Fig. 3and
). This response was greater than the sum of the
quantitative comparable separate responses obtained with comparable
amounts of Fab fragments ( p < 0.005).
Figure 3:
Changes in intracellular free
Ca after cross-linking of bsAb
Fc
RIIxFc
RIII. Indo-1-loaded neutrophils were preincubated for
5 min at 37 °C with: A, bsAb Fc
RIIxFc
RIII (10
µg/ml); B, bsAb Fc
RIIxFc
RIII (5 µg/ml);
C, IV.3 Fab and 3G8 Fab (both at 5 µg/ml); or D,
IV.3 Fab and 3G8 Fab (both at 2.5 µg/ml). The cells were then
washed and transferred to a cuvette. Cross-linking ( arrow) was
performed with GAM F(ab`)
at a concentration of 15
µg/ml ( A and C) or 8 µg/ml ( B and
D). Curves A and C are representative for
six independent experiments, curves B and D are
representative for three independent
experiments.
Comparison of the
bsAb response (obtained with 10 µg of bsAb/ml) with the combined
FcR response with the same amounts of Fab fragments (5 µg/ml
of each Fab fragment) showed that the peak value of the bsAb response
was consistently higher ( Fig. 3and ): an increase
of 32 ± 12% (mean ± S.E., n = 6) was
observed ( p < 0.05). However, the bsAb response occurred
more rapidly as compared to the combined Fc
R response, but also
appeared to be more transient. Apparently, the formation of
Fc
RIIxFc
RIII complexes by the bsAb prior to cross-linking
does have an influence on the characteristics of the final response.
The Synergistic Ca
To
investigate the source of the CaResponse Is
Mainly Derived from Extracellular Ca
in the various
responses described above, changes in indo-1 fluorescence were
investigated in the presence of EGTA (to prevent Ca
influx) or Mn
(to indirectly measure
Ca
influx). The peak levels of
[Ca
]
reached under the
conditions previously designated as the simultaneous homotypic Fc
R
response and the combined Fc
R response were lowered to a mean
level of 240 and 234 nM Ca
, respectively, in
the presence of EGTA, indicating that extracellular Ca
contributes profoundly to the peak values reached (
Fig. 4
and ). The separate responses via Fc
RIIa
and Fc
RIIIb were only slightly decreased in the presence of EGTA
(). Furthermore, in neutrophils loaded with the
intracellular Ca
chelator, BAPTA-AM, especially the
response to combined heterotypic and homotypic cross-linking was not
completely abrogated (Fig. 4). This might be explained by a
substantial contribution of extracellular Ca
to this
response overriding the buffer capacity of the BAPTA-loaded cells. In
contrast, the response after cross-linking of Fc
RII or Fc
RIII
alone was completely abrogated in BAPTA-loaded cells (data not shown).
Figure 4:
Effect of Ca chelation
on changes in [Ca
] induced by Fc
R
cross-linking. The upper figure depicts ( A) the simultaneous
homotypic Fc
R response and ( B) the combined Fc
R
response without chelation of Ca
, the middle figure
depicts these responses with chelation of extracellular Ca
by EGTA and the lower figure depicts these responses with
chelation of intracellular Ca
by BAPTA. Loading with
indo-1 and BAPTA was performed as described under ``Materials and
Methods.'' Subsequently, the neutrophils were preincubated with
antibodies ( A) exactly as described in the legend to Fig. 1 or
( B) exactly as described in the legend to Fig. 2.
Cross-linking ( arrow) was performed with: A,
streptavidin (10 µg/ml) and GAM F(ab`)
anti-Fc domains
(15 µg/ml); or B, GAM F(ab`)
(15 µg/ml) in
the presence of extracellular Ca
( upper and
lower figure) or without extracellular Ca
in
the presence of 1 mM EGTA ( middle figure). Curves
shown are representative for three to five independent
experiments.
More direct evidence for an effect of heterotypic FcR
cross-linking on influx of extracellular Ca
was
obtained by Mn
quenching experiments, in which
extracellular Mn
enters the cells via Ca
channels
(25) . In our experiments, an emission wavelength
of 446 nm was used, which made the fluorescence of indo-1-loaded
neutrophils insensitive for changes in
[Ca
]
(data not shown).
Combined homotypic and heterotypic Fc
R cross-linking, induced
after preincubation with both Fc
R antibodies together or with the
bsAb, resulted in a sharp decrease in indo-1 fluorescence, i.e. a high rate of Mn
influx (Fig. 5, A and B). Under conditions of simultaneous homotypic
cross-linking of both Fc
R, the rate of Mn
influx
was much lower (Fig. 5 C). After cross-linking of
Fc
RII or Fc
RIII alone (Fig. 5, D and
E), Mn
influx appeared to be only slightly
higher than in unstimulated cells (Fig. 5 F). The
enhancement of Mn
influx was not observed upon
co-cross-linking of Fc
RIIa and MHC class I or of Fc
RIIIb with
MHC class I (data not shown).
Figure 5:
Effect of FcR cross-linking on
Ca
influx as measured by
Mn
-dependent quenching of indo-1 fluorescence. Indo-1
emission was measured at the Ca
-independent
wavelength of 446 nm as explained in the text. The indo-1-loaded
neutrophils were preincubated for 5 min at 37 °C as indicated
below, washed, resuspended in incubation medium with 0.2
µM CaCl
, and transferred to a cuvette.
Subsequently, 0.5 mM MnCl
was added ( first
arrow) and cross-linking was performed ( second arrow). To
completely quench indo-1 fluorescence with Mn
,
digitonin (10 µM) was added ( third arrow).
A, combined Fc
R response: preincubation with IV.3 Fab (10
µg/ml) + 3G8 Fab (10 µg/ml), cross-linking with GAM
F(ab`)
(15 µg/ml). B, bsAb response:
preincubation with bsAb (10 µg/ml), cross-linking with GAM
F(ab`)
(15 µg/ml). C, simultaneous homotypic
Fc
R response: preincubation with IV.3 (10 µg/ml) + 3G8
F(ab`)
-biotin (10 µg/ml), cross-linking with GAM
F(ab`)
anti-Fc domains (15 µg/ml) + streptavidin
(10 µg/ml). D, Fc
RII response: preincubation with
IV.3 Fab (10 µg/ml), cross-linking with GAM F(ab`)
(15
µg/ml). E, Fc
RIII response: preincubation with 3G8
Fab (10 µg/ml), cross-linking with GAM F(ab`)
(15
µg/ml). F, preincubation without Fc
R antibodies,
cross-linking with GAM F(ab`)
(15 µg/ml). Curves are
representative for two to five independent
experiments.
Heterotypic Clustering of Fc
The
results depicted above indicated that especially upon heterotypic
cross-linking of FcR Induces a Higher
Depletion of Intracellular Ca
Stores
RIIa and Fc
RIIIb, Ca
influx from the extracellular environment is induced. In several
cell types (including neutrophils), the depletion of intracellular
stores can activate Ca
influx
(30, 33) . To assess the depletion of Ca
stores under the various conditions of Fc
R cross-linking,
stimulations were performed in the presence of EGTA. After 3 min
stimulation, ionomycin was added to mobilize all Ca
that had remained in the stores (). Especially after
heterotypic cross-linking of Fc
R the response to ionomycin was
very poor, indicating that under these conditions depletion of
Ca
stores had occurred to a much higher degree than
after homotypic simultaneous cross-linking or the separate Fc
R
cross-linking. Hence, an increased store depletion might cause the
Ca
influx observed after heterotypic Fc
R
cross-linking.
Immune Complexes Also Induce Ca
The
ability of immune complexes to bind to both FcInflux Characteristic for the Synergistic Response
RIIa and
Fc
RIIIb renders it likely that the synergism we observed is
relevant for physiological Fc
R stimulation. To test this
hypothesis, neutrophils were stimulated by heat-aggregated IgG. The
[Ca
]
increase induced
by heat-aggregated IgG could be partially blocked by either
anti-Fc
RII Fab or anti-Fc
RIII Fab (data not shown),
indicating involvement of both Fc
receptors in this response.
Moreover, heat-aggregated IgG induced a significant Mn
influx (Fig. 6) that was inhibited by either
anti-Fc
RII Fab or anti-Fc
RIII Fab (Fig. 6), indicating
a role for both Fc
receptors in inducing this response. This
conclusion was further supported by the observation that neutrophils
from an Fc
RIII-negative donor showed hardly any Ca
influx upon stimulation with heat-aggregated IgG as compared to
control neutrophils (Fig. 7), also indicating a contribution of
the Fc
RIIIb in this process.
Figure 6:
Effect of heat-aggregated IgG on
Ca influx as measured by Mn-dependent quenching of
indo-1 fluorescence. Indo-1 emission was measured at the
Ca
-independent wavelength of 446 nm as explained in
the text. Neutrophils were diluted five times in incubation medium
without Ca
before being transferred to the cuvette.
Subsequently, 0.5 mM MnCl
was added ( first
arrow) and stimulation with heat-aggregated IgG (1 mg/ml) was
performed ( second arrow) with preincubation of 3G8 Fab (10
µg/ml) ( A) or IV.3 Fab (10 µg/ml) ( B) or
without preincubation ( C). To completely quench indo-1
fluorescence with Mn
, digitonin (10 µM)
was added ( third arrow). Curves are representative
for two independent experiments.
Figure 7:
Effect of heat-aggregated IgG on
Ca influx in Fc
RIIIb-nega-tive neutrophils as
measured by Mn
-dependent quenching of indo-1
fluorescence. Indo-1 emission was measured at the
Ca
-independent wavelength of 446 nm as explained in
the text. Neutrophils were diluted five times in incubation medium
without Ca
before being transferred to the cuvette.
Subsequently, 0.5 mM MnCl
was added ( first
arrow) and stimulation with heat-aggregated IgG (1 mg/ml) was
performed ( second arrow) in Fc
RIIIb-negative neutrophils
( A) or control neutrophils ( B). The response of the
control neutrophils was representative for the neutrophils of five
different donors. To completely quench indo-1 fluorescence with
Mn
, digitonin (10 µM) was added
( third arrow).
Heterotypic Cross-linking of Fc
To
investigate whether heterotypic cross-linking of FcRIIa and Fc
RIIIb
Induces a Synergistic Activation of the Respiratory Burst
RIIa and
Fc
RIIIb affects other neutrophil responses induced by Fc
R
ligation, activation of the respiratory burst was measured. Neither
anti-Fc
RII Fab, anti-Fc
RIII Fab, nor GAM F(ab`)
alone induced an increase in respiratory burst activity. The
homotypic cross-linking of both Fc
RIIa and Fc
RIIIb induced
some activation as shown before by Hundt et al.(4) .
The combined Fc
R response was significantly higher then the sum of
the homotypic Fc
R responses ( p < 0.0125)
(Fig. 8). The increase in the respiratory burst activity of the
combined Fc
R response was 2.04 ± 0.3 times higher than the
sum of the separate responses, indicating a synergistic increase in
respiratory burst activity upon heterotypic cross-linking of both
Fc
receptors on human neutrophils.
Figure 8:
Activation of the respiratory burst after
heterotypic cross-linking of FcRIIa and Fc
RIIIb on human
neutrophils. Neutrophils were loaded with 0.25 µM
1,2,3-dihydrorhodamine ( DHR). The cells were pretreated with
IV.3 Fab (5 µg/ml) or 3G8 Fab (5 µg/ml) or both IV.3 Fab and
3G8 Fab for 5 min at 37 °C, washed, and incubated with GAM
F(ab`)
(15 µg/ml) for 30 min, as indicated. As control,
no antibodies were added. For comparison neutrophils were treated for 2
min with 1 µM platelet-activating factor ( PAF)
and for 10 min with 1 µM
formyl-methionyl-leucine-phenylalanine ( fMLP). Fluorescence of
DHR was measured by flow cytometry. Values were given as mean
fluorescence ± S.E. of four independent
experiments.
Rs on human
neutrophils can transduce signals
(4, 12, 15) .
In the present study we have investigated the effects of possible
interactions between signaling via Fc
RIIa and Fc
RIIIb.
Changes in [Ca
]
were
used as indicator for signal transduction events. In considering
possible interactions between signal transduction via Fc
RIIa and
Fc
RIIIb, one should differentiate at least three situations.
First, a mono- or divalent ligation of one receptor, without triggering
a signal, might have a modulating effect on a specific stimulation via
the other receptor. Second, the signal transduction pathway activated
by homotypic cross-linking of one receptor might interact with the
pathway simultaneously triggered by homotypic cross-linking of the
other receptor. Third, heterotypic clusters of Fc
RIIa and
Fc
RIIIb might initiate a type of signal transduction that has
quantitative or qualitative properties distinct from that of homotypic
clusters.
response subsequently
elicited via the other receptor. This is in contrast to the proposal of
some investigators
(20, 21) of modulation of the
Ca
response elicited via Fc
RIIIb by monovalent
ligation of Fc
RIIa. In these studies aggregated IgG
(20) or insoluble immune complexes
(21) were used as
stimuli. Although these complexes were proposed to bind specifically to
Fc
RIIIb, a simultaneous binding to Fc
RIIa is difficult to
exclude due to the much lower expression of this receptor. This could
account for the observed inhibition by ligation of Fc
RII.
Rs is required to
achieve a full response of the neutrophil to immune complexes
(21) . Indications for a synergistic effect of simultaneous
activation via both Fc
Rs have been found in some studies
(4, 34) . However, the level at which this synergism
takes place has not been revealed.
RIIa to Fc
RIIIb is possible (
Fig. 2
and ). Simultaneous stimulation via both
receptors performed in a way that allowed only homotypic cross-linking
of both receptors to occur did not result in synergism, and even had an
inhibiting effect. Under those circumstances, the response did not
reach the sum of the separate homotypic responses ( Fig. 1and
). Additional evidence for the requirement of heterotypic
cross-linking of Fc
RIIa and Fc
RIIIb for the generation of a
synergistic [Ca
]
response was obtained from experiments with the bsAb
Fc
RIIxFc
RIII. The heterotypic cross-linking by bsAb also
induced a Ca
response much higher than obtained after
separate Fc
R cross-linking ( Fig. 3and ). The
observation that immune complexes induced hardly any Ca
influx in Fc
RIIIb-negative neutrophils, in contrast to
normal neutrophils, also indicated an important contribution of
Fc
RIIIb in the immune complex-induced Ca
influx
(Fig. 7).
RIIa with
Fc
RIIIb in heterotypic clusters leads to a response that is
quantitatively distinct from the result obtained after homotypic
cross-linking. Whether this synergistic effect of heterotypic
cross-linking is only a quantitative phenomenon, induced by more
extensive cluster formation, or also reflects qualitative changes in
the signal transduction elicited by heterotypic clusters, remains to be
elucidated. However, we did observe that under conditions of
heterotypic cross-linking, the influx of extracellular Ca
was especially increased. Experiments in the presence of EGTA
indicated that the individual Fc
RIIa and Fc
RIIIb responses
exhibit a slight dependence on Ca
influx from the
extracellular medium ( Fig. 4and ). With increasing
responses we observed an enhanced Ca
influx from the
extracellular medium, especially under conditions of heterotypic
cross-linking of Fc
R (Fig. 5). This enhanced Ca
influx is probably caused by enhanced store depletion, according
to the model of Putney
(33) . Our results show a higher degree
of intracellular Ca
mobilization upon heterotypic
clustering as compared to simultaneous homotypic cross-linking
(). Even with much higher concentrations of cross-linking
agents (up to 50 µg/ml streptavidin or Fc-specific GAM F(ab`)
instead of 10 and 15 µg/ml, respectively, as used in most
experiments) in the simultaneous homotypic response the level of store
depletion was always lower than for the heterotypic Fc
R response
(data not shown).
stores in the area around the phagosome has been
described supporting locally the induction of high Ca
concentrations
(35) . Possibly, accumulation of
Ca
stores to the site of cross-linked receptors under
conditions of heterotypic cross-linking contributes to the response.
Alternatively, the formation of heterotypic clusters of Fc
receptors might allow additional signals by trans-phosphorylations of
associated proteins or Fc
RII itself that are instrumental for the
synergistic response to occur
(36) .
RIIa and Fc
RIIIb also induced synergism in
functional responses of neutrophils. Synergism in Fc
R-mediated
phagocytosis has recently been described in literature
(37) . We
observed also synergistic activation of the respiratory burst
(Fig. 8). This seems in contrast to earlier studies of our group,
showing the oxidative burst to be normal in neutrophils from paroxysmal
nocturnal hemoglobinuria patients when stimulated with immune complexes
(2) . Blood cells from paroxysmal nocturnal hemoglobinuria
patients lack glycosylphosphatidylinositol-anchored proteins, such as
Fc
RIIIb on neutrophils. However, neutrophils from paroxysmal
nocturnal hemoglobinuria patients still express approximately 10% of
normal levels of Fc
RIIIb
(2) , which is still a reasonable
number as compared to the number of Fc
RIIa on the neutrophils.
This amount of Fc
RIIIb may still play a role in the heterotypic
interaction between Fc
receptors in neutrophils.
R on
human neutrophils greatly enhances Ca
responses by an
effect on Ca
influx and induces a synergistic
activation of the respiratory burst. In this way, heterotypic cluster
formation of Fc
R may be a requirement for full generation of
effector functions
(4, 34) . The ability of immune
complexes to evoke a Ca
influx suggests that this
synergism is physiologically relevant.
Table:
Peak increases in
[Ca]
after homotypic
cross-linking of Fc
R on human neutrophils
Table:
Synergism of the combined FcR response
R
response are shown together with the reference Fc
R responses
determined in the same experiments. The experiments were carried out as
described in the legend to Fig. 2 with antibody concentrations as
indicated in the table. Results given are the mean ± S.E. of the
number of experiments indicated between parentheses.
Table:
Characterization of BsAb
FcRIIxFc
RIII
RIIIb-transfected
CHO cells (CHO
cells) or wild type CHO cells
(CHO
cells) were incubated with BsAb
Fc
RIIxFc
RIII, aspecific mouse IgG1, IV.3 Fab, or 3G8
F(ab`)
as described under ``Materials and
Methods.'' After washing, the cells were incubated with GAM-FITC
(62.5 µg/ml), IV.3-FTTC (2.5 µg/ml), or 3G8
F(ab`)
-FITC (2.5 µg/ml) for 30 min at 4 °C. After
another washing step, fluorescence data were collected from 5000 cells
by flow cytometry. Results are the mean of two independent experiments.
Table:
BsAb response compared to the combined
FcR response
R response were
compared. The experiments were performed as described in the legends to
Figs. 3 and 4. Peak increases above resting values under the various
conditions of stimulation were calculated. Results given are the mean
± S.E. of the number of experiments indicated.
Table:
Effect
of extracellular Ca on changes in
[Ca
]
induced by
Fc
R cross-linking
R response,
the combined Fc
R response, and the separate Fc
R responses
were measured in the presence of 1 mM extracellular
Ca
or in the presence of EGTA as described under
``Materials and Methods.'' The cells were pretreated with
IV.3, 3G8 F(ab`)
-biotin, IV.3 Fab, or 3G8 Fab (each 10
µg/ml) or a combination of these antibodies for 5 min at 37 °C,
were washed and were incubated with streptavidine (10 µg/ml) or GAM
F(ab`)
(15 µg/ml), as indicated. Peak increases above
resting values under the various conditions of stimulation were
calculated. Results given are the mean ± S.E. of the number of
experiments indicated.
Table:
Effect of FcR cross-linking on the
depletion of intracellular Ca
stores
R response, the combined Fc
R
response, and the separate Fc
R responses were performed in the
presence of 1 mM EGTA as described under ``Materials and
Methods.'' After 3 min 1 µM ionomycin was added to
quantitate the amount of Ca
still present in the
stores. Concentrations of cross-linking agents were the same as
separate responses. Results given are the mean ± S.E. of the
number of experiments indicated.
R, the
receptors for IgG; GAM, goat anti-mouse immunoglobulin antibodies; mAb,
mouse monoclonal antibodies; bsAb, bispecific antibodies; PAGE,
polyacrylamide gel electrophoresis; [Ca
],
intracellular free Ca
concentration; PBS,
phosphate-buffered saline; FITC, fluorescein isothiocyanate; CHO,
Chinese hamster ovary; SPDP,
N-succinimidyl-3-(2-pyridyldithiol)propionate; BAPTA,
1,2-bis-( O-aminophenoxyl)ethane- N,N,N`,N`-tetraacetic
acid; MHC, major histocompatibility complex.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.