From the
Two tyrosine kinase-dependent pathways exist for activation of
the respiratory burst by polymorphonuclear leukocyte (PMN)
immunoglobulin G Fc receptors. Direct ligation of Fc
Binding of immune complexes to cell receptors for immunoglobulin
is a powerful stimulus to activation of phagocytes. Immune complex
binding to these cells leads to activation of effector functions of
host defense such as phagocytosis, secretion, cytokine synthesis, and
the production of toxic oxygen metabolites, which occurs as a result of
NADPH oxidase assembly
(1) . The cloning of several members of
the immunoglobulin Fc receptor family over the past several years has
tremendously enhanced understanding of the molecular mechanisms for the
functions of these immunologically important receptors. Identification
of receptors within the family which associate with a second
transmembrane protein, called the
In contrast, little is
known about the molecular mechanisms of signal transduction from the
PMN Fc
Recently, we have developed an assay system which allows a more
detailed investigation of the contribution of individual PMN Fc
Immune complexes are potent stimuli for activation of PMN.
Immune complex deposition and subsequent PMN activation is an important
part of the pathogenesis of serum sickness, the Arthus reaction, acute
glomerulonephritis, rheumatoid arthritis, and other idiopathic
inflammatory diseases. Although these are host-damaging diseases,
immune complex-mediated PMN activation also plays an essential role in
host defense against bacterial infection. It is clear that in vivo both host defense and host damaging aspects of the PMN-immune
complex interaction involve complement activation and deposition onto
the immune complexes
(36) . The major PMN receptor for the
complement deposited onto immune complexes is the leukocyte integrin
CR3. Therefore, to understand immune complex activation at a molecular
level requires understanding the roles for both Fc
In the current work,
we have studied signal transduction during the synergistic respiratory
burst and compared it with the signal transduction cascade activated by
direct ligation of Fc
By using PT, which
suppresses the respiratory burst from direct ligation of Fc
Although direct evidence that Fgr kinase
activity is involved in NADPH oxidase assembly is lacking, we believe
it very likely that Fgr has a role in generation of the respiratory
burst from direct ligation of Fc
Immobilized Fc
Ligation of Fc
Thus,
we hypothesize that the role for ligation of Fc
We are indebted to Dr. Donald Anderson for provision
of blood from a patient with LAD and Dr. Andrey Shaw for antibodies
against Fyn, Yes, Lck, and Src. We also thank various members of the
Brown laboratory for helpful comments during the execution of these
experiments.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RII activates
the respiratory burst, but ligation of the glycan
phosphoinositol-linked Fc
RIIIB does not. Instead, this receptor
and the integrin complement receptor CR3 synergize in activation of the
respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell
Biol. 125, 1407-1416). Here we show that direct ligation of
Fc
RII leads to activation and Triton X-100 insolubility of the Src
family kinase Fgr, without effect on the related myeloid Src family
member Hck. In contrast, adhesion of PMN via Fc
RIIIB leads to
activation and Triton X-100 insolubility of Hck but not Fgr. The
exclusive association of Fc
RIIIB with Hck activation and Triton
insolubility is not solely a result of its glycan phosphoinositol
anchor, since decay accelerating factor (CD55), another prominent
glycan phosphoinositol-anchored PMN protein, is associated with Fgr
insolubility to a greater extent than Hck. Ligation of decay
accelerating factor, with or without coligation of CR3, does not
activate the PMN respiratory burst. Coligation of Fc
RIIIB with
Fc
RII overcomes the pertussis toxin inhibition of
H
O
production in response to direct ligation of
Fc
RII. These data support the hypothesis that activation of Hck
upon Fc
RIIIB ligation has a role in generation of the synergistic
respiratory burst.
chain, and realization that the
chain is itself a member of a family of proteins known to be
involved in tyrosine kinase-mediated signal transduction has further
enhanced understanding of Fc receptor-mediated cell activation. The two
Fc
receptors expressed on polymorphonuclear leukocytes
(PMN),
(
)
Fc
RII and the glycan
phosphoinositol (GPI)-linked form of Fc
RIII (Fc
RIIIB) are
distinct within this family for their failure to associate with the
chain, suggesting alternative mechanisms for cell activation by
these receptors. Nonetheless, even Fc
RII appears to activate
tyrosine kinases and to be phosphorylated on tyrosine during immune
complex-mediated cell activation
(2) . Fc
RII has been found
to associate with specific Src family kinases, including Fgr in PMN,
and to activate syk kinase
(3, 4, 5) . Ligation
of Fc
RII leads to the tyrosine phosphorylation of multiple
cellular proteins, including phospholipase C
, Shc, and syk, in
addition to Fc
RII itself (6-8).
RIIIB. This receptor is unique among Fc receptors, since it
is expressed on the plasma membrane by a GPI anchor, rather than as a
transmembrane protein. It has no association with the
chain,
which associates with the transmembrane form of Fc
RIII expressed
in NK cells and macrophages, and has no intracytoplasmic domain for
direct association with cytosolic signal transduction cascades. Indeed,
whether Fc
RIIIB mediates signal transduction at all remains
controversial. Many effects of immune complexes on PMN appear to be
mediated exclusively by Fc
RII
(9, 10, 11) ,
although this is not universally the case
(10, 12) .
Cross-linking of Fc
RIIIB can lead to an increase in
intracytoplasmic Ca
([Ca
]
), although
the mechanism for this effect is unclear
(13) . Moreover,
GPI-linked proteins in several cell types have been associated with
membrane domains rich in Src family tyrosine kinases
(14) , and
evidence has been presented recently for an association of PMN
Fc
RIIIB with tyrosine kinase activity
(15, 16) .
Thus, the role for Fc
RIIIB in PMN activation remains uncertain.
receptors to signal transduction leading to generation of a respiratory
burst
(15) . We have used PMN adhesion to surfaces coated with
monoclonal antibodies (mAb) to individual receptors to assess their
contribution to cell activation. We have found that direct ligation of
Fc
RII leads to a respiratory burst, whereas direct ligation of
Fc
RIIIB does not. Instead, Fc
RIIIB cooperates with the PMN
integrin CR3 (Mac-1, CD11b/CD18) to generate what we have termed a
synergistic respiratory burst. In the synergistic respiratory
burst, the two membrane receptors have distinct roles. Ligation of CR3
immobilizes Fc
RII to the adherent plasma membrane by a
cytoskeleton-dependent mechanism, and ligation of Fc
RIIIB induces
appropriate tyrosine kinase activation in proximity to the immobilized
Fc
RII. Thus, Fc
RII is required in addition to CR3 and
Fc
RIIIB for the synergistic respiratory burst. In the current
work, we have examined the nature of the Src family kinases activated
by ligation of the two Fc
receptors. We have found that Fc
RII
and Fc
RIIIB immobilized on the adherent PMN surface by direct
ligation lead to the activation and Triton X-100 insolubility of
different Src family kinases. Fc
RII is associated with activation
and translocation of Fgr to the Triton-insoluble cell fraction; and
Fc
RIIIB is associated with Hck activation and translocation. The
exclusive association of Fc
RIIIB with Hck activation is not a
property of all GPI-linked proteins in PMN, since immobilization of
decay accelerating factor (DAF, CD55) leads primarily to Fgr activation
at the adherent membrane. Moreover, DAF cannot substitute for
Fc
RIIIB in synergistic activation of the respiratory burst. From
these data we conclude that ligation of Fc
RII and Fc
RIIIB
activate and translocate distinct Src family members in PMN. We
hypothesize that the functional consequence of the activation and
translocation of distinct kinases is the existence of two separate
signal transduction pathways for activation of the PMN respiratory
burst by immune complexes.
Antibodies
The following mAb were used in these
studies: IB4 (anti-CD18)
(17) , W6/32 (anti-HLA)
(18) ,
B6H12 (anti-CD47)
(19) , 3D9 (anti-CD35)
(20) , 3G8
(anti-CD16)
(21) , IH4 (anti-CD55)
(22) , OKM1
(anti-CD11b)
(23) , and IV.3 (anti-CD32)
(24) . IB4, 3G8,
and IV.3 IgG were purified from ascites using octanoic acid as
described
(25) . W6/32 and B6H12 IgG were prepared using an
Amicon Bioreactor (Amicon Inc., Danvers, MA) according to
manufacturer's instructions. SDS-PAGE of all purified IgG
preparations showed them to be >90% IgG. PY20 (anti-phosphotyrosine)
was purchased from Transduction Laboratories (Lexington, KY).
Polyclonal anti-Fgr was as described
(26, 27) or was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), as was
anti-Hck.
Buffers and Other Reagents
Phosphate-buffered
saline was from Biowhittaker, Walkersville, MD. Krebs-Ringer buffer
(KRPG) was 145 mM NaCl, 4.86 mM KCl, 1.22 mM
MgSO, 5.7 mM Na
HPO
, 0.54
mM CaCl
, and 5.5 mM glucose, pH 7.4.
Reaction mixture (RM) consisted of 37.5 µM scopoletin,
1.25 mM NaN
, 1.25 units/ml HPO in KRPG. Kinase
buffer was 40 mM Hepes, pH 7.5, 10 mM
MgCl
, 3 mM MnCl
, 0.1 mM
Na
VO
, 10% glycerol, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride. MBS buffer
was 25 mM Mes, pH 6.5, 0.5% Triton X-100, 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 5
mM diisopropyl fluorophosphate, 1 mM
Na
VO
, 1 mM iodoacetamide, 10 µg/ml
each of aprotinin, leupeptin, and pepstatin A. Other reagents were
obtained from standard sources as described previously
(28) .
PMN Isolation
Human PMN were isolated by dextran
sedimentation and two-step Hypaque density centrifugation as described
(29). PMN were obtained from a integrin-deficient
(leukocyte adhesion-deficient, LAD) patient followed at Baylor College
of Medicine. This patient has been characterized as having the complete
deficiency phenotype. Expression of Fc
RII and FcR
III on this
patient's cells was normal, whereas CR3 was undetectable (not
shown). The patient's blood and a normal control were transported
and the PMN prepared as described
(30) .
Preparation of Coated Plate and
H
96-Well tissue
culture plates (Costar, Cambridge, MA) were coated with protein A and
then mAb IgG essentially according to the method of Berton et
al.(31) , with modifications as described
(28) . The
microwell HO
Assay
O
assay was adapted from the method
of De la Harpe and Nathan
(32) , as modified by Berton et
al.(31) . Data were collected and analyzed exactly as
described previously
(28) . In each experiment, data were
averaged from triplicate wells, which generally varied from each other
by
10%. Unless otherwise stated, H
O
accumulation was measured after 60 min of PMN incubation in
wells.
Pretreatment of PMN
PMN at 2.5 10
cells/ml in KRPG were pretreated with 5 µg/ml of Fab or 2.5
µg/ml of F(ab`)
of various mAb at 4 °C for 15 min.
Without washing, PMN were added to antibody coated plates containing
RM
(28) . For treatment with pharmacologic agents, PMN were
preincubated with herbimycin at 10 µg/ml and then added to RM
containing the same concentration of the indicated agent. To pretreat
PMN with pertussis toxin, cells in KRPG were incubated with 2 µg/ml
of pertussis toxin at 37 °C for 75 min. In experiments examining
the effects of cytochalasin, PMN were pretreated with 5 µg/ml
cytochalasin D and added to RM containing no additional drug. In all
experiments, control PMN were incubated with an identical concentration
of nonaqueous diluent as the PMN receiving any drug.
``In Situ'' Tyrosine Phosphorylation
5
10
PMN in KRPG with 0.1 mM
Na
VO
were allowed to adhere to mAb-coated
six-well plates at 4 °C for 10 min and then at 37 °C for 15
min. The nonadherent cells were removed by washing with cold MBS
buffer. The adherent cells were extracted with 0.5% Triton X-100 in MBS
buffer on ice for 8 min, following which the plate was rinsed with
kinase buffer containing 0.1 mM Na
VO
and 0.2% Triton X-100 once. In situ tyrosine
phosphorylation was performed by adding 50 µCi of
[
-
P]ATP and 1 µM cold ATP in
500 µl of kinase buffer at room temperature for 15 min. For
immunoprecipitation, following washing with the kinase buffer three
times, the cell residuals were solubilized with solubilization buffer
(20 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1%
deoxycholate, 0.1% SDS, 10 µg/ml each of aprotinin, leupeptin, and
pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 2 mM
diisopropyl fluorophosphate, 1 mM EDTA, 1 mM
Na
VO
) at 4 °C for 60 min with shaking. The
lysates were diluted with solubilization buffer without SDS and then
precleared with protein A-Sepharose at 4 °C for 2 h. The precleared
lysate was incubated with anti-Fgr, -Hck, or -phosphotyrosine IgG
captured by protein A-Sepharose at 4 °C for 2 h. The Sepharose
mixture was washed once with solubilization buffer and then six times
in the same buffer without deoxycholate, SDS, and protease inhibitors.
The Sepharose-bound proteins were analyzed by SDS-PAGE as
described
(33) .
Immune Complex Kinase Assay
To determine whether
Fgr and Hck were activated by adhesion, PMN were allowed to adhere to
surfaces coated with various mAb for 15 min at 37 °C. After MBS
buffer extraction, supernatant was collected and the Triton
X-100-insoluble fraction solubilized in solubilization buffer as above.
Both the Triton X-100-soluble and -insoluble fractions were
immunoprecipitated with anti-Fgr or anti-Hck. Following
immunoprecipitation, the immune complexes were incubated with 5 µCi
of [-
P]ATP in 30 µl of kinase buffer at
room temperature containing 20 µg of poly(Glu,Tyr) (Glu:Tyr ratio
4:1, Sigma) for 15 min. The reaction was stopped by adding 6 µl of
10% phosphoric acid and centrifugation. The supernatants were spotted
onto Whatman 3MM cellulose filter paper (1.5
1.5 cm), and the
filters were washed with 10% trichloroacetic acid four times, 5
mM phosphoric acid, and 80% acetone once each. After
air-drying the filters, the
P incorporation was measured
by scintillation counting. Alternatively, the supernatants were loaded
into the sample bucket of a phosphocellulose unit (Pierce) and washed
according to the manufacturer's instructions. In experiments in
which the Triton X-100-insoluble cell fraction was immunoprecipitated
with PY20, cell residuals were incubated with kinase buffer containing
10 mM unlabeled ATP at room temperature for 15 min prior to
solubilization and immunoprecipitation.
Western Blot
Following PMN adhesion on mAbs and
bovine serum albumin-coated plates, the adherent cells were extracted
with 0.5% Triton X-100 in KRPG buffer with protease inhibitors on ice
for 5 min. The cell residuals were lysed by solubilization buffer with
0.2% SDS. The recovered lysates were precipitated by adding 5 volumes
of cold acetone and kept at -20 °C for 30 min. After
centrifugation at top speed in an Eppendorf centrifuge at 4 °C for
10 min, the pellets were washed with 80% acetone twice and allowed to
air-dry. The precipitated proteins were dissolved by SDS-sample buffer
with 5 mM -mercaptoethanol and 2 mM EDTA and
separated by SDS-PAGE, then transferred to polyvinylidene difluoride
(Millipore, Bedford, MA). The membranes were incubated overnight in
blocking buffer (PBS Tween and 3% bovine serum albumin) and then with
anti-Hck and anti-Fgr polyclonal IgG at 1:1000 at room temperature for
4 h. The membranes finally were incubated with horseradish
peroxidase-conjugated protein A and developed using the ECL
chemiluminescence kit (Amersham Corp.).
Fc
Our previous
studies have shown distinct roles for the two PMN FcRII or Fc
III Ligation in PMN Leads to
Activation of Distinct Src Family Tyrosine Kinases
receptors in
activation of the respiratory burst. PMN adhesion to an Fc
RII
ligand leads to a respiratory burst, whereas the GPI-linked
Fc
RIIIB is required for the synergistic respiratory burst, but is
incapable of activation of the NADPH oxidase on its own. Nonetheless,
our previous data demonstrate that both pathways are inhibited by
tyrosine kinase inhibitors and suggest that Fc
RIIIB ligation is
associated with tyrosine kinase activity
(15) . To begin to
understand tyrosine kinase function in the two pathways, we examined
activation of two predominant PMN Src family kinases, Fgr and Hck, upon
ligation of PMN Fc
receptors. As assessed by phosphorylation of
poly(Glu,Tyr), ligation of Fc
RII led to Fgr activation, whereas
ligation of Fc
RIIIB led to Hck activation
(Fig. 1A). Fgr activation has been associated previously
with Fc
RII ligation in PMN
(4) . Ligation of either HLA or
CR3 did not lead to a detectable increase in the activity of either
kinase. Total Fgr activity increased approximately 6.5-fold (average of
two determinations) in cells adherent via Fc
RII compared with
ligation of HLA, and total Hck activity increased more than 20-fold
upon Fc
RIII ligation (Fig. 1A). A small amount of
Fgr kinase activity could be detected in the Triton X-100-soluble
fraction in PMN incubated on the control surfaces. However, neither Fgr
nor Hck kinase activity was detected in the Triton X-100-insoluble
fraction from PMN adherent via HLA or CR3. After either Fc
RII or
Fc
RIIIB ligation, kinase activity was increased in both Triton
X-100 soluble and insoluble cell fractions. Thus, Fgr kinase activity
was stimulated by ligation of Fc
RII; Hck activity was stimulated
by ligation of Fc
RIIIB.
Figure 1:
Hck kinase activity is enhanced by
FcRIIIB ligation, whereas Fgr kinase activity is enhanced by
Fc
RII ligation. A, Hck activity (left) and Fgr
activity (right) were measured in the Triton X-100-soluble and
-insoluble fractions from PMN adherent to anti-Fc
RIIIB,
anti-Fc
RII, anti-CR3, or anti-HLA. Kinase activity was measured
using the substrate poly(Glu,Tyr) as described under ``Materials
and Methods.'' Shown is a single experiment, performed in
duplicate, representative of three. B, Hck (left) and
Fgr (right) protein were measured in the Triton X-100-soluble
and -insoluble cell fractions from cells adherent to anti-Fc
RIIIB
and anti-HLA (left) or anti-Fc
RII and anti-HLA
(right). 2
10
cell equivalents were used
as the starting material for Hck and Fgr immunoprecipitation from the
Triton X-100-soluble fraction; 10
10
cell
equivalents were used for the immunoprecipitations from the Triton
X-100-insoluble fraction. The data are from one of two similar
experiments. C, the relative ``specific activities''
of Triton X-100-soluble and -insoluble Fgr and Hck were compared for
cells adherent to anti-Fc
RIII (Hck) and to anti-Fc
RII (Fgr).
Relative specific activities were estimated by dividing kinase activity
by protein concentration estimated from densitometry of Western blots.
Specific activity of each kinase in the Triton X-100-soluble fraction
of PMN adherent to anti-HLA was set to 1. Fgr specific activities in
the Triton X-100-soluble and -insoluble fractions of PMN adherent to
anti-Fc
RII were 5.5 and 32, respectively; Hck specific activities
in the Triton X-100-soluble and -insoluble fractions were 33.5 and 230.
Specific activities of Fgr and Hck are not comparable with each other,
because different antibodies were used for the Western blots. The graph
depicts data averaged from two experiments.
Fc
Src family kinases often associate with the Triton
X-100-insoluble cytoskeleton upon activation, and Src-induced oncogenic
transformation may require cytoskeletal association
(34) ,
suggesting that important kinase substrates are encountered through
translocation to the cytoskeleton. Since Triton X-100 insolubility may
reflect this translocation to the cytoskeleton, we examined the
differences in the Triton X-100 solubility of Fgr and Hck upon receptor
ligation. PMN were adhered to mAb-coated surfaces, briefly solubilized
with Triton X-100, and the cell residue analyzed by SDS-PAGE and
Western blotting (Figs. 1B and 2). Hck was present in the
Triton X-100- insoluble cell fraction if, and only if, PMN were
adherent to anti-FcRII or Fc
RIII Ligation Leads to Association
of Distinct Src Family Kinases with the Triton X-100-insoluble Cell
Fraction
RIIIB (Fig. 2A). Whether or not
anti-CR3 also was present on the adherent surface made no difference
for Hck localization, even though respiratory burst activation only
occurs when both antibodies are present. Fgr was present in the Triton
X-100-insoluble fraction only when Fc
RII was ligated on the
adherent surface (Fig. 2B). Unlike Fc
RIIIB,
ligation of a different GPI-linked protein, DAF, led predominantly to
Fgr localization in the Triton X-100-insoluble residue. When cells were
adherent to anti-CR3 alone, no Fgr or Hck protein was detectable.
Specific activity of Fgr and Hck was estimated in both the Triton
X-100-soluble and -insoluble fractions after Fc
R ligation by
comparing kinase activity (Fig. 1A) to protein
concentration as estimated from the Western blots
(Fig. 1B). Interestingly, although active Fgr or Hck was
present in both Triton X-100-soluble and -insoluble compartments, the
specific activity of both kinases was 6-7-fold greater in the
Triton X-100-insoluble fraction after appropriate receptor ligation
(Fig. 1C). Presumably, this reflects the fact that not
all Triton X-100-soluble kinase has been activated by receptor
ligation.
Figure 2:
Hck and Fgr protein localization to the
Triton X-100-insoluble fraction upon Fc receptor ligation. The
Triton X-100-insoluble residue from PMN adherent to various surfaces
was solubilized and analyzed by SDS-PAGE and immunoblotting. The mAb
used to coat the surfaces to which the PMN were adherent before Triton
X-100 extraction and solubilization are indicated in each lane. Each
lane contains 10
10
PMN. A, immunoblot
with anti-Hck. B, immunoblot with anti-Fgr. Hck was detected
if and only if PMN were adherent to anti-Fc
RIIIB; Fgr was present
when PMNs were adherent to anti-Fc
RII or
anti-DAF.
Tyrosine Kinase Activity in the Triton X-100-insoluble
Fraction after Ligation of Fc
To examine
the phosphorylation patterns in the Triton X-100-insoluble fractions
after FcRII or Fc
RIIIB
RII or Fc
RIIIB ligation, an in situ kinase
assay was performed after extraction of adherent PMN with Triton X-100.
After addition of [
P]ATP to the Triton
X-100-insoluble residue adherent to the immune complex-coated surfaces,
phosphotyrosine-containing proteins were examined by
immunoprecipitation of the products of the in vitro kinase
assay with anti-phosphotyrosine mAb, PY20, after solubilization with a
more stringent detergent (1% Triton X-100, 1% deoxycholate, 0.1% SDS).
As shown in Fig. 3, phosphotyrosine-containing proteins with
molecular mass of
55-60 kDa were the major products of the
in vitro kinase assay when PMN were adherent to either an
anti-Fc
RII- or an anti-Fc
RIIIB- coated surface. Under the
same conditions, these phosphotyrosine-containing proteins were not
detected from PMN adherent to anti-HLA- or anti-CD47-coated surfaces.
Importantly, no tyrosine kinase activity was found in PMN adherent to
an anti-CR3-coated surface (Fig. 3), even though Fc
RII is
present in the Triton X-100-insoluble material
(15) , and CR3
participates in the synergistic respiratory burst. No reproducible
kinase activity was found in anti-Fc
RII or anti-Fc
RIIIB
immunoprecipitates from unstimulated, nonadherent PMN (data not shown).
Figure 3:
Kinase activity associates with the Triton
X-100-insoluble PMN fraction on engagement of Fc receptors. PMN
adherent on mAb-coated surfaces were extracted with 0.5% Triton X-100
in MBS buffer with protease inhibitors for 8 min. Following an in
situ kinase assay (see ``Materials and Methods''),
phosphotyrosine-containing proteins were immunoprecipitated with
anti-phosphotyrosine mAb, PY20, and then analyzed by
SDS-PAGE.
The autophosphorylated Triton X-100-insoluble material also was
immunoprecipitated with monospecific antibodies against several Src
family kinases. No radiolabeled proteins were immunoprecipitated by
antibodies to Yes, Fyn, Src, or Lck from PMN adherent to any surface
(data not shown). Phosphorylated proteins from lysates of PMN adherent
to an anti-FcRIIIB coated surface were specifically
immunoprecipitated by anti-Hck, but not by anti-Fgr (Fig. 4). As
described previously
(26, 35) , the autophosphorylated
Hck appeared as a doublet. Conversely, a phosphorylated band was
immunoprecipitated by anti-Fgr, but not by anti-Hck from the lysate
from an anti-Fc
RII-coated surface. When autophosphorylated lysates
from an anti-HLA coated surface were precipitated by both anti-Hck and
anti-Fgr, no phosphorylated bands were identified, consistent with the
minimal kinase activity associated with the Triton X-100-insoluble
material remaining from PMN adherent to this substrate (see
Fig. 1A and 3).
Figure 4:
Hck
and Fgr associated with the Triton X-100-insoluble cell fraction after
FcRIIIB or Fc
RII ligation have kinase activity. Following an
in situ kinase reaction using [
P]ATP on
the Triton X-100-insoluble fractions from PMN adherent to various
surfaces (Coating Ab), immunoprecipitation was performed on
the products of the kinase reaction with anti-Fgr, anti-Hck, or
anti-phosphotyrosine (PY20) (IP
Ab).
Hck Is the Predominant Triton X-100-insoluble Tyrosine
Kinase Associated with Fc
To determine whether Hck was the predominant
tyrosine kinase associated with the Triton X-100-insoluble material
from FcRIIIB Ligation, and Fgr Is the
Predominant Triton X-100-insoluble Tyrosine Kinase Associated with
Fc
RII Ligation
RIIIB ligation and Fgr the predominant kinase associated
with Fc
RII ligation, poly(Glu,Tyr) phosphorylation was assayed.
When lysates from anti-Fc
RIII coated surface were
immunoprecipitated with anti-Hck, the anti-Hck immune complexes
displayed marked tyrosine kinase activity (Fig. 5). Minimal
tyrosine kinase activity could be immunoprecipitated by PY20 after
anti-Hck preclearing. In contrast, anti-Fgr precipitated almost no
poly(Glu,Tyr) phosphorylating activity from the PMN adherent via
Fc
RIIIB. When lysates from anti-Fc
RII-coated surface were
immunoprecipitated with anti-Fgr and anti-Hck, the anti-Fgr immune
complexes contained 80% of the tyrosine kinase activity. There was very
little detectable Hck tyrosine kinase activity from lysates from
anti-Fc
RII coated surfaces (Fig. 5). Immunoprecipitates with
PY20 after anti-Fgr preclearing contained minimal kinase activity
toward poly(Glu,Tyr). Thus, we concluded that Hck accounted for almost
all the Triton X-100-insoluble tyrosine kinase activity associated with
Fc
RIIIB ligation, and Fgr accounted for the kinase activity
associated with Fc
RII ligation. These data suggest that Hck
specifically associates with the Triton-insoluble cell fraction from
cells adherent via Fc
RIIIB, and Fgr kinase specifically associates
with the Triton-insoluble cell fraction following adhesion via
Fc
RII.
Figure 5:
Hck
and Fgr are the major tyrosine kinases associated with the Triton
X-100-insoluble fraction after FcR ligation. PMN were lysed with
Triton X-100 after adhesion to various surfaces, as described in the
legend to Fig. 4. Immunoprecipitations with anti-Hck, anti-Fgr, or
anti-phosphotyrosine were performed. Kinase activity in the
immunoprecipitates was determined using poly(Glu,Tyr) as substrate.
Activity is plotted as substrate-associated counts/min after a 15-min
reaction. Data are mean ± S.E. of three experiments. Hck
accounts for the tyrosine kinase activity from PMN adherent via
Fc
RIIIB, and Fgr accounts for the predominant kinase activity from
PMN adherent via Fc
RII or DAF.
To determine whether the apparent exclusive association
of Hck activity with FcRIIIB ligation was true for all GPI-linked
PMN proteins, PMN were allowed to adhere to anti-DAF-coated surfaces
and Triton X-100-insoluble kinase activity determined (Fig. 5).
Unlike PMN adherent to anti-Fc
RIIIB, cells adherent to anti-DAF
showed more Fgr than Hck activity.
Requirement for Both Fc
The signal transduction
involved in activation of the respiratory burst by direct ligation of
FcRIIIB and Fc
RII for
Synergistic Respiratory Burst Activation
RII or by synergy between Fc
RIIIB and CR3 differ in several
respects, including sensitivity to pertussis toxin (PT) and
cytochalasin D
(15) . Direct ligation of Fc
RII activates
H
O
production, which is inhibited by PT
(Fig. 6A); activation of the synergistic respiratory
burst is unaffected by PT. Because our previous studies suggested that
the role for CR3 in the synergistic respiratory burst was to immobilize
Fc
RII in proximity to Fc
RIIIB, we tested whether direct
ligation of Fc
RII and Fc
RIIIB together could activate the
synergistic respiratory burst, by taking advantage of the PT
sensitivity of the respiratory burst generated by direct ligation of
Fc
RII. PT inhibited the respiratory burst generated by ligation of
Fc
RII, but coligation of Fc
RIIIB with Fc
RII restored the
respiratory burst in the presence of PT (Fig. 6B).
Ligation of Fc
RIIIB specifically is required for generation of the
respiratory burst with Fc
RII in the presence of PT, since neither
coligation of DAF, another prevalent GPI-linked protein on PMN, nor of
HLA, could stimulate respiratory burst in combination with Fc
RII
in the presence of PT (Fig. 6B). Coligation of
Fc
RIIIB and DAF also failed to stimulate the respiratory burst
(not shown). Addition of Fab or F(ab`)
fragments of mAbs
against either Fc
RII or Fc
RIIIB specifically inhibited the
respiratory burst stimulated by coligation of Fc
RIIIB with either
Fc
RII or CR3 (Fig. 7A). mAb against CR3 inhibited
the PMN respiratory burst by coligation of Fc
RIIIB with CR3, but
had no effect on the respiratory burst generated by coligation of
Fc
RII and Fc
RIIIB in the presence of PT
(Fig. 7A). Thus, coligation of Fc
RII and
Fc
RIIIB in the presence of PT is a second form of the synergistic
respiratory burst in which the need for CR3 has been eliminated.
Figure 6:
Fc receptor activation of a PMN
respiratory burst through PT-inhibitable and PT-independent pathways.
The generation of H
O
by normal PMN and LAD PMN
adherent to wells coated with various mAbs is compared with (dark
bar) and without PT (light bar) treatment of the PMN. In
A the respiratory burst generated from normal PMN incubated
with various single mAb is plotted. In B is plotted the
respiratory burst generated from normal PMN incubated with combinations
of mAbs coating the wells. C plots the respiratory burst
generated by LAD PMN. Data in all panels is H
O
production at 60 min. Data from normal PMN are means ±
S.E. of three independent experiments, each performed in triplicate. A
single experiment (of two independent experiments, each performed in
triplicate) with LAD PMN is shown in C. PT inhibits the
respiratory burst from direct ligation of Fc
RII. Coligation of
Fc
RIIIB restores the respiratory burst in the presence of
anti-Fc
RII, even though ligation of Fc
RIIIB alone does not
lead to activation of H
O
production. Maximal
respiratory burst stimulated with 50 ng/ml PMA was 2.46 ± 0.42
nmol at 60 min.
Figure 7:
Effects of mAbs and pharmacologic reagents
in solution on PMN respiratory burst stimulated by surface-bound mAbs.
A, Effect of antibodies. PMN were preteated with 5 µg/ml
of Fab fragments (anti-FcRII) or 2.5 µg/ml of F(ab`)
fragments (anti-CD11b and anti-Fc
RIIIB). PMN were incubated
in wells coated with anti-Fc
RII or the combination of
anti-Fc
RIIIB and anti-CR3 in the absence of PT or the combination
of anti-Fc
RIIIB and anti-Fc
RII in the presence of PT.
H
O
production was measured in the presence and
absence of anti-receptor antibodies. Anti-CD47, which blocks the
integrin
-stimulated H
O
production (28), did not affect any Fc
R-dependent
respiratory burst (data not shown). B, effect of pharmacologic
reagents. PMN were pretreated with 2 µg/ml of pertussis toxin, 5
µg/ml of cytochalasin D, or both. Other PMN were pretreated with 10
µg/ml herbimycin A. These PMN were incubated in wells coated with
anti-Fc
RII, anti-Fc
RIII, and anti-CR3 as in A.
H
O
production was measured after 60-min PMN
incubation with the various surfaces. Control H
O
production, assessed for PMN incubated with diluents for the
various reagents, was identical to the buffer control in A (not shown). A single experiment performed in triplicate,
representative of three, is shown.
These data suggested that the only role for CR3 in the synergistic
respiratory burst was to immobilize FcRII on the adherent surface
of the PMN in proximity to the ligated Fc
RIIIB. To test this
hypothesis, we examined LAD PMN which genetically lack CR3. Coligation
of Fc
RIIIB with Fc
RII activated PT intoxicated LAD PMN
normally (Fig. 6C), even though these PMN lacked the
synergistic respiratory burst from adhesion to surfaces coated with
anti-Fc
RIIIB and anti-CR3
(15) . As a second test of the
hypothesis, we examined the effect of cytochalasin D on the synergistic
respiratory burst. Although the synergistic activation by adhesion to
anti-CR3 and anti-Fc
RIIIB is sensitive to cytochalasin
D
(15) , the PT-insensitive respiratory burst in response to
coligation of Fc
RII and Fc
RIIIB was not
(Fig. 7B). Since cytochalasin has been shown to inhibit
CR3-mediated immobilization of Fc
RII on the adherent PMN membrane
(15), this experiment proves that coimmobilization of Fc
RII and
Fc
RIIIB by antibodies overcomes the need for cytoskeletal assembly
in the synergistic respiratory burst.
receptors and
CR3. Although the role for CR3 in this activation process was
originally thought to be passive, merely increasing the interaction
between IgG and its cellular receptors
(37) , there is now good
evidence that in some circumstances CR3 ligation contributes actively
to signal transduction
(11, 38) .
RII. We have shown that the role for CR3 in
the synergistic respiratory burst is to immobilize Fc
RII in
proximity to ligated Fc
RIIIB. Indeed, activation of Fgr and Hck
through Fc
receptor ligation occurs normally in LAD PMN (data not
shown). Thus, unlike the respiratory burst induced by tumor necrosis
factor-
stimulation of PMN
(39) , there is no evidence that
CR3 or any other
integrin is involved in Fgr
activation through Fc
RII. In this sense, the initial postulate of
Ehlenberger and Nussenzweig
(37) that complement simply enhances
immune complex presentation to phagocyte Fc
receptors, is correct.
However, it is clear that signal transduction in the synergistic
respiratory burst is more complex, since it requires two different
Fc
receptors, with distinct roles for each.
RII, we
were able to reconstitute the synergistic respiratory burst in the
complete absence of CR3. This experiment proves our early hypothesis
that Fc
RII plays an essential role in the synergistic respiratory
burst, even when it is immobilized only by its association with CR3 and
not directly ligated
(15) . In this situation, we found no
tyrosine kinase activity associated with the immobilized Fc
RII. In
contrast, direct ligation of Fc
RII led to Fgr tyrosine kinase
activation and association of a significant fraction of the active
kinase with the Triton-resistant adherent cell membrane. An association
between Fc
RII and Fgr in PMN has been reported
previously
(4) . In our assay system at least, stable association
of Fgr with the Triton X-100-insoluble cytoskeleton requires direct
ligation of Fc
RII. Even unactivated Fgr protein is not associated
with the Triton X-100-insoluble cytoskeleton in the absence of direct
Fc
RII ligation. Presumably for this reason, Fc
RII immobilized
by association with ligated CR3 is unable to signal assembly of the
NADPH oxidase. Because of the nature of the assay, we do not know if
the Fc
RII-Fgr association we detect is direct or indirect. The
presence of Fgr in the Triton X-100-insoluble cell residue after
Fc
RII ligation may depend on other Triton-insoluble PMN proteins.
It is also possible that active Fgr associates with phosphorylated
tyrosine residues in the Fc
RII cytoplasmic
tail
(4, 5, 7) and has no direct association
with cytoskeletal proteins.
RII. The anti-Fc
RII-generated
respiratory burst is extremely sensitive to tyrosine kinase inhibitors
( Fig. 6and Ref. 15). Since a major difference between direct
ligation of Fc
RII with antibody (which is associated with
H
O
production) and its indirect immobilization
through CR3 ligation (which leads to no respiratory burst) is the
presence of Triton X-100-insoluble kinase-active Fgr, it is likely that
Fgr is involved in initiation of the respiratory burst after direct
Fc
RII ligation. This pathway for generation of the respiratory
burst is sensitive to PT. However, the association of active Fgr with
Fc
RII after direct ligation is unaffected by PT, and the in
situ kinase assay reveals no different phosphorylated proteins in
pertussis-intoxicated cells. Thus, the PT sensitivity of the signal
transduction cascade must result from inhibition at a step beyond Fgr
association with receptor and its activation.
RII
is required for a respiratory burst even in pertussis-intoxicated PMN.
Although Fc
RII is phosphorylated on tyrosine during Fc
receptor-mediated PMN activation
(8, 15) , it is unlikely
that this is the only phosphorylation essential for cell activation.
Fc
RII phosphorylation has been separated from signal transduction
for phagocytosis in PMN
(8) . Furthermore, phosphorylation of
Fc
RII itself in response to its direct ligation is unaffected by
PT, even though pertussis intoxication inhibits the respiratory
burst.
(
)
These data suggest that there is
another, unidentified, protein associated with Fc
RII which is
needed to signal assembly of the NADPH oxidase. A possible candidate is
syk, a kinase known to be involved in immune complex-mediated PMN
activation and known to be phosphorylated by Fc
RII
ligation
(6, 40) . However, a direct physical association
of syk with Fc
RII or other Triton-insoluble proteins has not been
demonstrated.
RIIIB is associated with Hck kinase
activation and translocation to the Triton X-100-insoluble cell
fraction. The existence of Hck in PMN is well described
(41) ,
but its specific role has been unclear. This exclusive association of
Fc
RIIIB ligation with Hck activation is not a property of all
GPI-linked PMN proteins, since DAF ligation is associated predominantly
with Fgr rather than Hck. This may explain why DAF cannot substitute
for Fc
RIIIB in the synergistic respiratory burst. However, other
differences, such as the association of extracellular domains of
Fc
RIIIB with specific membrane proteins
(42) , may also
contribute to the lack of equivalence between DAF and Fc
RIIIB in
the synergistic respiratory burst. Nonetheless, in PMN, whether a
particular Src family kinase is activated and becomes Triton insoluble
by ligation of a specific GPI-linked protein depends on more than
simply the mechanism of membrane attachment of the receptor.
RIIIB in the
synergistic respiratory burst is to bring Hck into proximity with
Fc
RII, when the Fc
RII has no associated kinase or has had its
kinase activation pathway blocked by PT (Fig. 8). This appears to
be an essential early step in activation of the synergistic respiratory
burst. Hck activation of the downstream effector pathway for assembly
of the NADPH oxidase is not affected by PT, providing an alternative
pathway for cell activation. Since Fc
RII immobilization is
required for the synergistic respiratory burst in the absence of
associated Fgr, it is tempting to speculate that the downstream target
for both Hck in the synergistic respiratory burst and Fgr in direct
ligation of Fc
RII is the same protein physically associated with
Fc
RII. Activation of this target by either a PT-insensitive
Hck-dependent pathway or a PT-sensitive Fgr-dependent pathway could
then lead to a final common pathway for NADPH oxidase assembly.
Figure 8:
Hypothetical signal transduction pathways
for Fc receptor activation of the PMN respiratory burst. Ligation
of Fc
RII alone activates a pathway leading to assembly of the
NADPH oxidase which is PT-sensitive. The synergistic pathway can be
activated by coligation of Fc
RIIIB with CR3 and is pertussis
toxin-insensitive. The role for CR3 in the synergistic respiratory
burst is to immobilize Fc
RII in proximity to Fc
RIIIB, and its
associated Hck kinase, by a cytoskeleton-dependent mechanism.
Fc
RII immobilized on the adherent surface of PMN by direct
ligation is associated with the tyrosine kinase Fgr, whereas Fc
RII
immobilized by CR3 ligation is unassociated with a kinase. This
suggests that the two pathways for respiratory burst activation, both
of which require immobilized Fc
RII, may be initiated by different
Src family kinases.
In
summary, we have shown that in PMN ligated FcRII and Fc
RIIIB
are associated with distinct Src family kinases. Fc
RII leads to
Fgr activation and Fc
RIIIB to Hck activation. The Hck-dependent
pathway initiates what we have termed the synergistic respiratory
burst. In the absence of pertussis toxin, this pathway for PMN
activation apparently requires three different receptors: CR3,
Fc
RII, and Fc
RIIIB. It is possible that immune complexes
deposited in tissue, by ligating Fc
RII and Fc
RIIIB
simultaneously, could activate the synergistic pathway in the absence
of complement. Indeed, the PMN respiratory burst in response to
insoluble immune complexes in vitro is
PT-insensitive
(43, 44) , consistent with activation of
the synergistic pathway. Moreover, pathologic immune complexes activate
complement
(36) , and complement deposition onto immune complexes
actually decreases, rather than increases, the efficiency of IgG
interaction with Fc
receptors
(45) . For this reason and
because there are about 10-fold more Fc
RIIIB than Fc
RII on
PMN, the most physiologically relevant interaction of immune complexes
with PMN is likely to be via CR3 and Fc
RIIIB; direct interaction
with the less abundant Fc
RII will likely be minimized by
complement deposition. These are precisely the conditions which lead to
the synergistic respiratory burst. We believe that the Hck-dependent
synergistic pathway for PMN activation is likely to be extremely
relevant to immune complex activation of PMN in vivo.
RII, IgG Fc receptor, type 2;
Fc
RIII, IgG Fc receptor, type 3; CR3, complement receptor type 3;
DAF, decay accelerating factor, CD55; mAb, monoclonal antibody; PT,
pertussis toxin; Mes, morpholinoethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.