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Receptor Signaling and Amplify Phagocytic Capacity1
Department of Medicine, Hospital for Special Surgery and New York Presbyterian Hospital, Graduate Program in Immunology, Weill Medical College of Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R) mediate internalization
of opsonized particles by human neutrophils (PMN) and mononuclear
phagocytes. Cross-linking of FcR leads to activation of protein
tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based
activation motifs (ITAMs) within FcR subunits, both obligatory early
signals for phagocytosis. Human PMN constitutively express two
structurally distinct FcR, FcRIIa and FcRIIIb, and can be
induced to express FcRI by IFN-. We have previously shown that
stimulation of PMN through FcRIIIb results in enhanced
FcRIIa-mediated phagocytic activity that is inhibited by catalase.
In the present study, we have tested the hypothesis that reactive
oxygen intermediates (ROI) have the capacity to regulate FcR
responses and defined a mechanism for this effect. We show that
H2O2 augmented phagocytosis mediated by
FcRIIa and FcRI in PMN and amplified receptor-triggered tyrosine
phosphorylation of FcR-associated ITAMs and signaling elements.
Generation of endogenous oxidants in PMN by cross-linking FcRIIIb
similarly enhanced phosphorylation of FcRIIa and Syk, a tyrosine
kinase required for phagocytic function, in a catalase-sensitive
manner. Our results provide a mechanism for priming phagocytes for
enhanced responses to receptor-driven effects. ROI generated in an
inflammatory milieu may stimulate quiescent cells to rapidly increase
the magnitude of their effector function. Indeed, human monocytes
incubated in the presence of stimulated PMN showed oxidant-induced
increases in FcRIIa-mediated phagocytosis. Definition of the role of
oxidants as amplifiers of FcR signaling identifies a target for
therapeutic intervention in immune complex-mediated tissue
injury. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R)3 provide a crucial link
between cellular and humoral components of the immune cascade.
Characterizing their function is critical to understanding
IgG-triggered effector events. For phagocytes, engagement of FcR by
opsonized microbes or immune complexes stimulates phagocytosis, the
release of inflammatory mediators, and generation of reactive oxygen
intermediates (ROI). To initiate these responses, cross-linking of
FcR activates intracellular signaling events analogous to those
induced by engagement of T and B cell Ag receptors. FcR aggregation
leads to phosphorylation and subsequent activation of tyrosine kinases
of the Src and Syk families, which become associated with the
immunoreceptor tyrosine-based activation motifs (ITAMs) within FcR
subunits (1, 2). For FcRIIa, a single chain receptor, the ITAM is
contained within cytoplasmic domain (3, 4). For other FcRs, such as
FcRI, the ITAM is present in associated FcR subunits (2, 3).
Activated tyrosine kinases phosphorylate the ITAM, as well as
downstream substrates that lead to intracellular Ca2+
concentration ([Ca2+]i) transients,
cytoskeletal changes, and ultimately transcriptional activation (5, 6).
The importance of phosphotyrosine accumulation to FcR function is
underscored by observations that mutations of the tyrosines within the
ITAM domains alter phagocytic capacity and inhibitors of protein
tyrosine kinases block FcR-stimulated responses (7, 8, 9, 10). Further
evidence of the critical role of tyrosine phosphorylation is derived
from studies of macrophages deficient in Syk, which have defective
FcR-mediated phagocytosis and signal transduction, and of
macrophages deficient in Src family kinases, which show poor Syk
activation and delayed FcR-mediated phagocytosis (11).
Tyrosine phosphorylation is regulated in part by the competing
activities of protein tyrosine kinases and phosphatases. Indeed, the
magnitude of FcR signaling and effector function is determined by
the interplay between these kinases and phosphatases (2, 12). Increased
kinase activity induced by overexpression of Syk or by inhibition of
tyrosine phosphatases by vanadate has been shown to potentiate FcR
signal transduction and phagocyte responses (13, 14). Conversely,
coligation of FcR with the protein tyrosine phosphatase CD45 is
associated with diminished FcR-triggered calcium mobilization,
respiratory burst, and degranulation (12, 15, 16, 17). Therefore, one would
predict that mediators that alter this balance to favor kinase activity
would amplify FcR-triggered functions.
Recent observations suggest that ROI serve as intracellular signaling
molecules and promote increased tyrosine phosphorylation in a wide
range of cells, including PMN, T lymphocytes, platelets, vascular
smooth muscle cells, and cardiac myocytes (18, 19, 20, 21, 22, 23, 24). Although ROI were
once regarded simply as toxic agents implicated in antimicrobial
defense, there is now considerable evidence that they act as intra- and
extracellular messengers. ROI have been shown to inhibit the activity
of certain tyrosine phosphatases, including CD45, and to induce the
phosphorylation and activation of Src and Syk family kinases (19, 23, 25, 26). In contrast, the enzymatic activity of SHP-1, another
phosphatase essential to monocytes and PMN, is not sensitive to
modulation by oxidants (27). In as much as tyrosine phosphorylation is
essential to FcR-triggered responses, and since phagocytes are
prodigious producers of ROI, we hypothesized that ROI could play an
important role in the regulation of FcR responses at sites of
inflammation through autocrine and paracrine effects.
Human PMN constitutively express two types of FcR: FcRIIa, the
predominant phagocytic receptor, and FcRIIIb, a glycosyl
phosphatidylinositol-linked protein. FcRI is expressed on
mononuclear phagocytes and its expression can be induced in PMN by
IFN- (2, 3, 4). We have previously shown that stimulation of PMN
through FcRIIIb or PMA leads to enhanced FcRIIa-mediated
phagocytic activity, and that this enhancement is inhibited by catalase
(28). Based on these data, we proposed a model of oxidant-dependent
activation of FcRIIa and FcRI. In the present study, we examine
the direct effects of oxidants on FcR function and signaling
capacity in human phagocytes. We show that H2O2
augments internalization mediated by FcR in PMN and monocytes, and
that endogenously and exogenously generated ROI amplify
receptor-triggered tyrosine phosphorylation of FcR-associated ITAMs
and signaling elements.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-FcRII mAb IV.3 Fab, anti-FcRIIIb mAb 3G8
F(ab')2 and anti-FcRI mAb 22.2 F(ab')2
fragments were obtained from Medarex (Annandale, NJ). Purified human
IgG2
myeloma protein was obtained from The Binding Site (Birmingham,
U.K.). Affinity pure F(ab')2 fragments of goat
anti-mouse IgG (GAM) were obtained from Jackson ImmunoResearch
(Westgrove, PA). Anti-FcRII receptor blotting mAb (II1A.5) was a
generous gift from Dr. Jurgen Frey (Universitat Bielefeld, Bielefeld,
Germany) (29). Rabbit polyclonal Abs to Syk were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Polyclonal Abs to the FcR
subunit were a gift from Dr. Jean-Pierre Kinet (Harvard University,
Boston, MA). Antiphosphotyrosine mAb 4G10 and PY-20 were purchased
from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz
Biotechnology, respectively. HRP-linked sheep anti-mouse and donkey
anti-rabbit Abs as well as the ECL Western blotting detection
reagents were obtained from Amersham (Arlington Heights, IL). Protein
G-Sepharose beads were obtained from Pharmacia Biotech (Uppsala,
Sweden). Aminotriazole (AT), hydrogen peroxide, catalase, and PMA were
purchased from Sigma (St. Louis, MO).
Cells
Leukocytes were isolated from the venous blood of healthy volunteers by centrifugation on a discontinuous two-step Ficoll-Hypaque gradient with specific gravities of 1.078 and 1.119 g/ml (30). PMN were isolated from the lower interface and washed in HBSS. Contaminating erythrocytes were lysed. Cells were resuspended to 5 x 106/ml in RPMI 1640 + 10% FCS. Mixed mononuclear cells were isolated from the upper interface, washed, and resuspended as above. Adherent monocyte monolayers were prepared from mixed mononuclear cells as described previously (31). Human monocytic cell lines U937 and THP-1 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% FCS.
For induction of FcRI, PMN were cultured overnight in medium (RPMI
1640 with 5% FCS and 0.7 mM 2-ME) supplemented with 400 U/ml of
IFN- (Genzyme, Cambridge, MA). For induction of FcRI and FcR
subunits, U937 cells were cultured for 4 days in medium supplemented
with 400 U/ml of IFN- (32). For activation by oxidants, cells were
pretreated with AT (20 mM) for 30 min at room temperature followed by
incubation with H2O2 (500 µM) for 10 min at
room temperature.
Phagocytosis assay
FcR-specific probes were prepared as previously described
(33). Briefly, bovine erythrocytes (E), human IgG2 (hIgG2) and
anti-FcRI mAb 22.2 F(ab')2 were biotinylated.
Erythrocytes were saturated with streptavidin, washed, and coated with
biotinylated Abs. E-hIgG2 and E-22.2 were labeled with PKH26
lipophilic dye as described (34), and washed and resuspended in
RPMI 1640 + 10% FCS to a final concentration of 1 x
108 erythrocytes/ml.
The capacity of PMN to internalize the target particles was measured using a flow cytometric assay (34). PMN (100 µl at 5 x 106 cell/ml) were mixed with E-hIgG2 or E-22.2 (125 µl at 1 x 108 E/ml). The cell mixture was centrifuged at 44 x g for 3 min at room temperature, incubated for 10 min at 37°C in RPMI 1640 + 20% FCS, followed by hypotonic lysis to remove noninternalized erythrocytes. Cells were washed three times in 1% BSA-PBS to remove lysed erythrocyte fragments. Quantification of phagocytosis of PKH26-labeled E-hIgG2 or E-22.2 by flow cytometry was performed using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a standard optical filter set as described (34). PKH26 fluorescence was detected in the FL2 channel and displayed on a logarithmic scale. The mean fluorescence intensity (MFI) for PKH26-labeled erythrocytes was standardized in each experiment. Increasing multiples of this MFI correspond to phagocytes with increasing numbers of internalized erythrocytes, as described (34). The phagocytic index (PI) was calculated by multiplying the percentage of cells that internalized PKH26-labeled erythrocytes (%P) and the MFI of phagocytes with internalized erythrocytes (PI = %P x MFI/100).
To study the capacity of stimulated PMN to alter monocyte phagocytic
function a 12-well format cell culture insert with 0.4 µM pore size
(Becton Dickinson, Franklin Lakes, NJ) was used. E-hIgG2 (125 µl at
1 x 108 E/ml) were added to wells with adherent
monocytes (
1 x 105 cells/well), allowed to settle
for 2 min, followed by the addition of medium to fill the well. PMN
(1.5/ml at 5 x 106/ml) treated with or without PMA
(20 ng/ml) were added to the upper chamber. After incubation for 15 min
at 37°C, phagocytosis by adherent monocytes was measured by light
microscopy as previously described (31). In preliminary experiments, it
was determined that PMA alone had no effect on the internalization of
E-hIgG2 by adherent monocytes.
FcRIIA genotyping
Genomic DNA was isolated using the Puregene DNA isolation kit
(Gentra Systems, Minneapolis MN), and assays to determine the genotype
of FcRIIA were performed as previously described (35).
Immunoprecipitations
Cells were suspended in buffer containing 125 mM NaCl, 5 mM KCl,
8 mM Na2HPO4, 2 mM
NaH2PO4, 5 mM glucose (pH 7.35) and opsonized
with the appropriate anti-FcR mAb fragments (5 µg/ml) for 10
min at room temperature, then washed and resuspended in the same buffer
supplemented with 1.09 mM CaCl2 and 1.62 mM
MgCl2. FcR aggregation was initiated by cross-linking
the surface-bound mAb with goat anti-mouse F(ab')2 (30
µg/ml) at 30°C for varying times. Cells (1–2 x
107 cells/lane) were then washed in ice cold buffer,
pelleted, and solubilized in lysis buffer (1% Nonidet P-40, 1% Triton
X-100, 10% glycerol, 70 mM NaCl, 50 mM NaF, 16 mM
Na2HPO4, 4 mM NaH2PO4,
5 mM EDTA, 0.4 mM Na3VO4, 10 µg/ml each
aprotinin, leupeptin, soybean trypsin inhibitor, and pepstatin A, and
500 µg/ml pefabloc (pH 7.4)) for 1 h at 4°C. The lysates were
centrifuged at 16,000 x g for 10 min at 4°C and the
supernatants were immunoprecipitated using specific Abs (0.5–1 µg
purified Ab) and protein G-Sepharose beads (15 µl) for 16 h at
4°C. The immune complexes were washed four times in PBS plus 1%
Nonidet P-40, mixed with 2x Laemmli buffer plus 2-ME, heated for 5 min
at 95°C, and separated on 10% SDS-PAGE. The samples were
electrophoretically transferred to nitrocellulose membranes, blocked in
PBS supplemented with 5% BSA, and analyzed for phosphotyrosine content
with 4G10 mAb (0.5 µg/ml) and PY-20 (1 µg/ml) and for protein
content with 1 µg/ml of the appropriate Ab followed by
peroxidase-conjugated second Ab. Proteins were visualized using the
enhanced chemiluminiscent detection (Amersham) and Kodak X-Omat
radiographic film (Eastman Kodak, Rochester, NY).
Quantitation of radiograms was performed by densitometry (Molecular Dynamics, Sunnyvale, CA). All measurements were normalized by subtracting the background value for each blot. Readings for individual phosphorylated bands (detected by antiphosphotyrosine mAbs) and nonphosphorylated protein bands (detected by the Abs for the specific proteins) were obtained. For comparison of extent of tyrosine phosphorylation under different experimental conditions, the ratios of values for phosphorylated to nonphosphorylated bands were calculated and indicated on the figures.
Statistical analysis
Experiments were performed in a matched-triplet design for
assessment of the effects of oxidants on the phagocytic capacity of
individuals with each of the three different FcRIIA genotypes.
Accordingly, in each experiment PMN from donors of different genotypes
were studied simultaneously. The data are displayed as mean ±
SEM. The effects of oxidant treatment were compared with control
conditions using a paired t test (two-tailed). A probability
of 0.05 was used to reject the null hypothesis that there is no
difference between the conditions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa-mediated phagocytosis in PMN and monocytes
Activation of PMN by cross-linking of FcRIIIb or with PMA leads
to a 2- to 4-fold increase in FcRIIa-mediated phagocytic activity
(28). We have shown that priming of FcRIIa is transferable to
resting PMN by supernatants from stimulated cells and that both the
direct and supernatant-mediated effects are inhibited by catalase (28).
To provide independent proof that it is the generation of oxidants that
amplify FcRIIa function, we treated PMN with
H2O2, a membrane-permeable ROI released at
inflammatory sites. Phagocytosis mediated by FcRIIa was probed with
an FcRIIa-specific native ligand, hIgG2 coupled to erythrocytes
(36). Treatment of PMN with H2O2 (500 µM) in
the presence of AT (20 mM), an inhibitor of catalase that can be
released from endogenous PMN stores and which metabolizes
H2O2, resulted in a 221 ± 22% increase
in E-hIgG2 internalization (p < 0.0003,
n = 35). The enhancing effect of
H2O2 was maximal at 500 µM (Fig. 1
, inset). Treatment of
E-hIgG2 with H2O2 did not alter their
internalization, indicating that the oxidants are acting on the
phagocytes rather than on the target particles. The kinetics of
phagocytosis of E-hIgG2 by PMN in the presence
H2O2 and AT were similar to those in untreated
cells (Fig. 1). Phagocytosis was initiated within 1 min of adding
E-hIgG2 and was completed by 10 min. At 1 min, the oxidant-treated
PMN had a nearly 2-fold increase in internalization as compared
with control PMN (PMNH2O2 vs
PMNcontrol: 43 ± 3 vs 26 ± 1 E-hIgG2 internalized/100
PMN, p = 0.02). This relative difference was maintained
at the peak phagocytic index, which was reached at 10 min under both
conditions (PMNH2O2 vs PMNcontrol:
91 ± 11 vs 45 ± 5, p = 0.015, n =
10). Thus, oxidant treatment increases FcRIIa phagocytic capacity
(number of E-hIgG2 internalized/PMN) without altering kinetics.
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RIIa has two codominantly expressed alleles, R131 and H131, which
differ structurally at amino acid position 131 in the second
extracellular domain and differ in their ability to bind human IgG2
(H131>>R131) (36, 37). If ROI act distal to receptor binding and alter
intracellular signaling, then H2O2 should
affect the function of both allelic variants of FcRIIa. To test this
possibility, we compared the effect of H2O2 on
PMN from healthy donors homozygous for R131 or H131 genes and
heterozygotes. Treatment of PMN with H2O2 (500
µM) in the presence of AT resulted in an increase in phagocytosis for
R131 homozygotes (227 ± 39%; p < 0.04,
n = 12), R131/H131 heterozygotes (263 ± 53%;
p < 0.02, n = 11), and H131/H131
homozygotes (176 ± 17%; p < 0.02, n
= 12) (Fig. 2). Oxidant-induced
enhancement of phagocytosis was evident for PMN expressing all three
genotypes, but the relative differences in E-hIgG2 internalization
among the alleles persisted in oxidant-activated PMN. These data are
consistent with oxidant-driven changes in signaling capacity, but they
do not exclude alterations in binding.
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RIIa function in
monocytes was demonstrated using a two-chamber apparatus. Adherent
monocytes were incubated in the presence of PMA-activated PMN or
resting PMN, and FcRIIa-mediated phagocytosis was probed with
E-hIgG2. In monocytes incubated with activated PMN, phagocytosis was
increased by 153 ± 26% compared with monocytes cultured with
resting PMN (p < 0.03, n = 4).
The effect of exposure to activated PMN was inhibited by 85% in the
presence of catalase. Thus, oxidant-induced activation of
FcRIIa can occur in resting "bystander" mononuclear
phagocytes.
Exogenous and endogenously generated oxidants induce
hyperphosphorylation of the FcRIIa ITAM in PMN
It has been previously shown that FcRIIa ligation leads to
receptor tyrosine phosphorylation that provides an obligatory early
signal for phagocytosis (6, 38). We sought to determine whether
endogenous or exogenously generated oxidants affect this process. In
PMN, enhanced phosphorylation of FcRIIa was observed following
receptor triggering in the presence of H2O2
(500 µM) and AT, the same conditions used for the functional studies
(Fig. 3A, lane 2).
Cross-linking FcRIIIb on PMN, a known trigger of endogenous ROI
generation (39), resulted in an even greater increase in
receptor-initiated tyrosine phosphorylation of FcRIIa (Fig. 3B, lane 4). In contrast, 3G8 F(ab')2
alone had no effect on FcRIIa (lane 3). The
addition of oxidants in the absence of FcRIIa cross-linking
similarly did not result in receptor tyrosine phosphorylation (data not
shown).
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RIIIb-mediated amplification of FcRIIa phosphorylation, we
repeated the FcRIIIb cross-linking experiments in the presence of
catalase (10,000 U/ml), which rapidly degrades
H2O2. Under these conditions,
hyperphosphorylation of FcRIIa in PMN stimulated with 3G8
F(ab')2 and goat anti-mouse F(ab')2 was
clearly reduced (Fig. 3
B, lane 5). Thus, both
enhanced FcRIIa phosphorylation and the associated augmented
phagocytic capacity are inhibited by catalase (28), indicating a key
role for endogenously generated oxidants.
Oxidants enhance tyrosine phosphorylation of Syk following
FcRIIa stimulation in PMN
The protein tyrosine kinase Syk is essential for FcR-mediated
phagocytosis (11). Upon cross-linking FcRIIa, Syk associates
with the phosphorylated ITAM and is itself phosphorylated on
tyrosine, resulting in its activation (6, 15). Hyperexpression of Syk
enhances phagocytosis by FcRIIa in a transfection system, suggesting
that increased activity of Syk can amplify the magnitude of
FcRIIa-mediated effector function (8). We hypothesized that
enhancement of phagocytosis by ROI was associated with increased
FcRIIa-triggered phosphorylation of Syk. To determine whether
endogenously generated oxidants increase the tyrosine phosphorylation
of Syk, PMN were stimulated by cross-linking FcRIIIb.
FcRIIa-initiated phosphotyrosine accumulation on Syk was markedly
increased in the cells pretreated with 3G8 F(ab')2 and
goat anti-mouse F(ab')2 (Fig. 4A, lane 3). This
enhancement was blocked by catalase, demonstrating its oxidant
dependence (lane 4).
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RIIa-mediated phosphorylation of Syk, PMN were incubated under the
same conditions as those used in the studies of phagocytosis. In the
presence of 500 µM H2O2, FcRIIa clustering
resulted in amplified and accelerated Syk phosphotyrosine accumulation.
As shown in Fig. 4B, Syk was more rapidly and more intensely
phosphorylated, followed by earlier dephosphorylation in
oxidant-treated PMN. In the presence of AT and
H2O2 without receptor cross-linking, there was
no evidence of tyrosine phosphorylation Syk (data not shown). In
experiments with human mononuclear phagocyte derived THP-1 cells, we
obtained similar evidence for H2O2-induced
increases in phosphorylation of FcRIIa ITAM and Syk (Fig. 5). These
results demonstrate that oxidant-induced activation of FcRIIa is not
cell-type specific, and occurs in both mononuclear phagocytes and PMN.
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and Syk
following FcRI stimulation and augment FcRI-mediated phagocytosis
To see whether oxidants modify signal transduction by other FcR
family members, we studied FcRI, the high affinity receptor for IgG
expressed constitutively on mononuclear phagocytes
(2, 3, 4). Phagocytosis by FcRI requires
the presence of the ITAM-bearing FcR subunit, which is tyrosine
phosphorylated upon receptor cross-linking. Like FcRIIa, stimulation
through FcRI leads to tyrosine phosphorylation and activation of Syk
(15, 32, 40, 41). To determine whether oxidants amplify this tyrosine
phosphorylation triggered by FcRI, we studied U937 monocytic cells
cultured with IFN- to increase FcRI expression. Consistent with
the findings of Durden and coworkers (40, 42), we observed that
nonphosphorylated Syk is constitutively associated with FcRI in
resting IFN--treated U937 cells (Fig. 6, lane 1). After
cross-linking FcRI with anti-FcRI mAb 22.2
F(ab')2 and goat anti-mouse F(ab')2, FcR
and FcR-associated Syk undergo tyrosine phosphorylation (lane
2). In U937 cells pretreated with AT followed by
H2O2 (500 µM) this process was markedly
amplified (lane 3). A similar oxidant-induced increase in
Syk phosphorylation was evident in lysates from U937 cells
immunoprecipated with anti-Syk Ab and immunoblotted with
anti-PY Abs.
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RI signaling in PMN, cells
were cultured with IFN- (400 U/ml) overnight. Under these conditions
there was a consistent and marked increase in FcRI expression
documented by staining with mAb 22.2 (43). In contrast, FcRIIIb
expression decreased, preventing experiments to examine the effects of
FcRIIIb-initiated ROI generation on FcRI function. We, therefore,
studied the effects of exogenous oxidants on FcRI signaling in PMN.
In the presence of H2O2 (500 µM), FcRI
cross-linking resulted in accelerated and markedly increased tyrosine
phosphorylation of FcR (Fig. 7A). Under these conditions,
FcR phosphorylation was augmented at 15 s, peaked at 30 s,
and was almost completely reversed by 1 min, whereas in PMN triggered
by FcRI without exogenous oxidants there was a much smaller and
slower increase in tyrosine phosphorylation.
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RI triggered signaling, we
examined the possibility that phagocytosis mediated by FcRI might
also be modulated by ROI. We selected PMN for these experiments because
of their robust phagocytic response compared with U937 cells. PMN
cultured with IFN- were treated with AT and
H2O2 (500 µM) or control medium and
FcRI-mediated phagocytic function was probed with erythrocytes
coupled to anti-FcRI 22.2 F(ab')2. Treatment with
oxidants increased phagocytic capacity by 157 ± 17%
(n = 7, p = 0.006) (Fig. 7B). These results indicate that ROI provide a common
mechanism to enhance both FcRI and FcRIIa signaling and amplify
effector function. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa and FcRI, and that
exogenously or endogenously generated oxidants amplify tyrosine
phosphorylation of FcR-associated ITAMs and signaling elements,
defining a mechanism for such enhanced function. ROI secreted by PMN
within an inflammatory milieu may thus stimulate quiescent cells to
rapidly increase the magnitude of their effector functions. Our
experiments showing that oxidants derived from activated PMN augment
"bystander" monocyte FcRIIa function underscore this point.
Several other lines of evidence support a role for oxidants as
modulators of FcR function. We have previously reported that in PMN
FcRIIa-mediated phagocytic function is augmented following
cross-linking of FcRIIIb, a receptor capable of production of ROI,
and that this enhancement is blocked in the presence of catalase (28).
ROI have similarly been implicated in the amplification of PMN FcR
phagocytic function by PMA (44). Patients with chronic granulomatous
disease (CGD), characterized by genetic defects in the NADPH oxidase
system that result in markedly diminished generation of ROI, have
served as clinical paradigms that establish the importance of oxidants
in phagocyte function (45). PMN from such patients show impaired PMA
and cytokine-dependent amplification of FcR-mediated internalization
(44), emphasizing the possibility that increased FcR responsiveness
is mediated by products of the respiratory burst. However, the
mechanism for these effects is not understood and the relative
sensitivity of the FcR isoforms to oxidant-induced modulation is
unknown.
The importance of tyrosine phosphorylation in FcR-mediated
internalization, taken together with observations that oxidants
influence phosphotyrosine accumulation, led us to systematically
examine the influence of endogenously generated and exogenously added
oxidants on the proximal events of FcRIIa signaling and on the
essential catalytic molecule that is recruited to its cytoplasmic
domain. Our results show that in the presence of
H2O2 there is increased phosphorylation of the
ITAM of FcRIIa following receptor triggering in both PMN and
mononuclear phagocytes. This phosphorylated ITAM functions as a
scaffold to recruit and organize effector molecules. Endogenous
generation of ROI initiated by FcRIIIb triggering induced an even
greater enhancement of FcRIIa phosphorylation in PMN, which may be
related to higher and more sustained intracellular oxidant levels.
Indeed, catalase inhibition of such hyperphosphorylation confirms its
oxidant-dependence. Our results are at variance with those recently
reported by Green et al. (46), showing that tyrosine phosphorylation of
cross-linked FcRIIa was diminished when co-cross-linked with
FcRIIIb in Jurkat T cells transfected with human receptors. While
their findings are not directly comparable with ours, obtained in fresh
human PMN which are efficient sources of ROI, Green et al. argue for
multiple mechanisms of cooperation between FcRIIa and FcRIIIb.
Although the ITAM motifs in FcRIIa and FcRI-associated FcR
subunit differ, phosphorylation of the FcR ITAM is similarly
amplified following receptor cross-linking in the presence of
H2O2, consistent with our observations of
rapidly increased FcRI-mediated phagocytic capacity under identical
conditions. These results suggest that ROI provide a common mechanism
to enhance FcR signaling and thereby amplify effector functions.
The initial step, ITAM phosphorylation, leads to the recruitment,
phosphorylation and activation of Syk, which then phosphorylates
downstream signaling targets. We focused on Syk because it has been
found to be a required element in FcR-mediated phagocytosis in
studies of macrophages from Syk-deficient mice (11). Transfected cells
expressing an FcRIII-Syk chimera can internalize particles that
cross-link FcRIII, indicating that Syk kinase is sufficient for
initiating cytoskeletal coupling and phagocytosis (47). Moreover,
alterations in Syk expression modify efficiency of phagocytosis (8, 38). Our data show that ROI increase the rate and magnitude of
FcRIIa-triggered phosphorylation of this critical kinase in PMN.
Inhibition of FcRIIIb-stimulated Syk hyperphosphorylation in PMN in
the presence of catalase emphasizes the importance of oxidant
generation as a means for synergism of FcRIIa and FcRIIIb. That
H2O2 enhanced Syk phosphorylation triggered by
FcRIIa was confirmed in mononuclear phagocytes. Thus, we propose
increased phosphorylation of this critical signaling element to be a
common mechanism to increase phagocytic efficiency. Previous workers,
using different conditions, have shown that ROI activate Syk, but they
did not consider the effects of oxidants on receptor-initiated
signaling. Brumell et al. (19) demonstrated phosphorylation and Syk
kinase activation in PMN which were electropermeabilized, vanadate
treated, and stimulated with GTP-S to activate NADPH oxidase.
Shieven et al. (23) showed Syk activation in lymphocytes exposed to 10
mM H2O2, a 20-fold higher concentration than
used in our experiments. Exposure of phagocytes to 500 µM
H2O2, in the absence of FcR cross-linking,
did not induce tyrosine phosphorylation of Syk. Our studies establish
the precedent that ROI, at nontoxic concentrations, modify
receptor-triggered signaling pathways in viable human phagocytes and
alter receptor-mediated function.
The means by which ROI increase tyrosine phosphorylation is not clear.
It has been suggested that endogenous or exogenous oxidants can promote
tyrosine phosphorylation by combined activation of kinases and
inhibition of phosphatases (18, 19, 26, 48). Tyrosine phosphatases may
be inactivated by oxidants that target critical cysteine residues in
their catalytic domains (49, 50, 51). As a consequence, constitutive
autophosphorylation and stimulation of kinases, which is no longer
offset by phosphatase activity, results in accumulation of
phosphotyrosine. Indeed, CD45, a known inhibitor of FcRIIa signaling
in PMN, is susceptible to inactivation by oxidants (26, 52). That CGD
PMN have diminished inhibition of CD45 tyrosine phosphatase activity in
response to activation of NADPH oxidase and show impaired PMA-induced
amplification of FcR function provides indirect support for this
mechanism of oxidant-induced modulation of FcR signaling (26, 44).
The accelerated dephosphorylation of Syk in PMN exposed to
H2O2 indicates that not all protein tyrosine
phosphatase activity is blocked by oxidants.
Our observations provide the basis for a better understanding of the
regulation of FcR function at sites of inflammation. Perhaps more
importantly, our data indicate a mechanism for priming phagocytes for
enhanced responses to receptor-driven effects. ROI generated in an
inflammatory milieu act in an autocrine and paracrine manner to rapidly
amplify the effector potential of FcR on phagocytes by altering the
signal transduction. Indeed, for FcRIIa, exposure to oxidants
enables uptake of an IgG2-opsonized particle by FcRIIa-R131
homozygotes, albeit to a lesser extent than that of other FcRIIa
genotypes, and thus allows removal of IgG2-opsonized microbes and
immune complexes despite relatively low binding capacity. Hence for
antimicrobial defense, ROI-initiated increases in phagocytosis are
protective. The importance of the interplay between ROI and FcR in
host defense is underscored by the recent report that the risk for
immune-mediated complications of CGD is associated with FcR allelic
polymorphisms (53).
In contrast, at sites of immune complex deposition, such as the kidney
in systemic lupus erythematosus, amplification of FcRI- or
FcRIIa-triggered release of inflammatory mediators may directly
promote tissue injury. Indeed, in the absence of PMN influx, renal
injury is attenuated in murine models of autoimmune glomerulonephritis
(54). Alternatively, FcR-driven phagocyte-derived ROI may act as
second messengers to increase platelet aggregation, vascular smooth
muscle cell proliferation, and mesangial cell proliferation (20, 21, 55, 56), all characteristic findings in diffuse proliferative
glomerulonephritis. Our data showing that oxidants from activated PMN
amplify monocyte FcR function support this paracrine mechanism.
These findings, along with recent evidence that FcR-deficient mice
are protected from autoimmune glomerulonephritis (57, 58), underscore
the importance of identifying the factors which modulate the efficiency
of FcR function. Definition of the role of oxidants as amplifiers of
FcR signaling provides a novel target for therapeutic intervention
in immune complex-mediated tissue injury.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jane E. Salmon, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021; E-mail address:
3 Abbreviations used in this paper: FcR, receptors for Fc portion of IgG in human cells; ROI, reactive oxygen intermediates; ITAM, immunoreceptor tyrosine-based activation motif; PMN, neutrophils; AT, aminotriazole; CGD, chronic granulomatous disease; E, bovine erythrocytes; E-hIgG2, E coated with human IgG2; FcRI, 72-kDa high affinity receptor for Fc portion of IgG; FcRIIa, 40-kDa receptor for Fc portion of IgG; H131, allelic variant of FcRIIa that binds human IgG2 (histidine at amino acid 131); FcR, subunit of FcR; PI, phagocytic index; PY, phosphotyrosine; R131, allelic variant of FcRIIa that does not bind human IgG2 (arginine at amino acid 131).
Received for publication November 10, 1998. Accepted for publication March 29, 1999.
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) for 10 min at room temperature and then combined with E-hIgG2.
The phagocytic index was measured at the indicated times by flow
cytometry as described in Materials and Methods
(n = 4–10 experiments for each time point).
Inset, PMN treated with AT and
H2O2 at concentrations ranging from 10 µM to
2 mM for 10 min were incubated with E-hIgG2 for 10 min. Phagocytic
index was determined by flow cytometry. Data are expressed as percent
control PI (PIH2O2 -
PIcontrol/PIcontrol). Values represent
mean ± SEM of five experiments.
) or AT (20 mM) and H2O2 (500
µM) (
) and incubated with E-hIgG2 for 10 min. The phagocytic index
was determined by flow cytometric analysis. Data represent mean ±
SEM of 12 experiments. Control vs H2O2:
*, p = 0.04; **, p =
0.02.
