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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 383-391
Physical and Functional Association of Fc R With Protein Tyrosine
Kinase Lyn
By
Heinz Gulle,
Aysen Samstag,
Martha M. Eibl, and
Hermann M. Wolf
From the Institute of Immunology, University of Vienna, Vienna,
Austria; and Immuno AG, Vienna, Austria.
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ABSTRACT |
In this report, we show that the Src family nonreceptor protein
tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor
(Fc R) in the monocytic cell line THP-1. Receptor aggregation and
subsequent immunoprecipitation of receptor complexes with huIgA
adsorbed to nitrocellulose particles shows that Lyn associates with
FcR by a mechanism sensitive to short treatment with the Src
family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc
receptor (Fc R) in THP-1 cells was unaffected by short treatment with
the PTK inhibitor. Cross-linking of FcR induced tyrosine
phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK
activity. Unexpectedly, in vitro kinase assays showed that FcR
aggregation-induced tyrosine phosphorylation of Syk did not result in
upregulation of Syk activity. Despite the lack of enhanced Syk kinase
activity, downstream signaling after FcR cross-linking was
functional and induced the release of significant amounts of
interleukin-1 receptor antagonist and interleukin-8. The induction of
cytokine release was completely blocked by PP1, thus confirming the
biological significance of the association of Lyn with aggregated
FcR. Our data show that early signal transduction after FcR
cross-linking as well as FcR-mediated activation of cellular
effector functions depends on Src family kinase activity. The
Src-family PTK involved in FcR-mediated tyrosine phosphorylation
appears to be Lyn, which coprecipitated with aggregated FcR
complexes.
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INTRODUCTION |
IgS OF THE IgA ISOTYPE prevail over other
Ig isotypes in the mucosal compartment, where they play a critical role
in protection against environmental challenges. In addition to
functions dependent on specific antibody activity, such as
neutralization of bacterial toxins and inhibition of attachment of
pathogenic microorganisms to the mucosal epithelium, IgA appears to be
a regulatory molecule capable of modulating immune and inflammatory
responses. The immunoregulatory functions of IgA seem to be confined to
interaction of IgA with receptors for the Fc portion of IgA expressed
on the surface of cells of the immune system, such as monocytes,
macrophages, neutrophils, or T cells, leading to upregulation or
downregulation of inflammation or immunologic reactivity under certain
conditions.1-3 CD89, the FcR of phagocytic cells, has
been defined as a 55-to 75-kD glycoprotein on monocytes/macrophages and
neutrophils, whereas a more heavily glycosylated form is expressed on
eosinophils.4,5 The cDNA sequence of FcR predicts a
transmembrane protein with two Ig-like extracellular domains, a
19-amino acid membrane spanning region, and a 41-amino acid cytoplasmic
tail.6 Genetic characterization of the human gene encoding
CD89 indicates that FcR represents a distantly related member of the
Ig receptor gene family.7
Transmembrane signal transduction induced by aggregation of receptors
specific for the Fc portion of IgG or IgE (Fc RI) has been
extensively studied over the past several years. Like multisubunit antigen receptors, such as B-cell and T-cell receptor, FcRI and FcR lack intrinsic kinase activity and, therefore, depend on the
association with nonreceptor protein tyrosine kinases (PTKs). Two
families of PTKs have been shown to interact with these receptors, and
a sequential activation pathway model was proposed
recently.8,9 Current evidence suggests that Src family
kinases, eg, Lyn, are activated in the initial events after FcR or
FcR cross-linking. Lyn has been described to be associated with
FcRI in mast cells and FcRs I and II on the surface of monocytic
cell lines, and cross-linking of these receptors upregulates, more or
less, the activity of the kinase.9-14 Interestingly,
binding of Lyn to some receptors seems to occur constitutively in the
absence of a stimulatory signal, underlining its potential as
first-step kinase.9,13,15 Activation of Lyn is followed by
tyrosine phosphorylation of conserved sequences present on the signal
transducing subunits of the Fc receptors.16 Phosphorylation
of these immunoreceptor tyrosine-based activation motifs (ITAMs) is
neccessary for binding of the PTK Syk, a member of the second family of
tyrosine kinases, which leads to further downstream signaling events,
and ultimately results in cellular effector functions.17,18
The importance of Syk in FcRI- and FcR-mediated signal
transduction is reflected by its presence in aggregated FcR complexes on various myloid cell lineages.9,19-21 Association of Syk
with receptor subunits occurs via its two Src homology 2 domains (SH2), which contribute cooperatively to the high-affinity binding, and depends on tyrosine phosphorylation of ITAM sequences.10,22 Direct interaction of Syk with further downstream molecules involved in
the moblization of intracellular calcium and the activation of Ras has
been described, suggesting a role in at least two signal transduction
cascades.17,23-25 Additionally, cellular effector functions, such as FcRI-mediated degranulation of mast cells and
FcR-mediated phagocytosis, depend on the presence of functional Syk
kinase.26,27
As compared with the Fc receptors for IgG or IgE, much less is known
about the events involved in FcR-mediated signal transduction. Cross-linking of FcR results in immediate tyrosine phosphorylation of celluar proteins, although known signaling motifs, such as the ITAM
domain, are missing from the cytoplasmic tail of its ligand-binding
-chain.6,7,28 Recently, association with a specialized
signaling subunit, FcR -chain, has been described in U937 monocytic
cells and this interaction was necessary for functional transmembrane
signal transduction in a transfected B-cell line.28,29
However, little is known about the PTKs responsible for FcR-mediated
tyrosine phosphorylation of cellular proteins. A preliminary report
described interaction of Syk with this receptor in U937 monocytic
cells, but the Src family PTKs involved in FcR signaling have not
been identified so far.30
We describe here PTKs engaged in transmembrane signaling of FcR in
the monocytic cell line THP-1. To investigate receptor-associated molecules, we cross-linked and subsequently immunoprecipitated FcR
complexes with the natural ligand, huIgA, adsorbed to nitrocellulose (NC) particles. Furthermore, these particles were used to study the
effect of FcR cross-linking on downstream cellular functions, such
as the release of cytokines. Our results show that, comparable to other
FcRs, aggregation of FcR recruits PTK Lyn. However, the mechanism of
kinase/receptor interaction appears to be different for FcR and
FcR.
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MATERIALS AND METHODS |
Cells.
The THP-1 monocytic cell line was obtained from the American Type
Culture Collection (ATCC; Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum
(FCS; Hyclone Lab, Logan, UK), 2 mmol/L L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (GIBCO BRL, Paisley, UK;
complete medium). Cell density was maintained between 5 × 105 and 2 × 106/mL.
Reagents and antibodies.
Highly purified human serum IgA (huIgA) and IgG (huIgG) preparations
were kindly provided by Dr Y. Linnau (Immuno AG, Vienna, Austria).31 The 4-hydroxy-3-nitrophenacetyl-specific
chimaeric huIgA antibody, clone JW393A, was purchased from Serotec
(Kidlington, UK). Anti-FcR (anti-CD89) monoclonal antibody (MoAb),
clone My43 (IgM isotype), was obtained from Medarex, Inc (Annandale,
NJ), and anti-CD14 MoAb, clone Mo2 (IgM isotype), was obtained from Coulter-Immunotech (Instrumentation Laboratory, Vienna, Austria). Polyclonal rabbit antibodies specific for Lyn, Syk, and Hck were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Antiphosphotyrosine MoAb, clone 4G10, was obtained from Upstate Biotechnology Inc (Lake Placid, NY), and rabbit anti-huIgA
F(ab )2 fragments (F2RahuIgA) was obtained
from Chemicon International, Inc (Temecula, CA). Rabbit polyclonal IgG
was purchased from Sera-Lab Limited (Crawley Down, UK). Horseradish
peroxidase-labeled sheep antimouse and donkey antirabbit antibodies,
[-32P]ATP (specific activity, 1.11 TBq/mmol), and the
enhanced chemoluminescence assay (ECL) were from Amersham (Little
Chalfont, UK). Dimethylsulfoxide was obtained from Sigma-Aldrich
Handels Ges.m.b.H. (Vienna, Austria), and Staph aureus
(Pansorbin cells) were from Calbiochem-Novabiochem Corp (La Jolla, CA).
Bovine serum albumine fraction V (BSA) was from Serva (Heidelberg,
Germany).
Preparation of Ig-adsorbed nitrocellulose particles.
NC particles were prepared and coated with human Ig preparations as
described.14 Briefly, NC sheets (Schleicher and Schuell, Dassel, Germany) were dissolved in dimethylsulfoxide and particles were
precipitated by dropwise addition of 1.5 vol of double-destilled H2O with constant mixing on a shaker. The particles were
washed three times with double-destilled H2O and the
resuspended pellet was transferred to Petridishes and dried at 50°C
on an Eppendorf Thermomixer (Engelsdorf, Germany). Prewetted NC
particles (2.5 mg; by weight, 5 mg correspond to 1 cm2 of
unprocessed NC sheets) were incubated for 2 hours at 4°C with 2 mg/mL of huIgA or huIgG or with 0.2 mg/mL chimaeric huIgA in phosphate-buffered saline (PBS) with constant shaking at 1.4 × 1,000 rpm. The NC was pelleted at 14,000g for 10 seconds and
free protein binding sites were blocked for 30 minutes at 37°C with PBS containing 5% BSA and 0.05% sodium azide on a Thermomixer. NC
particles were then washed three times with RPMI 1640 medium without
FCS and directly used for cell activation and subsequent immunoprecipitation of aggregated FcRs.
Cell stimulation and isolation of whole lysate proteins.
THP-1 cells were washed and resuspended in RPMI 1640 medium without FCS
to a density of 1 × 107/mL. Aliquots of 5 × 105 cells were transferred into 1.5-mL reaction tubes,
incubated for 30 minutes at 37°C, and stimulated with antibodies
for 5 minutes at 37°C. NC particles adsorbed with human Ig
preparations were used at a concentration of 1 mg of coated particles
per tube (dry weight). FcR-specific MoAb My43 (cell culture
supernatant) was added 1:2 to the cell suspensions, and the anti-CD14
MoAb Mo2 was used at a concentration of 5 µg/mL. The activation
reaction was stopped by addition of 500 µL of ice-cold PBS containing
1 mmol/L sodium orthovanadate (Na3VO4),
followed by brief centrifugation and immediate lysis of cells for 20 minutes on ice in Nonidet P-40 lysis buffer (20 mmol/L Tris/HCl, pH
7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium azide, 1 mmol/L
Na3VO4, 1 mmol/L sodium molybdate,
Na2MoO4, 2 mmol/L phenylmethanesulfonyl
fluoride [PMSF], 5 mmol/L EDTA, and 10 µg/mL of proteinase
inhibitors aprotinin and leupeptin). Nuclei and cellular debris were
removed by centrifugation for 10 minutes at 14,000g and soluble
proteins were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on 8% polyacrylamide gels under reducing
conditions.
Immunoprecipitation and in vitro kinase assays.
THP-1 cells were washed and resuspended in RPMI 1640 medium to a
density of 1.5 × 108/mL. Aliquots of 7.5 × 106 or 1 × 107 cells were transferred
into 1.5-mL reaction tubes and cells were activated for 5 minutes with
human Ig-adsorbed NC particles (concentrations of NC particles are
given in the respective figure legends) as described above. Cell
activation was stopped by addition of 0.9 mL of Brij 96 (polyoxyethylene 10 oleyl ether) lysis buffer (20 mmol/L Tris/HCl, pH
7.5, 150 mmol/L NaCl, 1% Brij 96, 0.1% sodium azide, 1 mmol/L
Na3VO4, 1 mmol/L
Na2MoO4, 2 mmol/L PMSF, 1 mmol/L MgCl2, and 10 µg/mL of proteinase inhibitors aprotinin
and leupeptin) and 20 U/mL of Dnase I (Boehringer Mannheim GmbH,
Mannheim, Germany). The lysed cells were incubated for 20 minutes on
ice to precipitate the NC particles. Supernatants were discarded, and
the particles were afterwards washed five times with Brij 96 lysis
buffer and precipitated on ice as described. Subsequently,
immunocomplexes were eluted with sample buffer and separated by
SDS-PAGE on 8% to 12% polyacrylamide gradient gels under nonreducing
conditions.
Syk kinase was precipitated from cell lysates of 1.5 × 107 THP-1 cells, which have been stimulated for 5 minutes
with My43 or Mo2 as described and lysed in Nonidet P-40 lysis buffer
without EDTA. To reduce unspecific binding of proteins to Syk-specific antibody, lysates were incubated on ice for 1 hour with 5 µg/mL of
rabbit polyclonal IgG followed by two successive treatments for 30 minutes at 4°C with 50 µL of a 10% Staph aureus slurry. Lysates were then incubated on ice for 1 hour with 5 µg/mL anti-Syk rabbit polyclonal antibody or the same amount of control rabbit IgG.
Antibody-bound molecules were precipitated by addition of Staph
aureus cells, and immunocomplexes were washed three times with
lysis buffer and once with 25 mmol/L HEPES, pH 7.2, containing 150 mmol/L NaCl, 0.1% Nonidet P-40, and 5 mmol/L MnCl2.
Pelleted Staph aureus cells were resuspended in 90 µL kinase
buffer (25 mmol/L HEPES, pH 7.2, 5 mmol/L MnCl2) and one
third of the slurry was used for in vitro kinase assay. The rest of
each sample was pelleted and bound proteins were eluted with reducing
sample buffer followed by separation on 8% to 12% polyacrylamide
gradient gels. To determine the PTK activity of Syk, immunocomplexes
were incubated for 15 minutes at 30°C in the presence of 10 µCi
[-32P] ATP. Bound proteins were then eluted with
reducing sample buffer and resolved on 8% to 12% gradient gels. Syk
kinase activity was visualized by autoradiography of dried gels.
Immunoblot analysis.
Electrophoretically separated proteins were transferred onto NC sheets
and the transfer efficiency was examined by staining with 0.5% Ponceau
S. Free protein binding sites were blocked by incubation of immunoblots
in Tris-buffered saline, pH 7.5, containing 0.1% Tween-20 and 1%
nonfat dry milk (Bio-Rad Lab, Hercules, CA). Tyrosine phosphorylation
of cellular proteins was probed with MoAb 4G10 followed by HRP-labeled
sheep antimouse antibody. After removal of excess antibody by washing
with TBST, specific binding was visualized by ECL. For reprobing of
immunoblots, phosphotyrosine-specific antibody was removed by treatment
with 100 mmol/L glycine/HCl, pH 2.5 (3 times for 20 minutes), on a
shaker. Reprobing with rabbit polyclonal antibodies specific for Syk,
Lyn, or Hck was performed after blocking of glycine-treated immunoblots
with TBST containing 1% nonfat dry milk. Specific binding was detected
by ECL.
Induction of cytokine release.
THP-1 cells were cultured for 40 hours in complete medium in 6-well
tissue culture plates (Macro Tray 635 TC; Greiner und Söhne,
Kremsmünster, Austria; 3 × 106 cells in a total
volume of 2 mL per well) in the presence of calcitriol
(1,25-dihydroxyvitamin D3; BIOMOL Research Lab, Plymouth Meeting, PA;
final concentration, 80 nmol/L) and recombinant human interferon-
(IFN-; Genzyme, Cambridge, MA; final concentration, 100 U/mL). The
cells were then washed and further incubated (3 × 106
cells/mL/well) in 24-well tissue culture plates (Becton Dickinson Labware, Lincoln Park, NJ) for 24 hours in the presence of complete medium alone or medium containing soluble monomeric huIgA (at a
concentration of 100 µg/mL, if not indicated otherwise) or huIgA adsorbed onto nitrocellulose particles (IgA-NC, where the amount of
huIgA-particles added to the cultures corresponded to an huIgA concentration of 100 µg/mL, if not indicated otherwise). As a control, BSA-adsorbed NC-particles were added in the same dilution to
parallel cultures. Cells were cultured in complete medium alone or in
complete medium containing polymyxin B (PMB; Sigma-Aldrich Handels
Ges.m.b.H., Vienna, Austria; final concentration, 10 µg/mL) or PP1
(Calbiochem-Novabiochem Corp, La Jolla, CA); if not indicated otherwise, PP1 was added at a concentration of 100 µmol/L. After 24 hours of incubation in a CO2 incubator at 37°C in
humidified air, cell culture supernatants were aspirated and
centrifuged for 3 minutes at 9,000g, and cytokine release was
examined using commercially available enzyme-linked immunosorbent assay
(ELISA) kits for tumor necrosis factor- (TNF-), interleukin-1
(IL-1), and IL-8 (IL-1-EASIA, TNF-alpha-EASIA, and IL-8-EASIA;
Medgenix Diagnostics, Fleurus, Belgium) or IL-1ra (Quantikine Human
Interleukin 1 Receptor Antagonist Immunoassay; R&D Systems,
Minneapolis, MN). Results are expressed as nanograms per milliliter or
as a percentage of control relative to the cytokine release observed in
THP-1 cells stimulated in the absence of PTK inhibitor.
Statistical analysis.
Results of the determination of cytokine release are expressed as the
mean ± SEM of repeated experiments performed on different days.
Statistical evaluation of the observed huIgA-mediated induction of
cytokine release by calculating the differences between more then two
study groups was performed with the nonparametric Kruskal-Wallis one-way ANOVA by ranks or the Newman-Keuls multiple comparisons test.
For statistical evaluation of the difference between two study groups,
the nonparametric Mann-Whitney U-test or the Student's t-test
for paired samples were used, as appropriate. P < .05 was considered a statistically significant difference.
| |
RESULTS |
Cross-linking of FcR induces tyrosine phosphorylation of cellular
proteins in THP-1 cells.
To study transmembrane signaling after FcR triggering, we
cross-linked the receptor with the MoAb My43, or huIgA adsorbed to NC
particles, on the surface of the human monocytic cell line THP-1. Both
stimuli induced tyrosine phosphorylation of an identical set of
cellular proteins, with the exception of an unidentified 115-kD
protein, which was phosphorylated exclusively by cross-linking of
FcR with NC particles adsorbed with the natural ligand, huIgA (Fig 1A, lanes 2 and 6 through 10). In
contrast, treatment of cells with the CD14-specific MoAb Mo2, with
monomeric huIgA, or with NC particles adsorbed with BSA failed to
induce tyrosine phosphorylation, indicating that induction of signaling
was specific for FcR ligation and that signal transduction required
FcR cross-linking (Fig 1A, lanes 1, 3, and 11). Unexpectedly,
cross-linking of monomeric huIgA with F(ab)2
fragments of a specific antibody failed to induce tyrosine
phosphorylation of cellular proteins, suggesting the need for extensive
FcR aggregation by multivalent ligand (Fig 1A, lane 4).
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| Fig 1.
Activation of THP-1 cells with MoAb My43 and huIgA
induces tyrosine phosphorylation of cellular proteins. THP-1 cells (5 × 105/sample) were activated at 37°C for 5 minutes by
the addition of 5 µg/mL Mo2 (anti-CD14, lane 1), My43 cell culture
supernatant 1:2 (anti-CD89, lane 2), 100 µg/mL huIgA (lane 3), 100 µg/mL huIgA + 100 µg/mL F2RAhuIgA (lane 4), 100 µg/mL
F2RAhuIgA (lane 5), or NC particles (1 mg/sample) incubated in
PBS containing different concentrations of huIgA, 3 mg/mL (lane 6), 1 mg/mL (lane 7), 0.33 mg/mL (lane 8), 0.11 mg/mL (lane 9), and 0.037 mg/mL (lane 10), and NC particles incubated in PBS containing 5% BSA
only (lanes 11 and 12). Cellular proteins were isolated, separated by
SDS-PAGE, and transferred to NC sheets as described in the experimental section. Immunoblotting of tyrosine-phosphorylated proteins was performed with the MoAb 4G10 followed by development with ECL as
described (A). The immunoblot was then stripped and reprobed with a
rabbit polyclonal anti-Syk antibody (lanes 1 through 11 in [B]) or
control rabbit polyclonal IgG (lane 12 in [B]).
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The PTK Syk is a major target in FcR-mediated tyrosine
phosphorylation.
Among the targets that became phosphorylated after cross-linking of
FcR was a predominant protein band of 75 kD. Because involvement of
Syk, a PTK of similar molecular mass, has been shown for various FcRs,
we wanted to clarify whether the 75-kD band corresponded to
p72syk. As shown in Fig 1B, rabbit polyclonal IgG specific
for human Syk recognized a single protein band of identical molecular
mass. To further characterize the effect of FcR-mediated signaling on this PTK, we immunoprecipitated Syk kinase from resting and stimulated THP-1 cells and performed anti-PY immunoblot analysis and in
vitro kinase assay. FcR cross-linking with MoAb My43 induced extensive tyrosine phosphorylation of Syk protein (6-fold increase in
phosphotyrosine; Fig 2A, lane 3), whereas
treatment of cells with the MoAb Mo2 (also of the IgM isotype) had no
effect (Fig 2A, lane 2). Although FcR aggregation after stimulation
with My43 induced Syk tyrosine phosphorylation, the enzymatic activity of precipitated Syk was unchanged (Fig 2C). Anti-Syk immunoblot analysis shows that an equal amount of Syk was immunoprecipitated from
resting and FcR-activated cells (Fig 2B).
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| Fig 2.
Syk is a major target of FcR-induced PTK activity.
THP-1 cells were activated at 37°C for 5 minutes by the addition of
5 µg/mL Mo2 (lanes 1 and 2), My43 cell culture supernatant 1:2 (lane 3), and RPMI 1640 cell culture medium only (lane 4). Lysates from 1.5 × 107 cells were immunoprecipitated with a rabbit
polyclonal anti-Syk antibody (lanes 2 through 4) or control rabbit
polyclonal IgG (lane 1). Two thirds of each immunoprecipitate were
separated by 8% to 12% SDS-PAGE, transferred to NC sheets, and
immunoblotted with an antiphosphotyrosine antibody (A). The immunoblot
was afterwards stripped and reprobed with Syk-specific antibody (B).
One third of each immunoprecipitate was used for in vitro kinase assay, as described in the Materials and Methods; separated by 8% to 12%
SDS-PAGE; and autophosphorylated Syk kinase was detected on dried gels
using autoradiography (C).
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The Src family kinase Lyn coprecipitates with FcR.
The results shown above prompted us to investigate whether PTKs
associate with FcR. Therefore, we adsorbed huIgA to NC particles and
used these complexes for cross-linking and subsequent
immunoprecipitation of aggregated FcR. Several
tyrosine-phosphorylated proteins were present in the FcR-specific
immunoprecipitate, whereas no proteins were found with NC particles
coated with BSA or two different preparations of human myeloma IgM
(Fig 3A).14 Among the proteins that
coprecipitated with the receptor was a notable double band of 56 and 58 kD. Similar migration patterns have been described for the Src family
kinases Hck and Lyn.32-34 To further characterize the
double band, we stripped the immunoblot in low pH buffer and reprobed
it with antibodies specific for human Hck or Lyn. Our results show that
the anti-Lyn antibody reacted with protein bands of identical molecular
mass, whereas no proteins were detected with the Hck-specific antibody
(Fig 3B, and data not shown). Additionally, Lyn kinase was also present
in receptor complexes immunoprecipitated with NC particles adsorbed
with the MoAb My43, but not with MoAb Mo2 (data not shown).
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| Fig 3.
Lyn coimmunoprecipitates with aggregated FcR. THP-1
cells (7.5 × 106/sample) were activated at 37°C for 5 minutes with 2 mg of NC particles adsorbed with BSA only (lane 1) or
huIgA (lane 2). Immunoprecipitation, separation of precipitated
proteins by 8% to 12% SDS-PAGE, and antiphosphotyrosine immunoblot
analysis of separated proteins were performed as described before (A).
The immunoblot was then stripped and reprobed with a rabbit polyclonal
anti-Lyn antibody (B).
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To exclude that precipitation of Lyn kinase was due to specific
antibody activities present in the huIgA preparation, a chimaeric huIgA
antibody specific for the hapten NP (4-hydroxy-3-nitrophenacetyl) was
used to immunoprecipitate FcR. Interaction of this hapten-specific antibody with THP-1 cells depends entirely on its huIgAFc domain and
is, therefore, restricted to ligation of FcR. Anti-PY analysis shows
that proteins of 55, 56, and 58 kD were present in the
receptor-specific immunoprecipitates using either huIgA or chimaeric
huIgA adsorbed to NC particles
(Fig 4A, lanes 1 and 2, respectively). Again, protein bands of identical molecular mass were
recognized in both immunoprecipitates by reprobing of the immunoblot
with the Lyn-specific antibody (Fig 4B).
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| Fig 4.
Immunoprecipitation of FcR depends on the Fc domain of
huIgA. THP-1 cells (1 × 107/sample) were activated at
37°C for 5 minutes with 2.5 mg of NC particles adsorbed with huIgA
(lane 1), chimeric huIgA (lane 2), or BSA only (lane 3).
Immunoprecipitation, separation of precipitated proteins by 8% to 12%
SDS-PAGE, and antiphosphotyrosine immunoblot analysis of isolated
proteins were performed as described. Immunoblot analysis of tyrosine
phosphorylated proteins (A). Reprobing of the same immunoblot with
Lyn-specific antibody (B).
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Tyrosine kinase inhibitor PP1 differentially affects association of
Lyn with FcR and FcR.
Treatment of RBL-2H3 cells with the Syk-specific inhibitor piceatannol
has been shown to partially inhibit FcR-mediated protein tyrosine
phosphorylation, but to completely block downstream cellular responses.35 The molecule responsible for tyrosine
phosphorylation of the remaining phosphorylated proteins appeared to be
the Src family kinase Lyn, which is constitutively associated with the FcR -chain and, hence, may function upstream of Syk.9
We used the Src family-selective inhibitor PP1 to study the role of Src
kinases in FcR-mediated protein tyrosine
phosphorylation.36 Activation of THP-1 cells in the
presence of 100 µmol/L PP1 abolished FcR-mediated protein tyrosine
phosphorylation of whole lysate proteins, including Lyn and Syk (data
not shown). PP1 blocked FcR- and FcR-mediated tyrosine
phosphorylation of Lyn kinase in a dose-dependent way and
phosphorylation of Lyn was completely abolished at a concentration of
100 µmol/L (Fig 5A, lanes 3 and 7). Interestingly,
association of Lyn with FcR was sensitive to short-term treatment
with PP1 and completely blocked at 100 µmol/L. However, interaction
of Lyn with FcR was not affected by PP1 (Fig 5B, lanes 1 through 3 and 5 through 7, respectively). These results support the hypothesis
that association of Lyn with FcR or FcR differs in the need of
Src kinase activity before and/or after receptor aggregation.
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| Fig 5.
Association of Lyn with FcR is sensitive to treatment
with PP1. THP-1 cells (1 × 107/sample) were incubated at
37°C for 30 minutes in RPMI 1640 cell culture medium only (lanes 1, 4, and 5) and cell culture medium containing 10 µmol/L of PP1 (lanes
2 and 6) or 100 µmol/L of PP1 (lanes 3 and 7). Subsequently, cells
were activated at 37°C for 5 minutes with 2.5 mg of NC particles
adsorbed with huIgA (lanes 1 through 3), huIgG (lanes 5 through 7), or
BSA only (lane 4). Immunoprecipitation, separation of precipitated
proteins by 8% to 12% SDS-PAGE, and antiphosphotyrosine
immunoblot analysis of isolated proteins were performed as described in
Materials and Methods (A). Reprobing of the same immunoblot with
Lyn-specific antibody (B).
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Cross-linking of FcR with huIgA adsorbed to NC particles induces
IL-1ra and IL-8 release in THP-1 cells.
In a previous study, we showed that stimulation of human mononuclear
cells or isolated human monocytes with huIgA induces the production of
IL-1ra, a naturally occurring inhibitor of IL-1 activity previously
shown to be produced by monocytes/macrophages in response to FcR
stimulation or LPS.37,38 The present results confirm and
extend these previous findings by showing that cross-linking of FcR
by huIgA adsorbed onto nitrocellulose particles induces IL-1ra release
in THP-1 cells (Fig 6A). In
addition, stimulation of THP-1 cells by FcR cross-linking induces
the production of IL-8 (Fig 6B), an 8-kD chemokine and angiogenic
factor previously shown to be produced by cells of the
monocyte/macrophage lineage, epithelial cells, or endothelial cells in
response to lipopolysaccharide (LPS), FcR cross-linking, or
cytokines such as TNF-.39,40
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| Fig 6.
Src family PTK activity is required for induction of
IL-1ra and IL-8 in THP-1 cells after cross-linking of FcR by
huIgA-adsorbed NC particles. THP-1 cells (3 × 106
cells/mL/well) were stimulated for 24 hours with huIgA adsorbed to
nitrocellulose particles (IgA-NC); parallel cultures were incubated in
the presence of corresponding concentrations of soluble huIgA or
BSA-adsorbed nitrocellulose particles (NC). In (A) and (B), cells were
cultured in complete medium (Med) or in complete medium containing PMB
(final concentration, 10 µg/mL) or PP1 (final concentration, 100 µmol/L). In (C), THP-1 cells were stimulated for 24 hours with huIgA
adsorbed to NC particles (final concentration of huIgA, 100 µg/mL) in
the presence of PP1 at the indicated concentrations. Release of IL-1ra
and IL-8 was examined in cell-free supernatants as described in
Materials and Methods. Results are expressed as nanograms per
milliliter (mean ± SEM of 3 experiments [A] or 6 experiments [B])
or (C) as a percentage of control (mean ± SEM of 3 experiments)
relative to the cytokine release observed in cells stimulated with
huIgA-particles in the absence of PP1 (cytokine release, in nanograms
per milliliter, mean ± SEM: IL-1ra, 7.99 ± 0.84; IL-8, 1.41 ± 0.04). (*) Statistically significant difference between cultures
stimulated with huIgA-NC and cells cultured in the presence of huIgA or
NC alone (both in the presence or absence of polymyxin B, 10 µg/mL;
P<0.05, Newman-Keuls multiple comparisons test; P = .004, analysis of variance). (#) Statistically significant difference
as compared with cultures containing soluble huIgA or NC particles
alone (P < .01, Kruskal-Wallis test for comparison of more
then two samples). (x) Statistically significant difference as compared
with cells stimulated with huIgA adsorbed to NC particles in the
absence of PP1 (P = .01, Mann-Whitney U test). (+)
Statistically significant inhibition of cytokine release after
stimulation with huIgA adsorbed to NC particles (P < .025, Student's t-test for paired samples).
|
|
Induction of cytokine release by particle-bound huIgA was
dose-dependent and became statistically significant even at a
relatively low concentration of particle-adsorbed huIgA (ie, 100 µg/mL). In contrast, levels of IL-1ra and IL-8 produced by cells
cultured in the presence of the same amount of BSA-adsorbed
nitrocellulose particles were only slightly elevated as compared with
background cytokine release, indicating that induction of cytokine
release by particle-bound huIgA was specific and mediated through
triggering of FcR. Cross-linking of FcR by particle-bound ligand
was required for induction of IL-1ra and IL-8 release, because
stimulation of THP-1 cells with soluble monomeric huIgA at the same
concentration had no effect on cytokine production, although flow
cytometric analysis confirmed strong binding of soluble huIgA to the
cells under these conditions (data not shown). In contrast to phorbol 12-myristate 13-acetate (PMA), which stimulates production
of IL-8, IL-1ra, TNF-, and IL-1 by directly activating protein kinase C independent of surface receptor triggering, particle-adsorbed huIgA specifically induced IL-1ra and IL-8 release accompanied by only
very low levels of TNF- or IL-1 release (TNF- release, in
nanograms per milliliter, mean ± SEM [n]: BSA-adsorbed particles, 0.29 ± 0.04 [9]; huIgA-coated particles, 0.76 ± 0.14 [9];
PMA [100 ng/mL], 3.98 ± 1.07 [5]; IL-1, in nanograms per
milliliter, mean ± SEM [n]: BSA-adsorbed particles, 0.12 ± 0.02 [9]; huIgA [100 µg/mL]-coated particles, 0.82 ± 0.22 [9]; PMA [100 ng/mL], 8.45 ± 1.66 [5]; ratio IL-1ra/IL-1:
huIgA coated onto NC particles, 28 ± 7; PMA: 5 ± 1;
P = .00135, Mann-Whitney U-test). Furthermore, particle-adsorbed huIgA triggered cytokine release even under conditions in which endotoxin-induced cytokine release was
blocked by the addition of polymyxin B (data not shown), thus
indicating that FcR cross-linking and LPS stimulate cytokine
release in THP-1 cells through different mechanisms (Fig 6A and B).
Induction of IL-1ra and IL-8 release after cross-linking of FcR by
multivalent ligand requires Src family protein tyrosine kinase
activity.
In the present study, we show that signaling events after FcR
cross-linking involve association of the PTK Lyn with FcR and
tyrosine phosphorylation of proteins involved in signal transduction such as Syk. The results presented in Fig 6B and C show that Src family
PTK activity is essential for FcR-mediated activation of cellular
functions, because the Src family-specific inhibitor PP1 abolished the
huIgA-mediated induction of IL-1ra and IL-8 release in THP-1 cells.
PP1-induced inhibition of cytokine production was dose-dependent over a
3-log range, with almost complete inhibition of cytokine release at a
concentration of 100 µmol/L of PP1 (Fig 6C).
| |
DISCUSSION |
In the present report, we show that, in the monocytic cell line THP-1,
the Src family kinase Lyn physically associates with FcR after
receptor aggregation by multivalent ligand. The association of Lyn with
FcR was found in immunoprecipitates of the native ligand of the
receptor, huIgA, or an MoAb of the IgM isotype directed against the
ligand-binding site of FcR, thus excluding precipitation of
FcR-bound Lyn. Furthermore, precipitation of Lyn kinase due to
specific antibody activities was excluded by the use of a chimeric huIgA consisting of a hapten-specific murine F(ab)2
component and the Fc part of human IgA.
Our approach, to cross-link and immunoprecipitate FcR with
huIgA-adsorbed NC particles, does not allow us to study whether Lyn is
constitutively associated with FcR in unstimulated cells. However,
the sensitivity of this interaction to short treatment with the PTK
inhibitor PP1 argues in favor of an aggregation-induced association
dependent on immediate kinase activity. PP1 has been shown to
selectively inhibit Src family PTKs, including Lck, Fyn, Src, and Hck,
at nanomolar concentrations, whereas no effect on the kinase activity
of ZAP-70, a member of the Syk-family of PTKs, was observed even in the
presence of 100 µmol/L of the inhibitor.36 Furthermore,
Amoui et al41 recently showed that Lyn in vitro kinase
activity was highly sensitive to PP1, whereas Syk activity was not
influenced by the inhibitor. The selectivity of PP1 provides, therefore, strong evidence that the PTK regulating FcR/Lyn
association is a Src family kinase, probably Lyn itself.
Presently, we do not know how Lyn interacts with FcR at the
molecular level, whereas various mechanisms have been described for
recruitment of Lyn to other FcRs expressed on myeloid cells, such as
FcRI, FcRI, and FcRII.9,12,13 However, the
dependence on Src kinase activity suggests that the target region on
FcR for Lyn binding may be a tyrosine-containing sequence motif. In this case, association of Lyn should occur to molecules other than the
Ig-binding chain, because of the lack of tyrosine residues in the
cytoplasmic tail of the receptor.6 Additionally, binding must be of considerable strength, because FcR/Lyn complexes did not
readily dissociate in 1% Nonidet P-40 lysis buffer (data not shown).
Recently, FcR has been described to associate with the ITAM-containing FcR chain in U937 monocytic cells, and association of Lyn kinase and chain has been shown in the same cell
line.13,28 However, association of Lyn with ITAM-containing
receptor subunits such as the FcR chain or the FcRI chain
has been described to occur constitutively and does not depend on
activation-induced phosphorylation of tyrosine
residues.9,13 In contrast to FcR, interaction of Lyn
with FcR was sensitive to treatment with PP1 (Fig 5), suggesting
that other mechanisms than binding to the FcR-associated chain
account for the observed association of Lyn kinase with FcR.
Coimmunoprecipitation of Lyn with the Ig-binding chain of FcRI
has been shown in monocytic cells lines under conditions in which chain dissociates from the receptor
complex.11,13 Like FcR, the cytoplasmic
tail of the ligand-binding chain of FcRI is devoid of
tyrosine-containing signaling motifs and functional signal transduction
depends on ITAM-bearing receptor subunits.42 Direct binding
of Lyn to the chain of FcR implies a more active role of the
Ig-binding subunit in signal transduction processes induced by receptor
aggregation than generally assumed. Recently, Hou et al43
showed that the membrane-proximal amino acids in the cytoplasmic tail
of the FcRIII chain are necessary for cell activation. In their
report, tyrosine phosphorylation of cellular proteins was considerably
impaired in cell lines transfected with a mutant chain that lacked
the respective amino acids, although association of FcRIII with  chain was not affected. These results suggest, therefore, a role for
the membrane-proximal amino acids of the chain in recruiting
nonreceptor PTKs to aggregated FcRIII. The purpose of the
membrane-proximal amino acids of the FcR chain is presently
unkown. However, they could also play a role in signal transduction,
for instance by recruiting Lyn to the aggregated receptor or by
stabilizing Lyn/FcR complexes.
Sequential activation of different PTKs appears to be a general feature
of multisubunit antigen receptors and FcRs of hematopoietic cells.8 In myeloid cells, involvement of members of the Src and Syk families of PTKs in transmembrane signaling after FcR or
FcR aggregation has been described.11,13,15,19,20,44 In
our study, tyrosine phosphorylation of Syk kinase increased sixfold
after FcR cross-linking, whereas baseline in vitro kinase activity
of Syk was unchanged (Fig 2). The observed lack of increased tyrosine
kinase activity after FcR cross-linking appears to be in conflict
with previous reports showing that tyrosine phosphorylation of Syk,
following aggregation of FcRI in U937 monocytic cells or FcRI in
mast cells, was accompanied by extensive upregulation of in vitro
kinase activity of Syk.21,35 Furthermore, Ueland et
al30 previously reported that aggregation of FcR in U937 cells resulted in increased tyrosine phosphorylation of Syk as well as
upregulation of its in vitro kinase activity. Discrepancies between
these previous findings and the results of the present report with
regard to enhancement of Syk in vitro kinase activity could be due to
distinct characteristics of the different cell lines studied. In
contrast to U937 cells, aggregation of FcRI or FcRII in THP-1
cells has been shown to result in a tremendous induction of Syk
tyrosine phosphorylation but only weak (2- to 3-fold) upregulation of
its in vitro kinase activity.45 In our study, considerable
baseline kinase activity of Syk could be found in THP-1 cells and
recent evidence indicates that downstream signaling can proceed despite
the lack of demonstrable upregulation of Syk kinase activity.
Aggregation of Syk/CD16 chimeric proteins on the surface of the
CD8+ cytolytic T-cell line WH3 was sufficient to allow
induction of specific cytolysis of target cells, although kinase
activity of Syk was unchanged after Syk/CD16
cross-linking.46 The finding that cross-linking of FcR
on THP-1 cells by huIgA bound to nitrocellulose particles is capable of
inducing the release of IL-1ra and IL-8 indicates that downstream
signaling events after FcR triggering are intact despite the lack of
significant FcR-mediated upregulation of Syk kinase activity.
Induction of IL-1ra and IL-8 release could be completely inhibited by
the Src family-specific PTK-inhibitor PP1, thus confirming the
biological significance of the newly described association of Lyn with
FcR after receptor cross-linking. The lack of detectable Hck kinase,
which has been shown to associate with FcRs in THP-1 cells,
additionally emphazises the crucial role of Lyn for FcR-induced
signaling processes.11,12 Although no other Src family
kinases have been described to be involved in signal transduction via
FcRs in THP-1 cells, involvement of Src kinases apart from Lyn cannot
be completely ruled out. Aggregation of FcR induced the release of
considerable amounts of IL-1ra, a cytokine with potent
anti-inflammatory properties in vitro and in vivo, as well as
IL-8.47 Although IL-8 was initially described as a
proinflammatory neutrophil chemotactic factor secreted by lipopolysaccharide-stimulated mononuclear cells, IL-8 has recently been
shown to play a more complex role in the regulation of the inflammatory
response, eg, by exerting a wide range of modulatory effects on
neutrophil-endothelial adhesive interactions.39,40 Our
finding that cross-linking of FcR by particle-bound huIgA induces
the release of IL-1ra and IL-8 confirms and extends previous studies
suggesting that huIgA, in addition to the protective functions mediated
by specific antibody activity, plays an active role in the regulation
of inflammatory responses by interacting with the FcR on cells of
the monocyte lineage.31,37,48
| |
FOOTNOTES |
Submitted September 4, 1997;
accepted October 24, 1997.
Address reprint requests to Heinz Gulle, PhD, Institute of Immunology,
University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Eleonore Gschaider and Astrid Lehner for expert
technical assistance and Paul Breit for excellent photographic assistance.
| |
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