Vol. 183, No. 2, 1992 March 16. 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 789-796
TYROSINE PHOSPHORYLATION AND ITS POSSIBLE ROLE IN SUPEROXIDE PRODUCTIONBY HUMANNEUTROPBILS STIMULATEDWITHFMLP ANDIgG
T. Kusunoki,
H. Higashi, M. Mayumi
S. Hosoi, D. Hata, K. Sugie* and H. Mikawa
Department of Pediatrics, Kyoto University Hospital, Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606. Japan *Department of Late Effect Studies, Radiation Kyoto University, Yoshida-konoecho, Sakyo-ku,
Received
January
31,
Biology Center, Kyoto 606, Japan
1992
SUMMARY: Superoxideproduction ____.
by human neutrophilsstimulated with FMLP and solubleaggregated human IgG wereinhibitedin a dose dependent manner by two kinds of tyrosine kinase inhibitors, erbstatin and gen&tein. Superoxide production stimulated with surface bound IgG, however, was scarcely inhibited either inhibitor. Protein tyrosine phosphorylation studies with by immunoblotting revealed specific tyrosine phosphorylatin of a 40 Kd protein by soluble aggregated and surface bound IgG, and that of a 39 Kd protein, as well as the 40 Kd protein, by FMLP. These were all inhibited by the tyrosine kinase inhibitors. These data suggest that superoxide production induced by FMLP and soluble aggregated IgG are, at least in part, tyrosine kinase dependent, but the tyrosine kinases and/or substrates of tyrosine kinases involved may be different. In addition, tyrosine kinase independent pathways are also suggested to be involved in superoxide production by stimulation with SurfaceboundIgG. e 1992*caclem1c Pres*, Inc.
It
is
production Recently,
well
known
by human FMLP-induced
on PTx-sensitive have suggested
that
there
neutrophils
are
depending
0,. production
G proteins
of IgG (or soluble
on the nature immune
different
pathways
on differences
kinases
of Fc~ receptor-mediated
in stimuli
(3-S). Other 0;.
O,m
(1, 2).
induce
activation
vWnonsz FMLP, formyl-methionyl-leucyl-phenylalanine; ___fF= . toxin: PMA, phorbol-12-myristate-13-acetate; r pertussis buffered salt solution containing Car+ and Mgi*.
reports
production
of the IgG (6, 7), and that soluble
complexes)
for
has been shown to be dependentnotonly
but also on tyrosine
that the sensitivity
PTx is dependent
several
of neutropbils
to
aggregates through
O;m, superoxide; HBSS, Hanks'
Vol.
a
183, No. 2, 1992
PTx-sensitive
complexes)
BIOCHEMICAL
pathway,
induce
02- production
the role of tyrosine yet clear,
tyrosine
Fcr receptors
while
kinases
involvemsntoftyrosinekinases -the
the
bound
through
in Fey receptor-mediated
depending
involvement
(or
insoluble
immune
pathway.
Although
02- production
is not
has been shown to occur when neutrophil
by soluble
aggregates
kinases
of different
of IgG (B), suggesting In the
in On- production
with FMLP,solubleaggregatedIgGand
pasaible
IgG
a PTx-insensitive
intheprocess
role of tyrosine
stimulated
surface
phosphorylalion
are crosshnked
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
study,
we
by human neutrophils
surfacebound activation
present
the
pathways
IgG,and of 0~
showed
production
on the stimuli.
MATEFUALS AND METHODS
For human IgG soum, pH4-treatx~l intact immunoglobulin (Sanglopor, Sankyo) was used. Soluble aggregates of IgG were formed by excess of dimethyl covalent crosslinking with the use of a 30-molar suberimidate (Nacalai Co.) as previously desuibed (9), and the soluble oligotners were separated by fractionation on Sephacryl S300 (Pharma&a) columns. FMLP, cytochrome C, B El, PMA and superoxide dismutase werepurchased from Sigma. GenisWn was purchased from Funakoshi. ErbsWtin was a generous gift of Dr.M.Imoto, Keio University, Japan. MATERIALS:
METHODS: Hepadnbed peripheral blood was collected from healthy adult volunteers. Neutrophils were isolated by established procedures (10) and suspendedinHBSS. Oa- production was measured by the super-oxide dismutase inhibitable reduction of ferriqtochrome C (11). Briefly, neutrophils at lxlO@/ml in HBSS containing lmg/ml cytochrome C were incubated fortheindi&ed periods at 37oc in maxisoap tubes (Nunc Co.) coated with IgG or in BSA coated tubes containing soluble aggregated IgG, FMLP with cytochalasin B, or PMA. IgG was -ted tothetubes by addinglmlof appropriateconcentration of IgGtothe tubes and incubating them at 40c overnight. Any remaining sites were blocked by BSA (1%). The blank contained all of these components plus 25 ug/ml supemxide dismutnse. Upon completion of the incubation, the tubes were centrifuged at 4oC for 5 minutes, and the optical absorbance of the supematant was determined at 550 nm. The amount of 0~ produced was calculated as previously described (ll), and the results were expressed as nmol 02-/106 cells. Tyrosinephosphorylalion studiesweredonewithimmunoblottingasdescribed by Gomex-Cambronero et a2 (4) with some modifications. AppxoximaWy 5x106 oells in 400 ~1 HBSS were incubated with various stimuli for the indiated periods at 37oC. At the end of the incubation, cells were peBeted by centrifugation, lysed by 100 ~1 ice-cold lysis buffer containing 1% NP-40, 20 mM Tris (pH B.O), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 3 wg/ml aprolinin, and 0.2 mM sodium orthovanadate (Na3V0,), and kept on ice for 10 minutes. Insoluble materials were removed by centafugation at 15,OOOxg at 40c for 10 minutes, and 100 ~1 of the supernatant was mixed with 25 d of 5xSDSgel sample buffer, boiled for 5 minutes, and electrophoresed on a 10% polyacrylamide gel. The proteins were then transferred toanitrocellulose she.&Tyr&nephospborylatedproductsweredetElrminedwith amnity-Purified 790
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AND
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RESEARCH
COMMUNICATIONS
phosphotymsine-s-c rabbit antibodies, prepared as described previously (12). The specificity of the anti-phosphotyrosine anma was amfimwd by inhibition of the binding with an excess cd phosphatymsine (data not shown). A (ICN), and the bands of The sheet was then incubated with [ “511-protein phosphotyrosine-Wntaining proteins were visualized by autoradiography. In SXXTE ex periments, densities af the phosphotyrosines were measured by densitrxnetry.
RESULTS Human
boundIgG
neutrophils
stimulated
generatzd
with either
aggregated
IgG or surface
0~ inadose-dependentmanner(Figs.land
of 300 wg/ml for soluble
aggrega@d
and an incubation
of 60 minutes
conditions
soluble
time
for the following
2),reachinga
IgG and of 5 ug/ml for
inhibition
both
studies.
for surface bound
stimuli
were selected
For FMLP, a dose of
IgG
as the 1
PM and
anincubationtimeof15minuteswereselected. We studied genktein,
which
pmduction response
the
in
effects
of two tymsine
have
difEerent
zzqxmse
to the
mechanisms various
.
Surface
0
Aggegated
a
Control
Bound
inhibited
30
45 TIME
60
(min.)
02- production
by human neutrophils.
(1 ml) in maxisoaptulxspmmatedwithIgG
(5 Ug/ml, 1 ml) or BSA (1%). Soluble aggregated IgG (300 pg/ml) was added to
BSA-coatxsd tubes. Cells wew 02. productionwasmeasumd.
incubated
for the indicated
791
02
IgG (Figs. 3A and 3B).
.
7
Figure 1. Time course aE IgGinduced placed
of them
aggregated
and
(13, 14), on O2
IgG
INCUBATION
were
Both
erbstalin
IgG
15
(IxlO8/mZ)
inhibitors.
of inhibition
stimuli.
of the azlls to FMLP and soluble
Neutmphils
kinase
periods
at 370C and
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
//.(.I I
I
37 75 Aggregated
Figure 2. Dose were placed axicentraiions concentraiions for 60 minutes
A
60
I
I
150 300 IgG Wml)
response of IgG-induced 02m produdion. Neutrophils (1 ml) in maxisoap tubes precoated with the of IgG (1 ml) (A) or in BSA-coated tubes to which the of soluble aggregated IgG were added (B). Cells were at 370C, and 0~ production was measured.
Control
Cells
Erbstatin Treated
Cells
B
r
60 1
z
55
9
25
q
Control
C~IIS
Genistein Treated
Cells
(lx106/ml) indicated indicated incubated
r
-E sz
20
6 2 15 0 E
FMLP
Aggregated Surface Bound kG kG
TT
FMLP
PMA
Aggregated Surface Bound kG kG
PMA
F!iguna 3. Effect of tyrosine kinase inhibitors on neutrophil 02. production. Neutrcphils (1~107/ml) were incubated with or without erbstaiin (10 fig/ml) for 60 minutes, or geni&ein (50 Ug/ml) for 30 minutes at 370C. The cells were diluted to l~lOs/ml with HBSS and placed (1 ml) in maxisoap tubes precoated with IgG (5 ug/ml) or BSA. To some BSA-coated tubes, FMLP (1vM) with cytochalasin B (5 pg/rnl), soluble aggregated IgG (300 gg/ml) or PMA (100 rig/ml) was added. They were again incubated for 60 minutes at 370C, and OSproductionwasmeasured. 792
Vol.
183,
No.
A loo
a 0 .
.
BIOCHEMICAL
2, 1992
AND
B
FMLP Aggregated IgG Surface Bound IgG
100
ERBSTATIN
_
RESEARCH
D 0 0
20
10
5
BIOPHYSICAL
COMMUNICATIONS
FMLP Aggregated IgG Surface Bound kG
50
25 GENISTEIN
(pg/ml)
100
(pglml)
Figure 4. Dose dependence of the effect of tyrnsine kinaae inhibitors on Neutrophils (lxlO~/ml) were incubated with or neutrophil Or- produtin. without the indicated amounts of either erbstatin for 60 minutea, or genistein for 30 minutes at 370C. The cells were incubated as described in the legend for Figure 3, and O,- production was measured.
The e&act
was dose-dependent,
aggregated
less efficient
phosphorylation
of
constitutively
any
stimulus
phosphorylated
phosphotyrosine
antibodies
was specifically
by either
in response to each stimulus
was
(Fig. 5). Under the conditions of the assay,
some of 50-60 Kd proteins were constitutively absence
affected
the
(Figs. 3 and 4).
in neutrophils
next examined with immunoblotig
tyrosine
response to soluble
bound IgG were scarcely
even at the highest concentration
Tyrosine
the
in
IgG than in response to FMLP (Figs. 4A and 4B). By contrast,
responses to PMA and surface inhibitor
but
and
in
proteins
tyrosine
the
phosphorylated
presence
were confirmed
of
by all three
erbstatin.
by using other
(data not shown). In addition, phosphorylated
even in These anti-
a 40 Kd protein
kinds of stimulation,
FMLP, soluble aggregated IgG, and surface bound IgG, though phosphorylationof the protein induced erbstalin
by
induced by soluble aggregated FMLP and surface
considerably
all stimulus
inhibited
(% inhibition
densitometry, phosphorylatlon
by
IgG was much weaker than those
bound IgG. tyrosine
Preincubation
phosphorylation
FMLP and surface
was 50% and 758, respectively). of a 39 Kd protein,
of the cells with of this protein
by
bound IgG, determined
by
FMLP induced additional tyrosine
which was almost completely inhibited 793
by
Vol.
183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ABCDEFGH
FigurE? 5. Tyrosine phosphorylation stimulated by FMLP or IgG in human neutrophils. Neutrophils (1~107/ml) were incubated with (l3, D, F, H) or without (A, C, E, G) erbstatin (10 ug/ml) for 60 minutes. The cells were then diluted to 1x106/ml with HESS and stimulated with FMLP (1 PM) (A, B) for 1 minute, soluble aggregated IgG (1.5 mg/ml) (C, D) or surface hound IgG (5 ug/ml) (E, F) for 5 minutes at 370C. Control, unstimulatid, cells (G, H) were incubated for 5 minutes. Tyrosine phosphorylation was determined with immunoblotting as described in Materials and Methods.
erbstatin
(Fig.
preincubated
Similar
5).
with genistein
phosphotyrosines
results
were
obtained
when
the
cells
were
(data not shown). The 40 Kd and 39 Kd protein
were seen 1 to 5 minutesafter
stimulation atthelatestand
almost disappeared 30 minutes after stimulation
(data not shown).
DISCUSSION __----___
An increase in tyrosine
phosphorylation
of a number of neutrophil
protein
was shown to occur by various stimuli (15). In the present study, we compared three kinds of stimuli that induce O)m production
of neutrophil
tyrosine
analysis of protein
kinase inhibitors
and the immunoblotting
by the use of tyIY&ne
phosphorylation. The fact that FMLP-induced tyrosine important
kinase inhibitors
Og. production suggests that
role in FMLP-induced
02- production
was almost completely inhibited tyrosine
Oe- production,
phosphorylaU.on
as previously
plays
described
by an (5).
induced by soluble aggregated IgG was also inhibited in a dose-
dependent manner by these inhibitors, was scarcely inhibited. soluble aggregated
whilethatinduced
by surface bound IgG
These results suggest that 02- production
IgG is, at least in part,
tyrosine
that several possible mechanisms may be involved 794
induced by
kinase dependent,
in Or- production
but
induced by
Vol.
183,
surface
No.
2, 1992
bound
IgG.
IgG, tyrosine strong
to
induced soluble
aggregated kinase
tyrosine
on the nature
stimulated
besides
proteins.
be attributed stimulus (20-26)
to the
of
constitutive
tyrosine
genistein;
tyrosine
inhibited
far
more
strongly
that tyrosine
kinase
dependent
FMLP, soluble
aggregated
the
reports
(5, El), but
that
kinases
and
roles
of
previous tyrosine
elucidated whether
whether these
the process
of 0.
50-60
IgG, which
be
kinases
in different
39 Kd protein
was
proteins
are
These
data
by stimulation
with
is consistent
with
It
stimuli
product to
and/or
different.
Kd
sensitivity
40 Kd protein.
the tyrosine
phosphorylated
specifically
some
in their
are induced
bound
may
by all three
are
of
of the
the
signals
the 40 Kd proteins
specifically
substrates
remains
are
actually
to
identical
be and
involved
in
production.
Neutrophil
activation
physiologically
very
or large insoluble
of
IgG and surface
their
FMLP,
of
IgG .
to be a degradation
phosphorylation that
bound
co-activation bound
phosphorylation
G
of FCY reCX?ptOrS
possible
of the difference
than
Ptx-sensitive
and surface
kinds
by
seems
of tyrosine
of 40 Kd protein
is not likely
because
boundIgG
the role
surface
39 Kd protein
The 39 Kd phosphoprotein
and
with
phosphorylation
that
case,
the
because
bound IgG. Therefore,
aggregated
and/or
did
by FMLP which
by surface
different
IgG
than
unlikely
of IgG, as with
by stimulation
tyrosine
erbstatin
seems
this
is the
soluble
(7, 16-19)
of the 40 Kd phosphoprotein
suggest
between
bound
phosphorylation
induced
depends
addition,
Surface
as much as did surface
in OL - production
aggregated
may become too
the O>- produdion
If this
that
in
inhibit
independent.
molecules
and,
tyrosine
could
each
by soluble
inhibitors.
inhibitors
might
We demonstrated
the
2 and 5). However,
kinase
COMMUNICATIONS
phosphorylation
and stronger
(6, 7). The difference
some adhesion
by
RESEARCH
induced
IgG (Figs.
involved
by
BIOPHYSICAL
but tyrosine
sufficiently
in O,.m response
stimulated
stimuli
dependent,
phosphorylation
IgG stimulation
AND
may be, as that
more Oim production
to be tyrosine
proteins
it
be inhibited
the main pathway
kinases
First,
kinase
the tyrosine induced
BIOCHEMICAL
by important
surface
bound
process,
immune complexes,
IgG can be regarded
because which 795
tissue
as a patho-
bound immune
complexes
may use the same activation
pathway
Vol.
183, No. 2, 1992
as the
surface
neutrophils insights
in into
BIOCHEMICAL
bound viva
IgG,
Further
are studies
Fey receptor-mediated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
presumably of these neutrophil
the
dominant
mechanisms activation
activators will provide
of new
in viva
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