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

Vol.

183,

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BIOCHEMICAL

2, 1992

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

REFERENCES 1. Rossi, F. (1986) Biochim.Biophys.Acta 853, 65-89. 2. Robinson, J.M., Heyworth, P-G., and Badwey, J.A.(1990) Biocbim.Biophys.Acta 1055, 55-62. 3. Huang, C-K., Laramee, G.R., and Casnellie, J.E. (1988) Biochem.Biophys. Res.Commun. 151, 794-801. 4. Gomez-Cambronero, J., Huang, C-K., Bonak,V.A., Wang,E., Casnellie, J.E., Shiraishi, T., and Shaa& Ramadan. (1989) Biochem.Biophys.Res.Commun. 162, 1478-1485. 5. Naccache, P-H., Gilbert, C., Caon, A.C., Gaudry, M., Huang, C-K., Bonak, V.A., Umezawa, K., and McCall, S.R. (1990) Blood 76, 2098-2104. 6. Blackburn, W.D., Jr., and Heck, L.W. (1988) Biochem.Biophys.Res.Commun. 152, 136-142. 7. Crockett-Torabi, E., and Fantone, J.C. (1990) J.Immunol. 145, 3026-3032. 8. Connely, P.A., Farrell, CA., Merenda, J.M., Conklyn, M.J., and Showell, H.J. (1991) Biochem.Biophys.Res.Commun. 177, 192-201. 74, 9. Segal, D.M., Taurog, J-D., and Metzger, H. (1977) Proc.Natl.Acad.Sci. 2993-2997. lO.Tsuruta, S., Ito, M., and H. Mikawa. (1990) FEBS letters 268, 241-244. ll.Newburger, P.E., Chovaniec, M.E., and Cohen, H.J. (1980) Blood 55, 85-92. 12.Lane, P.J.W., Ledbetter, J.A., McConnell, F.M., Draves, K., Deans, J., Schieven, G-L., and Clark, E.A. (1991) J.Immunol. 146, 715-722. 13.Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 5592-5595. 14.Imoto. M., Umezawa, K., Isshiki, K., Kunimoto, S., Sawa, T., Takeuchi, T., and Umezawa, H. (1987) J. Antibiotics 40, 1471-1473. 15.Berkow. R.L., and Dodson, R.W. (1990) Blood 75, 2445-2452. 16.Huizinga, T.W.J., van Kemenade, F., Koenderman, L., Dolman, K.M., von dem Borne, A.E.G.K., Tetteroo, P.A.T., and Roos, D. (1989) J.Immunol. 142, 2365-2369. 17Ximberly, R.P., Ahlstrom, J.W., Click, M.E., and Edberg, J.C. (1990) J.Exp.Med. 171, 1239-1255. 18.Huizinga, T.W.J., Dolman, K-M., van der Linden, N.J.M., Kleijer, M., Nuijens, J.H., von dem Borne, A.E.G.K., and Roos, D. (1990) J.Immunol. 144, 1432-1437. 19.S&non, J.E., Brogle, N.L., Edberg, J.C., and Kimberly, R.P. (1991) J.Immunol. 146, 997-1004. 20.Dahinden CA., Fehr, J., and Hugh, T.E. (1983) J.Clin.Invest. 72, 113121. Zl.Nathan, C.F. (1987) J.Clin.Invest. 80, 1550-1560. 22.Nathan C.F. (1989) Blood 73, 301-306. 23.Nathan, C.F., Surimal, S., Farber, C., Sanchez, E., Kabbash, L., Asch, A., Gailit, J., and Wright, S.D. (1989) J.Cell.Biol. 109, 1341-1349. 24Xabanas, C., La&, P., Mollinedo, F., Lopez-Rivas, A., Sanchez-Madrid, F., and Bernabeu, C. (1989) ImmunoLLett. 20, 193-198. 25.Sud.ina, G.F., Tatarintsev, A-V., Koshkin, A.A., Zaitsev, S-V., Fedrov, N.A., and Varfolomeev, S.D. (1991) Biochim.Biophys.Acta 1091, 257-260. 26.Gresham, H-D., Graham, I.L., Anderson, D-C., and Brown, E.J. (1991) J.Clin.Invest. 88, 588-597.

796

Tyrosine phosphorylation and its possible role in superoxide production by human neutrophils stimulated with FMLP and IgG.

Superoxide production by human neutrophils stimulated with FMLP and soluble aggregated human IgG were inhibited in a dose dependent manner by two kind...
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