Báo cáo Y học: The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils doc

Vignais Laboratoire de Biochimie et Biophysique des Syste`mes Inte´gre´s UMR 5092CEA-CNRS-UJF, De´partement Re´ponse et Dynamique Cellulaires, CEA-Grenoble, France In a previous study, t

The S100A8/A9 protein as a partner for the cytosolic factors

of NADPH oxidase activation in neutrophils

Jacques Doussiere, Farid Bouzidi and Pierre V Vignais

Laboratoire de Biochimie et Biophysique des Syste`mes Inte´gre´s (UMR 5092CEA-CNRS-UJF), De´partement Re´ponse

et Dynamique Cellulaires, CEA-Grenoble, France

In a previous study, the S100A8/A9 protein, a Ca2+- and

arachidonic acid-binding protein, abundant in neutrophil

cytosol, was found to potentiate the activation of the redox

component of the O2 generating oxidase in neutrophils,

namely the membrane-bound flavocytochrome b, by the

cytosolic phox proteins p67phox, p47phox and Rac

(Doussie`re J., Bouzidi F and Vignais P.V (2001) Biochem

Biophys Res Commun 285, 1317–1320) This led us to check

by immunoprecipitation and protein fractionation whether

the cytosolic phox proteins could bind to S100A8/A9

Fol-lowing incubation of a cytosolic extract from nonactivated

bovine neutrophil with protein A–Sepharose bound to

anti-p67phox antibodies, the recovered immunoprecipitate

con-tained the S100 protein, p47phox and p67phox Cytosolic

protein fractionation comprised two successive

chromato-graphic steps on hydroxyapatite and DEAE cellulose,

fol-lowed by isoelectric focusing The S100A8/A9 heterodimeric

protein comigrated with the cytosolic phox proteins, and

more particularly with p67phox and Rac2, whereas the

isolated S100A8 protein displayed a tendancy to bind

to p47phox Using a semirecombinant cell-free system of

oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2 In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration

of cytosolic phox proteins Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian The propensity of S100A8/A9 to bind the cytosolic phox pro-teins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b

Keywords: NADPH oxidase activation; superoxide O2; neutrophils; phox proteins; S100A8/ A9 protein

The heterodimeric Ca2+-binding protein S100A8/A9, also

referred to in the literature as MRP8/MRP14, is expressed

constitutively in large amounts in neutrophils and

mono-cytes [1–3], where it plays a role in the activation process

(reviewed in [4]) and adhesion [5] The S100A8/A9 protein

might also serve as a reservoir and a carrier of arachidonic

acid [6–8] This latter finding is all the more noteworthy as

arachidonic acid is currently used in cell-free system to

activate the O2 generating NADPH oxidase, an enzymatic

complex responsible for the microbicidal function of

neutrophils and macrophages [9] In its activated form,

the NADPH oxidase complex is composed of a

membrane-bound flavocytochrome b and proteins of cytosolic origin,

called phox (for phagocyte oxidase) factors of oxidase

activation or cytosolic phox proteins, namely p67phox,

p47phox and Rac (reviewed in [10]) An additional cytosolic

protein p40phox has been found to copurify with p47phox

and p67phox and the three cytosolic factors appear to form

an activation complex [11] The O2 generating oxidase activity can be measured in cell-free system after a few minutes of preincubation of flavocytochrome b (or a membrane fraction enriched in flavocytochrome b) w ith p67phox, p47phox and Rac in the presence of arachidonic acid The preincubation step is required for the transition of the flavocytochrome from a dormant state to an active state [12] In an early study of the bovine S100A8/A9 (called at that time p7/p23) [13], it was found by an immunofluores-cent approach that in resting neutrophils the S100A8/A9 protein was evenly distributed within the cytoplasm and that after activation of the cells by phorbol ester, it concentrated under the plasma membrane, together with the cytosolic phox proteins A similar behaviour was reported in the case of human neutrophil S100A8 [14] We recently reported that the S100A8/A9 protein potentiates the NADPH oxidase activation in bovine neutrophils [15]

We indirectly arrived at this finding by the study of the effect

of phenylarsine oxide (PAO) on the membrane and cytosolic protein components that participate in oxidase activation in the cell-free system Incubation of free or membrane-bound flavocytochrome b with PAO resulted in

a decrease in oxidase activation [16] In contrast, the activating potency of neutrophil cytosol treated by PAO on the oxidase activity of flavocytochrome b was significantly

Correspondence to J Doussiere, DRDC/BBSI, CEA-Grenoble,

17 rue des Martyrs, 38054 Grenoble cedex 9, France.

Fax: + 33 4 38 78 51 85, Tel.: + 33 4 38 78 34 76,

E-mail: jdoussiere@cea.fr

Abbreviations: PMSF, phenylmethanesulfonyl fluoride.

(Received 14 February 2002, revised 30 April 2002,

accepted 17 May 2002)

enhanced [16] Using a PAO affinity chromatography

column, we identified in neutrophil cytosol the S100A8/A9

protein as the PAO target responsible for the increased

oxidase activation [15] Here we report

immunoprecipita-tion and protein fracimmunoprecipita-tionaimmunoprecipita-tion experiments that suggest that

the S100A8/A9 protein interacts with the cytosolic factors

of oxidase activation and more preferentially with p67phox

We also report a study, in a cell-free system, of the effects of

S100A8/A9 on the kinetics of oxidase activation

M A T E R I A L S A N D M E T H O D S

Chemicals

NADPH, GTPcS, leupeptin, bestatin and aprotinin were

from Roche, horse heart cytochrome c, arachidonic acid,

hydroxyapatite, phenylmethanesulfonyl fluoride (PMSF)

and pepstatin were from Sigma DEAE-cellulose was from

Whatman Ampholines pH 3–10 were from Pharmacia

Biological preparations

A particulate fraction enriched in plasma membranes was

prepared by centrifugation on a discontinuous sucrose

gradient of a sonicated homogenate of bovine neutrophils in

NaCl/Pi [12] The saline buffer (2.7 mM KCl, 136.7 mM

NaCl, 1.5 mM KH2PO4 and 8.1 mM Na2HPO4, pH 7.4)

was supplemented with antiproteases: 0.1 mM phenyl

methyl sulfphonyl fluoride, leupeptin (1 lgÆmL)1),

pep-statin (1 lgÆmL)1), bestatin (1 lgÆmL)1) and aprotinin

(1 lgÆmL)1) The heme b content of neutrophil membrane

was determined by spectrophotometry [12] From the heme

content, the amount of flavocytochrome b was deduced

assuming the presence of two hemes per flavocytochrome b

[17] The recombinant cytosolic proteins, p47phox, p67phox

and Rac2, prepared as described [18] were kindly provided

by M C Dagher UMR 5092 CEA-Grenoble

The heterodimer S100A8/A9 was purified from bovine

neutrophil cytosol, as described previously [15] The stained

gel following SDS/PAGE did not show visible traces of

protein contaminant [15] The identity of S100A8 was further

ascertained by amino-acid sequencing, using Edman

degra-dation As S100A9 has a blocked N-terminal amino acid,

analysis of the protein was carried out by mass spectrometry

The protein band corresponding to S100A9 in the gel,

following SDS/PAGE, was excised, and washed 3 times

successively by 25 mMammonium bicarbonate pH 8.0, and

50% acetronitrile/50% 25 mM ammonium bicarbonate,

pH 8.0 A final wash with pure water was performed before

complete dehydratation in a vacuum dryer In gel tryptic

digestion was performed for 4 h at 37C in 10 lL of 25 mM

ammonium bicarbonate pH 8.0 with 0.5 lg of trypsin A

sample (0.5 lL) of the digestion supernatant was spotted

onto the MALDI sample probe on top of a dried 0.5 lL

mixture of 4 vol solution of saturated

a-cyano-4-hydroxy-transcinnamic acid in acetone and 3 vol of nitrocellulose

dissolved in acetone/isopropanol 1 : 1 (v/v) Dried samples

were rinsed by placing a 5-lL vol of 0.1% trifluoroacetic

acid on the matrix surface After 30 s, the liquid was blown

off by pressurized air MALDI mass spectrum of the

peptide mixture was obtained using an Autoflex mass

spectrometer (Bruker Daltonik) Peptide masses were

assigned and used for database searching (Swiss Prot) using

the program MS-Fit at the University of California San Francisco (http://prospector.ucsf.edu/) Eight peptides with masses ranging from 715.4 to 2184.9 were found to fully match with peptide fragments corresponding to the se-quence of the bovine S100A9, previously called p23 [13] and also referred as calgranulin B In addition, the mass spectrum indicated the absence of protein contaminant that could have comigrated with S100A9 during SDS/PAGE

In cell-free assays carried out at 20C, Rac2 was precharged in GTPcS by incubation at 20C in the presence of 15 lM GTPcS and 4 mMEDTA Incubation was terminated after 10 min by the addition of 10 mM MgSO4 Antisera against the gp91phox component of flavocytochrome b and against the S100A9 component of the S100A8/A9 heterodimeric protein were kindly provided

by G Brandolin UMR 5092 CEA-Grenoble and by

M.-J Stasia CHU-Grenoble, respectively The S100A9 antibody was able to interact with the nondissociated heterodimeric protein S100A8/A9 S100A9 antibodies were purified from the antiserum by sodium sulfate fractionation Other antisera directed against p67phox and p47phox were obtained from M.-C Dagher Rac1 and Rac2 antibodies were obtained from Santa Cruz The phox proteins and the S100A8/A9 heterodimer were immunodetected after incubation with goat anti-(rabbit IgG) Ig coupled to peroxidase The bound peroxidase was revealed by a luminescence method using the ECL kit from Amersham Protein concentration was assayed with the bicinchonic acid reagent (BCA) (Bio-Rad) using bovine serum albumin as standard Arachidonic acid was dissolved in ethanol and stored as a stock solution, at a concentration of 200 mM Protein fractionation of bovine neutrophil cytosol Protein fractionation was performed on cytosolic extracts of nonactivated bovine neutrophils obtained by centrifugation

at 140 000 g for 1 h of sonicated homogenates of bovine neutrophils in NaCl/Pi supplemented with antiproteases Crude cytosol (100 mg protein) was chromatographed successively on a hydroxyapatite column (7 cm· 2 cm) equilibrated in 20 mM Hepes pH 7.5, and on a DEAE cellulose column (20· 1 cm) equilibrated in the same buffer Elution from the first column (2.4 mL fraction) and the second column (3 mL fractions) was by linear gradients of 0–0.3 M potassium phosphate and 0–0.5M NaCl in 20 mMHepes, respectively Before application to the DEAE cellulose column, eluted fractions from the hydroxylapatite column were diluted twice with Hepes buffer supplemented with antiproteases Isoelectric focusing

of relevant fractions from preceding chromatographies was carried out in a sucrose gradient (5–40%) supplemented with 0.3% ampholines pH 3–10 The applied voltage was

1500 V for 20 h Fractions of 2.5 mL were recovered and analyzed for protein content The presence of S100A9, p47phox, p67phox and Rac in eluted fractions at each of the three steps of the protein fractionation was determined by immunoblotting

Immunoprecipitation assay The cytosolic extract from nonactivated bovine neutrophils was incubated for 45 min with 40 lL of protein

A–Sepharose incubated before hand with 30 lL of

anti-p67phox sera After three 1-mL washes in NP40 buffer, the

immunocomplex was solubilized in Laemmli

depolymeri-zation buffer and subjected to SDS/PAGE, using a 15%

polyacrylamide gel Proteins were transferred onto a

nitrocellulose membrane The membrane was subjected to

Western blotting using antisera to p67phox, p47phox, Rac2

and S100A9

Assay of NADPH oxidase activity after

oxidase activation

The dormant NADPH oxidase of neutrophil membranes

was activated by mixing neutrophil plasma membranes and

the recombinant cytosolic phox proteins, p67phox,

p47phox, GTPcS-loaded Rac2, MgSO4 and an optimal

amount of arachidonic acid determined for each assay of

oxidase activation [12] The rate of O2 production by the

activated NADPH oxidase was calculated from the rate of

the superoxide dismutase-inhibitable reduction of

ferricy-tochrome c (100 lM) in NaCl/Pisupplemented with 300 lM

NADPH at 20C More than 98% of cytochrome c

reduction was sensitive to superoxide dismutase NADPH

oxidase activity was also assayed by polarographic

meas-urement of the rate of O2 uptake at 20C w ith a Clark

electrode at a voltage of 0.8 V All experiments were carried

out at least twice

R E S U L T S

Coimmunoprecipitation and copurification

of S100A8/A9 and the cytosolic factors

of oxidase activation

The specificity of antibodies mostly used in the present study

(anti-p67phox, anti-p47phox, anti-Rac2 and anti-S100A9)

was ascertained by Western blotting of bovine neutrophil

cytosol (Fig 1, tracks a–d) Antibodies to p67phox,

p47phox and S100A9 were used to analyse by Western

blotting cytosolic proteins recovered by

immunoprecipita-tion with anti-p67phox antibodies (see Materials and

methods) The immunoblot shown in Fig 1, track e,

demonstrated the presence of p67phox, p47phox as well

as S100A9 in the immunoprecipitate

Interaction of S100A8/A9 with the cytosolic phox

proteins was corroborated by protein fractionation As

illustrated in Fig 2, the fractionation experiment comprised

three steps that differ in their principle, namely two

successive chromatographies on hydroxyapatite and DEAE

cellulose, followed by isoelectric focusing The first step

involved a chromatography of crude cytosol of bovine

neutrophils on a hydroxyapatite column The column was

eluted by a linear gradient of potassium phosphate

(0–0.3M) in Hepes buffer (Fig 2A) The S100A8/A9

protein and the cytosolic phox proteins were

immunode-tected by immunodot blot with antibodies, directed against

S100A9, p67phox, p47phox and Rac In the case of Rac, we

used a mixture of anti-Rac1 and anti-Rac2 sera A small

amount of p47phox came off by washing with Hepes

Elution of the hydroxylapatite column by the phosphate

gradient yielded two distinct pools of proteins of interest

The first one (HTP I fractions 14–17) contained the bulk of

S100A8/A9 and a small, but significant portion of p67phox,

p47phox and Rac The S100A8/A9 was in large excess with respect to the cytosolic phox proteins, the disproportion in these two categories of proteins reflecting probably their disproportion in crude neutrophil cytosol In fact S100A8/ A9 represents 10% to 20% of the cytosolic protein content

of bovine neutrophil [13], compared to less than 0.5% for p47phox and p67phox [19] The second pool (HTP II fractions 18–21) consisted of the remainder of S100A8/A9, accompanied by a majority of p67phox, p47phox and Rac Analysis of the protein distribution in the two HTP pools by SDS/PAGE followed by Coomassie blue staining revealed a discrete number of proteins, including S100A8 and S100A9 proteins (insert, Fig 2A) In the HTP I pool, the two components of the S100A8/A9 complex appeared to be present in nearly equal amounts, based on SDS/PAGE and staining by Coomassie blue In contrast, in the HTP II pool the relative amount of the S100A9 protein still detectable by immunodot blot was noticeably lower than that of S100A8 suggesting dissociation of the heterodimer S100A8/A9 Rac was uniformly distributed in the two HTP pools in contrast

to p67phox and p47phox that were more concentrated in the second HTP pool than in the first one

The two HTP pools were further processed separately After dilution with Hepes buffer, the proteins of the HTP I pool were subjected to chromatography on DEAE cellulose (Fig 2B) The column was washed with Hepes and then eluted with a linear gradient of NaCl (0–0.5M) in Hepes About half of p47phox was recovered in fractions 14–16, at concentrations of NaCl below0.15M, in the virtual absence

of p67phox, Rac and S100A8/A9 The following fractions 17–20 eluted between 0.15Mand 0.25M NaCl contained most of the S100A8/A9 and p67phox proteins associated with Rac and the remainder of p47phox At concentrations

of NaCl higher than 0.25M, the remainder of S100A8/A9 eluted in the absence of the cytosolic phox proteins Fractions 17–20 were assembled to be further processed

Fig 1 Coimmunoprecipitation of the cytosolic phox proteins p67phox, p47phox andRac, andthe S100A8/A9 protein The proteins from nonactivated bovine neutrophil cytosol, recovered from the immuno-complex (see Materials and methods), were resolved by SDS/PAGE, transferred onto nitrocellulose and detected with specific antisera to p67phox, p47phox and S100A9 (track e) Hc and Lc indicate the positions of the heavy and light chains of IgG Tracks a–d correspond

to the Western blotting of p47phox, p67phox, Rac2 and S100A9 following SDS/PAGE of neutrophil cytosol.

by isoelectric focusing Aliquots of proteins not retained on

DEAE cellulose (NR), fractions 17–20 (DEAEI) and

fractions 23–25 (DEAEII) were subjected to SDS/PAGE

Coomassie blue staining of the gel shows an enrichment

of the DEAEI fraction in the two components of the

S100A8/A9 protein with molecular masses of 7 kDa and

23 kDa and the disappearance of a 42–43 kDa protein that

was recovered in the DEAEII fraction and was most likely

actin (insert, Fig 2B)

Isoelectric focusing of the DEAEI cellulose fraction was

carried out in a 5–40% sucrose gradient supplemented with

ampholines pH 3–10 (Fig 2C) The bulk of S100A8/A9

and also that of p67phox focused between pH 7.7 and 6.2

(fractions 18–24) These fractions contained only a minor

amount of p47phox The protein pattern was characterized

by a major peak (FII) with a mean pI value of 6.7 (fractions

21–24), and a shoulder (FI) (fractions 18–20) with a mean pI

value of 7.4, where Rac was concentrated It is noteworthy

that the pI values of p67phox and p47phox deduced from

the isoelectrofocusing experiment are significantly different

from the theoritical pI values calculated for the isolated

protein, namely 5.9 for p67phox and 9.1 for p47phox This

is readily explained by the association of these proteins in a complex As in the preceding fractionations, eluted proteins were analyzed by SDS/PAGE, followed by Coomassie blue staining At this stage of the protein fractionation the two components of the S100 protein, S100A8 and S100A9 appeared to be the two major proteins (insert a, Fig 2C) Separate immunodot blots carried out with anti Rac1 and anti Rac2 antibodies revealed that more than 90% of Rac was the Rac2 isoform, essentially concentrated in the F1 pool (insert b, Fig 2C) P40phox that accompanied S100A8/A9, p67phox, p47phox and Rac in the DEAE cellulose eluates focused at a position corresponding to a mean pI of 5.0, which corresponds to that of its free form, apart from S100A8/A9 and p67phox

All the preceding fractionation experiments were conduc-ted with the HTP I pool The same procedure was applied to the HTP II pool Chromatography on DEAE cellulose allowed the separation of a fraction which came off with the washing medium (nonretained fraction NR) and contained most of p47phox, about the third of the S100 protein and a

Fig 2 Copurification of the cytosolic phox proteins, p67phox, p47phox andRac, andthe S100A8/A9 protein At each step of the fractionation experiment, the presence of S100A9, p67phox, p47phox and Rac1/2 in the eluted fractions was analyzed on 2 lL aliquots with specific antisera by dot blotting (bottom of the figure) In all panels, the inserts correspond to SDS/PAGE of fractions of interest followed by Coomassie blue staining, except the insert b in (C) which corresponds to dot blots of fractions FI and FII with antibodies to Rac1 and Rac2 The positions of S100A9 (apparent mass 23 kDa) and S100A8 (apparent mass 8 kDa) on the gels are indicated by arrows (insert in the following panels) (A) Chroma-tography of neutrophil cytosol (100 mg protein) on hydroxyapatite Absorption at 280 nm was recorded Two sets of eluates, differing by their relative contents in S100A8/A9 and p47phox, identified by immunodot blot, were assembled in pools, HTP I and HTP II B, Chromatography of the HTP I pool (20 mg protein) on DEAE cellulose (cf Materials and methods) Absorption at 280 nm was recorded Nonretained eluates (NR) and eluates corresponding to the highest concentrations of S100A9 (DEAE I fraction) were pooled separately (C) Isoelectric focusing of the DEAE

I fraction (B) (see Materials and methods) Eluted fractions were analyzed for protein content by the BCA technique Fractions corresponding to the shoulder in the elution pattern (F I) and the peak (F II) were pooled The presence of Rac1 and Rac2 in the two fractions was detected by immuno-dot blotting (insert b) (D) Chromatography of the HTP II pool (13 mg protein) on DEAE cellulose Same procedure as in (B) Absorption at 280 nm was recorded Two sets of eluates of interest were recovered, corresponding to nonretained proteins (NR) and to eluates enriched in S100A8/A9 (DEAE III fraction) (E) Isoelectric focusing of the NR pool (D) Same procedure as in (C).

small amount of p67phox (Fig 2D) This fraction was

devoided of Rac The remaining S100 protein, associated

with p67phox and Rac (DEAE III fraction) but not with

p47phox, was eluted with an NaCl gradient between 0.20M

and 0.38M NaCl Analysis by SDS/PAGE followed by

Coomassie blue staining (insert Fig 2D) showed that the

NR fraction was enriched in S100A8 accompanied by traces

of S100A9, still immunodetectable by dot blot, whereas the

DEAE III fraction contained the two components of the

heterodimer S100A8/A9 The DEAE III fraction was

characterized by an enrichment in S100A8/A9 and p67, like

the DEAE I fraction (panel B); it was not further processed

The NR fraction was subjected to isoelectric focusing

(Fig 2E) About half of p47phox (Fraction FIII)

comigrat-ed with the S100 protein and focuscomigrat-ed between pH 7.3 and

pH 6.2, i.e in a zone of pH quite remote from the highly

basic pI of free p47phox The S100 protein present in

fraction FIII consisted essentially of the S100A8 component

as shown by SDS/PAGE (insert, Fig 2E) with traces of

S100A9 revealed by immunoblot Rac was not detectable in

this fraction The remainder of p47phox free of other

proteins focused at pH of about 9.5, close to the theoritical

pI value, 9.1, of the isolated p47phox in accordance with

the large excess of basic residues in the molecule

The three-step fractionation described above led to the

following conclusions 1 Among the cytosolic factors of

oxidase activation, p67phox in association with Rac exhibits

the higher propensity to interact with the heterodimeric

S100A8/A9 protein as shown by their comigration from

the HTP I pool to the isoelectric focusing step (Fig 2A–C)

2 In contrast, in the HTP II pool (Fig 2A) the heterodimer S100A8/A9 was partly dissociated, and in the derived fractions (fraction NR, Fig 2D, and fraction FIII, Fig 2E) the S100A8 subunit comigrated preferentially with p47phox In summary, the fractionation experiment corro-borated the results of the coimmunoprecipitation experi-ment In addition, it suggested some subtle dynamics in the organization of a complex between S100A8/A9 and the cytosolic factors of oxidase activation which were substan-tiated by experiments on the effect of S100A8/A9 on the kinetics of elicited oxidase activity to be described below

Kinetics parameters of the potentiating effect of S100A8/A9 on oxidase activation in cell-free system The effect of S100A8/A9 purified to homogeneity from bovine neutrophil cytosol [15] on the kinetics of oxidase activation was investigated through the use of a semi-recombinant cell-free system It was first ascertained that S100A8/A9 added to neutrophil membranes in the absence

of cytosol was unable to promote oxidase activation (not shown) The activation medium consisted of a membrane fraction enriched in flavocytochrome b, arachidonic acid and the recombinant cytosolic factors of oxidase activation The cytosolic factors p47phox and GTP-cS-loaded Rac2 were added to the medium in a 10-fold excess with respect to the membrane-bound flavocytochrome b and the amount

of p67phox was varied as shown in Fig 3A In the absence

Fig 3 Effect of the relative concentrations of the cytosolic phox proteins and S100A8/A9 in the activation medium on the elicited NADPH oxidase activity (A) Effect of varying the molar ratio of p67phox to p47phox and Rac2 in the absence of S100A8/A9 on the elicited oxidase activity Oxidase activation and elicited oxidase activity were performed in a 96-well microtiter plate In each well p67phox (amounts varying from 2 pmol to

90 pmol), p47phox (10 pmol) and GTPcS-preloaded Rac2 (10 pmol) in 40 lL NaCl/P i were incubated for 10 min at 20 C with neutrophil membranes (4 lg protein corresponding to 1 pmol of heme b) Each well contained a different amount of arachidonic acid ranging from 0 to

7 lmolÆmg membrane protein)1 Oxidase activity was initiated by addition of NADPH and cytochrome c in NaCl/P i (200 lL) at the final concentrations of 300 l M and 100 l M , respectively Cytochrome c reduction was followed at 550 nm and recorded for 3 min A complementary experiment carried out in the presence of 10 lg of SOD showed that more than 98% of the reduction of cytochrome c was inhibited by SOD, therefore corresponding to the production of O 2 The oxidase activity was expressed in mol of O 2 generated per s and per mol of membrane-bound flavocytochrome b The traces correspond to the different amounts of p67phox present in the activation medium: j 2 pmol, 5 pmol, m 10 pmol,

20 pmol, d 40 pmol, n 70 pmol, + 90 pmol (B) Effect of the presence of S100A8/A9 in the activation medium on the elicitated oxidase activity Same conditions as in (A) Curve d corresponds to the control in the absence of S100A8/A9 (experiment shown in (A) carried out with 40 pmol of p67phox) Curve shows the effect of S100A8/A9 (64 pmol) added in the activation medium.

of S100A8/A9, the elicited oxidase activity attained a

maximal value (about 100 mol of O2/s/mol

flavocyto-chrome b, assuming two hemes b per flavocytoflavocyto-chrome b)

for a molar ratio of p67phox to p47phox and Rac2 of 7 The

peak of activity at the optimal concentration of arachidonic

acid (1 lmol of arachidonic acid per mg membrane protein)

for the lowconcentrations of p67phox had a tendancy to be

replaced at high concentrations of p67phox by a plateau

At saturating concentrations of p67phox, the plateau of

activity corresponded to a broad range of arachidonic acid

concentrations extending from 1 to 2.5 lmolÆmg membrane

protein)1 At a nonsaturating concentration of p67phox

(molar ratio of p67phox to p47phox and Rac of 4), S100A8/

A9 enhanced the elicited oxidase activity by more than 40%

(Fig 3B), with a shift of the enzyme activity from 86 mol

O2 per s per mol flavocytochrome b to 126 mol O2 per s

per mol flavocytochrome b, i.e to a higher value than that

measured at saturating concentrations of p67phox in the

absence of S100A8/A9 (100 mol O2 per s per mol heme b

(Fig 3A) In addition, the shape of the activity curve as a

function of the concentration in arachidonic acid was

different when S100A8/A9 was present in the activation

medium In the presence of S100A8/A9, a well defined peak

of activity could be observed for a concentration of

arachidonic acid of 1.2 lmolÆmg membrane protein)1 This

suggested that S100A8/A9 was able to overcome a

limita-tion in the full expression of oxidase activalimita-tion, possibly

through the control of an appropriate organization of the

cytosolic factors favoring their productive interaction with

the membrane-bound flavocytochrome b As S100A8/A9 is

a Ca2+binding protein, the effect of 1 mMCa2+was tested

No modification of the enhancement of oxidase activation

by S100A8/A9 was observed It is possible that, due to its high affinity for Ca2+, S100A8/A9 had a full charge of bound Ca2+

To check whether the potentiation of oxidase activation

by S100A8/A9 was due to a change in affinity of the flavocytochrome b for NADPH and O2or to an increase

in its turnover, we measured both the rate of O2 production (Fig 4A) and that of O2 uptake (Fig 4B) at optimal concentrations of arachidonic acid and at different concentrations of NADPH and O2 The double reciprocal plots of the elicited oxidase activity vs NADPH or O2 concentration shows that the S100A8/A9 protein enhanced the turnover of flavocytochrome b, but not its affinity for NADPH and O2

We pursued the exploration of the kinetic parameters of oxidase activation by measuring the elicited oxidase activity after incubation of neutrophil membranes with increasing concentrations of the phox cytosolic proteins and S100A8/A9 The molar ratio of p67phox to p47phox and Rac was maintained at a value of 4, i.e a nonsaturating concentration with respect to p47phox and Rac2 (cf Fig 3A) The concentration of flavocytochrome b was maintained at a fixed value, and the molar ratio of the cytosolic phox protein (p67phox taken as reference) to flavocytochrome b was varied up to 40 (Fig 5) Optimal arachidonic acid concentration was carefully determined for all experimental conditions Inspection of the direct plots of the elicited oxidase activity indicated an enhancing effect of

Fig 4 Kinetic parameters of elicitedNADPH oxidase after activation in the presence or absence of S100A8/A9 Membranes from bovine neutrophils (290 lg protein equivalent to 110 pmoles of flavocytochrome b) were incubated at 20 C with p67phox (1480 pmol), p47phox (370 pmol), GTPcS-loaded Rac2 (370 pmol) and arachidonic acid (1.2 lmol/mg membrane protein) in a volume of 450 lL (d) A parallel incubation was carried out in the presence of 2500 pmol of S100A8/A9 (s) Following incubation, 10 lg protein aliquots were withdrawn for measurement of O 2 generation by the superoxide dimutase inhibitable reduction of cytochrome c (A), and 100 lg protein aliquots for measurement of O 2 uptake with a Clark electrode (B) The rate of O 2 production expressed in lmol generated per min and per mg of membrane protein was measured in a spectro-photometric cuvette filled with 2 mL of NaCl/P i supplemented with 100 l M cytochrome c and different concentrations of NADPH In the case of

O 2 uptake, the O 2 concentration of the medium was lowered to 80–90 l M by controlled bubbling of nitrogen, and O 2 uptake was initiated by addition of NADPH at the saturating concentration of 300 l M The rates of O 2 uptake expressed in lmol of O 2 consumed per min and per mg of membrane protein was deduced from the slopes of the tangents to the oxygraphic trace, at different concentrations of O in the medium.

S100A8/A9 on the rate of O2 production (curves a to e,

Fig 5) This effect was more marked at higher

concentra-tions of the phox cytosolic proteins in the activation medium

In the absence of S100A8/A9, the reciprocal plots shown in

the insert of Fig 5 clearly departed from linearity above a

threshold concentration of the cytosolic phox proteins for

which the elicited oxidase activity was about half of the

theoretical maximal activity When S100A8/A9 was present

together with the phox cytosolic proteins, the reciprocal plots

become more linear Linearity increased with the increase in

the molar ratio of S100A8/A9 to the cytosolic phox proteins

Taking p67phox as reference for the cytosolic phox proteins,

it ensues that, at a molar ratio of S100A8/A9 to p67phox of

about 2, the kinetics of the elicited oxidase were virtually

linear In brief, in the absence of S100A8/A9, nonmichaelian

kinetics were observed for the rate of production of O2 by the membrane-bound flavocytochrome b activated by increas-ing amounts of the phox cytosolic proteins Addition of S100A8/A9 renders the kinetics michaelian The fact that michaelian kinetics are attained, using a ratio of S100A8/A9

to p67phox as lowas 2, suggests interaction between the two proteins within a complex

Effect of S100A8/A9 on the time course

of oxidase activation NADPH oxidase activation in a cell-free system is a process which at room temperature reaches its maximum after several minutes at 20C In the following experiment, we determined the effect of S100A8/A9 on the time course of oxidase activation For technical convenience, the time course of oxidase activation was analyzed by measuring the elicited oxidase activity of membrane-bound flavocyto-chrome b in terms of O2uptake in an oxygraphic cell The cytosolic phox proteins (p67phox, p47phox and GTPcS-loaded Rac2 present in a molar ratio of 3/1/1) were left in contact for 5 min at 20C with or without the S100A8/A9 protein (molar ratio of S100A8/A9 to p67phox adjusted to a value of 3.3) It should be noted that, like in the experiment

of Fig 3, the concentration of p67phox was not saturating with respect to p47phox and Rac2 Then the membrane fraction was added, immediately followed by arachidonic acid at the optimal concentration of 1.5 lmolÆmg)1 of membrane protein (insert A, Fig 6) The elicited oxidase activities at given times were measured as rates of O2uptake deduced from the slopes of the recorded curve of O2 concentration in the oxygraphic cuvette The oxidase activity values were plotted as a function of the period of time that elapsed from the addition of arachidonic acid During the first minute, the O2 uptake was faster in the absence of S100A8/A9 than in its presence However, after

30 s, the increase in O2uptake slowed down in the absence

of S100A8/A9 whereas in its presence, it remained more sustained, which resulted in the intersection of the two representative curves We reasoned that, in the presence of the cytosolic phox proteins and arachidonic acid, the inactive flavocytochrome b (Fbi) is transformed into the active catalyst (Fba), and that the elicited oxidase activity measured here by the rate of O2uptake depends directly on the concentration of Fba It followed that the oxidase activity (v) should reach a maximal value (V) when the totality of the flavocytochrome (Fbt) is activated On the basis of this hypothesis, the first order equation

v¼ V (1–e–kt) was used to describe the rate of flavocyto-chrome b activation In the case of preincubation of the cytosolic phox proteins with S100A8/A9, the experimental data fitted well with the theoretical curve derived from the above first order reaction, yielding a V-value of 300 nmol

O2uptake per min and per mg of membrane protein, and a rate constant k of 0.012 s)1 (Thick line in Fig 6) In contrast, in the absence of S100A8/A9, the experimental curve could not be fitted with a single first order reaction curve (thin line in Fig 6) However, a good fit was found by using the sum of two first order equations, the first one being characterized by a k-value of 0.112 s)1and a V of

110 nmol O2per min and per mg of membrane protein, and the second by a k-value of 0.008 s)1and a V of 111 nmol O2 per min and per mg of membrane protein (insert B, Fig 6)

Fig 5 Potentiation of neutrophil oxidase activation by S100A8/A9

depends on the concentration of the cytosolic phox proteins The oxidase

activation step was carried out in a 96-well microtiter plate Fixed

amounts of neutrophil membranes (8.7 lg protein corresponding to

2 pmol flavocytochrome b) were incubated for 10 min at 20 C w ith

different amounts of cytosolic phox proteins (p67phox, p47phox and

Rac2 being in a molar ratio of 4/1/1) For the sake of simplicity, only

the molar ratio of p67phox to flavocytochrome b is given in abscissa.

Oxidase activation was carried out in the presence of different fixed

amounts of S100A8/A9 [0 (a), 0.25 (b), 0.48 (c), 1.00 (d) and 2.06 (e)

mol/mol 67phox] In all cases, the optimal amount of arachidonic acid

w as determined and used to analyze the effect of S100A8/A9 on

oxidase activation After an incubation of 10 min at 20 C, the oxidase

activity was initiated by the addition of 100 l M cytochrome c and

300 l M NADPH in NaCl/P i (final volume 200 lL) The figure shows

the direct plots of the elicited oxidase activity (expressed in lmol of O 2

generated per min and per mg of membrane protein) at increasing

amounts of phox proteins, taking as reference p67phox and at different

fixed amounts of S100A8/A9 (a to e) (insert, reciprocal plots of the

data).

It is noteworthy that the sum of these two rates, i.e.

221 nmol O2per min and per mg of membranes protein is

lower than the rate of O2uptake measured in the presence of

S100A8/A9, namely 300 nmol O2per min and per mg of

membrane protein These results led us to hypothesize that

in the absence of S100A8/A9 the cytosolic phox proteins are

organized in at least two pools probably in slow equilib-rium, one of which is much more efficient than the other in oxidase activation The effect of S100A8/A9 would be to bind and rearrange the totality of the cytosolic phox proteins in a complex capable of activating optimally flavocytochrome b Association of S100A8/A9 with the cytosolic phox proteins might however, bring some steric constraint, so that oxidase activation during the first minute proceeds more slowly in the presence of S100A8/A9 than its absence (Fig 6) As the maximal oxidase activity measured

in the presence of S100A8/A9 was significantly greater than

in its absence, we can tentatively conclude that S100A8/A9 enhances the recruitment of the cytosolic phox proteins to the membrane-bound flavocytochrome b and (or) stabilizes their interaction

D I S C U S S I O N

In this study, we used two different approaches to explore the mechanism by which S100A8/A9 enhances the NADPH oxidase activation In the first approach, we demonstrated the propensity of S100A8/9 to interact with the cytosolic phox proteins, using coimmunoprecipitation and protein fractionation The fractionation experiment pointed to the preferential association of the S100A8/A9 heterodimer with p67phox The primacy of p67phox in oxidase activation has been recently emphasized [20,21] We therefore propose that the set of the cytosolic phox proteins is associated with the heterodimer S100A8/A9 via p67phox, and that this associ-ation is determinant in the potentiassoci-ation of oxidase activa-tion On the other hand, the physiological meaning of the association of p47phox with the S100A8 protein separated from its S100A9 partner (Fig 1E) remains to be assessed

In the second approach, we analyzed the effect of S100A8/A9 on the kinetics of oxidase activation The oxidase activity elicited in the cell-free system was the reflect

of the extent of oxidase activation The experiment of Fig 4 shows that S100A8/A9 enhances the turnover of the flavocytochrome b, not its affinity for NADPH or O2 This effect is in line with an increase in the productive interaction

of the cytosolic phox proteins with flavocytochrome b Exploration of the kinetics of oxidase activation (Figs 3 and 5) revealed also that, above a treshold concentration of the cytosolic phox proteins and in the absence of S100A8/ A9, the elicited oxidase activity does not followmichaelian kinetics Thus, at relatively high concentrations, the cyto-solic phox proteins appear not to interact productively any more with their target sites on flavocytochrome b in the absence of S100A8/A9 This observation corroborates the fact that, in absence of S100A8/A9, the time course of oxidase activation may be fitted by the sum of two first order kinetics (Fig 6) When the cell-free medium was supplemen-ted with S100A8/A9, michaelian kinetics were restored and

an homogeneous first order reaction was found for the production of O2 Through its association with cytosolic phox proteins, the S100A8/A9 protein might act as a scaffold protein, that favors the organization of the phox proteins in a reactive complex and helps this complex to interact in a productive manner with flavocytochrome b to activate its dormant oxidase activity It might also simply favor the delivery of bound arachidonic acid to the oxidase complex The S100A8/A9 heterodimer specifically binds long-chain unsaturated fatty acids and, most particularly, arachidonic

Fig 6 Effect of S100A8/A9 on the time course of oxidase activation.

Oxidase activity was measured by the rate of O 2 uptake with a Clark

electrode (cf Materials and methods) The cytosolic factors (CF)

p67phox (420 pmol), p47phox (140 pmol) and GTPcS-preloaded

Rac2 (140 pmol) were incubated together with 2.5 m M MgSO 4 , 15 l M

GTPcS and 300 l M NADPH for 5 min before addition of neutrophil

membranes (115 lg protein corresponding to 14 pmol of

flavocyto-chrome b), final volume 1.5 mL Oxidase activation was initiated

by addition of arachidonic acid (1.5 lmolÆmg membrane protein)1)

immediately after addition of membranes (insert A) and O 2 uptake

resulting from the elicited oxidase activity was recorded The rates of

O 2 uptake (lmol of O 2 consumed per min and per mg of membrane

protein) were calculated as in Fig 4 A parallel assay was run under

similar conditions except that S100A8/A9 was present with the phox

proteins during the 5 min incubation preceding the addition of

neu-trophil membranes (molar ratio of S100A8/A9 to p67phox, 3) When

oxidase was activated in the presence of S100A8/A9, the experimental

rate values plotted against the period of time elapsing from addition of

arachidonic acid (d) could be fitted with a theoretical curve (thick line)

derived from a first order reaction from which a maximal rate value

and a rate constant could be calculated (see text for details) In the

absence of S100A8/A9, the experimental rate values (s) could not be

fitted with a curve (thin line) derived from a first order reaction The fit

was however, possible by combining two first order reaction curves

(insert B), from which maximal rates and rate constants were

calcu-lated (see text for details).

acid in a calcium-dependent manner, whereas the S100A8

or S100A9 homodimers lack this property [6–8] Data

obtained through different experimental approaches suggest

that arachidonic acid is an in vivo activator of the NADPH

oxidase Through the use of a model of cytosolic

phos-pholipase A2 (cPLA2) deficient phagocyte-like cells, it was

demonstrated that cPLA2-generated arachidonic acid is

essential for the activation of NADPH oxidase [22] It was

also reported that the large subunit of flavocytochrome b,

gp91phox, constitutes an arachidonic acid-activated proton

channel (reviewed in [23,24]) In addition, it is noteworthy

that arachidonic acid at lowconcentration induces the

transition of the heme iron of flavocytochrome b from an

inactive hexacoordinated form to a pentacoordinated form

capable of binding O2[25] In a previous study on oxidase

activation carried with neutrophil membranes and crude

cytosol [12], we found that in the presence of relatively high

concentrations of arachidonic acid, the increase in the

turnover of NADPH oxidase depended on the amount of

the cytosolic fraction present in the cell-free system, in other

words of the amount of cytosolic phox proteins capable of

binding to flavocytochrome b The S100A8/A9 present in

crude cytosol and artificially loaded with arachidonic acid

was probably beneficial to this process Because of its high

concentration in neutrophils, the S100A8/A9 protein is

effectively a potential reservoir of arachidonic acid of high

capacity Its translocation to the plasma membrane [13]

together with p67phox, p47phox and Rac at the onset of the

oxidase activation would be consistent with the delivery of

arachidonic acid to the membrane-bound

flavocyto-chrome b

The physiological significance of the effect of S100A8/A9

on the NADPH oxidase is attested by our previous finding

that phenylarsine oxide (PAO) added at lowconcentrations

to neutrophils was able to potentiate oxidase activation by

phorbol ester Through the use of a photolabeled derivative

of PAO, the protein S100A8/A9 was identified as the target

responsible for the enhanced oxidase activation In the

present study, we show that S100A8/A9 interacts with the

cytosolic phox proteins, and we describe some of the

S100-dependent modifications of the kinetics of the elicited

oxidase The primary effect of S100A8/A9 in cell-free

system was to overcome a limitation in the full expression of

oxidase activation at subsaturating concentrations of the

cytosolic phox proteins In other words, S100A8/A9 does

not basically alter the functioning of the NADPH oxidase

It essentially modulates some of the kinetic features of

this enzyme In this context, it is interesting to note that

S100A8/A9 which is present at the concentration of 3 mMin

neutrophils [3] is virtually absent in cells like resident

macrophages that nevertheless are able to mount an efficient

respiratory burst [26] A different molecular environment in

the two types of cells might be responsible for this apparent

lack of correlation

Components of the neutrophil cytoskeleton, namely actin

and coronin have been reported to interact with the

cytosolic phox proteins, the b actin with p47phox [27] and

coronin with p40phox [28] Most significantly,

abnormali-ties in O2 production have been found in neutrophils of a

patient carrying a mutation in nonmuscle actin [29] These

data and ours on S100A8/A9 strongly suggest that there

exists a complex array of interactions between the classical

phox components of the oxidase complex and other

molecular protein species in phagocytic cells The function

of these ancillary species might be to regulate the kinetics of the production of O2 in the respiratory burst and to segregate activated oxidase complexes in the phagosomal membrane

A C K N O W L E D G E M E N T S

We thank Dr J Willison for careful reading of the manuscript and Mrs Bournet-Cauci for excellent secretarial assistance, Dr Marie-Claire Dagher for the gift of recombinant cytosolic phox proteins, Drs Anne-Christine Dianoux and Marie-Jose´ Stasia for anti S100A9 antibodies, and Dr Je´roˆme Garin for protein analysis This work was supported by funds from the Centre National de la Recherche Scientifique, the Commissariat a` lEnergie Atomique, the Universite´ Joseph Fourier-Grenoble I, and partially by a research grant from the Association pour

la Recherche sur le Cancer (9996).

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