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

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 5092 CEA-CNRS-UJF), De
´
partement Re
´
ponse
et Dynamique Cellulaires, CEA-Grenoble, France
In a previous study, the S100A8/A9 protein, a Ca
2+
-and
arachidonic acid-binding protein, abundant in neutrophil
cytosol, was found to potentiate the activation of the redox
component of the O
2
–
generating oxidase in neutrophils,
namely the membrane-bound flavocytochrome b,bythe
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 O
2
.Inthe
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 O
2
–
;
neutrophils; phox proteins; S100A8/ A9 protein.
The heterodimeric Ca
2+
-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 O
2
–
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 O

2
–
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)with
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:+33438785185,Tel.:+33438783476,

E-mail:
Abbreviations: PMSF, phenylmethanesulfonyl fluoride.
(Received 14 February 2002, revised 30 April 2002,
accepted 17 May 2002)
Eur. J. Biochem. 269, 3246–3255 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03002.x
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 fractionation 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.
MATERIALS AND METHODS
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/P
i
[12]. The saline buffer (2.7 m
M
KCl, 136.7 m

M
NaCl, 1.5 m
M
KH
2
PO
4
and 8.1 m
M
Na
2
HPO
4
,pH7.4)
was supplemented with antiproteases: 0.1 m
M
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 m
M
ammonium bicarbonate pH 8.0, and
50% acetronitrile/50% 25 m
M
ammonium bicarbonate,
pH 8.0. A final wash with pure water was performed before
complete dehydratation in a vacuum dryer. In gel Ôtryptic
digestionÕ was performed for 4 h at 37 °Cin10 lLof25 m
M
ammonium bicarbonate pH 8.0 with 0.5 lgoftrypsin.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 ( 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 20 °C, Rac2 was
prechargedinGTPcS by incubation at 20 °Cinthe
presence of 15 l
M
GTPcSand4m
M
EDTA. Incubation
was terminated after 10 min by the addition of 10 m
M
MgSO
4
. 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
A. C. Dianoux UMR 5092 CEA-Grenoble and
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 m
M
.
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/P
i
supplemented with antiproteases.
Crude cytosol (100 mg protein) was chromatographed
successively on a hydroxyapatite column (7 cm · 2cm)
equilibrated in 20 m
M
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 0–0.5
M
NaCl in 20 m
M
Hepes, 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 lLofprotein
Ó FEBS 2002 S100A8/A9 is a partner of p67phox in neutrophils (Eur. J. Biochem. 269) 3247
A–Sepharose incubated before hand with 30 lLofanti-
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, MgSO
4
and an optimal
amount of arachidonic acid determined for each assay of
oxidase activation [12]. The rate of O
2
–
production by the
activated NADPH oxidase was calculated from the rate of
the superoxide dismutase-inhibitable reduction of ferricy-
tochrome c (100 l
M
)inNaCl/P
i
supplemented with 300 l
M
NADPH at 20 °C. 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 O
2
uptake at 20 °CwithaClark
electrode at a voltage of 0.8 V. All experiments were carried
out at least twice.

RESULTS
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
(0–0.3
M
) 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 (0–0.5
M
) in Hepes.
About half of p47phox was recovered in fractions 14–16, at
concentrations of NaCl below 0.15
M
, in the virtual absence
of p67phox, Rac and S100A8/A9. The following fractions
17–20 eluted between 0.15
M
and 0.25
M
NaCl contained
most of the S100A8/A9 and p67phox proteins associated
with Rac and the remainder of p47phox. At concentrations
of NaCl higher than 0.25
M
, 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 and Rac, and the 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.

3248 J. Doussiere et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 and Rac, and the 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).
Ó FEBS 2002 S100A8/A9 is a partner of p67phox in neutrophils (Eur. J. Biochem. 269) 3249
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 0.20
M
and 0.38
M
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 focused 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 lLNaCl/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 2pmol,
5pmol,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.
3250 J. Doussiere et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of S100A8/A9, the elicited oxidase activity attained a

maximal value (about 100 mol of O
2
–
/s/mol flavocyto-
chrome b, assuming two hemes b per flavocytochrome 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 low concentrations 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
O
2
–
per s per mol flavocytochrome b to 126 mol O
2
–
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 O
2

–
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 activation, 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
aCa
2+
binding protein, the effect of 1 m
M
Ca
2+
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 Ca
2+
, S100A8/A9 had a full charge of
bound Ca
2+
.

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 O
2
or to an increase
in its turnover, we measured both the rate of O
2
–
production (Fig. 4A) and that of O
2
uptake (Fig. 4B) at
optimal concentrations of arachidonic acid and at different
concentrations of NADPH and O
2
. The double reciprocal
plots of the elicited oxidase activity vs. NADPH or O
2
concentration shows that the S100A8/A9 protein enhanced
the turnover of flavocytochrome b, but not its affinity for
NADPH and O
2
.
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 elicited NADPH 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
.TheratesofO
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
2
in the medium.
Ó FEBS 2002 S100A8/A9 is a partner of p67phox in neutrophils (Eur. J. Biochem. 269) 3251
S100A8/A9 on the rate of O
2
–
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 O
2
–
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 low as 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 20 °C. 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 O
2
uptake 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 20 °C 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 O
2
uptake
deduced from the slopes of the recorded curve of O
2
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 O
2
uptake was faster in the
absence of S100A8/A9 than in its presence. However, after
30 s, the increase in O
2
uptake 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 O
2

uptake 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
v ¼ 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
O
2
uptake 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
)1
and a V of
110 nmol O
2
per min and per mg of membrane protein, and
the second by a k-value of 0.008 s
)1

and a V of 111 nmol O
2
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 °Cwith
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
amountsofS100A8/A9[0(a),0.25(b),0.48(c),1.00(d)and2.06(e)
mol/mol 67phox]. In all cases, the optimal amount of arachidonic acid
wasdeterminedandusedtoanalyzetheeffectofS100A8/A9on
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).
3252 J. Doussiere et al. (Eur. J. Biochem. 269) Ó FEBS 2002
It is noteworthy that the sum of these two rates, i.e.
221 nmol O
2
per min and per mg of membranes protein is
lower than the rate of O
2
uptake measured in the presence of
S100A8/A9, namely 300 nmol O
2
per 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.
DISCUSSION

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 potentiation 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 O
2
.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 follow michaelian
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 O
2
–
. 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)couldnotbe
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).
Ó FEBS 2002 S100A8/A9 is a partner of p67phox in neutrophils (Eur. J. Biochem. 269) 3253
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 low concentration induces the
transition of the heme iron of flavocytochrome b from an
inactive hexacoordinated form to a pentacoordinated form
capable of binding O
2
[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 low concentrations
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 m
M
in
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 O
2
–
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 O
2
–
in the respiratory burst and to
segregate activated oxidase complexes in the phagosomal
membrane.
ACKNOWLEDGEMENTS
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

´
roˆ me Garin for protein analysis. This work was supported by
funds from the Centre National de la Recherche Scientifique, the
Commissariat a
`
lÕEnergie 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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