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: jdoussiere@cea.fr 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 (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 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. 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(1997) Cytosolic phox proteins interact and regulate the assembly of coronin in neutrophils. J. Cell Sci. 110, 3071–3081. 29. Nunoi, H., Yamazaki, T., Tsuchiya, H., Kato, S., Malech, H.L., Matsuda, I. & Kanegasaki, S. (1999) A heterozygotous mutation of b actin associated with neutrophil dysfunction and recurrent infection. Proc. Natl Acad. Sci. USA 96, 8693–8698. Ó FEBS 2002 S100A8/A9 is a partner of p67phox in neutrophils (Eur. J. Biochem. 269) 3255 . was further ascertained by amino-acid sequencing, using Edman degra- dation. As S10 0A9 has a blocked N-terminal amino acid, analysis of the protein was. whether the potentiation of oxidase activation by S10 0A8 /A9 was due to a change in affinity of the flavocytochrome b for NADPH and O 2 or to an increase in