J Biol Chem, Vol. 274, Issue 46, 32672-32679, November 12, 1999

The Two Calcium-binding Proteins, S100A8 and S100A9, Are Involved in the Metabolism of Arachidonic acid in Human Neutrophils^*

Claus Kerkhoff^§, Martin Klempt^¶, Volkhard Kaever, and Clemens Sorg

From the  Institut für Experimentelle Dermatologie, 48149 Münster and  Institut für Molekularpharmakologie, Medizinische Hochschule, 30623 Hannover, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81-84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prosta- glandin E₂, thromboxane B₂, leukotriene B₄) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The two myeloid-related proteins, MRP8 and MRP14, belong to the S100 family of calcium-binding proteins (for review, see Refs. 1-4). They are composed of two distinct EF-hands flanked by hydrophobic regions at either terminus and separated by a central hinge region, and they form a heterodimeric complex in a Ca²⁺-dependent manner. Heizmann and Schäfer (5) proposed a new logical nomenclature for these genes based on the physical arrangement of the genes encoding the S100 family on chromosome 1q21. According to this nomenclature, MRP8 and MRP14 are referred to as S100A8 and S100A9, respectively.

Both proteins are expressed in circulating neutrophils and monocytes but are absent in normal tissue macrophages and lymphocytes (6-9). Under chronic inflammatory conditions, such as psoriasis and malignant disorders, they are also expressed in the epidermis (10-12). S100A8 and S100A9 are predominantly localized in the cytoplasm. Upon elevation of the intracellular calcium level they are translocated from the cytosol to cytoskeleton and to plasma membrane (13). At a later time point, they appear as noncovalently associated S100A8/A9 heterodimers on the surface of monocytes (14). The mechanism by which the S100A8/A9 heterodimer penetrates the plasma membrane and how the S100A8/A9 protein complex is anchored into the cell membrane, remains unclear since both proteins lack a transmembrane region. Upon the activation of protein kinase C S100A8/A9 heterodimers are released from human monocytes by a novel secretion pathway that is energy-consuming and depends on an intact microtubule network (15, 16). The presence of enhanced S100A8/A9 levels in sera from patients suffering from a number of inflammatory disorders including cystic fibrosis, rheumatoid arthritis, and chronic bronchitis may indicate an extracellular role for S100A8/A9 (17-19). Recently, Newton and Hogg (20) showed that S100A9 stimulates neutrophil adhesion, and S100A8 reversed the stimulatory effect probably by the formation of the heterodimer. However, the study fails to identify the cell type by which S100A9 is released independently from S100A8.

In areas of acute inflammation, polymorphonuclear leukocytes, expressing the membrane-associated heterodimer S100A8/A9, are the predominant cell type. These cells have been shown to release high amounts of tumor necrosis factor- and interleukin-1, indicating that S100A8/A9 surface expression is restricted to activated or recruited phagocytes (7). These phagocytes perform several host defense functions, such as phagocytosis of invading microorganisms and cell debris, release of proteolytic enzymes, and generation of reactive oxygen metabolites. In addition, they release a number of arachidonic acid-derived eicosanoids, which amplify or perpetuate the acute inflammatory response. This subset of phagocytes is present in acute but absent in chronic inflammatory disorders (14). These findings have led to the assumption that S100A8 and S100A9 affect leukocyte trafficking and display a propagating role in inflammatory responses. Although there are a number of hypotheses, the exact functions of both proteins remain unknown.

Recently, we and others showed that S100A8/A9 heterodimers specifically bind (poly)unsaturated fatty acids in a calcium-dependent manner (21, 22), making them a good candidate for mediating effects of polyunsaturated fatty acids in a calcium-dependent fashion. Therefore, the present study was performed to consider the role of S100A8 and S100A9 in the arachidonic acid (AA)¹ metabolism of neutrophils (for review, see Ref. 23). We investigated the specificity of the S100A8/A9 protein complex toward various fatty acids and AA-derived eicosanoids and its role upon the AA release. We found that S100A8/A9 may serve as an intermediate intracellular reservoir for AA, thereby probably modulating the activities of AA-metabolizing enzymes. Alternatively, the secreted S100A8/A9·AA complex may have an extracellular role whereby S100A8/A9 serves as shuttle protein to reach the target cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of S100A8 and S100A9 from Human Neutrophils-- Human neutrophils were prepared from leukocyte-rich blood fractions ("buffy coat") according to Müller et al. (24). S100A8 and S100A9 were purified as described by van den Bos et al. (25) with some modifications. Prior to use, the proteins were rechromatographed by anion-exchange chromatography using a UnoQ column (Bio-Rad).

Incorporation of [¹⁴C]AA into HL-60 Cells-- Incorporation of [¹⁴C]AA into HL-60 cells was performed analogously to Kerkhoff et al. (26) with some modifications. For differentiation into a neutrophil-like phenotype, HL-60 cells were cultured at a density of 1 × 10⁶ cells/ml in the presence of 1.25% (v/v) Me₂SO for 72 h. Differentiated HL-60 cells were counted, and viability was monitored by trypan blue exclusion. It was always more than 95%. The cells were harvested by centrifugation at 400 × g for 10 min at room temperature and washed three times with RPMI 1640 supplemented with 0.1% (w/v) fatty acid-free bovine serum albumin (BSA).

AA·BSA complexes were formed by the addition of 25 µl of [¹⁴C]AA (specific activity 56 mCi/mmol) to 228 µl of AA (1 mM in ethanol). The solution was dried under N₂, and the fatty acids were resuspended in 2.5 ml of Hepes-buffered RPMI 1640 supplemented with 1% (w/v) fatty acid-free BSA and sonicated at 4 °C for 5 min at 20 watts. The Me₂SO-treated cells were adjusted to a cell density of 2 × 10⁷ cells/ml in Hepes-buffered RPMI 1640/0.1% (w/v) fatty acid-free BSA and labeled at 37 °C for 2 h by the addition of the [¹⁴C]AA·BSA complex (final arachidonate concentration of 10 µM). At the end of labeling cells were washed three times with RPMI 1640/0.1% (w/v) fatty acid-free BSA. For lipid analysis the lipids were extracted according to the method of Bligh and Dyer (27) and analyzed by high performance thin layer chromatography as described by Kerkhoff et al. (26). Typical value range for incorporated [¹⁴C]AA was 100,000 dpm/5 × 10⁶ cells. Under these conditions, more than 90% of the total radioactivity was incorporated into cellular lipids.

For stimulation experiments, 5 × 10⁶ HL-60 cells/900 µl were added to 100 µl of 10 µM A23187, 100 nM PMA, or a combination of A23187/PMA in polypropylene tubes and incubated at 37 °C for different time intervals as indicated. The stimulation was terminated by placing samples on ice followed by centrifugation in an Eppendorff centrifuge at 14,500 rpm for 2 min at 4 °C. Aliquots of the supernatants were used for determination of AA release and immunoprecipitation as described above.

Immunoprecipitation of S100A8/A9-- The S100A8/A9 heterodimer-specific monoclonal antibody 27E10 was purified from hybridoma supernatants using protein G-coupled Sepharose as described by the manufacturer (Amersham Pharmacia Biotech). The immunoprecipitation experiments were performed according to Roth et al. (28) with some modifications. Briefly, aliquots of the cell supernatants were subjected to preadsorption by incubation for 1 h after the addition of 100 µl/ml rabbit IgG (Calbiochem), followed by incubation for 1 h after the addition of 100 µl/ml protein G-Sepharose fast flow (Amersham Pharmacia Biotech). After centrifugation for 10 min at 14,000 × g, 1 µg/ml nonspecific mouse IgG1 or 1 µg/ml monoclonal antibody 27E10 were added to the supernatants and incubated for 1 h. Protein G-Sepharose (30 µl/ml) was added, and samples were further incubated for 1 h. Sepharose was collected by centrifugation, and the supernatants were discarded. The pellets were resuspended with 0.1% SDS and transferred to scintillation vials, and radioactivity was determined after the addition of 5 ml of scintillation liquid in an LKB 1211 Rackbeta counter.

For immunoprecipitation cytosolic proteins (10 µg) were preincubated with 1 µM [³H]AA in the presence of calcium for 1 h at 0 °C, and increasing concentrations of 27E10 were added. After incubation for 1 h at 0 °C, antigen-antibody complex was removed by protein G-Sepharose, washed three times with Lipidex binding assay buffer, and radioactivity was determined. In the supernatant, protein-bound fatty acids were separated from nonbound fatty acids using the Lipidex assay (see below).

Fatty Acid Binding Assay-- Binding of [³H]AA to proteins was carried out as described earlier (29, 30) with some modifications. Instead of Lipidex-1000 from United Technologies Packard, the hydroxyalkoxypropyl dextran type VI (Sigma) was used, which has identical properties to Lipidex-1000 (31). Nonspecific adsorption of proteins to the surface of the 1.5-ml microcentrifuge tubes was prevented by coating the tubes with poly(propylene glycol), average M_r 4,000. Briefly, in an assay volume of 250 µl, 100 pmol of the proteins were incubated in buffer containing 20 mM Tris-HCl, pH 7.4 and 0.01% (w/v) Triton X-100 together with [³H]AA at concentrations as indicated for 1 h at 37 °C in the presence and absence of 5 mM CaCl₂. From these samples 50-µl aliquots were used for determination of actual ligand concentration by means of liquid scintillation counting. Then the mixtures were cooled down for 10 min on ice, and 150 µl of a 50% (v/v) Lipidex suspensions were added. After mixing, the samples were further incubated on ice for 60 min. Equilibrium binding of the fatty acid both to the proteins and to Lipidex was completed within the period of the assay (data not shown). Subsequently the samples were centrifuged at 12,000 × g for 5 min at 0 °C, and 100-µl aliquots of the supernatants were counted after the addition of 4 ml of scintillation fluid. Each value was corrected against a blank determined under identical conditions without protein. The determination of the actual ligand concentration in the tubes before the addition of the Lipidex suspension revealed no significant loss of AA via precipitation. The amount of Lipidex necessary to bind 95% of the AA added was derived from titration experiments in the absence of the protein (data not shown).

Displacement experiments were performed using a fixed concentration of 1 µM [³H]AA, 1 nmol of S100A8/A9, and increasing concentrations of the nonlabeled competitors in the presence of 5 mM CaCl₂. The radiolabeled ligand and the competitors were added simultaneously to the proteins.

The calcium dependence of the AA binding to the proteins was investigated by the addition of increasing concentrations of calcium to 0.5 µM [³H]AA and 100 pmol of the proteins in a total volume of 1 ml. The calcium concentrations were measured by atomic adsorption spectrophotometry.

Gel Filtration Analysis of S100A8 and S100A9-- Before injection into a Superdex-75 column, the proteins (100 µg) were preincubated for 5 min at 37 °C in the absence and presence of 5 mM calcium. The protein elution profiles were obtained by UV absorption at 280 nm, and the different peaks were collected and analyzed by SDS-PAGE. For the analysis of arachidonic acid binding, the proteins (100 µg) were preincubated with 50 pmol of [³H]arachidonic acid (specific activity, 2,800 cpm/pmol) in the presence of 5 mM calcium for 1 h at 25 °C before size-exclusion chromatography (Superdex-75 column). The different peaks were collected, and the radioactivity was determined using a LKB 1211 Rackbeta counter.

Protein Determination-- Determination of protein content was performed according to Smith (32), using BSA as the standard. The concentrations of the purified proteins were accurately determined using the extinction coefficient of 0.998 for S100A8, 0.526 for S100A9, 0.762 for S100A8/A9, or 1.4 M¹ cm¹ for 27E10 at 280 nm, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

S100A8/A9 Exclusively Binds Arachidonic Acid in a Ca²⁺-dependent Manner-- Using proteins either from human or murine origin, two groups have shown independently that S100A8/A9 specifically binds fatty acids in a Ca²⁺-dependent manner. Both studies differ in so far as the protein complex formed by the purified proteins from human keratinocytes binds unsaturated as well as polyunsaturated fatty acids with comparable affinities (22). In contrast, the heterodimer of murine recombinant S100A8 and S100A9 specifically binds arachidonic acid and shows only little affinity toward oleic acid (21). Therefore, we purified S100A8 and S100A9 from human neutrophils and investigated their specificity toward various fatty acids. The S100A8 and S100A9 proteins from human neutrophils were purified and renatured as described under "Experimental Procedures." The native proteins were used in different combinations to analyze whether the individual components of the heterodimer and/or the S100A8/A9 protein complex were able to specifically bind AA. Protein-bound AA was discriminated from unbound fatty acid by the Lipidex assay. We found that both S100A8 and S100A9 were not able to bind the fatty acid either in the absence or presence of calcium. Exclusively, the native S100A8/A9 heterodimer showed fatty acid binding capacity in the presence of calcium (Fig. 1, A and B). The binding of [³H]AA to the S100A8/A9 protein complex was prevented by the addition of EDTA, although the presence of EDTA does not destabilize the protein complex once being formed (33).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Analysis of the fatty acid binding properties of purified S100A8, S100A9, and S100A8/A9. The purified and renatured proteins were incubated at different combinations with 1 µM [3H]AA in the absence (A) or presence (B) of 5 mM calcium. For determination of the calcium-induced AA binding to the protein complex (C), S100A8/A9 (2.4 µg) was incubated with 0.5 µM [3H]arachidonic acid and increasing concentrations of calcium as indicated. The protein-bound fatty acids were separated from nonbound fatty acids using the Lipidex assay. The bars represent data from three independent experiments with duplicate determinations ±S.D.

Next, we investigated the molar concentrations of free Ca²⁺ required for the [³H]AA binding. As shown in Fig. 1C, the S100A8/A9 showed significant AA binding at 0.2 µM calcium, and the AA binding capacity reached a plateau for values greater than 1 µM calcium. An IC₅₀ value of about 0.5 µM calcium was calculated, indicating that the calcium concentration required for AA binding to the S100A8/A9 complex is within the physiological range.

Then a binding isotherm was obtained for the S100A8/A9 heterodimers using increasing amounts of [³H]AA. The binding of AA to S100A8/A9 in the absence of calcium was defined as nonspecific binding. Arachidonic acid was bound by the S100A8/A9 protein complex in a saturable manner (Fig. 2), and the corresponding Scatchard plot revealed a single class of binding sites for AA with a K_D of 0.2 µM (Fig. 2 insert) and a stoichiometry of 0.4 mol of fatty acid/mol of S100A8/A9 heterodimer.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Binding isotherm of AA to S100A8/A9. S100A8/A9 (2.4 µg) was incubated with increasing concentrations of [3H]AA in the absence and presence of calcium, and the protein-bound fatty acids were separated from nonbound fatty acid using the Lipidex assay. The KD for AA was calculated to be 0.2 µM (see the inset). Shown are means of duplicate determinations ±S.D. , total binding; , nonspecific binding; , specific binding.

This low value might be due either to the existence of S100A8/A9 isoforms, which did not display fatty acid binding capacity, or to the calcium-induced formation of higher order species of S100A8/A9 heteromers. Therefore, we performed size-exclusion chromatography followed by SDS-PAGE analysis to investigate the association state of the S100A8/A9 protein complex. As shown in Fig. 3, in the absence of calcium the elution profile showed only one peak corresponding to 34-kDa proteins. The monomeric proteins could not be detected. Upon the addition of calcium, the elution profiles showed two peaks corresponding to 34- and 48-kDa proteins, respectively. Then the proteins were incubated with radiolabeled AA in the presence of calcium and analyzed by size-exclusion chromatography. The different peaks were collected, and the radioactivity was determined in these fractions. It is obvious from Table I that the radiolabeled AA comigrates with both protein complexes, indicating that both protein complexes display AA binding capacity.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Size-exclusion chromatography with S100A8 and S100A9 in the absence and presence of calcium. Before injection into a Superdex-75 column, the proteins (100 µg) were preincubated for 5 min at 37 °C in the absence and presence of 5 mM calcium. The lines represent the protein elution profiles measured at 280 nm. The molecular mass standard contained albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa). The different peaks were collected and analyzed by SDS-PAGE. Fractions containing S100A8 and S100A9 are indicated by arrows. dotted line, molecular mass standard; dash line, S100A8 and S100A9 in the absence of calcium; solid line, S100A8 and S100A9 in the presence of calcium

To answer the question whether S100A8/A9 exclusively binds AA, we performed binding and competition studies with various fatty acids as well as AA metabolites. Purified S100A8/A9 was incubated with 1 µM [³H]AA and a final concentration of 5 mM calcium in the presence of increasing concentrations of the various competitors as indicated. As shown in Fig. 4B, binding of [³H]AA to the S100A8/A9 protein complex was competed in a concentration-dependent manner by increasing concentrations of AA and -linolenic and -linolenic acids, whereas saturated fatty acids, such as palmitic and stearic acids, and the monounsaturated fatty acid oleic acid were poor competitors (Fig. 4A). In addition, none of the tested AA metabolites were able to displace AA from the S100A8/A9 protein complex (Fig. 4A). It is of interest to note that the arachidonic trifluoromethyl ketone (AA-COCF3) as well as 15-hydroxyeicosatetraenoic acid, which did not compete for AA binding to S100A8/A9, exhibit structural similarity to AA. Analogous experiments with [³H]oleic acid as ligand revealed that the ligand was bound by S100A8/A9. However, there was no displacement by increasing concentrations of unlabeled oleic acid (in the concentration range 0-20 µM). Therefore, this binding was regarded as nonspecific binding to S100A8/A9 (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Displacement of [ 3H]AA from S100A8/A9 by nonlabeled fatty acids and eicosanoids. A, S100A8/A9 (24 µg) was incubated with 1 µM [3H]AA in the presence of 20 µM competitor as indicated. The data for displacement by the various competitors are expressed as % deviation from control. B, S100A8/A9 (24 µg) was incubated with 1 µM [3H]AA and increasing concentrations of arachidonic acid (), -linolenic acid (), -linolenic acid (), and oleic acid (). Details of the experiment are described under "Experimental Procedures."

S100A8/A9 Represents the Major AA-binding Protein in Human Neutrophils-- Because of their unusual abundance in the human neutrophilic cytosol (approximately 40-50% of the cytosolic protein) (34), we assessed the AA binding capacity in the cytosolic fraction. Cytosolic proteins from human neutrophils were prepared as described under "Experimental Procedures," and increasing protein concentrations were incubated with 1 µM [³H]AA in the absence and presence of calcium. The amount of protein-bound AA was determined using the Lipidex assay, which is widely used for the measurement of affinity constants for the binding of fatty acids to fatty acid-binding proteins. Apparently, no AA binding capacity was present in the cytosolic fraction from human neutrophils in the absence of calcium. By the addition of calcium, the formation of S100A8/A9 heterodimers was induced, and consequently, there was a protein concentration-dependent increase of the AA binding capacity in the cytosol (Fig. 5A). In analogous experiments, lymphocytic cytosol was used as the control because S100A8 and S100A9 are absent in lymphocytes. Thus, an AA binding capacity was not present either in the absence or in the presence of calcium (Fig. 5B).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Analysis of [3H]AA binding capacity in the cytosol of human neutrophils (A) and lymphocytes (B). Increasing concentrations of cytosolic protein either from neutrophils or lymphocytes were incubated with 1 µM [3H]AA in the absence () or presence of 5 mM calcium (), and the protein-bound fatty acids were separated from nonbound fatty acid using the Lipidex assay. For immunoprecipitation (C), cytosolic proteins from human neutrophils (10 µg) were incubated with 1 µM [3H]AA in the presence of calcium, and after addition of increasing concentrations of 27E10, the antigen-antibody complex was removed by protein G-Sepharose. The protein-bound [3H]AA was found in the antigen-antibody complex (). In the supernatant the protein-bound fatty acids were separated from nonbound fatty acid using the Lipidex assay (). The bars represent data from three independent experiments with duplicate determinations ±S.D.

Next, aliquots of the cytosolic proteins from human neutrophils (10 µg) were incubated with 1 µM [³H]AA in the presence of calcium to induce formation of S100A8/A9. Then increasing amounts of the human S100A8/A9-specific monoclonal antibody 27E10 were added, and the antigen-IgG complex was removed by protein G-coupled Sepharose. The supernatants were applied to a Lipidex assay to determine the amount of the remaining protein-bound fatty acids. It is obvious from Fig. 5C that the radiolabeled ligand was removed from the binding solution with increasing concentrations of 27E10 antibodies, indicating a strong interaction between AA and the S100A8/A9 protein complex. Correspondingly, the amount of AA bound by S100A8/A9 was decreased in the binding solution with increasing concentrations of 27E10 antibodies.

S100A8/A9 Secreted from Neutrophil-like HL-60 Cells Binds PLA₂-released AA-- It has been shown that S100A8/A9 is released from human monocytes after stimulation of protein kinase C (16). Therefore, we investigated (i) whether S100A8/A9 was also secreted from neutrophil-like HL-60 cells and (ii) whether the released AA was bound by the secreted S100A8/A9 protein complex.

Neutrophil-like HL-60 cells were incubated for different time intervals with [¹⁴C]AA·BSA complexes. Lipid analysis revealed that AA was incorporated into membrane lipids in a time-dependent manner. The majority of AA (greater than 60%) was incorporated into cellular phospholipids, whereas less than 30% were incorporated into triglycerides. The proportions of fatty acid incorporation into other cellular lipids such as cholesterol esters and mono- and diacylglycerols were found to be negligible. The amount of nonesterified fatty acid was determined to be less than 5% of the total radioactivity incorporated in the cells (data not shown).

Arachidonic acid release was rapidly induced by the Ca²⁺ ionophore A23187, the phorbol ester PMA, or a combination of both agents as indicated in Fig. 6A. AA release was time-dependent and linear over 1 h (Fig. 6A). Both A23187 and PMA stimulated AA release to a comparable content. Maximal AA release was achieved with a combination of A23187 and PMA. S100A8/A9 heterodimer secretion from neutrophil-like HL-60 cells was induced by PMA or the combination of PMA and A23187, but not by A23187. This result was confirmed in analogous experiments in which S100A8/A9 were labeled with [¹⁴C]leucine (data not shown). Therefore, aliquots of the cell supernatants were applied to immunoprecipitation with the monoclonal antibody 27E10. The immunoprecipitate of the cell supernatant of PMA-stimulated neutrophil-like HL-60 cells contained significant amounts of the radiolabeled AAs, whereas the amount of radiolabeled AA in the immunoprecipitate of the cell supernatant of A23187-stimulated neutrophil-like HL-60 cells was found to be negligible. It is obvious from Fig. 6B that the S100A8/A9 secretion coincides in time with the AA release. The maximum of radiolabeled AA in the immunoprecipitate was achieved in the cell supernatant of neutrophil-like HL-60 cells stimulated with the combination of A23187 and PMA (Fig. 6B). Neutrophil-like HL-60 cells have an exceedingly high capacity to release AA, and only little of the liberated AA is converted into eicosanoids. In addition, a low level of immunodetectable cyclooxygenase-2 was expressed in unstimulated neutrophilic-like HL-60 cells. The cyclooxygenase-2 protein expression was induced by both agents within hours (data not shown). Therefore, the radiolabeled AA in the immunoprecipitate is considered to present nonmetabolized AA bound by the secreted S100A8/A9 protein complex. Whether AA and S100A8/A9 protein complex were secreted either in a concerted or independent manner remains unclear.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Release of AA and S100A8/A9 from neutrophil-like HL-60 cells. Neutrophil-like HL-60 cells (5 × 106 cells/ml) were prelabeled with 10 µM [14C]AA·BSA complex and then stimulated with 10 nM PMA, 1 µM A23187, or a combination of both agents at 37 °C for different time intervals as indicated. Aliquots of the cell supernatant were used either for the determination of the released AA (A) or for immunoprecipitation (B) as described in Fig. 5. Shown are the means of triplicate determinations ±S.D from a representative experiment. , control; , PMA; , A23187; , PMA/A23187. Details of the experiment are described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The investigation of the AA binding properties by using purified S100A8 and S100A9 from human neutrophils revealed that exclusively the S100A8/A9 protein complex binds AA in a calcium-dependent manner (Fig. 1, A and B). The estimated calcium concentration required to induce fatty acid binding was within the physiological range (Fig. 1C). The fact that the individual components of the protein complex were unable to bind fatty acids either in the absence or in the presence of calcium leads to the assumption that docking of the two subunits creates an asymmetric fatty acid-binding site located at the interface between the subunits. In preliminary studies, different molar ratios of S100A8 and S100A9 were used in the presence of calcium to induce protein complex formation followed by the determination of AA binding capacity. They indicate that the protein complex that is able to bind AA consists of equal moles of S100A9 and S100A8.² This result is in accordance with other studies (33).

The binding studies revealed that arachidonic acid binding was saturable and had specific binding characteristics. A K_D of 0.2 µM and a stoichiometry of 0.4 mol of fatty acid/mol of S100A8/A9 heterodimer was derived from Scatchard analysis (Fig. 2). Klotz (35) proposed that Scatchard analysis of ligand is only valid if an S-shaped curve for binding is obtained. This was not possible in the present investigation due to the limited binding capacity of the Lipidex material. Thus, the actual number of fatty acid molecules bound by the protein complex may be overestimated as outlined by Klotz (35). On the other hand, it is worth mentioning that upon calcium binding, conformational changes lead to the exposure of hydrophobic surfaces. Therefore, the amount of protein-bound AA may be underestimated in the Lipidex assay, although the microcentrifuge tubes were precoated with poly(propylene glycol) to minimize nonspecific adsorption. Both limitations should be taken into account but do not change the interpretation that a part of the S100A8/A9 complex was able to bind AA (Fig. 2).

Alternatively, the calculated stoichiometry is based on the assumption that the multimer displaying the AA binding capacity represents a heterodimer. However, whether S100A8/A9 represents a heterodimer or an oligomer containing S100A8 and S100A9 at equimolar concentrations is still in debate. We performed gel filtration with the native proteins followed by SDS-PAGE analysis (Fig. 3). In the absence of calcium the monomeric proteins could not be detected, indicating a strong interaction of S100A8 and S100A9 also in the absence of calcium. This assumption is also confirmed by the finding that the S100A8/A9 complex formation is not interfered by Ca²⁺-chelation (33). The heterodimeric protein complex showed an apparent M_r of 34 kDa in accordance to Siegenthaler et al. (22). However, several biophysical determinations confirm that the heterodimer has a M_r of 24 kDa (33, 36). Thus, the heterodimeric protein complex is assumed to show an unusual migration in size-exclusion chromatography. In addition to the heterodimer, a protein complex with a M_r of 48 kDa was analyzed after the addition of calcium, and radiolabeled AA comigrated with both protein complexes, indicating that both complexes display AA binding capacity (Table I). The 48-kDa protein is assumed to represent a tetrameric protein complex consisting of two molecules of S100A8 and two molecules of S100A9. The existence of the tetramer is confirmed by a recently published study from our group by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (36).

In addition, both S100A8 and S100A9 have some post-translational modifications (33, 36), which may influence complex formation as well as fatty acid binding properties. However, phosphorylation of S100A9 within purified S100A8/A9 protein complexes did not affect the AA binding capacity (data not shown).

The competition studies clearly indicate that exclusively polyunsaturated fatty acid are bound by the protein complex in a saturable and reversible manner, whereas saturated fatty acids, such as palmitic and stearic acids, and the monounsaturated oleic acid were poor competitors. The S100A8/A9 protein complex showed the highest specificity toward arachidonic acid. In addition, the S100A8/A9 complex did not show any affinity for AA-derived eicosanoids (Fig. 4, A and B). These assays give insights into the nature of the fatty acid binding site. The affinity was significantly influenced by (i) the polar heads, (ii) the number of the double bonds, and (iii) their position within the fatty acid molecule. The identification of this binding pocket is currently under investigation in our laboratory. The specificity toward polyunsaturated fatty acids as well as the reversibility of the binding to the protein complex excludes the possibility that merely a solvation of insoluble fatty acid calcium salts occurs. These results also argue against a coordination of the fatty acid by one of the calcium ions of the S100A8/A9 complex. In contrast to our results, Siegenthaler et al. (22) found that S100A8/A9 shows specificity toward various fatty acids. This finding may be due to the fact that they used purified proteins from human keratinocytes for their binding and competition studies.

In the present study we could show for the first time that the S100A8/A9 protein complex represents the only AA binding capacity in the neutrophilic cytosol. This finding is corroborated by the facts that (i) there was only a low capacity for AA binding in the neutrophilic cytosol in the absence of calcium (Fig. 5A); (ii) the AA binding capacity in the cytosol was increased in a protein concentration-dependent manner after the addition of calcium, which induces the formation of S100A8/A9 protein complexes (Fig. 5A); and (iii) the AA binding capacity in the cytosol was depleted by immunoprecipitation with the S100A8/A9 protein complex-specific monoclonal antibody 27E10 (Fig. 5C). Furthermore, our conclusion is supported by the finding that members of the fatty acid binding protein family are not expressed in human neutrophils (37). Activated neutrophils, the predominant cell type present in areas of acute inflammation, release arachidonic acid as well as arachidonic acid-derived eicosanoids into the plasma where they may amplify or perpetuate the acute inflammatory response. Our novel finding that the S100A8/A9 protein complex represents the exclusive AA binding capacity in the neutrophilic cytosol together with their unusual abundance in human neutrophilic cytosol (34) clearly indicate that S100A8/A9 complexes play an important role in the AA metabolism of neutrophils. The calcium-induced binding of AA points to a role of S100A8/A9 in the mobilization, metabolization, or release of AA.

Therefore, the neutrophil-like HL-60 cells (for review, see Ref. 38) were used to study the release of AA as well as the secretion of S100A8/A9 heterodimers (Fig. 6). Arachidonic acid release was induced by either an increase of the intracellular calcium level by the calcium ionophore A23187 or an activation of protein kinases by the phorbol ester PMA (39-41). S100A8/A9 was secreted from neutrophil-like HL-60 cells by PMA, whereas the calcium ionophore A23187 was ineffective to induce the S100A8/A9 secretion. This finding was confirmed by the metabolic labeling of de novo-synthesized S100A8/A9 with [¹⁴C]leucine (data not shown), indicating that, in addition to monocytes as described by Rammes et al. (16), neutrophils also secreted the protein complex after activation of protein kinase C. The immunoprecipitation experiments of the cell supernatants revealed that (i) the secreted S100A8/A9 protein complex binds fatty acids and (ii) the majority of the simultaneously released AA was bound by the protein complex. Furthermore, the time courses of both AA release and S100A8/A9 secretion were found to be similar (Fig. 6). In contrast, the stimulation with A23187 led to a rapid release of AA but not to secretion of S100A8/A9. Consequently, no significant amounts of radiolabeled fatty acids were determined in the immunoprecipitate. Still, the functional consequences of AA release either in the absence of S100A8/A9 secretion (as induced by calcium ionophore) or in parallel to S100A8/A9 secretion (as induced by PMA) remain unclear. The S100A8/A9 secretion from human neutrophils is also induced by the chemoattractants formylmethionylleucylphenylalanine and complement 5a (42), indicating that this mechanism is functional following receptor-dependent activation of cells.

It is worth mentioning that apart from phosphorylation of S100A9, also oxidative modification of the protein complex by hypochlorite did not affect the AA binding capacity (Table II). The latter finding is of interest, since upon PMA stimulation the protein complex is secreted into an oxidative environment, and the proteins contain oxidative-sensitive cysteines. Additionally, it has been shown that oxidative modification affects the chemotactic activity of murine S100A8 (43). The monomeric and chemotactic S100A8 are transformed into nonchemotactic S100A8 homodimers by hypochlorite oxidation-driven disulfide linkage. The oxidation is suggested by the authors to provide a mechanism limiting excess infiltration of leukocytes and terminating the progression of acute inflammation. The analysis of the nature of the secreted AA-binding protein complex is currently under investigation in our laboratory.

The S100A8/A9·AA complex may have an extracellular role. For example, it has been shown that endothelial cells utilize both endogenous and exogenous AA for transcellular production of thromboxane (44). Therefore, it can be speculated that the AA thus liberated is taken up by bystander cells to be metabolized to eicosanoids, representing a particular transcellular pathway for AA metabolism. In addition, it has been shown that AA affects leukocyte adhesion by suppressing the expression of adhesion molecules on endothelial cells (45-47), activates NADPH oxidase (48-50), and exerts direct effects on phagocyte H⁺ and Ca²⁺ ion flux (51-54). These actions may contribute to propagation of inflammatory processes, and the secreted AA-S100A8/A9 complex may here be involved, probably as the shuttle protein to reach target cells.

Alternatively, S100A8/A9 represents the intracellular AA reservoir. It is likely that resting neutrophils (as well as other inflammatory cells) allow only relatively low concentrations of cellular AA to accumulate. The intracellular concentration of AA is under the control of various acyltransferases that rapidly incorporate the fatty acid into particular glycerolipids followed by a slower remodeling into other glycerolipids (for review, see Ref. 23). The mobilization of esterified AA from cellular sources and the subsequent metabolism of this AA into oxygen-containing metabolites (eicosanoids) is the key regulatory event in most inflammatory cells. Its mobilization is induced via phospholipase(s) by an increase of calcium or by protein phosphorylation through mitogen-activated protein kinases (for review, see Refs. 55 and 56). However, the mechanism by which arachidonic acid as well as eicosanoids, once biosynthesized, leave the producer cell to reach their target cells is still poorly understood. In addition, arachidonic acid is liberated from cellular phospholipids, but the activities of the AA-metabolizing enzymes are detectable within hours after stimulation (57-59). This finding was confirmed by cell stimulation experiments. Both PMA and A23187 led to a time-dependent increase in the level of immunodetectable cyclooxygenase-2 (data not shown). In parallel, the enhanced calcium level also induces S100A8/A9 protein complex formation and its AA binding capacity. Thus, it could be envisioned either that the AA·S100A8/A9 complex may function as an intermediate reservoir or that the translocation of S100A8/A9 is accompanied by AA transport as suggested by Roulin et al. (37). Further investigations have to elucidate the exact nature of the protein complex which exhibits AA binding capacity as well as its role in the biological events caused by eicosanoids. They will provide further insights into the molecular mechanisms of inflammatory responses.

	ACKNOWLEDGEMENTS

We thank Dr. F. Schönlau and Dr. W. Nacken for critical reading of the manuscript, Dr. A. von Eckardstein and Alois Roetrige (Institut für Arterioskleroseforschung, Universität Münster) for gel filtration experiments, and Dr. B. Reichel for calcium determination (Geologisch-Paläontologisches Institut, Universität Münster). The excellent technical assistance of Anneliese Gassen and Heike Hater is appreciated.

	FOOTNOTES

^* This work was supported by grants from Ke-1-1-II/97-9 of Innovative Medizinische Forschung and from Teilprojekt C15 of Interdisziplinäres Zentrum für Klinische Forschung of Westfälische-Wilhelms- Universität Münster.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Institut fur Experimentelle Dermatologie, von-Esmarch-Str. 56, 48149 Münster, Germany. Tel.: 049-251-8356584; Fax: 049-251-8356549; E-mail: kerkhoc@uni-muenster.de.

^¶ Present address: Institüt für Physiologie und Biochemie der Ernähung, Bundesanstalt für Milchforschung, 24121 Kiel, Germany.

² C. Kerkhoff, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES