The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils.

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, prostaglandin E(2), thromboxane B(2), leukotriene B(4)) 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.     

Evidence for the involvement of the unique C-tail of S100A9 in the binding of arachidonic acid to the heterocomplex S100A8/A9

Protein complexes formed by S100A8 and S100A9 represent the only AA-binding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism. The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids. The present study was undertaken to characterize the fatty acid binding site within the protein complex. Deletions at both termini and point mutations of different basic amino acids especially within the extended C-terminal tail of human S100A9 were introduced. The S100A9 mutant proteins were then analyzed with respect to protein-protein interaction (GST pull down-assay and yeast two-hybrid system) and functional properties (arachidonic acid and calcium binding). The data give strong evidence that the unique C-tail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxy-group is bound to the protein complex. The localization of the AA-binding site within the unique C-tail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins.     

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils.

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière 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. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, 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.     

Binding of arachidonic acid to myeloid-related proteins (S100A8/A9) enhances phagocytic NADPH oxidase activation

Activation of the O(2)(-) generating NADPH oxidase of phagocytes results from the assembly of the membrane-bound flavocytochrome b(558) with cytosolic proteins, p67(phox), p47(phox), and Rac. However, it has been recently reported that the arachidonic acid- and calcium-binding heterodimer S100A8/A9, abundant in neutrophil cytosol, influences the activation process. In a semi-recombinant system comprising neutrophil membranes, recombinant proteins, p67(phox), p47(phox), GTPgamma S-loaded Rac2, and arachidonic acid (AA), both the rate and the extent of the oxidase activation were increased by S100A8/A9, provided it was preloaded with AA. Binding of [(14)C]AA to S100A8/A9 was potentiated by recombinant cytosolic phox proteins and GTPgammaS, suggesting the formation of a complex, comprising oxidase activating proteins and S100A8/A9, with a greater affinity for AA. The rate constant of oxidase activation was not increased by AA-loaded S100A8/A9, whereas the maximal oxidase activity elicited was twice as high. AA-loaded S100A8/A9 increases oxidase activation probably by decreasing the deactivation rate.     

S100A8 and S100A9 in Cancer

S100A8 and S100A9 are Ca²⁺ binding proteins that belong to the S100 family. Primarily expressed in neutrophils and monocytes, S100A8 and S100A9 play critical roles in modulating various inflammatory responses and inflammation-associated diseases. Forming a common heterodimer structure S100A8/A9, S100A8 and S100A9 are widely reported to participate in multiple signaling pathways in tumor cells. Meanwhile, S100A8/A9, S100A8, and S100A9, mainly as promoters, contribute to tumor development, growth and metastasis by interfering with tumor metabolism and the microenvironment. In recent years, the potential of S100A8/A9, S100A9, and S100A8 as tumor diagnostic or prognostic biomarkers has also been demonstrated. In addition, an increasing number of potential therapies targeting S100A8/A9 and related signaling pathways have emerged. In this review, we will first expound on the characteristics of S100A8/A9, S100A9, and S100A8 in-depth, focus on their interactions with tumor cells and microenvironments, and then discuss their clinical applications as biomarkers and therapeutic targets. We also highlight current limitations and look into the future of S100A8/A9 targeted anti-cancer therapy.     

Zinc binding reverses the calcium-induced arachidonic acid-binding capacity of the S100A8/A9 protein complex

Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF-hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein-protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.     

The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation

Since no data are available concerning fatty acid (FA) transport in neutrophils we studied the presence of possible FA carriers. The kFA-p34 complex, composed of S100A8 and S100A9, has been implicated in the intracellular transport of arachidonic acid and its precursors in human keratinocytes. Here, we show that FA-p34 is the major FA carrier in human neutrophils (nFA-p34). The complex is highly expressed in resting neutrophils (2.65% of cytosolic proteins) and translocates to the membrane fraction upon stimulation with opsonized zymosan. Comparison of purified nFA-p34 with kFA-p34 shows that both complexes are composed of nearly the same subunits and possess similar binding properties for oleic acid. Densitometrical analyses of 2D gels show that n and kFA-p34 contain twice as much S100A8 and S100A9 suggesting an estimated stoichiometry of (S100A8)2S100A9. A method is described allowing to distinguish n and kFA-p34 from S100A8/S100A9 homo- and heteromer complexes that are devoid of FA-binding properties. After solvent extraction, we find by GC analysis linoleic acid as major endogenous ligand of purified kFA-p34. Our results suggest that nFA-p34, might be involved in the shuttling of unsaturated FA between the cytosol and the plasma membrane of neutrophils.     

The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca(2+)-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.     

The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment

Background/aimS100A8/A9 and myeloid cells in the tumor microenvironment play an important role in cancer invasion and progression, and the effect of tumor-infiltrated myofibroblasts on myeloid cells in the tumor microenvironment is relatively unknown. Accordingly, we investigated the role of myofibroblasts in the upregulation of S100A8/A9 as well as in the differentiation of myeloid cells in the colorectal cancer (CRC) microenvironment.Materials and methodsTo investigate the interactions among cancer cells, myofibroblasts, and inflammatory cells in the microenvironment of CRC, we used 10 CRC cell lines, 18CO cells and THP-1 cells, which were co-cultured with each other or cultured in conditioned media (CM) of other cells. Expression of S100A8/A9 was evaluated via Western blot, immunohistochemical staining and immunofluorescence. The secreted factors from the cell lines were analyzed using cytokine antibody array. Flow cytometry analysis was performed to analyze the differentiation markers of myeloid cells.Results18CO CM induced increased expression of S100A8/A9 in THP-1 cells. Increased expression of S100A8/A9 was noted in inflammatory cells of the peri- and intra-tumoral areas, along with myofibroblasts in colon cancer tissue. S100A8/A9-expressing inflammatory cells also exhibited CD68 expression in colon cancer tissue, and 18CO CM induced differentiation of THP-1 cells into myeloid-derived suppressor cells (MDSCs) or M2 macrophages expressing S100A8/A9. Significant amounts of IL-6 and IL-8 were detected in 18CO CM, compared to those in both controls and THP-1 CM, and tumor-infiltrated myofibroblasts expressed IL-8 in colon cancer tissue. Finally, neutralizing antibodies to IL-6 and IL-8 attenuated 18CO CM-induced increased expression of S100A8/A9.ConclusionsThe upregulation of S100A8/A9 in tumor-infiltrated myeloid cells could be triggered by IL-6 and IL-8 released from myofibroblasts, and myofibroblasts might induce the differentiation of myeloid cells into S100A8/9-expressing MDSCs or M2 macrophages in the CRC microenvironment.     

S100A8 and S100A9 in human arterial wall. Implications for atherogenesis

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.     

Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.     

S-nitrosylated S100A8: novel anti-inflammatory properties

S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

S100A8 and S100A9—oxidant scavengers in inflammation

S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys₄₂ residue. Met₇₈(O) and Trp₅₄(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp₅₄(+16). Evidence for oxidation of the single Cys₃ residue in recS100A9 HOCl was weak; Met₆₃, Met₈₁, Met₈₃, and Met₉₄ were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met₆₃, Met₈₁, and Met₉₄ were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.