Computational Deciphering of the Role of S100A8 and S100A9 Proteins and Their Changes in the Structure Assembly Influences Their Interaction with TLR4, RAGE, and CD36.

S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.

Toward an understanding of the conformational plasticity of S100A8 and S100A9 Ca2+-binding proteins.

S100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wildtype full-length S100A8 and S100A9 subunits in biologically relevant homo- and heterodimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies.

S100A9-Targeted Cowpea Mosaic Virus as a Prophylactic and Therapeutic Immunotherapy against Metastatic Breast Cancer and Melanoma.

Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease.

Development of anti-thrombotic vaccine against human S100A9 in rhesus monkey.

In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we reported an antiplatelet vaccine against S100A9 in mice, but the use of Freund's adjuvant and the difference in amino acid sequences in epitopes between mice and humans were problematic for clinical use. Here, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys with Alum adjuvant. First, we assessed several candidate epitopes and selected 102 to 112 amino acids as the suitable epitope, which could produce antibodies. When this peptide vaccine was intradermally injected into 4 cynomolgus monkeys with Alum, the antibody against human S100A9 was successfully produced. Anti-thrombotic effects were shown in two monkeys in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey. Additionally, the anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects of produced antibodies. Prolongation of bleeding time was not observed in vaccinated monkeys. Although further studies on increasing the effect of vaccine and safety are necessary, this vaccine will be a promising approach to improve adherence to antiplatelet drugs in clinical settings.

Pathogenic Roles of S100A8 and S100A9 Proteins in Acute Myeloid and Lymphoid Leukemia: Clinical and Therapeutic Impacts.

Deregulations of the expression of the S100A8 and S100A9 genes and/or proteins, as well as changes in their plasma levels or their levels of secretion in the bone marrow microenvironment, are frequently observed in acute myeloblastic leukemias (AML) and acute lymphoblastic leukemias (ALL). These deregulations impact the prognosis of patients through various mechanisms of cellular or extracellular regulation of the viability of leukemic cells. In particular, S100A8 and S100A9 in monomeric, homodimeric, or heterodimeric forms are able to modulate the survival and the sensitivity to chemotherapy of leukemic clones through their action on the regulation of intracellular calcium, on oxidative stress, on the activation of apoptosis, and thanks to their implications, on cell death regulation by autophagy and pyroptosis. Moreover, biologic effects of S100A8/9 via both TLR4 and RAGE on hematopoietic stem cells contribute to the selection and expansion of leukemic clones by excretion of proinflammatory cytokines and/or immune regulation. Hence, the therapeutic targeting of S100A8 and S100A9 appears to be a promising way to improve treatment efficiency in acute leukemias.

S100A9-targeted tobacco mosaic virus nanoparticles exhibit high specificity toward atherosclerotic lesions in ApoE-/- mice.

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE-/- mice.

Oxidative Post-translational Modifications Accelerate Proteolytic Degradation of Calprotectin.

Oxidative post-translational modifications affect the structure and function of many biomolecules. Herein we examine the biophysical and functional consequences of oxidative post-translational modifications to human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer, calgranulins A/B oligomer). This abundant metal-sequestering protein contributes to innate immunity by starving invading microbial pathogens of transition metal nutrients in the extracellular space. It also participates in the inflammatory response. Despite many decades of study, little is known about the fate of CP at sites of infection and inflammation. We present compelling evidence for methionine oxidation of CP in vivo, supported by using 15N-labeled CP-Ser (S100A8(C42S)/S100A9(C3S)) to monitor for adventitious oxidation following human sample collection. To elucidate the biochemical and functional consequences of oxidative post-translational modifications, we examine recombinant CP-Ser with methionine sulfoxide modifications generated by exposing the protein to hydrogen peroxide. These oxidized species coordinate transition metal ions and exert antibacterial activity. Nevertheless, oxidation of M81 in the S100A9 subunit disrupts Ca(II)-induced tetramerization and, in the absence of a transition metal ion bound at the His6 site, accelerates proteolytic degradation of CP. We demonstrate that native CP, which contains one Cys residue in each full-length subunit, forms disulfide bonds within and between S100A8/S100A9 heterodimers when exposed to hydrogen peroxide. Remarkably, disulfide bond formation accelerates proteolytic degradation of CP. We propose a new extension to the working model for extracellular CP where post-translational oxidation by reactive oxygen species generated during the neutrophil oxidative burst modulates its lifetime in the extracellular space.

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site.

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca(II) ions on the structure and Ni(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni(II)-bound CP-Ser [oligomer of S100A8(C42S) and S100A9(C3S)] crystals obtained in the absence and presence of Ca(II). We present a 2.1 Å resolution crystal structure of Ni(II)-bound CP-Ser and compare this structure to a reported Ni(II)- and Ca(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) J. Am. Chem. Soc. 139, 8828-8836]. This analysis reveals conformational changes associated with coordination of Ca(II) to the EF-hands of S100A9 and that Ca(II) binding affects the coordination number and geometry of the Ni(II) ion bound to the His3Asp site. In contrast, negligible differences are observed for the Ni(II)-His6 site in the absence and presence of Ca(II). Biochemical studies show that, whereas the His6 site has a thermodynamic preference for Ni(II) over Zn(II), the His3Asp site selects for Zn(II) over Ni(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space.

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)-Dependence and Its Ability to Chelate Multiple Nutrient Transition Metal Ions.

Calprotectin (CP) is an abundant host-defense protein that contributes to the metal-withholding innate immune response by sequestering nutrient metal ions from microbial pathogens in the extracellular space. Over the past decade, murine models of infectious disease have advanced understanding of the physiological functions of CP and its ability to compete with microbes for essential metal nutrients. Despite this extensive work, murine CP (mCP) has not been biochemically evaluated, and structural and biophysical understanding of CP is currently limited to the human orthologue. We present the reconstitution, purification, and characterization of mCP as well as the cysteine-null variant mCP-Ser. Apo mCP is a mS100A8/mS100A9 heterodimer, and Ca(II) binding causes two heterodimers to self-associate and form a heterotetramer. Initial metal-depletion studies demonstrate that mCP depletes multiple first-row transition metal ions, including Mn, Fe, Ni, Cu, and Zn, from complex microbial growth medium, indicating that mCP binds multiple nutrient metals with high affinity. Moreover, antibacterial activity assays show that mCP inhibits the growth of a variety of bacterial species. The metal-depletion and antibacterial activity studies also provide evidence that Ca(II) ions enhance these functional properties of mCP. This contribution provides the groundwork for understanding the similarities and differences between the human and murine orthologues of CP and for further elucidation of its biological coordination chemistry.

Insights from Ion Binding Site Network Analysis into Evolution and Functions of Proteins.

Many biological phenomena can be represented as complex networks. Using a protein binding site comparison approach, we generated a network of ion binding sites on the scale of all known protein structures from the Protein Data Bank. We found that this ion binding site similarity network is scale-free, indicating a network in which a few ion binding site scaffolds are the network hubs, and these are connected to hundreds of nodes, whereas the vast majority of nodes have only a few neighbors. Enrichment and statistical analysis of the network components and communities yielded insights into underlying processes from the functional and the structural perspective. Largest components and communities were observed to be closely related to basic metabolic processes and some of the most common structural folds, which, from the evolutionary point of view, indicates that they may be the oldest ones. Further, we derived the first comprehensive map of ion interchangeability, based on binding site similarity. Several highly interchangeable protein-ion binding site pairs emerged (e.g., Ca2+ and Mg2+ ), as well as structurally distinct ones. The constructed network of ion binding site similarities will aid in understanding the general principles of protein-ion binding sites structure, function and evolution. We demonstrate potential uses of the network on proteins involved in cancer development and immune response, where individual ions play prominent roles in disease development.

Recurrent ECSIT mutation encoding V140A triggers hyperinflammation and promotes hemophagocytic syndrome in extranodal NK/T cell lymphoma.

Hemophagocytic syndrome (HPS) is a fatal hyperinflammatory disease with a poorly understood mechanism that occurs most frequently in extranodal natural killer/T cell lymphoma (ENKTL). Through exome sequencing of ENKTL tumor-normal samples, we have identified a hotspot mutation (c.419T>C) in the evolutionarily conserved signaling intermediate in Toll pathway (ECSIT) gene, encoding a V140A variant of ECSIT. ECSIT-V140A activated NF-κB more potently than the wild-type protein owing to its increased affinity for the S100A8 and S100A9 heterodimer, which promotes NADPH oxidase activity. ECSIT-T419C knock-in mice showed higher peritoneal NADPH oxidase activity than mice with wild-type ECSIT in response to LPS. ECSIT-T419C-transfected ENKTL cell lines produced tumor necrosis factor (TNF)-α and interferon (IFN)-γ, which induced macrophage activation and massive cytokine secretion in cell culture and mouse xenografts. In individuals with ENKTL, ECSIT-V140A was associated with activation of NF-κB, higher HPS incidence, and poor prognosis. The immunosuppressive drug thalidomide prevented NF-κB from binding to the promoters of its target genes (including TNF and IFNG), and combination treatment with thalidomide and dexamethasone extended survival of mice engrafted with ECSIT-T419C-transfected ENKTL cells. We added thalidomide to the conventional dexamethasone-containing therapy regimen for two patients with HPS who expressed ECSIT-V140A, and we observed reversal of their HPS and disease-free survival for longer than 3 years. These findings provide mechanistic insights and a potential therapeutic strategy for ENKTL-associated HPS.

Calprotectin Pegylation Enhanced Its Physical and Structural Properties.

Calprotectin is member of the S-100 protein family with a wide plethora of intra-and extracellular functions. Anticancer activities, antimicrobial effects and being a qualified disease marker are among the compelling features of this protein to be used as a pharmaceutical agent. However, there are several impediments to applications of protein pharmaceuticals including: proteolytic degradation, short circulating half-life, low solubility and immunogenicity. Pegylation is a common bioconjugation polymer capable of overcoming these drawbacks. Recombinant expression and purification of calprotectin along with its pegylation would result in enhanced pharmaco-dynamic and pharmacokinetic properties. Our florescence spectroscopy and far Ultraviolet-optical density results indicate that pegylation altered the physical and structural properties of the calprotectin to become in a more stable and functionally active state. Due to enhanced pharmacodynamic and pharmacokinetic properties of the calprotectin via pegylation, this study would pave the way for better in vitro and in vivo validations of calprotectin applications in medical practice.

Blocking the interaction between S100A9 and RAGE V domain using CHAPS molecule: A novel route to drug development against cell proliferation.

Human S100A9 (Calgranulin B) is a Ca(2+)-binding protein, from the S100 family, that often presents as a homodimer in myeloid cells. It becomes an important mediator during inflammation once calcium binds to its EF-hand motifs. Human RAGE protein (receptor for advanced glycation end products) is one of the target-proteins. RAGE binds to a hydrophobic surface on S100A9. Interactions between these proteins trigger signal transduction cascades, promoting cell growth, proliferation, and tumorigenesis. Here, we present the solution structure of mutant S100A9 (C3S) homodimer, determined by multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain via NMR spectroscopy. CHAPS is a zwitterionic and non-denaturing molecule widely used for protein solubilizing and stabilization. We found out that CHAPS and RAGE V domain would interact with mS100A9 by using (1)H-(15)N HSQC NMR titrations. Therefore, using the HADDOCK program, we superimpose two binary complex models mS100A9-RAGE V domain and mS100A9-CHAPS and demonstrate that CHAPS molecules could play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support the conclusion that CHAPS inhibits the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.

[The Role of Calcium in the Conformational Changes of the Recombinant S100A8/S100A9].

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.

Influence of storage conditions on MALDI-TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations.

Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI-TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at -20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP-3, S100A8, full-length S100A9 and its truncated form were detected after 3 months at -80°C. The alterations found in the "stored GCF" profile not only may affect the pattern-based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at -80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen.

Effect of poly and mono-unsaturated fatty acids on stability and structure of recombinant S100A8/A9.

Recombinant pET 15b vectors containing the coding sequences S100A8 and S100A9 are expressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. The structural changes of S100A8/A9 complex are analyzed upon interaction with poly/mono-unsaturated fatty acids (UFAs). The thermodynamic values, Gibbs free energy and the protein melting point, are obtained through thermal denaturation of protein both with and without UFAs by thermal scanning of protein emission using the fluorescence spectroscopy technique. The far-ultraviolet circular dichroism spectra show that all studied unsaturated fatty acids, including arachidonic, linoleic, alpha-linolenic and oleic acids, induce changes in the secondary structure of S100A8/A9 by reducing the α-helix and ß-sheet structures. The tertiary structure of S100A8/A9 has fluctuations in the fluorescence emission spectra after the incubation of protein with UFAs. The blue-shift of emission maximum wavelength and the increase in fluorescence intensity of anilino naphthalene-8-sulfonic acid confirm that the partial unfolding is caused by the conformational changes in the tertiary structure in the presence of UFAs. The structural changes in S100A8/A9 and its lower stability in the presence of UFAs may be necessary for S100A8/A9 to play a biological role in the inflammatory milieu.

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.

Manganese and microbial pathogenesis: sequestration by the Mammalian immune system and utilization by microorganisms.

Bacterial and fungal pathogens cause a variety of infectious diseases and constitute a significant threat to public health. The human innate immune system represents the first line of defense against pathogenic microbes and employs a range of chemical artillery to combat these invaders. One important mechanism of innate immunity is the sequestration of metal ions that are essential nutrients. Manganese is one nutrient that is required for many pathogens to establish an infective lifestyle. This review summarizes recent advances in the role of manganese in the host-pathogen interaction and highlights Mn(II) sequestration by neutrophil calprotectin as well as how bacterial acquisition and utilization of manganese enables pathogenesis.

Secretion of S100A8, S100A9, and S100A12 by Neutrophils Involves Reactive Oxygen Species and Potassium Efflux.

S100A8/A9 (calprotectin) and S100A12 proinflammatory mediators are found at inflammatory sites and in the serum of patients with inflammatory or autoimmune diseases. These cytoplasmic proteins are secreted by neutrophils at sites of inflammation via alternative secretion pathways of which little is known. This study examined the nature of the stimuli leading to S100A8/A9 and S100A12 secretion as well as the mechanism involved in this alternative secretion pathway. Chemotactic agents, cytokines, and particulate molecules were used to stimulate human neutrophils. MSU crystals, PMA, and H2O2 induced the release of S100A8, S100A9, and S100A12 homodimers, as well as S100A8/A9 heterodimer. High concentrations of S100A8/A9 and S100A12 were secreted in response to nanoparticles like MSU, silica, TiO2, fullerene, and single-wall carbon nanotubes as well as in response to microbe-derived molecules, such as zymosan or HKCA. However, neutrophils exposed to the chemotactic factors fMLP failed to secrete S100A8/A9 or S100A12. Secretion of S100A8/A9 was dependent on the production of reactive oxygen species and required K(+) exchanges through the ATP-sensitive K(+) channel. Altogether, these findings suggest that S100A12 and S100A8/A9 are secreted independently either via distinct mechanisms of secretion or following the activation of different signal transduction pathways.