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Proinflammatory S100A12 Can Activate Human Monocytes via Toll-like Receptor 4 ¨ ken3, Toni Weinhage1, Georg Varga1, Thomas Vogl3, Dirk Foell1,2, Helmut Wittkowski1, Christoph Kessel1, Aloys Lu ¨ rk5, Marieke A. D. van Zoelen6, Faekah Gohar1, Geetha Srikrishna7, Timo Wirth1, Dorothee Viemann4, Per Bjo Matthias Kraft8, and Johannes Roth2,3 1 Department of Pediatric Rheumatology and Immunology, University Children’s Hospital Muenster, Muenster, Germany; 2Interdisciplinary Center of Clinical Research and 3Institute of Immunology, University of Muenster, Muenster, Germany; 4Department of Pediatric Pulmonology, Allergology, and Neonatology, Hannover Medical School, Hannover, Germany; 5Active Biotech, Lund, Sweden; 6Department of Experimental Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 7Sanford-Burnham Medical Research Institute, La Jolla, California; and 8Department of Internal Medicine A, University of Greifswald, Greifswald, Germany

Rationale: S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. Objectives: We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. Methods: Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. Measurements and Main Results: S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. Conclusions: Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis. (Received in original form September 7, 2012; accepted in final form April 6, 2013) Supported by grants from the German Research Foundation (DFG Fo 354/3-1 and SFB1009) and the German Ministry for Education and Science (BMBF 01GM08100), and by funding from the European Union’s Seventh Framework Program under EC-GA No. 305266 (MIAMI). Author Contributions: D.F. designed the work, supervised experiments, analyzed data, wrote the manuscript, and drafted the paper. D.F. and C.K. performed and analyzed secretion experiments, immunohistochemistry, and immunoassays. H.W. and D.V. performed and analyzed microarray studies. H.W. and C.K. performed PCRs, immunoassays, blocking experiments, and migration studies. A.L. performed PCRs, blocking experiments, and transfections. C.K., T. Weinhage, G.V., F.G., T. Wirth, and T.V. helped with protein and antibody purification, transfections, and PCRs. P.B. performed and analyzed Biacore studies. M.A.D.v.Z. contributed with the LPS challenge in healthy control subjects. G.S. provided gb3.1 antibody and helped with RAGE production. M.K. included patients with sepsis. J.R. supervised the study design and experiments. Correspondence and requests for reprints should be addressed to Dirk Foell, M.D., Department of Pediatric Rheumatology and Immunology, University Children’s Hospital Muenster, Roentgenstr. 21, D-48149 Muenster, Germany. E-mail: dfoell@ uni-muenster.de This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 187, Iss. 12, pp 1324–1334, Jun 15, 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201209-1602OC on April 23, 2013 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

S100A12 is a calcium-binding protein expressed and released by human granulocytes. Its concentrations rise during inflammation and correlate to inflammatory activity. A proinflammatory role has been ascribed, but the exact mechanism by which human S100A12 activates monocytes is unclear. What This Study Adds to the Field

Human S100A12 is overexpressed during clinical and experimental sepsis. It is a novel endogenous TLR4 ligand facilitating inflammatory monocyte activation in a cross-talk with activated granulocytes. This makes S100A12 a proinflammatory amplifier of early inflammatory processes that are involved in sepsis.

Keywords: granulocytes; pattern recognition receptors; innate immunity; receptor for advanced glycation end products; inflammation

Sepsis is among the most challenging clinical conditions, characterized by the excessive production of inflammatory mediators causing systemic inflammation and collateral tissue damage that can lead to multiorgan failure and death. Exogenous and endogenous stimuli can act together, leading to an overwhelming immune response to initial triggers that are often of infectious origin. In this context, innate immunity indemnifies our host defense but can also promote deleterious autoinflammatory processes. Granulocytes, monocytes, and macrophages secrete numerous humoral factors targeting the cellular networks of immunity (e.g., cytokines or chemokines). Additional effectors are released by activated or damaged cells under conditions of cell stress and may operate extracellularly as damage associated molecular pattern proteins (DAMPs) (1, 2). Cells expressing pattern recognition receptors, (e.g., the multiligand receptor for advanced glycation end products [RAGE] or Toll-like receptors [TLRs]) are activated during stimulation not only by pathogen-associated molecular patterns but also by endogenous DAMPs, including S100 proteins (3–5). This may serve as a mechanism initiating inflammation both as a host defense response to invading pathogens and as a reaction to selfmolecules (6–8). S100 proteins are calcium-binding proteins with divergent tissue expression (9). S100A12 (calgranulin C) is expressed in significant amounts almost exclusively by granulocytes (10). It is found at high concentrations in pulmonary tissue and bronchoalveolar

Foell, Wittkowski, Kessel, et al.: S100A12 as Proinflammatory TLR4 Ligand

lavage fluid in acute respiratory distress syndrome, in which neutrophils and monocytes/macrophages are among the most abundant cell types (11–15). S100A12 has been implicated in a novel inflammatory axis involving RAGE as a receptor transducing inflammatory signals (1, 16). The description of the proinflammatory S100A12-RAGE axis was based on the initial finding that S100A12 extracted from bovine lungs bound to purified murine RAGE. Furthermore, RAGE-dependent cell activation by S100A12 was established primarily by showing that its effects could be blocked by soluble RAGE (sRAGE). Bovine S100A12 stimulated cells including murine BV2 macrophages in vitro and induced inflammation in mice in vivo (5). However, cross-species experiments with S100A12 are not easy to interpret; although in a number of publications S100A12 effects in mice were studied to establish the proinflammatory S100A12/RAGE axis (5, 17), it is now accepted that rodents do not express S100A12 (18, 19). Human S100A12 has been described to enhance proinflammatory responses of human endothelial cells and mast cells (11, 15, 16, 20). The exact extracellular functions of S100A12, however, are not yet clear. As S100A12-expressing granulocytes and circulating monocytes represent the cellular constitutes of our first line defense, it is likely that S100A12 effects have a prominent role in recruitment and activation of these cells during early inflammatory events. Monocyte activation is complex, and, furthermore, differentiation into different macrophage subphenotypes results in partially opposing effector functions ranging from pro- to antiinflammatory responses (21). In light of the pleiotropic effects of S100A12, the broad spectrum of reported RAGE involvement, and the fundamental role of monocyte activation during sepsis, we set out to analyze S100A12provoked activity of human monocytes. In the present study we demonstrate that human S100A12 is overexpressed during clinical and experimental sepsis and promotes inflammatory activation of monocytes by activating TLR4. Some of the results of these studies have been previously reported in the form of an abstract (22–25).

METHODS Human Samples and Cells All studies were approved by the scientific and ethics committees of the University of Muenster (Germany), the University of Greifswald (Germany), and/or the Academic Medical Center Amsterdam (The Netherlands). Written informed consent was obtained from all subjects or their relatives. Patients. Seventeen subjects with sepsis were included (7 women, 10 men; median age, 53 yr; range, 22–76 yr). From all patients, serum was obtained on the day sepsis was diagnosed, and occurrence of multiorgan dysfunction syndrome (MODS) was registered on Day 3. LPS challenge. Eight healthy men (22.6 6 0.6 yr) were studied after intravenous injection of Escherichia coli LPS at a dose of 4 ng/kg. Plasma was obtained before as well as up to 24 h after challenge. Healthy control subjects. Thirty healthy subjects (16 women, 14 men; median age, 33 yr; range, 18–63 yr) were included for the determination of normal S100A12 serum concentrations. Human cells. Human single-donor granulocytes were obtained from healthy volunteers (10). For stimulations before microarray analyses, peripheral blood monocytes were obtained from single healthy donors as previously described (26). For other experiments, mixed donor monocytes were isolated from buffy coats by density gradient centrifugation. Cell lines. HEK293 cells stably expressing TLR4, CD14, and MD2 (HEK-TCM) and control vector transfected cells (Invivogen, San Diego, CA) were used, next to HEK293 cells stably transfected with full-length RAGE (HEK-RAGE) or control vector as described before (27). Additional details on patients, samples, and cells are provided in the online supplement.

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Reagents and Immunoassays Reagents. LPS-free recombinant tag–free human S100A12 (recS100A12) was expressed in E. coli. Native S100A12 was isolated from human granulocytes as previously described. Recombinant sRAGE was purified from HeLa cells according to a recently described protocol (28). The following regents were purchased: E. coli LPS (055:B5; Sigma, St. Louis, MO), E. coli LPS (lot G; United States Pharmacopeial Convention, Rockville, MD), recombinant RAGE-Fc (R&D Systems, Minneapolis, MN), staurosporine (Alexis Biochemicals, Farmingdale, NY), LPS-EKmsbB (Invivogen, San Diego, CA), HTA125 (Abcam, Cambridge, UK), advanced glycation end products (AGEs) (SigmaAldrich, Deisenhofen, Germany), and HMGB1 (HMGBiotech, Milan, Italy). Assays. S100A12 was measured by a double sandwich ELISA system established in our laboratory as reported previously (14). IL-6 and IL-8 were determined by Pelikine compact ELISA (Sanquin, Amsterdam, The Netherlands); tumor necrosis factor (TNF)-a and IL-1b were determined by ELISA from BD Biosciences (La Jolla, CA). Additional details on reagents and assays are provided in the online supplement.

Cell Activation and Receptor Binding Studies Granulocyte stimulation. Cells were activated with different stimuli to induce release of S100A12. Immunohistochemistry was performed as described previously (13). Induction of necrosis and apoptosis. Apoptosis was induced by staurosporine or anti-FAS (Beckmann Coulter). For induction of necrosis, cells were treated with 75% double distilled water (ddH2O) or ultraviolet irradiation. Monocyte stimulation. Monocytes were incubated 4 hours with 10 mg/ml S100A12 in at least three independent experiments. As a control stimulus, LPS was used at 10 ng/ml. In some experiments, inhibitors of ligandreceptor interaction were used: TLR4-antagonist LPS-EKmsbB (1 mg/ml), anti-TLR4 HTA125 (1 mg/ml), anti-glycan clone gb3.1 (20 mg/ml), or sRAGE (20 mg/ml). Surface plasmon resonance binding assays. The interactions of S100A12 with TLR4-MD2 and RAGE were analyzed on a BIAcore 3,000 system (GE Healthcare, Uppsala, Sweden) using the label-free surface plasmon resonance technology. Activation of HEK293 cells. Wild-type HEK293, HEK-RAGE, or HEK-TCM cells were left untreated or were stimulated with 20 mg/ml S100A12 in the presence or absence of sRAGE (50 mg/ml) as well as with HMGB1 (10 mg/ml), AGEs (20 mg/ml), or LPS (100 ng/ml). Additional details on receptor binding and cell activation studies are provided in the online data supplement.

Microarrays and Functional Analyses DNA microarray hybridization. RNA preparation, sample preparation, and hybridization to Affymetrix (Santa Clara, CA) Human Genome 133 Plus 2.0 Gene Chip arrays for microarray analysis were performed as described previously (29). Statistical analysis of microarray data. S100A12-treated versus untreated monocytes were compared, and detection as well as change calls were assigned as previously described (29, 30). Genes with a consistent change call were further analyzed using Expressionist Suite software (GeneData, Basel, Switzerland) as described earlier. Quantitative real-time polymerase chain reaction. RNA was analyzed in duplicate. Gene expression was analyzed using specific primers obtained from MWG Biotech (Ebersberg, Germany) using QuantiTect SYBR Green PCR Kits (Qiagen, Hilden, Germany). Detection was performed using an ABI PRISM 7900 sequence analyzer (Applied Biosystems, Foster City, CA). Analysis of migration. Monocyte migration assays were performed as previously described, using 5-mm pore size Transwell filters (Costar, Bodenstein, Germany) and leukotriene B4 (100 nM LTB4, Cayman Chemicals, Ann Arbor, MI) as chemoattractant (31). Additional details on microarrays and functional analyses are provided in the online supplement.

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RESULTS S100A12 Expression during Severe Sepsis and Release from Activated Granulocytes

S100A12 serum concentrations were significantly elevated at onset of severe sepsis in patients with early MODS (n ¼ 6; median, 1,045 ng/ml; range, 195–2,000 ng/ml) compared with sepsis without MODS (n ¼ 11; median, 230 ng/ml; range, 20–960 ng/ml) and healthy control subjects (n ¼ 30; median, 65 ng/ml; range, 12–150 ng/ml), although this should be interpreted with caution due to the small patient numbers (Figure 1A; see Figure E1 and Table E1 in the online supplement). S100A12 correlated with C-reactive protein (r ¼ 0.57; P , 0.05) but not with white cell

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count (r ¼ 0.18; P ¼ 0.50) or neutrophils (r ¼ 0.16; P ¼ 0.53) (Figure E1). In experimental sepsis, levels of circulating S100A12 protein in individuals who were challenged with LPS revealed kinetics that explain the demonstrated accuracy as a biomarker of inflammation (Figure 1B). We next tested in vitro whether the overexpression of S100A12 in sepsis was a consequence of an enhanced protein expression in granulocytes that are exposed to proinflammatory stimuli. Although the capacity of these cells to contribute to inflammation by up-regulating gene transcription is generally believed to be weak, granulocytes increased S100A12 mRNA expression with a peak at 8 hours after stimulation (Figure 1C). Even more pronounced was the release of the protein from activated granulocytes on in vitro stimulation

Figure 1. S100A12 expression during sepsis and release by activated granulocytes. (A) S100A12 serum concentrations are significantly elevated in patients at onset of severe sepsis compared with healthy control subjects (n ¼ 30). Moreover, patients who developed early multiorgan dysfunction syndrome (MODS, n ¼ 6) have significantly higher levels of S100A12 compared with patients with sepsis without this complication (n ¼ 11). Horizontal lines indicate medians, error bars show interquartile ranges (*P , 0.05; **P , 0.01). (B) Increased release of S100A12 after in vivo stimulation. Human volunteers (n ¼ 8) were challenged intravenously with LPS (4 ng/kg). There was an onset of flu-like symptoms after 3 hours, which resolved within 24 hours. In parallel, S100A12 plasma concentrations increased significantly after challenge. Mean 6 SEM (*P , 0.05; **P , 0.01). (C) Granulocytes up-regulate S100A12 expression when activated. Granulocytes were purified and stimulated with IL-1b (10 U/ml), tumor necrosis factor (TNF)-a (5 ng/ml), or LPS (100 ng/ml). S100A12 RNA expression was analyzed at different time points by quantitative real-time polymerase chain reaction, normalized to glyceraldehyde-3-phosphate dehydrogenase, and plotted in relation to nonstimulated controls. Mean with error bars showing SEM (*P , 0.05). (D) Increased release of S100A12 after in vitro stimulation. Purified granulocytes were stimulated with TNFa (5 ng/ml), LPS (100 ng/ml), or phorbol myristate acetate (PMA) (100 nM) for up to 4 hours. A specific sandwich ELISA was used to quantify S100A12 in culture supernatants (*P , 0.05; **P , 0.01). Data are from three independent experiments (mean 6 SEM).


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Figure 2. Active and passive S100A12-release from granulocytes. (A, B) Purified granulocytes were stimulated with phorbol myristate acetate (PMA) (100 nM) (A) or left untreated (B). After fixation, immunohistochemical staining was used to localize S100A12. In stimulated granulocytes, S100A12 is translocated toward the plasma membrane. Although this shift was observed as a robust phenomenon, a quantification of the effect was not possible. (C) Granulocytes were stimulated with tumor necrosis factor (TNF)-a (5 ng/ml) or PMA (100 nM) for indicated time points and treated with monensin (10 mg/ml), staurosporine (1 nM), nocodazole (2 mg/ml), or forskolin (10 nM). In all experiments L-lactate dehydrogenase activity was not significantly affected. (D–F) Purified granulocytes were challenged for up to 4 hours to induce either necrosis (25–75% double-distilled H2O; 0.1–10 mM H2O2) or apoptosis (Stsp ¼ staurosporine 400 mM; anti-FAS 5 mg/ml; ultraviolet [UV] light, 20 min). Cells were stained with annexin VFITC and 7AAD and analyzed by flow cytometry for apoptosis (D) and/or necrosis (E). (F) Release of S100A12 into the culture supernatant was quantified by ELISA. Data in C–F are from three independent experiments (mean 1 SEM).

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(Figure 1D). Although not formally proven, these data indicate that the S100A12 protein rise in vivo is not simply a bystander phenomenon due to higher numbers of circulating neutrophils but rather linked to an up-regulation and secretion from activated granulocytes in the context of inflammatory responses. Alternative Secretory Pathways and Passive Release Increase Extracellular S100A12

S100A12 lacks a signal peptide for secretion via the classical route involving the endoplasmic reticulum and the Golgi apparatus. Immunostainings of granulocytes suggested that the cytoplasmic protein translocates toward the plasma membrane in activated cells (Figures 2A and 2B). To assess possible routes of secretion, granulocytes were stimulated with TNF-a or phorbol

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myristate acetate for 30 or 60 minutes and treated with blockers for different intracellular transportation pathways. The inhibitor of Golgi-trafficking monensin did not affect S100A12 release. In contrast, inhibition of protein kinase activity (staurosporine) or inhibition of tubulin polymerization (nocodazole) blocked S100A12 release. Forskolin as a cAMP modulator did not induce S100A12 secretion (Figure 2C). Another characteristic of DAMPs is their passive release from necrotic cells. To study passive release of S100A12, purified granulocytes were either left untreated or challenged for 4 hours to induce either necrosis or apoptosis (Figures 2D–2F). Early apoptosis was induced to varying extents in staurosporine-, antiFas–, or ultraviolet light–treated cells as indicated by AnexV1 7AAD2 cell populations (Figure 2D; 25.3%, 20.4%, and 67.6%, respectively). Granulocyte necrosis induced by hypotonic shock

TABLE 1. TOP UP-REGULATED GENES IN MONOCYTES ON S100A12 STIMULATION Description Similar to RIKEN cDNA 4930457P18 Chemokine (C-C motif) ligand 20 Inhibin, b A (activin A, activin AB alpha polypeptide) Similar to immune-responsive gene 1 Nuclear receptor subfamily 5, group A, member 2 Norrie disease (pseudoglioma) Solute carrier organic anion transporter family, member 4A1 IL-1, a Chromosome 20 open reading frame 160 Solute carrier family 1 (glial high affinity glutamate transporter), member 3 Sphingosine-1-phosphate phosphatase 2 IL-6 (interferon, b2) ELOVL family member 7, elongation of long chain fatty acids (yeast) Tumor necrosis factor, a-induced protein 6 Hairy/enhancer-of-split related with YRPW motif 1 Gap junction protein, b2, 26kDa (connexin 26) Myosin IB Chemokine (C-C motif) ligand 23 KIAA0376 protein G protein-coupled receptor 31 EH-domain containing 1 Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) TNFAIP3 interacting protein 3 Tenascin C (hexabrachion) Musculin (activated B-cell factor-1) B-factor, properdin G protein-coupled receptor 43 CDC42 effector protein (Rho GTPase binding) 5 Putative lymphocyte G0/G1 switch gene Colony stimulating factor 3 (granulocyte) Mucolipin 2 Pentaxin-related gene, rapidly induced by IL-1b Adenylate kinase 3 Adenylate kinase 3-like 2 Pleckstrin homology domain containing, family C (with FERM domain) member 1 Chemokine (C-C motif) ligand 4 Homo sapiens, clone IMAGE:4133286, mRNA Transcribed locus, weakly similar to NP_775735.1 l(3)mbt-like 4 (Drosophila) [Homo sapiens] Hypothetical LOC387763 CD44 antigen (homing function and Indian blood group system) CDNA: FLJ20923 fis, clone ADSE00893 Tumor necrosis factor receptor superfamily, member 4 Early B-cell factor Adenosine A2a receptor Chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2)

Gene Symbol

Entrez

P Value

Fold Change 25

LOC123872 CCL20 INHBA LOC341720 NR5A2 NDP SLCO4A1 IL1A C20orf160 SLC1A3

AW663632 NM_004591 M13436 BG236136 AF146343 NM_000266 NM_016354 M15329 BF970287 BC022285

2.18435 3 10 0.001960299 0.000594067 0.004041633 0.000945162 0.00038906 0.001063534 0.000611211 0.02068992 0.001513068

SGPP2 IL6 ELOVL7

AW779536 NM_000600 AW138767

9.42047 3 1025 0.004005239 0.002865589

47.0183 43.77923 42.55163

TNFAIP6 HEY1 GJB2 MYO1B CCL23 KIAA0376 GPR31 EHD1 FLT

AW188198 NM_012258 M86849 BF432550 U58913 AA481137 NM_005299 AL579035 AA149648

0.001968954 0.003313315 0.000153431 0.000425799 0.02045774 1.24686 3 1025 0.000929176 2.05224 3 1025 0.000455616

41.76492 40.47276 40.3485 40.19905 39.9334 39.17511 37.97106 36.51133 35.19753

TNIP3 TNC MSC BF GPR43 CDC42EP5 G0S2 CSF3 MCOLN2 PTX3 AK3 AK3, AK3L2 PLEKHC1

NM_024873 NM_002160 AF060154 NM_001710 NM_005306 AW084544 NM_015714 NM_000759 AY083533 NM_002852 AI566130 AI653169 AW469573

0.002943991 0.001390216 0.001384845 0.006216734 0.003168672 0.000324896 0.000719905 0.000197316 0.001705715 0.001619394 6.6088 3 1025 0.002156772 0.008689795

32.99512 32.03719 31.26347 30.46245 30.32967 29.42337 28.62501 27.02029 25.82313 25.7641 25.55252 25.05864 25.04481

CCL4

NM_002984 BC016366 BF664545

0.000785006 0.000443746 0.01721469

24.78477 24.61909 23.86122

LOC387763 CD44

AW276078 U94903

0.01328909 0.006850048

23.63267 22.60912

TNFRSF4 EBF ADORA2A CXCL6

H22954 AJ277151 AF208502 NM_000675 NM_002993

0.000117179 0.000262543 0.00819636 0.000113433 0.00052255

22.5262 22.29149 21.36218 20.71766 20.4725

* Top 45 of a total of 745 significantly up-regulated genes (more than twofold).

341.5771 173.4552 149.9793 139.0322 126.1213 77.85728 74.77097 74.7457 69.27634 50.06431


(25–75% ddH2O) or radical stress (0.1–10 mM H2O2) also revealed apoptotic events (Figure 2D). However, exposure of granulocytes to 75% ddH2O as well as 1 and 10 mM H2O2 resulted in significant cellular necrosis (Figure 2E). Strikingly, S100A12 was released from exactly those cells (Figure 2F; Figure E2). Protein release from just apoptotic cells was significantly lower if at all detectable above the unstimulated control background. S100A12 Is a Strong Inducer of Proinflammatory Responses in Monocytes

The proinflammatory potential of S100A12 was confirmed on a genome-wide level in primary human monocytes. Using broad gene-expression profiling, a significant up-regulation of 745 genes on stimulation with native S100A12 was detected, with most induced genes linked to proinflammatory functions (Tables 1 and 2; Table E2). A total of 1,594 genes were significantly downregulated, with most suppressed genes linked to cell homeostasis (Table 3; Table E3), probably indicating cell stress. The pattern of monocyte stimulation clearly shows a proinflammatory activation of monocytes induced by human S100A12 (Tables 2 and 3). The induction of gene expression as shown by microarray data could be confirmed by independent methods, using quantitative real-time polymerase chain reaction (qRT-PCR) on selected immunologically relevant genes that were regulated in microarray analyses (Figure 3A). Furthermore, the induction of key cytokines and chemokines was confirmed on the protein level (Figure 3B). In addition to inflammatory response functions, chemotaxis was one of the functional groups that were overrepresented as a consequence of S100A12-induced genes; therefore, we also analyzed the migration as one key function of monocytes that is relevant under physiologic and pathophysiologic conditions. In transwell filter experiments, S100A12 induced a significant increase of LTB4-directed migratory activity of monocytes (Figure 3C). In addition, S100A12 revealed a direct chemotactic effect on primary human monocytes (Figure 3D). Both effects could be abrogated below significance by blocking TLR4, whereas blocking RAGE did not result in any (Figure 3C) or that pronounced decrease in migration (Figure 3D). S100A12 Binds to RAGE and TLR4

Previously published data showed that S100A12 is a ligand for RAGE, a receptor that has been described as a multiligand receptor linked to inflammatory cell-activation pathways. However, blocking the RAGE axis either by an anti-RAGE antibody or addition of sRAGE did not affect TNF secretion by recS100A12-stimulated monocytes in a significant manner (Figure 4A). Instead, blocking TLR4 as a putative second receptor either by the anti-TLR4 antibody HTA125 or the TLR4 antagonist LPS-EKmsbB abrogates S100A12-mediated monocyte stimulation. Due to these results, we investigated the binding of

1329 TABLE 3. FUNCTIONS OF S100A12-SUPPRESSED GENES Functional Groups GTPase activator activity Negative regulation of cell cycle Transferase activity Carbohydrate metabolism Intracellular signaling cascade Lysosome Metabolism Protein kinase cascade Catalytic activity Endocytosis

Gene Ontology

P Value

0005096 0045786 0016740 0005975 0007242 0005764 0008152 0007243 0003824 0006897

6.57 3 1026 0.00015 0.00032 0.00034 0.00066 0.00110 0.00116 0.00132 0.00142 0.00266

S100A12 to RAGE in comparison to TLR4 as another possible receptor using surface plasmon resonance analysis. Hence, native S100A12 was injected at three different concentrations over immobilized human RAGE-Fc fusion protein (Figure 4B) and murine TLR4-MD2 complexes (Figure 4C). Kinetic and affinity analysis of sensorgrams revealed binding of S100A12 to both RAGE and TLR4 with a relatively fast on-rate accompanied by a biphasic dissociation phase. As sensorgrams demonstrated poor fit to standard kinetic models, apparent affinity and maximal binding were calculated by plotting responses at steady-state against S100A12 concentration and fit of the dose–response curve to a one-site hyperbola model (Figure 4D). A twofold higher affinity (Kd, 0.92 and 1.89 3 1027 M) and a threefold higher response maximum (Bmax z 1.1 and 0.36 kRU) was demonstrated for S100A12 binding to TLR4 than to RAGE. Stimulatory Effects of S100A12 on Monocytes Differ from Those of LPS Alone

Presence of LPS in recombinant preparations of alarmins with proposed TLR-mediated signaling has to be assessed carefully. Therefore, we investigated gene expression responses in monocytes stimulated with native S100A12 or LPS alone. A comparison of the respective data revealed a common expression of some genes, such as TNF-a, IL-8, or MCP-1, regardless of whether monocytes were stimulated with either S100A12 or LPS. However, genes such as IL15RA, IL-7, and IL-18 in particular are significantly more strongly induced by S100A12 but not LPS (Figure 5A). In addition, TNF-a secretion by monocytes stimulated with recS100A12 depends on intact protein. Destroying the S100A12 structure by heat denaturation leads to an abrogation of signaling resulting in TNF-a release, whereas similar treatment of LPS alone does not show any effect. In addition, immunoprecipitation of S100A12 via preincubation with protein G–bound anti-human S100A12 polyclonal antibodies was successful in abolishing stimulatory effects of S100A12 by greater than 95%. As S100A12-mediated stimulation significantly differs from that of LPS alone and can be modulated by treatments affecting the presence of intact protein, a direct impact of the protein on TLR4 signaling can be proposed.

TABLE 2. FUNCTIONS OF S100A12-INDUCED GENES Functional Groups Inflammatory response Cell–cell signaling Chemokine activity Chemotaxis Immune response Antimicrobial humoral response Extracellular space Signal transduction Calcium ion homeostasis Antiapoptosis

Gene Ontology 0006954 0007267 0008009 0006935 0006955 0019735 0005615 0007165 0006874 0006916

P Value 2.81 8.50 9.26 5.64 7.46 9.32 1.07 2.73 3.11 5.35

3 3 3 3 3 3 3 3 3 3

10214 10210 10210 1029 1028 1027 1026 1025 1025 1025

S100A12-induced Monocytic Cell Activation Is TLR4 Dependent

Our surface plasmon resonance data suggest the binding of S100A12 to both RAGE and the TLR4-MD2 complex, with higher affinity for the latter. To confirm an independent role of TLR4 for S100A12-mediated proinflammatory signaling, we chose HEK293 cells transfected with respective constructs. This allows us to investigate the sole contribution of the distinct receptors TLR4 and RAGE to cellular signaling. HEKRAGE cells expressing membrane-bound RAGE on their

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Figure 3. Monocyte activation after stimulation with native S100A12. (A) The induction of a specific up- or down-regulation of gene expression as detected by microarrays could be confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) as second independent technique. (B) Up-regulation of IL-1b, IL-6, and IL-8 could also be confirmed on the protein level by specific ELISA systems measuring concentrations in supernatants of human monocytes stimulated by S100A12 for 24 hours (*P , 0.05; **P , 0.01). (C, D) The functional characteristics of S100A12-treated monocytes were assessed by analyses of migratory and chemotactic activity. (C) After purification, monocytes were treated for 24 hours with S100A12 (10 mg/ml) 6 Toll-like receptor 4 (TLR4)-blocker (LPS-EKmsbB) or anti–receptor for advanced glycation end products (RAGE) monoclonal antibodies (mAb) (gb3.1) or left untreated. Subsequently, cells were placed into the upper chamber of a transwell filter system. The lower chamber contained monocyte medium without or with LTB4 as chemotactic stimulus. After 4 hours, the number of cells that had migrated into the lower compartment was counted (ns ¼ not significant; *P , 0.05; **P , 0.01). (D) The direct chemotactic potential of S100A12 on primary human monocytes was tested. Cells were treated with TLR4-blocker (LPS-EKmsbB), anti-RAGE mAb (gb3.1), or left untreated for 24 hours and placed in the upper chamber of a transwell filter system. Medium in the lower chamber was supplemented with S100A12 at concentrations (in mg/ml) as indicated. Cell numbers that had migrated to the lower chamber were quantified after 4 hours (ns ¼ not significant; *P , 0.05; **P , 0.01). All data are from three independent experiments (mean 1 SEM).

surface do respond to the primary RAGE ligands’ AGEs but are unresponsive to recS100A12 (Figure 6A). Instead, HEK-TCM cells expressing fully functional TLR4-MD2 complexes and CD14 up-regulate the expression of proinflammatory genes as IL-8 or TNF-a (not shown) on stimulation with recS100A12 compared with mock-transfected HEK cells (Figure 6B). In line with the observations from monocyte stimulations, the effects of S100A12 could be blocked by inhibiting S100A12 binding to TLR4 by the anti-TLR4 antibody HTA125. The addition of sRAGE to HEK-TCM cultures did not affect S100A12triggered stimulation. Hence, although we could not find proof for biological relevance of RAGE or sRAGE for S100A12 binding in these assays, some modulating effect of S100A12 stimulation on primary monocytes is not excluded.

DISCUSSION We and others have demonstrated that granulocyte-derived S100A12 is elevated during early systemic inflammation (11,

14, 32, 33). Although our data presented here confirm that S100A12 may thus be a biomarker that correlates to disease severity and the risk of complications such as MODS, studies using higher case numbers or subgroup analyses (e.g., correlation to specific infections or complications) were beyond the scope of our study. Instead, the main goal of our work was to establish the role of S100A12 in early events of inflammatory cell activation that may lead to sepsis and organ damage. Multiple studies revealed a pivotal function of S100A12 as a proinflammatory alarmin or DAMP molecule. DAMP molecules are often characterized by the fact that they serve a physiological role while intracellular but act as proinflammatory danger signals when present extracellularly after active secretion or passive release. Exact intracellular functions of S100A12 are not known, but a role in calcium homeostasis has been proposed (34). Even more relevant may be extracellular effects, as S100A12 is expressed in significant amounts almost exclusively in granulocytes that infiltrate tissues in inflammatory conditions and are capable of depositing the protein in this context (6, 10,


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Figure 4. Effects of S100A12 on monocytes are not receptor for advanced glycation end products (RAGE)-dependent. (A) Monocytes were stimulated with recS100A12 (10 mg/ml) in the presence of RAGE-blocking anti-glycan antibody (clone gb3.1), soluble RAGE (sRAGE), anti–Tolllike receptor 4 (TLR4) antibody (HTA.125), or LPS-EKmsbB. Tumor necrosis factor (TNF)-a secretion was assayed by ELISA (ns ¼ not significant; *P , 0.05; **P , 0.01). Data are from three to five independent experiments (mean 1 SEM). (B) Sensorgrams obtained after injection of 50 (light gray), 100 (medium gray), and 200 (dark gray) nM native S100A12 (1.1, 2.1, and 4.2 mg/ml) over immobilized recombinant human RAGE/ Fc. (C) Corresponding sensorgrams for native S100A12 injected over immobilized murine TLR4-MD2. Sensorgrams were corrected for contribution of sample buffer (1 mM CaCl2 and 20 mM ZnCl2 in HEPES-buffered saline) to the signal. (D) Binding curves demonstrating satiable binding of S100A12 to both RAGE (triangles) and TLR4/MD-2 (squares). Steady-state responses were plotted versus concentration of S100A12 and data fit to a 1:1 model in GraphPad Prism. Affinity (Kd) and response maximum (Bmax) for S100A12 interaction with RAGE and TLR4 were calculated to 190 and 92 nM and 364 and 1,130 RU, respectively.

14, 35, 36). DAMPs have been supposed to be released with a delay in comparison with early released mediators. We now demonstrate that a DAMP such as S100A12 is released soon in response to granulocyte activation, which is a hallmark of sepsis. RAGE has been shown to transduce extracellular effects of S100A12 but also S100B, S100A4, S100A6, S100A11, S100A13, and S100P. However, a significant protein expression of RAGE is found only in the lung, whereas surface expression is weak on other human cells. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells (37). Recent studies showed that interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for signal transduction (38). It has been proposed that S100A12-induced effects are mediated by RAGE. In line with this, treatment with sRAGE significantly attenuated the increases in neutrophil infiltration, lung permeability, production of inflammatory cytokines, and nuclear factor-kB activation, as well as development of pathologic changes in ex vivo and in vivo studies (5, 17, 39). However, there is no doubt that cross-species studies performed with bovine S100A12 in murine models are hard to interpret, as there is no murine S100A12 (18, 19). Furthermore, beneficial effects of sRAGE are no final proof for RAGE receptor signaling as the main transducer of S100A12 effects, especially on cells that show a weak RAGE expression. Scavenger effects of sRAGE on S100A12 may block other receptor interactions as well. Importantly, studies with RAGE-null mice show that RAGE plays little role in adaptive immune responses (40). Although sRAGE reduced delayed-type hypersensitivity responses proposed to be mediated by S100A12, it was similarly suppressive in RAGE-

null mice, and undefined effects besides simply blocking cellsurface RAGE function were proposed. Others reported RAGE-independent pathways and also suggested an alternate receptor for S100A12 on mononuclear cells (20, 41). Among others, the extracellular effects of S100A12 include the induction of cytokines as well as chemotactic activity of neutrophils and macrophages (5, 42, 43), induction of neurite outgrowth (44), mast cell activation (20), and activation of endothelial cells (5, 11, 15). However, to date the interaction of human S100A12 with receptors on monocytes and their subsequent activation has not been investigated. The main source of S100A12 in humans is represented by granulocytes. Accumulation of both granulocytes as well as monocytes and also of the S100A12 protein has been demonstrated in inflammation. Hence, it appears a strikingly relevant question whether the S100A12 that is detectable extracellularly has direct effects on coinfiltrating monocytes that have a crucial function in directing further inflammatory processes (12–14, 45). Our results show that S100A12 is up-regulated and rapidly secreted by activated granulocytes and released from necrotic granulocytes in vitro in a manner that has been described as DAMP-typical (1). This leads to a rapid increase in local S100A12 concentrations, which is relevant in vivo and may explain the kinetics of S100A12 levels that are detectable in the human circulation (11, 35, 46). The release from granulocytes in circulation and especially during transendothelial diapedesis with infiltration of affected tissues can directly lead to elevation in the plasma/serum (as in experimental sepsis, shown in Figure 1B) but also result in a spill-over from affected tissues. A combination of both effects is probably relevant in established inflammation, such as in

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Figure 5. S100A12 activation of monocytes is independent of LPS but can be inhibited by Toll-like receptor 4 (TLR4)-blockade. (A) Gene expression of several genes up-regulated by native S100A12 according to microarray analyses was analyzed by quantitative real-time polymerase chain reaction, comparing mRNA expression after stimulation with either LPS (10 ng/ml) or recS100A12 (10 mg/ml). (B) Human monocytes were stimulated with LPS (10 ng/ml), recS100A12 (10 mg/ml), S100A12 (10 mg/ml) after endotoxin removal column or after immunoprecipitation (IP) with anti-S100A12 antibodies, heat-treated (958 C, 15 min) S100A12 or LPS. After 4 hours of incubation, tumor necrosis factor (TNF)-a was analyzed in the supernatant of cells via ELISA. All data are from three independent experiments (mean 1 SEM).

patients with sepsis. We also demonstrate that S100A12 induces a strong inflammatory activation of monocytes. The increased S100A12 concentrations that accumulate in inflamed tissue may thus contribute to an orchestrated amplification of inflammatory processes via cross-talk between granulocytes and monocytes. Monocytes express a number of innate immune receptors allowing their rapid responsiveness to a broad spectrum of stimuli. Pattern recognition receptors are especially important for sensing threats to the host from invading pathogens. However, these multiligand receptors, including TLRs and RAGE, are also capable of binding endogenous ligands that serve as danger signals indicating tissue stress and amplifying innate defense mechanisms. We show here that monocyte activation is amplified by endogenous S100A12. Although this protein is a ligand of both TLR4 and RAGE, the latter was found to be a modulating factor, whereas the signal transduction is TLR4 dependent. In addition, binding of S100A12 to RAGE is mediated by

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Figure 6. S100A12 exerts proinflammatory activation via interaction with Toll-like receptor 4. HEK293 wild-type cells (HEK) transfected with the empty plasmid HEK293 cells were compared with either cells stably transfected with an expression plasmid for receptor for advanced glycation end products (HEK-RAGE) (A), or with an expression plasmid for TLR4, CD14, and MD2 (HEK-TCM) (B). Cells were stimulated with 20 mg/ml recS100A12 in the presence or absence of soluble RAGE (sRAGE) (50 mg/ml), 10 mg/ml HMGB1, advanced glycation end products (AGEs) (20 mg/ml), or LPS (100 ng/ml). IL-8 mRNA expression in cell lysates was quantified by quantitative real-time polymerase chain reaction. All data are from three to five independent experiments (mean 1 SEM).

carboxylated glycans expressed on RAGE, and this glycan modification of RAGE is cell-type dependent (28). Because oligomerization of S100 proteins under nonreducing, high-Ca21 conditions found extracellularly appears to play a pivotal role in S100A12 biology, complex interactions of S100A12 as a ligand for cell surface receptors are not unlikely (28, 47). Opposing roles of RAGE and MyD88 signaling in liver resection–associated deleterious inflammatory responses have been described (48). Our data indicate that the modulatory effects of RAGE on monocytes may result in specific effects, as the activation of monocytes by S100A12 via TLR4 differs from that induced by LPS. It is interesting in this regard that some factors specifically up-regulated by S100A12 but not by LPS have relevant immune functions (Figure 5A). The interactions of IL-7, IL-15, IL15RA, and IL-18 are complex and only partially understood. However, these mediators are involved in the cooperation between the innate and adaptive immune systems (49–54). The observed dissociation between pathogen-associated molecular pattern and DAMP effects is thus an interesting observation; further analyses of related mechanisms may provide future insights into a pro- and antiinflammatory disequilibrium that is involved in sepsis. In conclusion, we introduce human S100A12 as a novel endogenous TLR4 ligand and establish its role as a mediator facilitating inflammatory monocyte activation in a cross-talk with activated granulocytes. This makes the protein a proinflammatory amplifier of early inflammatory processes that are involved in sepsis. Although


RAGE-dependent effects on other cell types, such as endothelial cells, or in other conditions involving rather chronic inflammatory processes cannot be excluded, the engagement of RAGE on monocytes is not necessary for S100A12-induced monocyte activation. Instead, sRAGE may have a modulating function on monocyte activation. It remains a matter of future studies whether this may serve as a function to temper chronic inflammatory reactions during further monocyte/macrophage differentiation.

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Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank Melanie Saers, Sabrina Fuehner, and Susanne Schleifenbaum for excellent technical assistance. They also thank Torsten Kucharzik (Lueneburg, Germany) for including patients with sepsis at the University Hospital Muenster and Marco E. Bianchi (Milano, Italy) for kindly providing HEK-RAGE cells and recombinant HMGB1.

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