The Journal of Immunology

S100A8 Induces IL-10 and Protects against Acute Lung Injury Yuka Hiroshima,1 Kenneth Hsu,1 Nicodemus Tedla, Yuen Ming Chung, Sharron Chow, Cristan Herbert, and Carolyn L. Geczy S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys42-Ala42 mutant S100A8 mutant was used to assess whether S100A8’s effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-kB activation via an IkBa/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys42-Ala42 S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury. The Journal of Immunology, 2014, 192: 2800–2811.

T

he inflammation S100 protein, S100A8, is considered proinflammatory because it triggers TLR4 (1) or the receptor for advanced glycation end products (RAGE) (2). S100A8 (also called CP-10 or MRP8) is an S100 calcium-binding protein often coexpressed with S100A9 (MRP14). The S100A8/ S100A9 heterocomplex is elevated in lesions and in the circulation of patients with many inflammatory diseases, including those with lung pathologies such as cystic fibrosis and acute respiratory distress syndrome (3, 4). S100A8 comprises ∼20% of the neutrophil cytosol, is constitutively expressed at low levels in monocytes, but generally is not present in tissue macrophages (5). It is induced in several cell types by inflammatory mediators or by oxidative stress (6). In murine macrophages, S100A8 is induced

Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia 1

Y.H. and K.H. contributed equally to this work.

Received for publication September 23, 2013. Accepted for publication January 12, 2014. This work was supported by National Health and Medical Research Council of Australia Grants 630647 and 1027189. Y.H. and K.H. performed the experiments; K.H. and C.L.G. designed the gene set to be analyzed. N.T. assisted with design of signaling experiments and provided advice. Y.M.C. provided S100 preparations for experiments. S.C. performed some immunohistochemistry. C.H. provided assistance and advice in handling the mice and harvesting lung and bronchoalveolar lavage fluid. C.L.G. conceived the experiments and prepared most of the manuscript. Y.H. and K.H. assisted with manuscript preparation. The PCR array data set presented in this article has been submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE52720) under accession number GSE52720. Address correspondence and reprint requests to Prof. Carolyn Geczy, Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: ALI, acute lung injury; BALF, bronchoalveolar lavage fluid; Dex, dexamethasone; GC, glucocorticosteroid; iNOS, inducible NO synthase; RAGE, receptor for advanced glycation end products; redox, reduction/ oxidation; ROS, reactive oxygen species; SAA3, serum amyloid A3. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302556

by TLR agonists in the absence of S100A9 (7, 8), and although some functions depend on heterocomplex formation, they can function independently (reviewed in Ref. 9). Murine S100A8 is a leukocyte chemoattractant, with mechanisms atypical of classical chemoattractants and properties similar to the anti-inflammatory mediator TGF-b (10). It provokes mild, transient leukocyte infiltration after i.p. or intradermal injection. S100A8 is a two-electron oxidant scavenger, and oxidation of the single Cys42 thiol residue can generate reactive oxygen species (ROS)–dependent changes in function (reviewed in Ref. 11). Novel adducts found in isolates from human asthmatic sputum (12) indicate that S100A8 protects against excessive oxidative stress generated by hypohalous acid oxidants. S100A8 also inhibits mast-cell activation by scavenging ROS required for activation of linker for activation of T cells; intranasal S100A8 administration suppressed acute murine asthma by reducing chemokines required for eosinophil infiltration into the lung and for mucous production (13). S100A8 gene induction in monocytes/macrophages is IL-10 dependent and enhanced by glucocorticosteroids (GCs) (14). It is prominent in lungs of mice treated with LPS and GCs (15). IL-10 is a potent immunosuppressive cytokine constitutively expressed at low levels in normal lung (16). In some airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, levels are less than in healthy subjects and decreases may contribute to pathogenesis (16, 17). In this study, we compared changes induced by S100A8 in murine lung after intranasal challenge, with those provoked by the TLR4 agonist, LPS. Unexpectedly, S100A8 did not cause overt inflammation but induced high levels of IL-10 mRNA. The protein was apparent only in lung airway epithelial cells and in bronchoalveolar lavage fluid (BALF). Importantly, in a model of acute lung injury (ALI) induced by LPS inhalation, S100A8 totally suppressed neutrophil infiltration and mast cell activation, and induction of proinflammatory and chemokine genes. Suppression was remarkably similar to that seen with dexamethasone (Dex),

The Journal of Immunology which directly induced S100A8 in alveolar macrophages. The Cys42-Ala42S100A8 mutant did not induce IL-10 or reduce mastcell activation, although suppression of LPS-induced mediators was only partially dependent on the reactive Cys residue, indicating ROS-dependent and -independent mechanisms. We propose a novel pathway in which S100A8, in cooperation with IL-10, downmodulates innate immune responses and may contribute to some anti-inflammatory actions of GCs.

2801

Specific pathogen-free female BALB/c mice (7–9 wk old; 19–20 g) from Australian BioResources (Moss Vale, Australia) were group housed in ventilated cages, exposed to a 12-h light/dark cycle, and provide autoclaved food and water ad libitum. Experimental procedures were performed according to ethics guidelines of the National Health and Medical Research Council of Australia with specific institutional approval of the Animal Care and Ethics Committee of the University of New South Wales (reference number 10/96B).

lungs of at least three mice per group. Because S100A8 is reported to initiate proinflammatory responses by ligating TLR4 and/or RAGE, a quantitative PCR array was developed to analyze 63 genes, selected to reflect potential acute inflammatory changes induced by ligation of these receptors, particularly cytokines and chemokines, CSFs and genes facilitating leukocyte transmigration (endothelial cell adhesion molecules and matrix metalloproteases), some genes involved in regulating reduction/ oxidation (redox), 3 housekeeping genes, and a nonamplification control (list is given in Supplemental Table I; primer sequences are available on request). Primer sets were validated as a single peak in melting temperature (Tm) calling analysis, with no secondary products evident. Primer sets were precoated into 384-well quantitative PCR plates (Roche) using an epMotion 5075 automated pipetting system and stored at 220˚C. Serum amyloid A3 (SAA3), tissue factor, and redox-related genes were measured in a second array. Nonsupervised hierarchical clustering of the entire data set is presented in clustergrams. The volcano plots graph the log2 of fold changes (DDCt method, significance set at 2.5-fold) and 2log10 of p values comparing test and control groups (Student t test, set at p , 0.05) for each gene. Clustergrams and volcano plots were generated by web-based software (SABiosciences). Data files for this experiment are lodged on Gene Expression Omnibus (accession no. GSE52720, http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE52720).

Assessment of putative proinflammatory effects of S100A8

ELISA

Recombinant murine S100A8 and the corresponding Cys42 to Ala42 mutant were expressed as GST-fusion proteins in Escherichia coli and generated as described previously (12, 18) and purified by C8- and C4-HPLC. Native S100A8 was stored in 45% acetonitrile, 0.1% trifluoroacetic acid at 280˚C under argon. Reconstitution, mass validation by mass spectrometry, and purity of S100A8 preparations were performed as described previously (12). Both were used at 10 mg/50 ml HBSS administered onto the nares for all experiments detailed. To assess direct effects, we sacrificed mice 1, 4, 6, 12, or 20 h postinhalation of S100A8, or 12 and 20 h postinhalation of Ala42S100A8. For comparison with Dex inhalation (used at 10 mg/50 ml HBSS in all experiments detailed; Sigma-Aldrich), lungs were harvested 6 and 12 h after administration.

IL-4, IL-6, IL-10, TNF-a, and CCL-2 levels in BALF were quantitated by ELISA according to manufacturer’s instructions (R&D Systems).

Materials and Methods Mice

Induction of inflammation by LPS and effect of S100A8 LPS (10 mg/50 ml PBS; E. coli Serotype 055:B5; Sigma-Aldrich) was administered onto the nares. Intranasal S100A8 was given 2 h before or 15 min after LPS, and Ala42S100A8 or Dex 2 h before LPS. Control mice received equal volumes of PBS and HBSS instead of LPS and S100A8, respectively. Mice were sacrificed 1 or 4 h after LPS inhalation. Portions of right lung were preserved in RNAlater (Ambion), stored at 4˚C until RNA was extracted. Portions of right lung were stored at 280˚C for Western blotting. Left lungs were fixed with 10% neutral buffered formalin.

Analysis of infiltrating cells A cannula (19G) was inserted into the trachea, and ice-cold PBS (1 ml 3 2) instilled into the lung; then BALF was collected. BALF was centrifuged (400 3 g, 7 min, 4˚C) to pellet cells, and supernatants were removed and stored at 280˚C. Cells resuspended in PBS were used to determine total numbers and for differential counts by staining with Diff-Quick Stain Set (Lab Aids, Narrabeen, Australia). At least 300 cells were counted microscopically.

RT-quantitative PCR array Total RNA was extracted from the lung by standard procedures using TRIzol Reagent (Invitrogen), genomic DNA removed with DNase (Turbo DNase; Ambion) before reverse transcription (1.5 mg) using SuperScript VILO Master Mix (Invitrogen). PCR amplification was carried out with LightCycler 480 SYBR Green I Master (Roche). Reactions were performed using the LightCycler 480 system (Roche) under standard cycle conditions. Reactions contained 23 SYBR Green master mixture, 4 ml diluted cDNA template or nonamplified controls, and 250 nM of each primer in a final volume of 10 ml. Expression of inflammatory genes was evaluated with the RT-quantitative PCR array. Relative quantities of mRNA in duplicate samples were obtained using the LightCycler 480 Software 1.5 and the Efficiency-Method. Gene expression was calculated with the web-based software package (SABiosciences), which automatically performs all DDCt-based fold-change calculations from the specific uploaded raw threshold cycle data. mRNA expression was normalized to the geometric mean of three housekeeping genes (HPRT, b-actin, and GAPDH), and cutoff threshold was set to 37 Cp (cross point) value. Data represent fold changes of mRNA relative to unstimulated samples (mean 6 SEM) from

Macrophage stimulation in vitro A murine alveolar macrophage cell line (MH-S; ATCC CRL-2019; American Type Culture Collection) was used to determine S100A8 induction by LPS and/or by Dex. Cells were cultured at 37˚C in 5% CO2 in air in RPMI 1640 (Invitrogen) supplemented with 127 U/ml penicillin, 127 mg/ml streptomycin (Sigma), and 10% heated (56˚C, 30 min) bovine calf serum (Invitrogen). Dex (1 mM) and/or LPS (1 ng/ml) was incubated with cells for 4, 10, and 24 h; cells were harvested; and S100A8, Ym1, arginase1, inducible NO synthase (iNOS), IL-10, and TNF-a mRNA levels were quantitated by quantitative RT-PCR. Alternatively activated alveolar macrophages were generated by incubating alveolar macrophages from naive mice for 48 h in RPMI 1640 with 10% FBS supplemented with M-CSF (20 ng/ml; eBioscience), IL-4, and IL13 (both 10 ng/ml; R&D Systems) as described previously (19). S100A8 and arginase-1 mRNA levels were quantitated by quantitative RT-PCR.

Western blot Lungs were homogenized in 10 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 100 mM pervanadate, 1% Nonidet P-40, and complete proteinase inhibitor mixture (Roche), centrifuged (800 3 g, 10 min, 4˚C) to remove debris and nuclei, and supernatants were removed and centrifuged at 17,000 3 g for 20 min at 4˚C. Total protein content of resulting supernatants was measured using BCA Protein Assay Kit (Thermo Scientific), standardized to BSA according to the manufacturer’s protocol. Protein (20 mg) was subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes (Millipore), and Western blotting was performed with the following Abs: rabbit monoclonal anti–phospho-MEK1/2 (Ser217/221, 1:1000 v/v), anti–phospho-p38 MAPK (Thr180/Tyr182, 1:1000 v/v), anti– phospho-Akt (Thr308, 1:1000 v/v) and anti-IkBa (1:1000 v/v), and mouse monoclonal anti–phospho-tyrosine (biotinylated, 1:2000 v/v), and rabbit polyclonal anti-MEK1/2 (1:1000 v/v), anti–phosho-Erk1/2 MAPK (Thr202/ Tyr204, 1:1000 v/v), anti–phospho-STAT3 (Tyr705, 1:1000 v/v), anti-STAT3 (1:1000 v/v; all from Cell Signaling Technology), anti–SHP-2 (1:1000 v/v; Millipore) and anti-SHIP (1:1000 v/v; Millipore), and goat polyclonal anti–SHP-1 (1:500 v/v; R&D Systems). Anti–b-actin (1:500 v/v, rabbit polyclonal; Thermo Scientific) was used to control for protein loading. After washing with 0.1% Tween 20 in TBS, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:2000 v/v; Bio-Rad Laboratories), rabbit anti-goat IgG (1:2000 v/v; Bio-Rad), or goat anti-biotin (1:2000 v/v; Vector Laboratories) at room temperature for 2 h, and reactivity was detected as described previously (20).

Paranitrophenyl phosphate phosphatase assay The general phosphorylation activity of lung lysates (10 mg total protein in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, and 1% w/v NP40) was measured by assessing the total phosphatase activity using paranitrophenyl phosphate as a substrate as described previously (21). Paranitrophenyl release was determined spectrophotometrically by measuring

2802 A405 nm, and absorbance was calculated as a ratio of enzyme activity relative to control.

S100A8 INDUCES IL-10 AND INHIBITS ACUTE LUNG INJURY anti-goat IgG secondary Ab (1:500 v/v; Dako). Images were taken by Olympus DP73.

Immunohistochemistry Formalin-fixed, paraffin-embedded lung tissue was sectioned (4 mm) and stained with H&E or with specific rabbit anti-S100A8, -S100A9 Abs (5 mg/ml) generated in-house as described previously (22); rabbit monoclonal anti–acetyl-histone H3, rabbit anti–acetyl histone H4 (both 1:500 v/v; Cell Signaling Technology) or normal rabbit IgG (5 mg/ml; Dako) were used after blocking. Secondary Ab was biotinylated goat anti-rabbit IgG (1:500 v/v; Dako), and reactivity was identified using VECTASTAIN ABC-AP Kit and Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratory); counterstaining was with Mayer’s hematoxylin (Sigma). IL-10 was detected using goat anti–IL-10 (10 mg/ml; R&D Systems) or normal goat IgG (5 mg/ml; Dako), with biotinylated rabbit

FIGURE 1. Direct effects of S100A8, Ala42S100A8, or Dex (all 10 mg) after intranasal administration to mice. (A) Total cell numbers and percentages of lymphocytes and neutrophils in BALF collected from control (HBSS), S100A8-treated mice (1, 4, 6, 12, 20 h after administration). Data are means 6 SD; n = 3–5 mice/group for each treatment. *p , 0.05, **p , 0.01, ****p , 0.0001 versus HBSS group determined by ANOVA followed by Holm–
´ k’s multiple-comparison test. (B) Clustergram of Sida the time course of mRNA expression of 63 genes expressed in lung tissue from control (HBSS; n = 5 mice), S100A8 (1, 4, 6, 12, 20 h after administration; n = 3–5 mice/group), and Ala42S100A8 (12, 20 h; n = 3 mice/group). (C) Clustergram of genes expressed in lungs from mice treated with Dex (n = 3 mice/group) for 6 or 12 h. (B and C) Data from nonsupervised hierarchical clustering of the entire data set were combined to display a heat map with dendrograms indicating coregulated genes in each group. Changes in expression of individual genes are shown in Supplemental Table I. (D) Protein levels of CCL-2 in BALF from mice treated with S100A8 or Ala42S100A8. Data are means 6 SD; n = 3 mice/group; no significant differences compared with HBSS control, determined by ANOVA followed by Bonferroni’s multiple-comparison test. (E) Anti-S100A8 immunoreactivity of lung sections from mice after inhalation of S100A8, Ala42S100A8, or Dex, as detailed in the Materials and Methods. Arrows indicate S100A8-expressing endothelial cells (S100A8 6 h), leukocytes positive for S100A8 (S100A8 6 h), or S100A8 (Dex 12 h). Sections are representative of three sections from each of three mice per group. Scale bars, 20 mm.

Mast-cell analysis Paraffin-embedded sections fixed with Mota’s lead acetate (1 g basic lead acetate, 50 ml ethanol, 50 ml water, 0.5 ml glacial acetic acid) for 5 min were stained with 0.5% toluidine blue in 0.7 N HCl for 2 h at room temperature, washed with water, air-dried, and mounted in dibutyl phthalate xylene. b-Hexosaminidase activity was determined as a measure of mast-cell degranulation. BALF (50 ml) was incubated with 50 ml 5 mM p-nitrophenyl N-acetyl-b-D-glucosaminide in 50 mM sodium citrate buffer (pH 4.5) at 37˚C for 2 h. Reactions were terminated with 200 ml 0.2 M glycine-NaOH, pH 10.6, and A405 nm determined spectrophotometrically, and values were reported as OD.

The Journal of Immunology

2803

Statistical analysis Comparisons of mRNA levels between groups were analyzed using a one
´ k’s multiple-comparisons test. way ANOVA in conjunction with Holm–Sida ELISA results were compared using a one-way ANOVA in conjunction with Bonferonni’s multiple-comparisons test, allowing for comparisons between each of the groups. Statistical significance was set at p , 0.05. GraphPad Prism 6.00 software was used for data analysis and preparation of graphs.

Results Direct effects of S100A8 administration into lung Expression profiles of genes after intranasal S100A8 administration over 1, 4, 6, 12, and 20 h and of Ala42S100A8 (12, 20 h) are shown in Fig. 1B, and of Dex (6, 12 h) in Fig. 1C. A cluster of endogenously expressed genes (including CCL-3, NOX-2, FGF-2, IL18, TLR4, CSF-3, and NF-kB) were reduced by S100A8 between 1 and 12 h but returned to almost baseline at 20 h. Although there were marked similarities in genes affected by S100A8 and Dex, these genes were not reduced by Dex or by Ala42S100A8, 6 or 12 h postinhalation. Interestingly, a second cluster shows mRNA profiles for some genes (e.g., TGF-b, ∼40-fold; IL-18, ∼10-fold; CCL-2, ∼12-fold) that were moderately increased by Dex, and by Ala42S100A8, earlier (12 h) than seen with S100A8 (20 h; Supplemental Table I). Although endogenous mRNA levels were low, all agents increased CCL-2 (∼11- to 22-fold) and CXCL-1 (∼4- to 8-fold) mRNAs, but CCL-2 levels in BALF were modestly, but not significantly, higher than control (Fig. 1D). Fig. 1A shows that total leukocyte numbers in BALF increased some 3fold, from 0.3 6 0.1 (control) to 0.9 6 0.2 3 106 (p , 0.01, n = 6) 6 h after S100A8 administration, mainly because of lymphocytes. At this time, CCL-4 mRNA was significantly elevated (Supplemental Table I), ∼3.3-fold more than control levels (p , 0.05), and this may have contributed to increased lymphocyte recruitment. Neutrophil numbers increased significantly only 20 h after S100A8 administration (1.4% of total cells, p , 0.0001 compared with control). Infiltration of S100+ myeloid cells within lung tissue was rare, and similar to controls (see Fig. 1E). Only a few genes, including MMP-2 and endothelin-1, were solely upregulated by Dex (Supplemental Table I). S100A8 was not constitutively expressed in murine lung, but mRNA levels increased 2-fold 1 h after administration (Supplemental Table I). At 4 h, airway lining epithelial cells and alveolar macrophages expressed high amounts, and levels increased over 12 h (Fig. 1E). Some endothelial cells expressed S100A8. As shown earlier (22), this heparin binding protein also apparently bound the extracellular matrix. In contrast, Ala 42 S100A8 only mildly upregulated S100A8 expression 12 h after administration, indicating a requirement for the ROS-reactive Cys42 residue (23, 24) for optimal induction. Dex only induced S100A8 in macrophage-like cells 12 h after treatment (Fig. 1E). Dex synergizes with LPS to induce S100A8 in elicited murine macrophages (14), but it directly elevated S100A8 mRNA in the alveolar macrophage cell line (MH-S) some 10-fold at 10 h (Fig. 2A) and 40-fold 24 h after stimulation (data not shown); LPS did not promote synergy. Dex also significantly elevated expression of mRNA for the markers of alternative activation (M2 macrophages), Ym1 and arginase-1, but as expected did not increase iNOS (M1 macrophages). IL-10 was elevated only in MH-S cells activated with LPS+Dex (Fig. 2A). Conversely, Dex suppressed TNF-a mRNA induced by LPS. Furthermore, S100A8 mRNA levels were increased some 100-fold in alternatively activated naive alveolar macrophages (M2 macrophages) in which arginase-1 mRNA was also significantly elevated (Fig. 2B). Results suggest distinct responses in different macrophage pop-

FIGURE 2. Dex upregulates S100A8 in alveolar macrophages. (A) Relative mRNA expression levels of S100A8, Ym1, arginase-1, and iNOS expressed at 10 h, and IL-10 and TNF-a at 4 h after incubation of MH-S alveolar macrophage cells with Dex (1 mM) 6 LPS (1 ng/ml). Data are means 6 SD; n = 3 separate experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 compared with control, determined by ANOVA followed by Bonferroni’s multiple-comparison test. (B) Relative mRNA expression levels of S100A8 and arginase-1 in naive lung alveolar macrophages preincubated with IL-4 and IL-13 to differentiate to the M2 phenotype. Data are means 6 SD; n = 3 separate experiments. *p , 0.05, **p , 0.01, compared with control, determined by Student t test.

ulations and confirm direct S100A8 induction by Dex in alveolar macrophages. The most profound change provoked by S100A8, but not by Ala42S100A8 or Dex, was in IL-10 mRNA, which increased 1611 6 535-fold (Fig. 3A), in lungs harvested 12 h after administration. IL-10 in BALF reflected these differences (Fig. 3B). Immunohistochemistry (Fig. 3C) confirmed strong IL-10 expression, seen almost exclusively in airway epithelial cells 12 h after S100A8; expression was weaker at 6 h, and airways of normal lung sometimes expressed low amounts. IL-10 was not apparent in alveolar macrophages or other cells. No increases in IL-10 above control levels were seen with Ala42S100A8 or Dex. S100A8 suppresses LPS-provoked ALI LPS inhalation is a widely used model of ALI. We first compared cytokines induced by LPS and by S100A8, and then determined whether S100A8 modulated LPS reactivity, as suggested previously (25). Fig. 4A shows marked neutrophil recruitment 4 h after

2804

S100A8 INDUCES IL-10 AND INHIBITS ACUTE LUNG INJURY

FIGURE 3. S100A8 induced IL-10 in airway epithelial cells. (A) Relative mRNA expression of IL-10 in lung tissue from mice treated with S100A8 (12, 20 h), Ala42S100A8 (12, 20 h), and Dex (12 h). Data are means 6 SEM; n = 3–5 mice/group in each experiment. ***p , 0.001 compared with HBSS control. (B) IL-10 in BALF from mice treated with S100A8, Ala42S100A8, or Dex. Data are means 6 SD; n = 3 mice/group. ****p , 0.0001 versus HBSS control. Significant differences were calculated as in Fig. 1. (C) Anti–IL-10 immunoreactivity of lung sections from mice treated with S100A8 (1, 6, 12 h), Ala42S100A8 (12 h), or Dex (12 h), as detailed in the Materials and Methods. Sections are representative of three lung sections from three mice per group. Scale bars, 50 mm.

LPS inhalation, whereas S100A8 alone had no effect. Remarkably, like Dex, S100A8 or Ala42S100A8 given 2 h before LPS reduced neutrophil numbers to almost baseline (Fig. 4A). S100A8 inhalation 15 min after LPS also significantly reduced neutrophil influx by ∼63%, suggesting that suppression was unlikely due to desensitization.

Neutrophils expressed high amounts of S100A8 mRNA/protein, and S100A8 mRNA levels increased some 7-fold 4 h after LPS inhalation (Supplemental Table II). Immunohistochemistry confirmed elevated numbers of S100A8+ myeloid cells with neutrophil morphology infiltrating the lungs of these mice. However, lungs from mice pretreated with S100A8 were indistinguishable

FIGURE 4. S100A8, Ala42S100A8, and Dex significantly reduced inflammation in ALI provoked by LPS. (A) Percentage of neutrophils in BALF collected 6 h after administration of HBSS+PBS (vehicle), HBSS+LPS, S100A8, S100A8+LPS, Ala42S100A8+LPS, or Dex+LPS (HBSS or S100A8 given 2 h before LPS), and LPS+S100A8 (given 15 min after LPS). Data are means 6 SD; n $ 5 mice/group for each treatment. ****p , 0.0001 versus the
´ k’s multiple-comparison test. (B) Anti-S100A8 immunoreactivity of lung sections from HBSS+LPS group determined by ANOVA followed by Holm–Sida mice treated with S100A8, Ala42S100A8, or Dex for 2 h, then LPS for 4 h, as detailed in the Materials and Methods. Sections are representative of three sections from each of five mice. Scale bars, 20 mm. (C) Numbers of intact/degranulating mast cells in lung sections after LPS, and S100A8, Ala42S100A8, or Dex pretreatments. Mast cells in six sections from equivalent areas of the lung from each of five mice per group were manually counted by a blinded operator. (D) b-Hexosaminidase levels in BALF from mice pretreated with S100A8, Ala42S100A8, or Dex. For (C) and (D), data are means 6 SD. *p ,
´ k’s multiple-comparison test (C, D). 0.05, **p , 0.01, ***p , 0.001 versus the HBSS+LPS group determined by ANOVA followed by Holm–Sida

The Journal of Immunology

2805

FIGURE 5. S100A8, Ala42S100A8, and Dex markedly suppress genes induced by LPS in lung. (A) Clustergram of mRNA expression of 63 genes expressed in lung tissue from mice after administration of HBSS+PBS, HBSS+LPS, S100A8+LPS (A8-2h+L), Ala42S100A8+LPS (A8A-2h+L), Dex+LPS (Dex-2 h+L), and LPS+S100A8 (given 15 min after LPS; L+A8+15 min; all harvested 4 h after LPS treatment) are presented. Nonsupervised hierarchical clustering of the entire data set was performed to provide a heat map with dendrograms indicating coregulated genes across groups and in individual samples. (B–E) Specific genes are listed in the plots. Differentially regulated mRNAs, with changes .2.5-fold, and p , 0.05 (indicated as a blue line), are shown in volcano plots, which arrange fold differences along the x-axis and statistical significance along the y-axis. (B) mRNAs differentially regulated in lungs from LPS-treated versus S100A8-pretreated mice (2 h before LPS), (C) LPS-treated versus Ala42S100A8-pretreated mice (2 h before LPS), (D) LPStreated versus Dex-pretreated mice (2 h before LPS), and (E) LPS-treated versus S100A8-posttreated groups (15 min after LPS); n $ 5 mice/group.

2806 from vehicle-treated mice, with no S100A8+ cells of any type, after LPS treatment (Fig. 4B). In contrast to S100A8 alone (Fig. 1E), S100A8 was not detected in airway epithelial cells of LPStreated mice, with or without S100A8 pretreatment, suggesting that LPS may override S100A8-mediated induction in these cells. In contrast, low numbers of S100A8+ myeloid cells were apparent in lungs from mice pretreated with Ala42S100A8. In contrast, and in keeping with our report showing increased S100A8 in alveolar macrophages and in BALF from lungs of mice pretreated with Dex before LPS (15), Dex pretreatment promoted strong S100A8 expression in alveolar macrophage-like cells; epithelial cells were weakly positive (Fig. 4B). S100A8 suppresses activation of mast cells (13). Significantly more degranulated than intact mast cells were apparent in lungs from mice treated with LPS, compared with those pretreated with S100A8 or Dex (Fig. 4C). In keeping with this, b-hexosaminidase levels (indicator of degranulation) in BALF from LPS-treated mice were significantly elevated, whereas pretreated samples were similar to controls (Fig. 4D). As expected (13), Ala42S100A8 did not reduce degranulation. S100A8 suppresses genes induced by LPS The clustergram (Fig. 5A) allows comparisons of expression levels of genes measured after LPS inhalation and all pretreatments. LPS strongly upregulated most chemokines, proinflammatory cytokines (IL-1b, TNF-a, IL-6), CSFs, and endothelial cell adhesion molecules (Fig. 5A, Supplemental Table II). In particular, the extremely high levels of CCL-2, CXCL-1, -2, -9, and -10 (469-

S100A8 INDUCES IL-10 AND INHIBITS ACUTE LUNG INJURY fold) mRNAs (all .37-fold above baseline) suggest these as principal mediators of leukocyte recruitment in this model. Elevation of VCAM-1 and ICAM-1 on the endothelium is essential for leukocyte transmigration, and mRNAs increased some 5-fold. S100A8 preparations suppressed LPS-induced genes and had marked similarities to inhibition by Dex (Fig. 5A). A volcano plot (Fig. 5B) comparing genes significantly increased by LPS with those reduced by S100A8 pretreatment shows significant suppression of 22 genes (p , 0.05 compared with the HBSS+LPS group). When S100A8 was administered 15 min after LPS, the same genes were reduced, 19 significantly (Fig. 5E), although inhibition was somewhat less. Fig. 5C and Supplemental Table II show that most genes induced by LPS were suppressed by Ala42 S100A8 pretreatment, and .2.5-fold reduction was significant for 24 genes (principally TNF-a, all chemokines, VCAM-1, ICAM-1, and CSFs), although the magnitude of suppression, particularly of genes that were highly elevated by LPS, was somewhat weaker. For example, S100A8 reduced CXCL-10 mRNA levels some 173. 5-fold, whereas suppression by Ala42S100A8 was 31.4-fold. The similarities and potencies of S100A8 and Dex are noteworthy (compare in Supplemental Table II, Fig. 5B, 5D). In addition, Fig. 6A shows the highly significant reductions in relative mRNA levels of the acute-phase reactant SAA3 that contributes to lung pathologies (26) by the three agents. However, Dex suppressed CCL-5 mRNA induction, whereas S100A8 did not, and inhibition of CCL-2 and IL-6 mRNAs by Dex was some ∼50% less than seen with S100A8 (see Supplemental Table II). Fig. 6B shows suppression of IL-6, TNF-a, and CCL-2 protein expression

FIGURE 6. S100A8 markedly suppressed mRNA levels of proinflammatory genes and proteins induced by LPS in lung. (A) Relative mRNA expression levels of SAA3 in lungs from mice treated with S100A8, Ala42S100A8, and Dex for 2 h and then LPS for 4 h. Data are means 6 SEM; n $ 5 mice/group/
´ k’s multiple-comparison test. (B) Cytokine levels treatment. ****p , 0.0001 versus HBSS+LPS group determined by ANOVA followed by Holm–Sida (means [pg/ml] 6 SD) in BALF from mice treated with S100A8, Ala42S100A8, and Dex for 2 h and then LPS for 4 h; n = 5–7 mice/group/treatment. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 determined by ANOVA followed by Bonferroni’s multiple comparisons. (C) Anti–IL-10 immunoreactivity of lung sections from mice treated with S100A8 or Dex for 2 h and then LPS for 4 h, as detailed in the Materials and Methods. Representative of three sections each from five mice. Scale bars, 20 mm. (D) Relative mRNA expression levels of IL-10 in lungs from mice treated with S100A8 and Ala42 S100A8 for 2 h and then LPS for 1 h. Data are means 6 SEM; n = 5 mice/group/treatment. *p , 0.05 versus HBSS+LPS group determined by ANOVA
´ k’s multiple-comparison test. followed by Holm–Sida

The Journal of Immunology by S100A8, Ala42S100A8, or Dex. In BALF from mice pretreated with S100A8, CCL-2 levels were significantly less than those induced by LPS (Fig. 6B). IL-10 mRNA/protein was increased by LPS (Fig. 6B, Supplemental Table II). Moreover, Fig. 6D shows that S100A8 pretreatment further elevated IL-10 mRNA induction in lungs harvested 1 h after LPS inhalation (p , 0.05 compared with HBSS+LPS group). However, all pretreatments significantly reduced IL-10 mRNA induction when lungs were harvested 4 h after LPS inhalation. IL-10 levels in BALF, harvested 4 h after LPS, were not significantly elevated by S100A8 pretreatment, although BALF from Ala42S100A8-treated mice contained levels similar to those induced by LPS (Fig. 6B). Immunohistochemistry indicated somewhat higher IL-10 expression in epithelial airway cells in lungs from LPS-treated mice, and this increased with S100A8, but not with Dex pretreatment (Fig. 6C). Again, IL-10 was most obvious in airway epithelial cells with little expression in alveolar macrophages or other cells. S100A8, Ala42S100A8, and Dex suppressed iNOS mRNA induced by LPS (Fig. 7A), although nitrite levels in BALF were not significantly altered (data not shown). We found no effect on arginase-1 (data not shown); arginase-2 mRNA was significantly decreased by the three agents (Fig. 7A). The NOX enzymes were not altered by S100A8 (data not shown), whereas metallothionein

FIGURE 7. S100A8 regulates mRNA levels of redox-related genes. (A) Relative mRNA expression levels of iNOS, arginase-2, heme oxygenase-1, and IDO in lung tissues from mice treated with S100A8, Ala42S100A8, or Dex for 2 h and then LPS for 4 h. Data are means 6 SEM, n $ 5 mice/ group/treatment. (B) Relative mRNA expression levels of metallothionein1 and -2 and glutathione peroxidase-2 in lungs from mice treated with S100A8 or Ala42S100A8 for 2 h and then LPS for 1 h. Data are means 6 SEM; n = 5 mice/group/treatment. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 versus HBSS+LPS determined by ANOVA followed by
´ k’s multiple-comparison test (A, B). Holm–Sida

2807 -1 and -2 mRNAs were significantly upregulated by S100A8 pretreatment in lungs harvested 1 h after LPS; glutathione peroxidase-2 was moderately increased by S100A8 (Fig. 7B), indicating likely positive effects on antioxidant defense. Unexpectedly, heme oxygenase-1, a gene downstream of the Nrf2 transcription factor that provides cytoprotection against lung injury, and IDO, an enzyme contributing to immunosuppression, were elevated by LPS but were both reduced by S100A8 or Dex pretreatment, suggesting that these were unlikely contributors to suppression. Pathways mediating S100A8 suppression Next, we assessed some proinflammatory pathways that may mediate S100A8 immunosuppression. It is important to note that because global changes were examined in whole lung tissue, these may not reflect pathways altered in individual cell types. Increases in total phosphatase activity in lung lysates could reduce signaling, but there were no apparent differences in samples harvested 1 h after LPS treatment (Fig. 8A), whereas activity in lungs from mice harvested 4 h after LPS was significantly less than control (∼50%; p , 0.05 compared with control lung; Fig. 8A). In contrast, phosphatase levels in lungs from mice pretreated with S100A8, Ala42S100A8, or Dex were similar to control values (Fig. 8A). S100A8 pretreatment did not significantly alter levels of SHP-1, SHP-2, or SHIP, phosphatases that regulate many signaling pathways (data not shown). Moreover, comparisons of phosphorylation levels of products of TLR4 signaling, namely, the MAPKs Erk1/2, MEK1/2, and p38 indicated no significant differences between the groups when lungs were harvested 1 h after LPS, although p38 was somewhat reduced by S100A8 pretreatment (Fig. 8B). However, in lungs harvested 4 h after LPS inhalation, p-Erk1/2 was elevated and p-MEK1/2 and p-P38 levels were significantly higher in samples from S100A8 pretreated mice, but not with Ala42S100A8 (Fig. 8C). This suggests a redox-sensitive response in which phosphorylation of these MAPKs was sustained by S100A8. Because STAT3 signaling mediates IL-10 (27) and S100A8 (28) induction, we examined p-STAT3 levels at the two time points but found no significant changes with pretreatments (Fig. 8D). We have found no suppression of LPS-mediated activation of macrophages by S100A8 in vitro, indicating that direct antagonistic effects via interaction with TLR4, CD14, or MD2 that may influence LPS receptor interactions are unlikely. PI3K activates Akt and this can inhibit NF-kB activation in some cells (29). S100A8, but not Ala42S100A8, pretreatment elevated p-Akt levels compared with LPS alone (Fig. 8C). NF-kB is a key transcription factor mediating upstream LPS signaling, and degradation of IkBa allows its nuclear translocation (30). S100A8 significantly reduced NF-kB mRNA levels induced by LPS, but increased inhibitory IkBa mRNA (Fig. 8E, left panels; p , 0.05 compared with LPS) and protein (Fig. 8E, right panel), indicating that S100A8 may limit NF-kB–mediated activation (31) by this mechanism. We found remarkable similarities in the pattern and potencies of suppression by S100A8 and Dex. GC increases histone acetylation to activate transcription of anti-inflammatory genes. Immunohistochemistry (Fig. 9) suggested that only Dex pretreatment promoted acetylation of histones 3 and 4, whereas LPS alone did not, but no overt acetylation was seen with S100A8.

Discussion Results reported in this study were unexpected, given that S100A8 is reported to bind TLR4 (1) or RAGE (2), although its interaction with RAGE is controversial (32). RAGE is highly expressed in

2808

S100A8 INDUCES IL-10 AND INHIBITS ACUTE LUNG INJURY

FIGURE 8. Pathways contributing to suppression of LPS response by S100A8. (A) Paranitrophenyl phosphate phosphatase in lung homogenates from mice pretreated with S100A8, Ala42S100A8, or Dex for 2 h and then with LPS for 1 or 4 h. Data are means 6 SD; n = 4–6 mice/group/treatment. *p , 0.05 versus HBSS+LPS. (B and C) Western blots and quantitative signal intensities of densitometry of bands corresponding to p-MEK1/2, p-Erk1/2, p-P38, and p-Akt in lung lysates from mice treated with S100A8 or Ala42S100A8 for 2 h and then LPS for (B) 1 or (C) 4 h. A representative blot is shown; densitometry is shown as means 6 SD of bands relative to MEK1/2 for p-MEK1/2 or to b-actin; all n = 3 individual lung lysates/group in two independent analyses. *p , 0.05 and **p , 0.01 determined by ANOVA followed by Bonferroni’s multiple-comparison test. (D) A representative Western blot and densitometry shown as means 6 SD of bands corresponding to p-STAT3, relative to STAT3, in lung lysates; n = 3 lysates/group in two independent analyses. (E) Relative mRNA levels of NF-kB and IkBa in lung tissues from mice treated with S100A8 and Ala42S100A8 for 2 h and then LPS for 1 (NF-kB) and 4 h (IkBa).
´ k’s multipleData are means 6 SEM, n $ 4 mice/group/treatment; *p , 0.05 versus HBSS+LPS group determined by ANOVA followed by Holm–Sida comparison test. Western blots show IkBa increases in lung lysates from mice pretreated with S100A8 or Ala42S100A8 for 2 h and then LPS for 1 h. Quantitative signal intensities are given as means 6 SD for densitometry of bands relative to b-actin, all n = 3 individual lung lysates/group in two independent analyses. *p , 0.05 determined by ANOVA followed by Bonferroni’s multiple-comparison test.

lung (33), and TLR4 plays an important role in ALI. In this article, we report, to our knowledge, the first in-depth study of effects of administering S100A8 to a healthy organ. We found little proinflammatory effect other than mild increases in a few genes that were induced to similar levels by Dex. Our early studies indicated

that murine S100A8 was a leukocyte chemoattractant and provoked a mild inflammatory response reminiscent of a delayedtype hypersensitivity reaction when injected intradermally (34). In this study, we show that S100A8 inhalation somewhat increased cell numbers in BALF from naive mice, principally

The Journal of Immunology

2809

FIGURE 9. S100A8 did not promote histone acetylation. Representative immunoreactivity of anti–acetyl-H3 (upper panels) or anti-H4 (lower panels) in lung sections from mice treated with S100A8 or Dex for 2 h, then LPS for 4 h, or with S100A8 or Dex alone for 6 h, as detailed in the Materials and Methods. Arrows indicate cells with positive immunoreactivity only in the Dex1 LPS-treated group. Representative of three sections from five mice/group/treatment. Scale bars, 20 mm.

infiltrating lymphocytes; neutrophils were only significantly elevated 20 h postinhalation. Importantly, and unlike LPS or Dex, S100A8 markedly upregulated IL-10, apparent in healthy airway epithelial cells, but not in other cell types (Fig. 3). Low-level IL-10 expression is seen in normal murine lung, primarily in bronchial and alveolar epithelial cells and macrophages (16), but little is known regarding its regulation. IL-10 mRNA levels were significantly elevated in S100A8-treated mice 1 h after LPS treatment, but increases were no longer apparent 4 h after LPS administration. IL-10 was detected in the airways of mice pretreated with S100A8 when harvested 4 h after LPS (Fig. 6C), at levels similar to those seen 6 h after S100A8 inhalation (Fig. 3C). The finding that S100A8 increased IL-10 in airway epithelial cells was unpredicted, because we have been unable to induce cytokines, or influence their induction by LPS or other TLR agonists, in monocytes/ macrophages stimulated with various S100A8 doses over a time course (K. Hsu and C. Geczy, unpublished observations) (20). IL10–deficient mice are more susceptible to airway inflammation in response to LPS than normal mice, and induced epithelial expression of human IL-10 attenuates this (35). We propose that IL10 partially contributed to S100A8-mediated suppression. Because IL-10 is essential for S100A8 induction in macrophages (7), and S100A8 also promoted its own expression in airway epithelial cells (Fig. 3C), results suggest a novel regulatory feedback loop to potentiate production of this important anti-inflammatory mediator in the lung. Cytokines generated during LPS-induced ALI in mice were recently profiled (36). Our gene array included mediators measured in that study, and results mirrored those reported at the protein level for cytokines produced 6 h after LPS inhalation. The roles of chemokines in neutrophil recruitment, particularly CXCL1, CXCL-2, and CCL-2 in LPS-mediated ALI are well documented and were strongly induced. We also found extraordinarily high levels of CXCL-10 mRNA. CXCL-10 is less well studied and LPS promoted a 469-fold increase in mRNA. CXCL-10 increases in lungs with acute respiratory distress syndrome mostly originating from infiltrating neutrophils that express a unique CXCR3 receptor, and CXCL10-CXCR3 autocrine activation perpetuates the oxidative burst and chemotaxis of inflamed neutrophils (37), and this may be a major contributor to ALI. Results in this study show that S100A8 is an unlikely TLR4 or RAGE ligand in the lung. S100A8 pretreatment before LPS inhalation, or 15 min later, reduced neutrophil recruitment and mastcell activation, and was immunosuppressive in magnitudes similar

to Dex. Dex induced S100A8 in alveolar macrophages, which also expressed Ym1 and arginase-1; these cells did not express TNF-a or iNOS, markers typical of M1 macrophages (38), and results indicate an anti-inflammatory phenotype. Moreover, naive alveolar macrophages differentiated to an M2 phenotype expressed S100A8 (Fig. 2B). Dex elevates S100A8 levels in lungs treated with LPS (15), and alveolar macrophage-like cells in lungs from mice pretreated with Dex followed by LPS inhalation were strongly S100A8+ (Fig. 4B). These findings led us to propose that S100A8 underlies some of the anti-inflammatory effects of GC, possibly without the adverse effects linked to histone acetylation of anti-inflammatory genes (Fig. 9), although this requires further analysis. Other studies indicate protective effects of S100A8 (39). i.v. injection into endotoxemic mice somewhat reduced neutrophil infiltration into organs, and markers of oxidative damage, and doubled survival rates, possibly via S100A8 binding to TLR4, an interaction proposed to interfere with protease activated receptor-2 cooperation with TLR4, thereby reducing the magnitude of the inflammatory response (25). The S100A8/S100A9 complex was also proposed to protect against LPS injury by directly binding and neutralizing several proinflammatory cytokines (IL-1b, IL-6, and TNF-a), thereby suppressing overproduction of NO (40). In contrast, we show induction of IL-10 by S100A8, reduced mastcell activation that can initiate vascular responses (41), and suppression of proinflammatory genes induced by LPS. S100A8 has a single, highly reactive Cys residue, and care was taken to ensure that the preparation used in this study was in the reduced form. We reported several oxidation products (11, 12, 24). S100A8 in BALF from mice 4 h after LPS treatment is predominantly disulfide-linked homodimer, not complexed with S100A9, confirming its oxidation in vivo, likely via peroxide (23). The free thiol was critical for IL-10 induction in the airways because the Cys-Ala42S100A8 mutant had little effect. Similarly, the mutant reduced LPS inflammation but was not as effective. The hinge domain of S100A8 (residues 43–56), located immediately after Cys42, is moderately active in leukocyte chemotaxis (34) and may contribute to the activity seen with the mutant (compare Fig. 5B, 5C). Interaction of the hinge region with a receptor such as the G protein–coupled receptor proposed by us (10) may allow the Cys residue to more efficiently modulate redox responses in close proximity to the TLR–receptor complex. ROS is essential for TLR4 activation and influences several signaling cascades, including protein tyrosine phosphatases, tyrosine kinases, and MAPKs (42); phagocytic ROS signaling is

2810 critical for promoting NF-kB–dependent acute inflammatory responses, particularly in the lung (43). Antioxidants such as Nacetylcysteine inhibit NF-kB activation provoked by aerosolized LPS (44), and N-acetylcysteine is used clinically, for example, to boost antioxidant defense in lungs of patients with chronic obstructive pulmonary disease (45). S100A8 significantly reduced NF-kB mRNA levels that increased with LPS (Fig. 8E), and significantly elevated inhibitory IkBa, indicating mechanisms that could limit NF-kB–mediated activation (31). S100A8 pretreatment also increased p-Akt, and because the PI3K pathway contributes to resolution of inflammation, and its sustained activation reduces TLR4-mediated gene induction (46), this pathway may also contribute. No changes in phosphorylation of MAPK, important in TLR4 signaling, were found in lungs from mice harvested 1 h after LPS, although activation may have occurred earlier. Unexpectedly, S100A8 elevated pMEK1/2 pERK1/2 and p38 MAPKs in lungs harvested 4 h after LPS. pERK mediates antioxidant protection in monocytoid cells challenged by oxidative stress (47), and a novel oxidant stress-activated pathway that is protective in ischemia is dependent on p38-MAPK and p-Akt (48). Thus, activation of these may constitute part of a survival pathway mediated by S100A8. S100A8 also reduced iNOS and increased transcription of the antioxidant metallothionein genes 1 h after LPS challenge, processes that could reduce generation of ROS and reactive nitrogen species. S100A8 reduces FcεRI-mediated mast-cell activation by scavenging ROS (13). Mast cells are critical for innate immune defense, initially by releasing preformed TNF-a, and respond directly to LPS (49). In this study, we show that mast-cell degranulation provoked by LPS was reduced to control levels by S100A8 (but not by Ala42S100A8) and by Dex, indicating that like Dex (50), S100A8 may tonically restrain mast-cell degranulation in vivo, thereby reducing inflammation. In summary, S100A8 promoted high IL-10 expression in lung airway epithelial cells in vivo and totally suppressed neutrophil infiltration in response to intranasal challenge with LPS by reducing induction of proinflammatory mediators and chemokine genes, reducing mast-cell activation and NF-kB activation, and activating Akt and MAPK pathways that may sustain protection against oxidative stress. Inhibition was not totally dependent on the reactive thiol residue in S100A8, indicating effects in addition to those on ROS signaling. There were marked similarities in suppression patterns seen with Dex. We propose that S100A8 acts in concert with IL-10, and may contribute to some immunosuppressive functions mediated by corticosteroids, to reduce inflammation in ALI.

Acknowledgments We thank Prof. Rakesh Kumar for advice in assessing histology, Prof. Mark Raftery for validating masses of S100A8 preparations by mass spectrometry, and Rylie Flesher for cDNA from M2-differentiated alveolar macrophages.

Disclosures The authors have no financial conflicts of interest.

References 1. Vogl, T., K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M. A. van Zoelen, W. Nacken, D. Foell, T. van der Poll, C. Sorg, and J. Roth. 2007. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat. Med. 13: 1042–1049. 2. Boyd, J. H., B. Kan, H. Roberts, Y. Wang, and K. R. Walley. 2008. S100A8 and S100A9 mediate endotoxin-induced cardiomyocyte dysfunction via the receptor for advanced glycation end products. Circ. Res. 102: 1239–1246.

S100A8 INDUCES IL-10 AND INHIBITS ACUTE LUNG INJURY 3. Lorenz, E., M. S. Muhlebach, P. A. Tessier, N. E. Alexis, R. Duncan Hite, M. C. Seeds, D. B. Peden, and W. Meredith. 2008. Different expression ratio of S100A8/A9 and S100A12 in acute and chronic lung diseases. Respir. Med. 102: 567–573. 4. Roth, J., S. Teigelkamp, M. Wilke, L. Gr€un, B. T€ummler, and C. Sorg. 1992. Complex pattern of the myelo-monocytic differentiation antigens MRP8 and MRP14 during chronic airway inflammation. Immunobiology 186: 304–314. 5. Foell, D., H. Wittkowski, T. Vogl, and J. Roth. 2007. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J. Leukoc. Biol. 81: 28–37. 6. Hsu, K., C. Champaiboon, B. D. Guenther, B. S. Sorenson, A. Khammanivong, K. F. Ross, C. L. Geczy, and M. C. Herzberg. 2009. Anti-infective protective properties of S100 calgranulins. Antiinflamm. Antiallergy Agents Med. Chem. 8: 290–305. 7. Endoh, Y., Y. M. Chung, I. A. Clark, C. L. Geczy, and K. Hsu. 2009. IL-10dependent S100A8 gene induction in monocytes/macrophages by doublestranded RNA. J. Immunol. 182: 2258–2268. 8. Xu, K., T. Yen, and C. L. Geczy. 2001. Il-10 up-regulates macrophage expression of the S100 protein S100A8. J. Immunol. 166: 6358–6366. 9. Goyette, J., and C. L. Geczy. 2011. Inflammation-associated S100 proteins: new mechanisms that regulate function. Amino Acids 41: 821–842. 10. Cornish, C. J., J. M. Devery, P. Poronnik, M. Lackmann, D. I. Cook, and C. L. Geczy. 1996. S100 protein CP-10 stimulates myeloid cell chemotaxis without activation. J. Cell. Physiol. 166: 427–437. 11. Lim, S. Y., M. J. Raftery, and C. L. Geczy. 2011. Oxidative modifications of DAMPs suppress inflammation: the case for S100A8 and S100A9. Antioxid. Redox Signal. 15: 2235–2248. 12. Gomes, L. H., M. J. Raftery, W. X. Yan, J. D. Goyette, P. S. Thomas, and C. L. Geczy. 2013. S100A8 and S100A9-oxidant scavengers in inflammation. Free Radic. Biol. Med. 58: 170–186. 13. Zhao, J., I. Endoh, K. Hsu, N. Tedla, Y. Endoh, and C. L. Geczy. 2011. S100A8 modulates mast cell function and suppresses eosinophil migration in acute asthma. Antioxid. Redox Signal. 14: 1589–1600. 14. Hsu, K., R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, and C. L. Geczy. 2005. Regulation of S100A8 by glucocorticoids. J. Immunol. 174: 2318–2326. 15. Bozinovski, S., M. Cross, R. Vlahos, J. E. Jones, K. Hsuu, P. A. Tessier, E. C. Reynolds, D. A. Hume, J. A. Hamilton, C. L. Geczy, and G. P. Anderson. 2005. S100A8 chemotactic protein is abundantly increased, but only a minor contributor to LPS-induced, steroid resistant neutrophilic lung inflammation in vivo. J. Proteome Res. 4: 136–145. 16. Bonfield, T. L., M. W. Konstan, P. Burfeind, J. R. Panuska, J. B. Hilliard, and M. Berger. 1995. Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 13: 257–261. 17. Takanashi, S., Y. Hasegawa, Y. Kanehira, K. Yamamoto, K. Fujimoto, K. Satoh, and K. Okamura. 1999. Interleukin-10 level in sputum is reduced in bronchial asthma, COPD and in smokers. Eur. Respir. J. 14: 309–314. 18. Iismaa, S. E., S. Hu, M. Kocher, M. Lackmann, C. A. Harrison, S. Thliveris, and C. L. Geczy. 1994. Recombinant and cellular expression of the murine chemotactic protein, CP-10. DNA Cell Biol. 13: 183–192. 19. Kurowska-Stolarska, M., B. Stolarski, P. Kewin, G. Murphy, C. J. Corrigan, S. Ying, N. Pitman, A. Mirchandani, B. Rana, N. van Rooijen, et al. 2009. IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. J. Immunol. 183: 6469–6477. 20. Chung, Y. M., J. Goyette, N. Tedla, K. Hsu, and C. L. Geczy. 2013. S100A12 suppresses pro-inflammatory, but not pro-thrombotic functions of serum amyloid A. PLoS ONE 8: e62372. 21. Monick, M. M., L. S. Powers, T. J. Gross, D. M. Flaherty, C. W. Barrett, and G. W. Hunninghake. 2006. Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase 1. J. Immunol. 177: 1636–1645. 22. McCormick, M. M., F. Rahimi, Y. V. Bobryshev, K. Gaus, H. Zreiqat, H. Cai, R. S. Lord, and C. L. Geczy. 2005. S100A8 and S100A9 in human arterial wall. Implications for atherogenesis. J. Biol. Chem. 280: 41521–41529. 23. Harrison, C. A., M. J. Raftery, J. Walsh, P. Alewood, S. E. Iismaa, S. Thliveris, and C. L. Geczy. 1999. Oxidation regulates the inflammatory properties of the murine S100 protein S100A8. J. Biol. Chem. 274: 8561–8569. 24. Raftery, M. J., Z. Yang, S. M. Valenzuela, and C. L. Geczy. 2001. Novel intraand inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation. J. Biol. Chem. 276: 33393–33401. 25. Sun, Y., Y. Lu, C. G. Engeland, S. C. Gordon, and H. Y. Sroussi. 2013. The antioxidative, anti-inflammatory, and protective effect of S100A8 in endotoxemic mice. Mol. Immunol. 53: 443–449. 26. Bozinovski, S., M. Uddin, R. Vlahos, M. Thompson, J. L. McQualter, A. S. Merritt, P. A. Wark, A. Hutchinson, L. B. Irving, B. D. Levy, and G. P. Anderson. 2012. Serum amyloid A opposes lipoxin A₄ to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease. Proc. Natl. Acad. Sci. USA 109: 935–940. 27. Murray, P. J. 2006. Understanding and exploiting the endogenous interleukin-10/ STAT3-mediated anti-inflammatory response. Curr. Opin. Pharmacol. 6: 379– 386. 28. Cheng, P., C. A. Corzo, N. Luetteke, B. Yu, S. Nagaraj, M. M. Bui, M. Ortiz, W. Nacken, C. Sorg, T. Vogl, et al. 2008. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J. Exp. Med. 205: 2235–2249. 29. Chaurasia, B., J. Mauer, L. Koch, J. Goldau, A. S. Kock, and J. C. Br€uning. 2010. Phosphoinositide-dependent kinase 1 provides negative feedback inhibition to

The Journal of Immunology

30.

31. 32.

33.

34.

35.

36. 37.

38. 39.

Toll-like receptor-mediated NF-kappaB activation in macrophages. Mol. Cell. Biol. 30: 4354–4366. Lawrence, T., M. Bebien, G. Y. Liu, V. Nizet, and M. Karin. 2005. IKKalpha limits macrophage NF-kappaB activation and contributes to the resolution of inflammation. Nature 434: 1138–1143. Ligeti, E., R. Cse´pa´nyi-Ko¨mi, and L. Hunyady. 2012. Physiological mechanisms of signal termination in biological systems. Acta Physiol. (Oxf.) 204: 469–478. Bjo¨rk, P., A. Bjo¨rk, T. Vogl, M. Stenstro¨m, D. Liberg, A. Olsson, J. Roth, F. Ivars, and T. Leanderson. 2009. Identification of human S100A9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides. PLoS Biol. 7: e97. Sukkar, M. B., M. A. Ullah, W. J. Gan, P. A. Wark, K. F. Chung, J. M. Hughes, C. L. Armour, and S. Phipps. 2012. RAGE: a new frontier in chronic airways disease. Br. J. Pharmacol. 167: 1161–1176. Lackmann, M., P. Rajasekariah, S. E. Iismaa, G. Jones, C. J. Cornish, S. Hu, R. J. Simpson, R. L. Moritz, and C. L. Geczy. 1993. Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10. J. Immunol. 150: 2981–2991. Garantziotis, S., D. M. Brass, J. Savov, J. W. Hollingsworth, E. McElvaniaTeKippe, K. Berman, J. K. Walker, and D. A. Schwartz. 2006. Leukocytederived IL-10 reduces subepithelial fibrosis associated with chronically inhaled endotoxin. Am. J. Respir. Cell Mol. Biol. 35: 662–667. Bosmann, M., N. F. Russkamp, and P. A. Ward. 2012. Fingerprinting of the TLR4-induced acute inflammatory response. Exp. Mol. Pathol. 93: 319–323. Ichikawa, A., K. Kuba, M. Morita, S. Chida, H. Tezuka, H. Hara, T. Sasaki, T. Ohteki, V. M. Ranieri, C. C. dos Santos, et al. 2013. CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin. Am. J. Respir. Crit. Care Med. 187: 65–77. Sica, A., and A. Mantovani. 2012. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122: 787–795. Sroussi, H. Y., Y. Lu, Q. L. Zhang, D. Villines, and P. T. Marucha. 2010. S100A8 and S100A9 inhibit neutrophil oxidative metabolism in-vitro: involvement of adenosine metabolites. Free Radic. Res. 44: 389–396.

2811 40. Ikemoto, M., H. Murayama, H. Itoh, M. Totani, and M. Fujita. 2007. Intrinsic function of S100A8/A9 complex as an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats. Clin. Chim. Acta 376: 197–204. 41. Lim, S. Y., M. Raftery, H. Cai, K. Hsu, W. X. Yan, H. L. Hseih, R. N. Watts, D. Richardson, S. Thomas, M. Perry, and C. L. Geczy. 2008. S-nitrosylated S100A8: novel anti-inflammatory properties. J. Immunol. 181: 5627–5636. 42. Ray, P. D., B. W. Huang, and Y. Tsuji. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 24: 981–990. 43. Zhang, W. J., H. Wei, Y. T. Tien, and B. Frei. 2011. Genetic ablation of phagocytic NADPH oxidase in mice limits TNFa-induced inflammation in the lungs but not other tissues. Free Radic. Biol. Med. 50: 1517–1525. 44. Rockse´n, D., B. Lillieho¨o¨k, R. Larsson, T. Johansson, and A. Bucht. 2000. Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation. Clin. Exp. Immunol. 122: 249–256. 45. Millea, P. J. 2009. N-acetylcysteine: multiple clinical applications. Am. Fam. Physician 80: 265–269. 46. G€unzl, P., K. Bauer, E. Hainzl, U. Matt, B. Dillinger, B. Mahr, S. Knapp, B. R. Binder, and G. Schabbauer. 2010. Anti-inflammatory properties of the PI3K pathway are mediated by IL-10/DUSP regulation. J. Leukoc. Biol. 88: 1259–1269. 47. Luchetti, F., M. Betti, B. Canonico, M. Arcangeletti, P. Ferri, F. Galli, and S. Papa. 2009. ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells. Free Radic. Biol. Med. 46: 339–351. 48. Herna´ndez, G., H. Lal, M. Fidalgo, A. Guerrero, J. Zalvide, T. Force, and C. M. Pombo. 2011. A novel cardioprotective p38-MAPK/mTOR pathway. Exp. Cell Res. 317: 2938–2949. 49. Masuda, A., Y. Yoshikai, K. Aiba, and T. Matsuguchi. 2002. Th2 cytokine production from mast cells is directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways. J. Immunol. 169: 3801–3810. 50. Coutinho, A. E., J. K. Brown, F. Yang, D. G. Brownstein, M. Gray, J. R. Seckl, J. S. Savill, and K. E. Chapman. 2013. Mast cells express 11b-hydroxysteroid dehydrogenase type 1: a role in restraining mast cell degranulation. PLoS ONE 8: e54640.