S100A8 Chemotactic Protein Is Abundantly Increased, but Only a

S100A8 Chemotactic Protein Is Abundantly Increased, but Only a Minor Contributor to LPS-Induced, Steroid Resistant Neutrophilic Lung Inflammation in Vivo Steven Bozinovski,†,# Maddalena Cross,§,# Ross Vlahos,‡,# Jessica E. Jones,†,# Kenneth Hsuu,O Philippe A. Tessier,⊥ Eric C. Reynolds,∇ David A. Hume,|,# John A. Hamilton,§,# Carolyn L. Geczy,O and Gary P. Anderson*,†,‡,# Lung Disease Research Group, Departments of Pharmacology and Medicine, The University of Melbourne, Parkville 3010 VIC Australia, Arthritis and Inflammation Research Center, Department of Medicine, Royal Melbourne Hospital, Centre for Molecular and Cellular Biology, Department of Biochemistry and Microbiology, The University of Queensland, Cooperative Research Center for Chronic Inflammatory Diseases, Inflammatory Diseases Research Unit, School of Medical Sciences,University of New South Wales, NSW 2065, Australia, Infectiology, CHUL Research Center, Laval University Quebec, G1V 4G2 Canada, and School of Dentistry, The University of Melbourne, VIC 3010, Australia Received September 24, 2004

Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFR transcripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo. Keywords: neutrophils • chemotaxis • inflammation • lung

Introduction Neutrophilic lung inflammation provides an essential host defense mechanism against diverse pathogenic organisms. This * To whom correspondence should be addressed. Fax: ++ 61 3 8 344 0241. Tel: ++ 61 3 8 344 8602. E-mail: [email protected]. † Lung Disease Research Group, Department of Pharmacology, The University of Melbourne. ‡ Lung Disease Research Group, Department of Medicine, The University of Melbourne. § Arthritis and Inflammation Research Center, Department of Medicine, Royal Melbourne Hospital. | Centre for Molecular and Cellular Biology, Department of Biochemistry and Microbiology, The University of Queensland. # Cooperative Research Center for Chronic Inflammatory Diseases O Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales. ⊥ Infectiology, CHUL Research Center, Laval University Quebec. 3 School of Dentistry, The University of Melbourne.

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host defense is broadly effective because the molecular armature of neutrophils comprises proteolytic, oxidant, and phagocytic killing mechanisms that can be fatal to many eukaryotic and prokaryotic pathogens and some viruses. However, neutrophilic inflammation is a pathological feature of many debilitating chronic inflammatory lung diseases1 such as Chronic Obstructive Pulmonary Disease (COPD),2 cystic fibrosis,3 severe asthma,4 and bronchiolitis.5 Neutrophilic inflammation with accumulation and activation in both tissue2,6 and lavage7 compartments of the lung is also a prominent feature of infectious exacerbations of these conditions.4,6,8-10 Once sequestered in close proximity to the alveolar walls, activated neutrophils release potent proteases that have the capacity to destroy interstitial matrix proteins, such as collagens and elastin, a process central to the pathogenesis of emphysema.11,12 Neutrophil products are also implicated in tissue damage to 10.1021/pr049829t CCC: $30.25

 2005 American Chemical Society

Steroid Resistant S100A8 in Lung Neutrophilic LPS

small airways and the airway mucosa. In these circumstances, neutrophil accumulation and activation may be detrimental to the host. Glucocorticosteroids are widely used in the treatment of diverse inflammatory lung diseases although their clinical efficacy varies markedly with the nature of disorder. Glucocorticosteroids are, for example, highly efficacious in suppressing eosinophilic inflammation in asthma13 whereas neutrophilic inflammation is less well controlled.14,15 A recent Cochrane Review indicated that the clinical efficacy of glucocorticosteroids in COPD is poor.16 Although very widely used, glucocorticosteroids are also poorly efficacious in infectious exacerbations of asthma and COPD even when administered systemically, resulting in only modest improvements in clinical outcomes such as hospitalization and lung function parameters,17,18 at the cost of considerable side effects.18 Neutrophilic inflammation in particular may not respond well to glucocorticoids, because steroids promote survival of this cell type.19,20 The weak clinical benefit observed in some studies may in part be associated with clearance of steroid-responsive inflammatory cells such as eosinophils,21 which are also recruited during an acute exacerbation,7,22 or of mast cells. Hence, there is a need for new therapies to target and dampen excessive neutrophilic accumulation. As neutrophils are implicated in the pathogenesis of steroidrefractory inflammatory lung diseases and their exacerbations, we sought to understand the molecular basis of this problem by applying molecular profiling methodologies in an in vivo model of intense acute neutrophilic lung inflammation.23-25 In the present study, we have exploited our recent observation that neutrophilic inflammation induced in the innate immune response triggered by high dose Gram-negative lipopolysaccharide (LPS, endotoxin) is refractory to suppression by saturating concentrations of the potent glucocorticosteroid, dexamethasone, in mice in vivo.25 We chose LPS since 30-50% of acute exacerbations of COPD have a positive sputum culture for bacteria including Gram-negative Haempohilus influenzae and Moraxella catarrhalis,26 LPS is a known exacerbant of asthma27 and Pseudomonas aeruginosa LPS is implicated in CF.28 Glucocorticosteroids exert their antiinflammatory effects by binding a cytoplasmic glucocorticosteroid receptor and forming a complex which positively or negatively regulates gene transcription by at least three mechanisms. Monomeric complexes may sequester inflammatory transcription factors such as AP-1 or NFκB (transrepression),29 dimeric complexes may activate positive or negative Glucocorticoid Response Elements (GREs) in upstream gene regulatory regions (transactivation)29 and glucocorticosteroids may also directly affect higher order transcriptional complex assembly or transcriptional machinery procession.29 As we observed that these complex gene regulatory mechanisms lead to high false positive and negatives in mRNA transcriptional profiling methods employing gene microarrays, we used in vivo proteomic profiling that focuses on the relative abundance of expressed proteins. Our approach demonstrates the feasibility of identifying steroid-refractory proteins generated in the lung in vivo and identified S100A8 as a candidate regulator of steroid-insensitive neutrophilic inflammation in the mouse.

Methods Animals. Specific pathogen-free male Balb/c mice (IAd) weighing 20-24 g, 6-7 weeks of age, were obtained from the

research articles Biological Research Facility (Perth, Australia). Animals were group housed at 20 °C on a 12 h day night cycle and were fed a standard diet of Purina mouse chow with water allowed ad libitum. All procedures were approved by the Animal Experimentation Ethics Committee of the University of Melbourne and conformed to international standards of animal welfare as specified in the National Health and Medical Research Committee (NHMRC) of Australia guidelines. Transnasal Instillation of LPS and Lung Processing. Inflammation was induced by instilling LPS (E. coli Serotype 026: B6, Sigma) into lungs of groups of 6-8 mice using a transnasal challenge as described.24 Briefly, 50 µL of solution was applied to the nares. As mice are obligate nose breathers this method uniformly instilled fluids through all lung regions. Mice were pretreated for 3 h with: saline (Sal), or dexamethasone (50 µg/ mouse Dex, tissue culture grade, Sigma). Mice were then challenged with Sal containing 10 µg LPS, the maximal tolerated dose, for specified times. Some mice were also injected i.p. with 2 mg of anti-S100A8 or anti-S100A9 (both rabbit IgG) 16 h before LPS challenge as previously described.30 For necropsy, mice were anaesthetized with ketamine/xylazine (15 mg/kg and 30 mg/kg, i.p., respectively). Inflammatory responses were measured by bronchoalveolar lavage (BAL) of the lungs via tracheotomy (SP30 Duran polyethylene tubing) proximal to the larynx and total/differential cells counts were performed as previously described.24 Following lavage, lungs were perfused free of blood via right ventricular perfusion with 5 mL warmed saline, rapidly excised en bloc, blotted and snap frozen in liquid nitrogen, ground to a fine powder and stored at -80 °C. Lung Fixation and Immunohistochemistry. Histology was performed as previously described, in separate groups of mice.31 Briefly, to ensure consistent morphological preservation of lungs, mice were anesthetized and then perfusion fixed via a tracheal cannula with 4% formaldehyde at exactly 200 mm H2O pressure. Fixed tissues were embedded in paraffin and sections stained with hematoxylin and eosin (H&E). Immunostaining of paraffin-embedded lung sections was performed using a specific S100A8 antibody and the ABC staining system (both from Santa Cruz) in accordance with the manufacturers’ instructions. Briefly, rehydrated sections were subjected to antigen unmasking by heat treatment and endogenous peroxidase activity quenched with 1% H2O2 in methanol. Sections were blocked for 1 h in normal blocking serum and primary antibody was incubated at a 1:200 dilution (1.5% blocking solution in PBS) overnight at 4 °C. Control sections were incubated in the absence of primary antibody. Sections were incubated with biotinylated secondary antibody and subsequent immunoperoxidase staining using diaminobenzidine (DAB) chromogen as a substrate as per manufacturers’ instructions. Sections were lightly counterstained with hematoxylin. 2D-PAGE and Protein Identification. A 100-mg portion of pooled lung tissue from eight mice was homogenized in 200 µL ice cold buffer containing; 20 mM Tris (pH 7.8), 10 mM EDTA and 1 mM DTT. Cytosols were prepared by centrifugation at 15000g for 10 min at 4 °C and protein assay performed as detailed.24 Samples were diluted to 10 mg/mL in the above buffer and 20 µL (200 µg) of sample was solubilized in a total volume of 185 µL containing; 9.0 M Urea, 4% CHAPS, 50 mM DTT, 0.002%bromophenol blue and 0.2% carrier ampholyte (pH 5-8, Bio-Rad). Samples were resolved in the first dimension using pre-cast 11 cm IPG strips (pH 5-8, Bio-Rad) by application to individual strips and overnight passive rehydration at Journal of Proteome Research • Vol. 4, No. 1, 2005 137

research articles room temperature. IPG strips were then subjected to isoelectric focusing for 30 000 V/h using a rapid ramp protocol performed at 20 °C. Strips were stored at -20 °C prior to resolution in the second dimension, which were performed using Criterion precast gradient gels (8-16%, Bio-Rad). Prior to running the second dimension, rehydrated strips were reduced in 1.5 M Tris (pH 8.8), 6 M Urea, 2% SDS, 20% glycerol, 2% DTT for 15 min, and alkylated in 1.5 M Tris (pH 8.8), 6 M Urea, 2% SDS, 20% glycerol, 2.5% iodacetamide for 15 min. Strips were briefly dipped into SDS-PAGE running buffer and loaded directly onto the pre-cast gels and resolved at 200V for 1 h at RT. Gel slabs were stained overnight with Bio-Rad Coomassie G-250 stain as per instructions. Stained gels were scanned using a Biorad GS710 scanner and TIFF images were imported into PDQuest Software version 7.1 (Bio-Rad) for quantitative analysis between treatment groups. After analysis of differentially expressed proteins, the Coomassie blue-stained spots were excised from the gel and washed in 500 µL 100 mM NH4HCO3/acetonitrile (1:1) for 1.5 h. Supernatants were removed and this step repeated. Gel pieces were dried in 200 µL acetonitrile for 5 min, supernatants removed and the gel pieces further dried using an integrated SpeedVac system (Thermo Savant) for 5 min and rehydrated in a minimal volume of 25 mM NH4HCO3, containing 0.5 mM CaCl2 and 12.5 ng/µL sequencing-grade modified trypsin (Promega). After rehydration (∼30 min) excess trypsin was removed and the gel pieces incubated at 37 °C for 16 h. Digested fragments were extracted from the gel pieces in 30 µL 20 mM NH4HCO3 followed by three extractions in 50 µL 5% formic acid/50% acetonitrile/45% water (v/v/v). The extracts were combined and dried in an integrated SpeedVac system (Thermo Savant). Peptide Mass Fingerprinting. Peptide Mass Fingerprinting was performed using a Voyager DE MALDI-TOF mass spectrometer (Applied Biosystems) in linear mode with a delay time of 100 ns and an acceleration voltage of 20 kV. Dried extracts were dissolved in 30% acetonitrile/1% formic acid. A 0.6-µL sample of 0.2% (w/v) nitrocellulose and saturated matrix in acetone was applied to the target plate and allowed to dry. A 0.3 µL sample of the peptide extract was then applied. After drying, the sample was washed twice with 4 µL 1% formic acid. The matrix used was R-cyano-4-hydroxycinnamic acid and calibration was performed externally with Bovine Serum Albumin (Sigma) and internally with the peptide fragments of trypsin. Once a positive identification had been made, internal standardization was performed using two prominent sample peaks. The obtained peptide mass fingerprint spectra were analyzed searching the Mascot search engine (www.matrixscience.com) based on the NCBI and Swiss-Prot protein database (taxonomy: mus) using the assumption that the peptides are monoisotopic, have undergone variable carbamidomethylation and are oxidized at methionine residues. Up to one missed trypsin cleavage was allowed. A mass accuracy tolerance of a maximum of 200 ppm was used for matching tryptic peptides. Probability bases Mowse score defined at -10*log(P), where P was the probability that the observed match was a random event were determined. All protein identifications were in the expected size range based on the position in the gel. Western Analysis. Cytosolic lung extracts (30 µg) were diluted in Laemmli sample buffer, denatured at 90 °C for 5 min and subjected to SDS-PAGE. Proteins were resolved on 10% gels at 200 V for 45 min and transferred onto Hybond PVDF membrane (Amersham) using Trans-blot SD transfer cell (Bio-Rad) 138

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at 10V for 40 min. PVDF membranes were incubated in blocking solution (TBS containing 5% [w/v] skim milk powder and 0.5% [v/v] Tween-20) for 1 h at room temperature. S100A8 expression was determined using specific antibody (Santa Cruz, Ca) by Western analysis as previously detailed.24 RNA Extraction and Quantitative Real Time PCR. Total RNA was isolated from 10 mg of ground lung tissue according to manufacturer instructions using the RNeasy kit (Qiagen). The purified total RNA prep was used as a template to generate first-strand cDNA synthesis using SuperScript II (Invitrogen) as previously described.24 Briefly, the reaction mix containing 1 µg of RNA was converted to cDNA, diluted 10-fold in sterile water and stored at -20 °C prior to amplification. Based on a 10-µL reaction volume performed in a 384-optical well plate, the master mixture was prepared from the TaqMan Universal Master Mix (Applied Biosystems) comprising AmpliTaq Gold DNA polymerase, Amperase UNG, dNTPs (dCTP, dGTP, dATP, and dUTP), passive ref 6-carboxy-rhodamine (ROX), MgCl2, and buffer components in amounts undisclosed by the manufacturer. Primers and probes for S100A8 were purchased from Applied Biosystems as a pre-optimized 20× stock (Assay on Demand) that is diluted to a final concentration of 900 nM forward and reverse primer and 250 nM FAM target probe. As an internal control, a 20× VIC labeled primer/probe for eukaryotic 18S rRNA (Applied Biosystems) optimized for multiplexing was measured for each individual reaction. A negative (no-template) control was included in every run. DNA was amplified with the TaqMan system using the default cycling parameters: heating at 50 °C for 2min; heating at 95 °C for 10 min followed by 40 cycles of a two-stage temperature profile of 95 °C for 15 s and 60 °C for 1 min. The cycle threshold (CT) value is the PCR cycle number (out of 40) at which the measured fluorescent signal exceeds a calculated background threshold identifying amplification of the target sequence value and is proportional to the number of input target copies present in the sample. Threshold cycle numbers were transformed using the ∆∆Ct and relative value method as described by Applied Biosystems and were expressed relative to 18S rRNA levels. ELISA. Quantification of S100A8 in the BAL washings from Balb/c mice were performed using a double-sandwich ELISA and rabbit polyclonal anti-S100A8 IgG as described32,33 using recombinant S100A8 (0.1-50 ng/mL) as standard. The lower limit of detection was 30 pg/mL. Statistical Analysis. Data are expressed as mean ( SEM for n observations. Statistical significance was determined using ANOVA followed by Dunnett’s multiple comparison test. P < 0.05 was considered to be statistically significant.

Results LPS Elicits an Intense Neutrophilic Lung Inflammation that is not Attenuated by Dexamethasone. Mice responded to instillation of LPS by acutely recruiting large numbers of neutrophils into the lung 24 h post challenge. As shown in Figure 1, neutrophils are prominent in the BAL (Figure 1A) and recruitment into this compartment was not suppressed by pretreatment with Dex. The antiinflammatory actions of glucocorticoids are attributed in part to suppression of NFκB activation via multiple actions: induction of IκBR,34,35 direct interaction between the glucocorticoid receptor and NFκB,34,35 and reduction of p65 translocation into the nucleus.35 We therefore assessed the efficacy of Dex pretreatment by determining gene expression (quantitative real time PCR) of two


Figure 1. Transnasally administered dexamethasone does not suppress LPS-induced neutrophilia. As shown in A, mice were pretreated for 3 h with vehicle (Saline) or 50µg/mouse dexamethasone (Dex) and then challenged with saline (clear bar) or 10µg LPS (black bar) to elicit an intense neutrophilic lung inflammation. BAL was performed at necroscopy at 24 h post challenge. Data are expressed as total neutrophils in BAL (mean ( SEM) for 8-10 mice per treatment group. No significant changes were observed between the Saline and Dex pretreated group, ANOVA/Dunnett’s test. As shown in B & C, mice were pretreated with Saline (9) or Dex (∆, 50 µg/kg for 3 h) and then challenged with LPS for the specified times. Total RNA was isolated from whole lung homogenates, reverse transcribed using random hexamers and subjected to Taqman PCR to detect GM-CSF and TNFR transcripts relative to 18S rRNA. Data are presented as a fold increase above Saline (time ) 0) group. #, p < 0.05; ANOVA/Dunnett’s test, significantly different from Saline and Dex group (n ) 4).

NFκB-regulated genes, TNFR and GM-CSF. Kinetic analysis of TNFR and GM-CSF gene expression revealed that both transcripts were maximally induced by LPS at 2 h, and this peak expression was suppressed in mice pretreated with Dex by 4050% (Figure 1B, 1C). Furthermore, we previously found that this dose of dexamethasone was sufficient to promote thymic atrophy when administered systemically.25 In addition, as shown below, we identified annexin (or lipocortin) as a Dex-

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Figure 2. Coomassie blue stained 2-DE gel of Balb/c mouse lung cytosol. Numbered spots in circles are proteins that were induced by LPS in a Dex resistant manner. Saline or Dex pretreated (3 h) mice were challenged with LPS for 2 h. Lung cytosols were prepared and subjected to 2-DE. LPS challenged gels were compared to reference Saline alone gel and integrated spot intensities were generated using PDQuest software. Labeled spots were tryptically digested; yielding masses subjected to MALDI/TOF-MS for submission to a peptide mass database for protein identification (see Table 1).

induced protein, consistent with reports that implicate annexin as one of multiple mechanisms which coordinate the broad antiinflammatory actions of glucocorticoids.36,37 Thus neutrophilic inflammation is not suppressed under conditions where dexamethasone regulates both the transcription and expression of steroid-responsive genes in vivo. 2D-PAGE Approach Identified Multiple Proteins that are Induced by LPS in a Dex Resistant Manner. Since most proteins that promote neutrophil migration are secreted chemotactic peptides, cytosolic extracts from lung homogenates were prepared and separated by 2D-PAGE. Representative images of the resultant Coomassie G250 gel patterns are shown in Figure 2, and each gel was matched to the reference sample (saline) to compare integrated spot intensities using PDQuest Journal of Proteome Research • Vol. 4, No. 1, 2005 139

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Table 1. Summary of the MALDI-MS Data Obtained from Mouse Cytosolic Lung Proteins Identified as LPS-Induced and Dex Resistant

spot no.

Sal

Sal/LPS

Dex/LPS

no. matched MALDI masses

0101 2004

100 100

1000 427

1078 650

9 5

54 73

2005

100

270

434

5

73

1003 2301

100 100

2785 210

8550 238

4 8

21 33

MALDI/TOF MALDI/TOF MS/MS MALDI/TOF MS/MS MALDI/TOF MALDI/TOF

5501 5203

100 100

526 272

580 531

4 8

9 35

MALDI/TOF MALDI/TOF

relative abundance (%)

sequence coverage (%)

software. This approach identified 258 spots that were successfully matched in all gels. Individual gels were then normalized relative to the total density in each image, and quantitative analysis based on a 2-fold cutoff identified six proteins that were induced by LPS treatment in a Dex resistant manner. Spot identification number and relative abundance of each protein are summarized in Table 1, along with peptide mass fingerprinting details. Of particular interest was spot 2004/2005 corresponding to S100A8 since it has been associated with neutrophilia and neutrophil migration in in vitro and in vivo models.30,38-42 Figure 2 shows increases in levels of components 2004 and 2005, i.e., S100A8, in lung homogenates of Dexprimed animals challenged with LPS. Analysis of tryptic peptides did not indicate any differences in primary amino acid sequence. The small differences in gel mobility of spots 2004 vs 2005 was attributed to post-translational modification, specifically oxidation of Met36 and Met73 in both components. No other changes such as phosphorylation, or evidence of oxidation by hypochlorite generation of covalent Cys-Lys sulfinamide bonds43 were observed. To validate the 2D-PAGE results, we next assessed expression of S100A8 in cytosolic extracts from individual mice by Western analysis using an antibody specific for this S100 protein (Figure 3). Here, we confirm that S100A8 expression in lung is elevated in LPS challenged mice and that S100A8 is actively secreted into lung lavage fluid. Consistent with the proteomic data (Table 1), pretreatment with Dex also augmented release of this protein above that seen in vehicle pretreated lungs. Localization of S100A8 Expression and Kinetic Analysis of its Transcript in Lung. S100A8 expression was examined by immunohistochemistry using paraffin-embedded lung tissue sections from saline or LPS-challenged mice (Figure 4). Salinetreated lungs show no evidence of inflammation and did not contain S100A8-positive cells. In contrast, lungs from LPSchallenged mice were inflamed as shown by the infiltration of polymorphonuclear leukocytes from adjacent blood vessels to the peribronchial region of the larger airways (H&E stain). Infiltrating mononuclear cells were not observed at this timepoint. Immunostaining for S100A8 identified strongly positive cells within the peribronchial region, and the absence of staining in control animals, in conjunction with their distinct morphological appearance, indicated that the immunopositive cells were neutrophils that had moved into inflamed lung tissue, consistent with the study of Thomas et al.44 At 4 h, S100A8-positive neutrophils were observed intravascularly in alveolar capillaries and notably, immediately subjacent to the epithelium. At 24 h, the point of peak neutrophil influx in BAL, S100A8-positive neutrophils were prominent in the alveolar 140


mass analysis

protein ID

accession no.

Rho GDI-β S100A8

NP031512 NP038678

S100A8

NP038678

unknown arachidonate 12-lipoxygenase (leukocyte-type) serum albumin annexin 1 (lipocortin 1)

P39654 P07724 NP034860

Figure 3. Validation of S100A8 expression by Western analysis. Mice pretreated for 3 h with saline or Dex were challenged with Veh (Saline) or LPS for 2 h. Following challenge, mice were killed and individual lung cytosolic extracts subjected to Western analysis using an S100A8 specific antibody. Immunoblots were quantitated by densitometry and intensities presented graphically as the percentage above Saline control group. #, p < 0.05; ANOVA/Dunnett’s test, significantly different from Saline group (n ) 6).

spaces. We did not observe S100A8 positive macrophages or positive staining of the epithelium above background, although, in vitro studies, have reported it to be induced by LPS in macrophages at later time points.33,45,46 In addition, we also stained for S100A9 at the 4 h time-point and observed a similar staining pattern of neutrophil staining as S100A8 (data not shown). We next performed kinetic analysis of S100A8 expression in whole lung extracts by quantitative real time PCR using specific primers (Figure 5A). Values were normalized to 18S rRNA and expressed relative to the group not challenged with LPS. S100A8 transcript levels were elevated in LPS treated lungs, peaking at 4 h and tapering off over the 24 h time-course. Pretreatment with Dex did not inhibit S100A8 transcription but promoted more rapid production of the LPS-induced transcript, as reflected by the 2 h peak. Furthermore, the decline in transcript expression at 24 h, relative to the maximal peak, strongly indicates that S100A8 was induced in response to endotoxin and not simply a reflection of neutrophil accumulation in the lung. Since S100A8 is abundant in the neutrophil cytosol, we


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Figure 4. Histological analysis of lungs and immunostaining for cellular expression of S100A8. Cross sections of lungs collected from mice challenged for 4 h with Saline (a,c,e) or with LPS (b,d,f) were stained with hemoatoxylin and eosin (a and b) or immunostained with conjugated secondary antibody alone (c and d) or S100A8 antibody (e and f). Note the polymorphonuclear leukocytes only in the LPS treated lungs corresponding with strong reactivity of these infiltrating cells using anti-S100A8. Original magnification × 400.

immunostained lung sections with this marker to identify the location and abundance of neutrophils (Figure 5B). Similar infiltration of immunopositive cells were observed in lungs 24 h after LPS challenge when compared to 4 h challenged sections (Figure 5B) suggesting de novo synthesis of S100A8 transcripts at the earlier time-point. S100A8 is Secreted into the BAL at Nanomolar Levels. S100A8 is secreted from stimulated monocytes and granulocytes47-49 and elicited murine macrophages46 and elevated levels are found in the BAL compartment of bleomycin-treated mice.50 Here, we measured the levels of S100A8 in the BAL of mice challenged with LPS by ELISA and, as shown in Figure 6, endotoxin proved to be a potent stimulus for the release of S100A8 under in vivo conditions. High levels of S100A8 (about 150 ng/mL) were detected in LPS treated mice and pretreatment with Dex did not inhibit release. Unlike total S100A8 content in whole lung homogenates assessed by Western analysis (Figure 3), secreted S100A8 in BAL was not released in a Dex augmented manner. Since Western analysis will

predominately detect compartmentalized S100A8 abundant in the neutrophil cytosol, this observation may implicate an alternative source of secreted S100A8 such as activated monocyte/macrophages or epithelium, which have been shown to release S100A8 in response to an inflammatory insult. Secreted S100A8 appeared to be monomeric as Western analysis of BAL for S100A8 under reducing and nonreducing conditions identified a single band at about 9kDa (data not shown). In other models of acute inflammation, heterodimeric complexes of S100A8 have been found at sites of inflammation, however we only observed monomeric S100A8 in the lung. Differential regulation of S100A8 vs S100A9 has previously been noted in response to LPS in vivo.30 Anti-S100A8 but not Anti-S100A9 Weakly and Incompletely Inhibits Neutrophilia Induced by Local Instillation of LPS. To establish the functional role of S100A8 following local endotoxin challenge, mice were pretreated with blocking antibodies to S100A8 or S100A9. Figure 7 shows that neutralizing S100A8, but not S100A9, significantly reduced total Journal of Proteome Research • Vol. 4, No. 1, 2005 141

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Figure 5. LPS induced S100A8 transcription in murine lung in a temporal manner that does not simply reflect neutrophil infiltration. As shown in A, mice were pretreated with Saline (9) or Dex (∆, 50 µg/kg for 3 h) and then challenged with LPS for the specified times. Total RNA was isolated from pooled (6-8 mice per time-point) whole lung homogenates, reverse transcribed using random hexamers and subjected to Taqman PCR to detect S100A8 transcript relative to 18S rRNA (represented are replicates of four per time-point). Data are presented as a fold increase above Saline (time ) 0) group. As shown in B, immunoperoxidase staining for cellular S100A8 in cross sections of lungs from either 4 h or 24 h LPS challenged mice identifies positive staining cells in the peribronchial region of the larger airways and sequestered in capillaries within the alveolar septa.

Figure 6. S100A8 is secreted into the BAL compartment in response to LPS. ELISA was used to measure endogenous secreted S100A8 in BALF of saline (Sal) or Dex pretreated mice that were challenged with LPS for 4 h prior to necroscopy. At this time point no detectable neutrophils apoptosis/necrosis was observed. Data were compared to the Saline alone group. #, p < 0.05; ANOVA/Dunnett’s test, (n ) 8).

neutrophil numbers s recruited into the lung BAL compartment by about 20%. The lack of S100A9 activity is consistent with our findings relating to the detection of only monomeric S100A8. Since multiple chemokines (MIP2, KC, and Gro-R) also mediate neutrophilia, the degree of inhibition observed after neutralization of S100A8 was surprisingly large although blockade did not result in complete suppression. Our data indicate for the first time that S100A8 is a steroid insensitive mediator of the lung inflammatory response to LPS contributing to the coordination of neutrophil recruitment, in vivo. 142


Figure 7. Blocking antibody to S100A8 partially suppressed neutrophil influx. Mice were injected i.p. with saline (SAL), purified rabbit IgG against S100A8 (anti-A8), S100A9 (anti-A9) or nonimmune serum (ISO) 16 h prior to transnasal challenge with 10 µg LPS. BAL was performed at necroscopy 24 h post challenge. Data are expressed as % inhibition of total neutrophils in BAL relative to the SAL group. Also, the number of mice per treatment, mean total neutrophil count in BAL and SEM are shown. A significant change was observed between the Saline and anti-A8 pretreated group, ANOVA/Dunnett’s test (#, p < 0.05).


Discussion The recruitment of polymorphonuclear leukocytes from circulation to the lung in response to endotoxin is coordinated by the complex and sequential release of cytokines, chemokines and proteases.25,51,52 Key chemokines involved in neutrophil mobilization have been identified and include IL-8 and its mouse homologues Macrophage Inflammatory Protein 2 (MIP2), KC and Growth Related Oncogene alpha (GroR). Such chemokines act through the CXC receptors (CXCR2) to promote chemotaxis and selective CXC receptor antagonists are completely effective at blocking neutrophil migration in response to these chemotactic peptides.53 However, the response to bacterial products is more intricate as CXCR2 receptor antagonists only partially block the recruitment of neutrophils in response to LPS,53 which is indicative of alternative mechanisms for leukocyte mobilization as a consequence of bacterial colonization. Conventional approaches used in efforts to suppress leukocyte accumulation to sites of inflammation utilize powerful glucocorticosteroids, such as methylprednisolone, fluticasone, beclomethasone and dexamethasone administered systemically or topically by inhalation. The systemic use of very high dose steroids in animal models of neutrophilia has been shown to exhibit some inhibitory effects,54,55 which are associated with the glucocorticosteroid sensitive secretion of CXC chemokines.55-57 However, the use of such high systemic doses in the clinical setting is undesirable due to adverse side effect including hastening death and worsening skeletal muscle dysfunction, bone weakening, bruising, growth suppression, induction of diabetes and neurological disorders. Here, we show that the topical lung administration of dexamethasone displays antiinflammatory actions including down-regulation of TNF-R and GM-CSF expression, as well as induction of annexin (lipocortin) in murine lung. Despite these actions, robust recruitment of neutrophils into endotoxin exposed lungs was observed. We therefore used a global 2D-PAGE proteomics approach to screen for novel molecules that regulate lung inflammation in a steroid refractory manner, identifying S100A8 as a likely candidate. S100A8 belongs to a family of low molecular weight calcium binding proteins involved in the regulation of calcium dependent intracellular processes.58 Despite the high homology between S100 members, S100A8 does exhibit nonredundant functions since targeted disruption of this gene, but not S100A9, is embryonic lethal.59 S100A8 is a prominent protein found in the cytosol of neutrophils. It is only constitutively expressed in circulating neutrophils and monocytes, and generally not present in tissue macrophages.58 Mouse and human S100A8 share 58% homology at the amino acid level. S100A8 was first identified as Cystic Fibrosis Antigen.60 Following cloning, it was realized that S100A8 belongs to a group of related proteins encoded on human chromosome 1q. The function of S100A8 has remained obscure. Recently, both mouse and human recombinant S100A8 were found to be major determinants of neutrophil accumulation in a murine air pouch model.42 S100A8 also forms a heterodimeric complex with S100A9 (S100A8/S100A9 heterodimer is known as calprotectin) and this complex exerts some distinct effects including inducing neutrophil adhesion to fibrinogen61 and arachidonic acid metabolism.62 More recently, calprotectin has been shown to promote IL-8 production in immortalized airway epithelial cells, although, it is not yet known if S100A8 alone triggers a similar response.63 Furthermore, calprotectin has been found to be

research articles elevated in the serum of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease64,65 and lung pathologies such as cystic fibrosis and chronic bronchitis.66,67 On the basis of our previous dose-response study25 we used a maximally tolerated dose of LPS delivered to the lung, a very intense stimulus, and observed approximately 20% suppression with antiS100A8 and no effect of blocking S100A9. In contrast, neutralization of S100A8 strongly suppressed neutrophil recruitment in a urate crystal model of gout41 and suppressed skin pouch LPS responses by 50%.30 Neutralization of S100A9 was also effective in these models. The relative lack of effectiveness of anti-S100A8 in our lung LPS model may relate to the intensity of the responses and relative surface areas for inhibition. The dose of LPS we used was the maximally tolerated dose- higher doses induce lung hemorrhage and the model is therefore particularly intense. The antibody was administered intraperitoneally, at the maximal dose technically possible, and may not have reached the high local concentrations needed to inhibit LPS instilled directly into the lung. It should be noted that the lung retains virtually all blood neutrophils in its microcirculation after LPS challenge and our results are therefore likely to somewhat underestimate the potential benefit of blocking S100A8 for lung inflammatory responses. These other studies also show that S100A8 plays a role in neutrophil migration to the inflamed tissue; however it appears to not influence neutrophil release from the bone marrow.30 The reason for the difference between lung and other tissues most likely related to the dual microcirculation present in the lung. In other tissues, neutrophil diapedesis occurs only after adherence to postcapillary venules. In contrast, neutrophils enter lung tissue and the alveolar airspace via both adherence to postcapillary venules, a process dependent on adherence to CD11/18 integrins and direct chemotaxis across the very thin alveolar capillary/epithelial barrier largely independently of integrin interactions.68 As the role of S100 proteins might vary with different innate immunity agonists, it will be of considerable interest to determine the specific role of S100A8 in neutrophil recruitment, including responses triggered by live pathogens in future studies. Our protein and transcriptional data identify S100A8 as a dexamethasone nonresponsive and LPS inducible protein. LPS induces S100A8 in murine macrophages ex vivo, with optimal expression around 16-24 h,46 although immunohistochemistry failed to locate any macrophage-associated S100A8 in vivo in our model (Figure 4). Importantly, we report the new finding that steroid resistant in vivo secretion of S100A8 occurs into BAL fluid within 4h of LPS challenge. All detectable S100A8 identified in immunohistochemistry studies was associated with neutrophils, suggesting they are the likely source of release S100A8. This release is not a passive consequence of neutrophil apoptosis/necrosis as we only detected viable neutrophils in the BAL as assessed by viability cell staining (data not shown) at this early time-point. This is also consistent with the identification of oxidized S100A8. Activated neutrophil derived oxidants, in particular hypochlorite, gradually promote formation of S100A8 covalent homodimers that loose their chemotactic activity.58 It might be speculated that an additional reason the contribution of S100A8 to neutrophils recruitment is less that observed in other mouse compartments could be rapid oxidative stress induced inactivation of S100A8 chemotactic activity in the lung. Given its newly identified chemotactic activity, S100A8 has the capacity to work in conjunction with Journal of Proteome Research • Vol. 4, No. 1, 2005 143

research articles conventional CXC chemokines to promote the effective recruitment of neutrophils into the lung in response to LPS. However, it is not known if S100A8 has any specific interactions with established neutrophil chemokines or proteins identified in this study such as 12-lipoxygenase. In conclusion, we found S100A8 to be a steroid refractory mediator of lung neutrophilia through the application of in vivo protein profiling methods. S100A8 might represent a potential therapeutic target for the treatment neutrophilic lung inflammation in conditions where glucocorticosteroid responses are suboptimal however its relatively weak overall affect mitigates against a major therapeutic utility. Our data further emphasizes the need for comprehensive in vivo functional profiling to understand the true biological significance of disease candidates identified using contemporary molecular profiling methods.

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