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ResponseCounter-Regulator of ProinflammatoryWAP Domain Protein, AMWAP, Acts as a The Novel Activated Microglia/Macrophage

Fuchshofer and Thomas LangmannEbert, Jan Van den Brulle, Heinz Schwer, Rudolf Marcus Karlstetter, Yana Walczak, Karin Weigelt, Stefanie

http://www.jimmunol.org/content/185/6/3379doi: 10.4049/jimmunol.0903300August 2010;

2010; 185:3379-3390; Prepublished online 13J Immunol 

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0.DC1http://www.jimmunol.org/content/suppl/2010/08/13/jimmunol.090330

Referenceshttp://www.jimmunol.org/content/185/6/3379.full#ref-list-1

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The Journal of Immunology

The Novel Activated Microglia/Macrophage WAP DomainProtein, AMWAP, Acts as a Counter-Regulator ofProinflammatory Response

Marcus Karlstetter,* Yana Walczak,* Karin Weigelt,*,† Stefanie Ebert,*

Jan Van den Brulle,‡ Heinz Schwer,‡ Rudolf Fuchshofer,x and Thomas Langmann*

Microgliosis is a common phenomenon in neurodegenerative disorders, including retinal dystrophies. To identify candidate genes

involved in microglial activation, we used DNA-microarray analysis of retinal microglia from wild-type and retinoschisin-deficient

(Rs1h2/Y) mice, a prototypic model for inherited retinal degeneration. Thereby, we cloned a novel 76 aa protein encoding

a microglia/macrophage-restricted whey acidic protein (WAP) termed activated microglia/macrophage WAP domain protein

(AMWAP). The gene consists of three exons and is located on mouse chromosome 11 in proximity to a chemokine gene cluster.

mRNA expression of AMWAP was detected in microglia from Rs1h2/Y retinas, brain microglia, and other tissue macrophages.

AMWAP transcription was rapidly induced in BV-2 microglia upon stimulation with multiple TLR ligands and IFN-g. The TLR-

dependent expression of AMWAP was dependent on NF-kB, whereas its microglia/macrophage-specific transcription was regu-

lated by PU.1. Functional characterization showed that AMWAP overexpression reduced the proinflammatory cytokines IL-6

and IL-1b and concomitantly increased expression of the alternative activation markers arginase 1 and Cd206. Conversely, small

interfering RNA knockdown of AMWAP lead to higher IL-6, IL-1b, and Ccl2 transcript levels, whereas diminishing arginase 1

and Cd206 expression. Moreover, AMWAP expressing cells had less migratory capacity and showed increased adhesion in

a trypsin-protection assay indicating antiserine protease activity. In agreement with findings from other WAP proteins, micro-

molar concentrations of recombinant AMWAP exhibited significant growth inhibitory activity against Escherichia coli, Pseudo-

monas aeruginosa, and Bacillus subtilis. Taken together, we propose that AMWAP is a counter-regulator of proinflammatory

microglia/macrophage activation and a potential modulator of innate immunity in neurodegeneration. The Journal of Immu-

nology, 2010, 185: 3379–3390.

Microglial cells, the resident phagocyte population of thenervous system, exert several important functions inimmune surveillance (1, 2) and neuronal homeostasis

(3, 4). In the healthy brain and the retina, ramified microglia serveas highly motile patrolling cells constantly surveying their mi-croenvironment (5). There is an ongoing debate whether residentmicroglia in the CNS and the retina are replenished by in situproliferation and/or recruitment of myeloid cells from the blood-stream (6–8). Accumulating evidence now suggests that experi-mental confounds including alterations of the blood-retina barriercould have biased earlier results from bone marrow chimeras (9).

Thus, parabiosis studies demonstrated that microglia in the healthyor experimentally damaged brain are poorly replenished from thebloodstream (10). Moreover, Mildner et al. (11) showed that irra-diation is essential for infiltration of circulating microglial pre-cursors into the brain in chimerism experiments.Microglia communicate with other glial cells and neurons that

regulate their activation status, their capacity for phagocytosis ofcellular debris, and secretion of neurotrophic factors (12). Keyregulatory signals from neurons and glia are transmitted to micro-glia cells through various soluble factors, including nucleotides(13), the chemokine fractalkine (14), TGF-b (15), and nerve growthfactor (16). Control of microglial activation and function is furtherregulated by direct neuron-microglia crosstalk, including Cd200/Cd200 receptor ligation (17) and complex formation of triggeringreceptors expressed on myeloid cells with different ligands onneurons (18).Activated microglia can elicit both protective and deleterious

functions. In theearly phaseofneurodegeneration,microglia evokedpotent tissue remodeling programs (4) and initiated repair mecha-nisms, such as glial scar formation (19). However, excessive orprolonged microglial activation in the CNS and the retina can leadto chronic inflammation with severe pathological side effects oftenresulting in irreversible neuronal loss (2, 20, 21). In eye research,various animal models have demonstrated that microglial activationis tightly associated with and mostly precedes retinal degenerationand photoreceptor apoptosis (22–24). Functional and phenotypiccharacterization of microglia populations in the diseased retinacould be of high relevance to reveal signaling events that triggertheir activation. However, like other tissue macrophages (25), act-

*Institute of Human Genetics and xInstitute of Human Anatomy and Embryology,University of Regensburg, Regensburg; ‡Sloning Bio Technology, Puchheim, Ger-many; and †Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

Received for publication October 8, 2009. Accepted for publication July 11, 2010.

This work was supported by grants from the German Research Foundation (FOR1075Project 4), the Elite Network of Bavaria, and the Pro Retina Foundation.

Address correspondence and reprint requests to Dr. Thomas Langmann, Institute ofHuman Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042Regensburg, Germany. E-mail address: thomas.langmann@klinik.uni-regensburg.de

The online version of this article contains supplemental material.

Abbreviations used in this paper: AMWAP, activated microglia/macrophage WAPdomain protein; CAPE, caffeic acid phenethyl ester; CHX, cycloheximide; CSPG-DS, chrondroitin-sulfate proteoglycan-disaccharide; DAMP, damage-associated mo-lecular pattern; Expi, extracellular peptidase inhibitor precursor; OHT, 4-hydroxy-tamoxifen; qRT-PCR, quantitative RT-PCR; Rho, rhodopsin; Rs1h2/Y, retinoschisindeficient; siRNA, small interfering RNA; SLPI, secretory leukoprotease inhibitor;WAP, whey acidic protein; wt, wild-type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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ivated microglia comprised a continuum of diverse functionalphenotypes with a broad spectrum of activation markers (26).Our laboratory has used large-scale transcriptional phenotyping

to identify characteristic gene signatures of LPS and chondroitinsulfate proteoglycan-disaccharide (CSPG-DS) stimulated BV-2microglia as clearly polarized cell populations (27). Moreover, wehave profiled primary retinal microglia from wild-type (wt) andretinoschisin-deficient (Rs1h2/Y) mice (28), a prototypic model forrapid retinal apoptosis and degeneration (29). In contrast to LPS-and CSPG-DS–treated microglia, expression profiles of retinalRs1h2/Y microglia indicated overlapping transcript clusters remi-niscent of both pro- and anti-inflammatory macrophage activation(28, 30, 31). Finally, several transcripts previously not linked tomicroglial activation have been identified in these genome-wideexpression studies, including high levels of the adaptor proteinSTAP-1 (32) and the uncharacterized whey acidic protein (WAP)motif-bearing protein AMWAP.WAP domain proteins have originally been described as low

m.w. proteins with “defensin-like” properties involved in immunehomeostasis (33). Their counter-regulatory role on inflammatorymediators is mainly ascribed to the antiprotease and antimicrobialactivities of theWAP domain (34–36). This 40–50 aamotif containseight conserved cysteins that form four defined disulfide bonds.Secretory leukocyte protease inhibitor (SLPI) and elafin are the beststudied WAP proteins in humans and mice (37). SLPI is constitu-tively produced at many mucosal surfaces and is also produced bylung epithelial cells, neutrophils, and macrophages (38). SLPI ex-pression in macrophages is induced by bacterial endotoxin leadingto the suppression of NO and TNF secretion (39). Elafin is mainlypresent in epithelia of the skin, oral cavity, vagina, and lung to fulfilldistinct antimicrobial and immunomodulatory functions (40).In this study, we identified a novel microglia/macrophage-restricted

WAP domain protein, AMWAP, in activated primary retinal microglia.AMWAP expression is rapidly induced by ligands for TLR2, -4,and -9 and IFN-g in the BV-2 cell line. AMWAP overexpression re-duces proinflammatory cytokine expression and concurrently in-duces markers for alternative macrophage activation. We thereforepropose that AMWAP is a novel modulator of microglial activa-tion in neurodegenerative disorders.

Materials and MethodsAnimals

Retinoschisin knockout (Rs1h2/Y) mice have been described previously(29) and C57BL/6 mice were purchased from Charles River Laboratories(Sulzfeld, Germany). Mice were kept in an air-conditioned barrier envi-ronment at constant temperature of 20˚C–22˚C on a 12-h light-darkschedule, and had free access to food and water. The health of the ani-mals was regularly monitored, and all procedures were approved by theUniversity of Regensburg animal rights committee and complied with theGerman Law on Animal Protection and the Institute for Laboratory AnimalResearch Guide for the Care and Use of Laboratory Animals, 1999.

Isolation and culture of primary cells

Retinal microglia were isolated and cultured as described earlier (28). Briefly,retinal tissue from wt and Rs1h2/Y mice at postnatal days 14 and 18 wasisolated from eye bulbs and purified from contaminating vitreous body andretinal pigment epithelium/choriocapillaris. Pools of four to six retinas eachwere cut into small pieces and incubated for 40min at 37˚C in 1ml PBSwith 1mg/ml collagenase type I (Sigma-Aldrich, St. Louis, MO), 0.3mg/ml DNase I(Roche, Basel, Switzerland), and 0.2 mg/ml hyaluronidase (Sigma-Aldrich).The cell suspension was filtered through a 70-mm cell strainer (BectonDickinson, Heidelberg, Germany). Cells were washed twice with 10 mlDMEM/10% FCS and finally subjected to Ficoll density gradient centrifu-gation for 20 min at 2000 rpm (690 g, without brake) in a Heraeus centrifugefor the isolation of mononuclear cells. The interphase was carefully removedand washed with 10 ml DMEM/FCS. The cells were cultured for 11 d in75-cm2 flasks containing DMEM/10% FCS supplemented with 50 ng/ml

recombinant human M-CSF (R&D Systems, Minneapolis, MN) and phasecontrast micrographs were takenwith a Nikon Eclipse TE2000-Smicroscope.

Brain microglia were isolated from the brains of 4-wk-old wt mice. Eachbrain was dissected, cut into small pieces in 520 ml PBS, and incubated for40 min at 37˚C with vigorous shaking (800 rpm) containing the samedissolving solution described previously. The single-cell suspension wasfiltered through a 70-mm cell strainer (Becton Dickinson), washed twicewith 10 ml DMEM/10% FCS, followed by centrifugation at 1600 rpm for5 min at room temperature. The pellet was resuspended in 10 ml DMEM/10% FCS supplemented with 50 ng/ml recombinant human M-CSF (R&DSystems) and cultured in 75 cm2 cell culture flasks. Nonadherent cellswere removed after 4 d by washing with culture medium. After 11 d ofculture, 100 ng/ml LPS was added where indicated and total RNA wasisolated after 24 h of stimulation.

Bone marrow macrophages were isolated from bone marrow of adult wtanimals. Femurs and tibias were dissected from the surrounding muscletissue and both ends were cut. Bone marrow was flushed with 2 ml DMEM/10% FCS with a 27 G syringe and cell clumps were separated by pipetting.The cell suspension was centrifuged for 10 min at 1200 rpm at roomtemperature. The supernatant was discarded and the pellet was dissolved in2 ml RBC lysis buffer (Sigma-Aldrich) and incubated for 7 min at roomtemperature. Then incubation was stopped with 5 ml DMEM/10% FCS,followed by centrifugation. The cell pellet was resuspended in 10 mlDMEM/10% FCS supplemented with 50 ng/ml recombinant M-CSF (R&DSystems). Cell culture medium was removed at days 4 and 8. After 10 d inculture, bone marrow macrophages were stimulated with 100 ng/ml LPSwhere indicated and total RNA was isolated after 24 h.

Dissected spleen tissue of adult wt animals was disintegrated by scis-sors in 520 ml PBS, passed through a wire mesh, and washed with 10 mlDMEM/10% FCS. Cells were filtered through a 40-mm cell strainer andprocessed as described previously for bone marrow macrophage isolation.

Immature bone marrow dendritic cells (DCs) were isolated as describedpreviously (41). For maturation, DCs were collected after 10 d, and resus-pended in culture medium containing 100 U/ml rmGM-CSF and 1 mg/mlLPS. Total RNAwas collected after 24 h of maturation.

Cell lines and reagents

RAW264.7 cells were received from the American Type Culture Collection(Manassas, VA) and BV-2 cells were kindly provided by Professor RalphLucius (Clinic of Neurology, Christian Albrechts University, Kiel, Ger-many). RAW264.7 macrophages were kept in DMEM supplemented with10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. BV-2 cellswere cultured in RPMI/5% FCS supplemented with 2 mM L-Glutamine and195 nM b-mercaptoethanol. Culture and treatment of tamoxifen-induciblePUER cells has been described elsewhere (42). LPS, IFN-g, and cyclo-heximide (CHX) were purchased from Sigma-Aldrich. PAM3CSK4 waspurchased from Invitrogen. The phosphorothioate CpG oligonucleotide (59-tccatgacgttcctgatgct-39) and control oligonucleotide (59-tccatgaggttcctgat-gct-39) were synthesized by Metabion (Martinsried, Germany). Caffeic acidphenethyl ester (CAPE) was purchased from Tocris Bioscience (Ellisville,MO). For activation experiments, cells were exposed to LPS, IFN-g, CpGoligonucleotides, or PAM3CSK4 in various doses for different time points.For inhibition of NF-kB and protein synthesis, BV-2 cells were pretreatedwith 15 mg/ml CAPE for 2 h or 5 mg/ml CHX for 1 h, respectively. Cellswere then washed with culture medium prior to stimulation with 50 ng/mlLPS, 1 mg/ml CpG oligonucleotides, or 1 mg/ml PAM3CSK4 for 4 h.

RNA isolation, reverse transcription, and 59-RLM-RACE

Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden,Germany). Purity and integrity of the RNAwas assessed on the Agilent 2100bioanalyzer with the RNA 6000 Nano LabChip reagent kit (Agilent Tech-nologies, Palo Alto, CA). The RNAwas quantified spectrophotometricallyand stored at280˚C. cDNAs were generated using the RevertAid H MinusFirst Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany).

To identify and clone full-length AMWAP transcripts, 59RNA ligasemediated RACE-PCR was carried out using RNA from RAW264.7 cellsactivated with 100 ng/ml LPS and the Ambion FirstChoice RLM-RACEkit according to the manufacturer´s instructions. AMWAP-specific reverseprimerswere designedon the incomplete database entryMm.24097. Thefirst59 RLM-RACE PCR was carried out with the AMWAP-specific reverseprimer 59-GGGCAGGATCCATCTCCT-39 and the 59RACE outer primer59-GCT GAT GGC GAT GAA TGA ACA CTG-39. The nested 59 RLM-RACE PCR was conducted with the AMWAP-specific reverse primer 59-TTT GCA GAC ATG ACC ACA GC-39 and the inner 59 RACE primer 59-CGC GGATCC GAA CAC TGC GTT TGC TGG CTT TGATG-39. PCRproducts were analyzed on 2% agarose gels and individual bands wereextracted, cloned into the pCR2.1TopoTAvector (Invitrogen,Carlsbad, CA)

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and sequenced. The full-length AMWAP sequence was submitted to Gen-Bank under accession no. FJ007372 (www.ncbi.nlm.nih.gov/Genbank).

Quantitative real-time RT-PCR

Relative transcript levels were assessed by amplifications of 50 ng cDNA ina 7900 HT real-time PCR detection system (Invitrogen Life Technologies,San Diego, CA). The 20 ml reaction volumes contained 13 TaqMan GeneExpression Master Mix (Invitrogen Life Technologies), 200 nM primers(Supplemental Table I) and 0.25 ml dual-labeled probe (Roche UniversalProbe Library). The PCR reaction parameters were as follows: 2 min 50˚Chold, 30 min 60˚C hold, and 5 min 95˚C hold, followed by 45 cycles of20 s 94˚C melt and 1 min 60˚C anneal/extension. The results were ana-lyzed using the DDCt method for relative quantification (43).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments of RAW264.7 cellscoupled to Affymetrix Promoter Arrays have been described elsewhere (44).For ChIP-PCR with microglia, 10 million BV-2 cells were treated with 1%formaldehyde for 15 min and lyzed with SDS, Empigen, and NP-40 (sup-plemented with 1 mM PMSF, 1 mg/ml aprotinin, and 1 mg/ml pepstatin A).Thenuclear pelletwas homogenizedby sonication twice at 30%amplitude for10 s. Immunoprecipitation was performed on the lysate with 2.5 mg of anti-PU.1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) anti–di-acetylated(K9 and K14) histone H3 (Upstate Biotechnology, Lake Placid, NY), anti-p300 clone RW128 (Upstate Biotechnology), anti-CBP (Upstate Biotech-nology), or anti-IgG Ab (Santa Cruz Biotechnology). After washing andelution steps, cross-links were reversed at 65˚C overnight. The immunopre-cipitatedDNAwas purified using theQIAquick PCR purification kit (Qiagen)and analyzedbyPCRusing the forwardprimer 59-CCCCTCGAGCTGGAAAAAGGAACCTGGTG-39 and the reverse primer 59-CCCAAGCTTTCATCC CCA CAG TGATCA AA-39 specific for the AMWAP proximal pro-moter region2114/+68.

Overexpression of AMWAP in BV-2 cells

The full-length coding sequence of mouse AMWAP was RT-PCR amplifiedfromRAW264.7RNAwith primer pair forward 59-CCCAGCTTCCCAACATGA AGA CAG CCA CA-39 and reverse 59-CCC CTC GAG TAA AAGACAGGAGTTTTGCAGAC-39. PCR products were sequenced and clonedinto the HindIII/XhoI site of a pCEP1.4 vector inframe with a C-terminalrhodopsin (Rho)-1D4 tag (45) to generate pAMWAP-1D4. A fusion protein ofmilk fat globule protein 8 with a C-terminal Rho-1D4 tag (pMFG-1D4) wascloned as positive control. The AMWAP-GFP-fusion protein (pAMWAP-GFP) was created by cloning the AMWAP open reading frame into thepcDNA/CT-GFP-TOPO vector using RT-PCR with primer pair forward 59-CCCGGTACCCCAACATGAAGACAGCCACA-39 and reverse 59-CCCGATATC TAAAGACAGGAGTTT TGCAGAC-39, containing restrictionsites for KpnI and EcoRV, respectively. BV-2 cells were transfected with 2 mgof endotoxin-free plasmid using trans-IT-LT1 transfection reagent (Mirus Bio,Madison,WI), according to the manufacturer’s recommendations. Stable BV-2 cell transformants expressing GFP or AMWAP-GFP were created by neo-mycin selection. Successful expression of GFP and AMWAP-GFP wasmonitored by GFP fluorescence.

Small interfering RNA knockdown of AMWAP in BV-2 cells

Target sequences for murine AMWAP were based on the longest transcriptobtained from the 59RLM-RACE-PCR (Accession no. FJ007372.1). Smallinterfering RNAs (siRNAs) were designed with the Ambion siRNA targetfinder and synthesized with the siRNA construction kit (Ambion, Austin,TX). AMWAP siRNA was synthesized using sense primer 59-AAAGG-ATCCATCTCCTGAGCACCTGTCTC-39 and antisense primer 59-AAT-GCTCAGGAGATGGATCCTCCTGTCTC-39. BV-2 cells were transfectedwith 33 nM AWMAP siRNA or scrambled siRNA (Ambion) using lipo-fectamine 2000 (Invitrogen). Twenty-four hours after transfection, cellswere stimulated with 5 ng/ml LPS for 24 h before isolation of total RNA.

Western blotting

Cells were grown in 10 cm dishes and lysed in 1 ml boiling lysis buffer(PBS, 1% SDS). Equal amounts of protein samples were separated withNuPAGE Bis-Tris 4–12% gels (Invitrogen) and subsequently transferred toa Hybond ECL membrane (Amersham Biosciences, Arlington Heights,IL). For determination of protein size, PageRuler prestained protein stan-dard (Fermentas) was used. The membranes were blocked in 5% milkpowder in PBS and probed with either anti–Rho-1D4mouse mAb (1:10000)(a generous gift of Robert S. Molday, Department of Biochemistry andMolecular Biology, Centre for Macular Research, University of British

Columbia, Vancouver, British Columbia, Canada) or anti–His-Penta mousemAb (Qiagen, 1:10000). A HRP-conjugated secondary Ab against mouse Ig(Cell Signaling Technology) and the ECL Plus system (Amersham Bio-sciences) were used for detection.

Immunocytochemistry

BV-2 cells were plated overnight on coverslips, fixed with 4% parafor-maldehyde for 10min at 37˚C, permeabilizedwith 0.2%TritonX-100, blockedwith 5% nonfat milk in 0.2% Triton X-100, and stained with anti–GFP-Ab(Abcam). Nuclei were stained with DAPI for 10 min (0.1 mg/ml in PBS, 496-diamidino-2-phenylindol, Molecular Probes, Eugene, OR). Coverslips weremounted on glass slides and visualized with an Axioskop 2 fluorescencemicroscope equipped with an Eclipse digital analyzer (Carl Zeiss).

Wound healing and transwell migration assays

Stably pAMWAP-GFP and pGFP expressing BV-2 cells were grown in 6-well plates as 80% confluent monolayers and were wounded with a sterilepipette tip. Cell migration into the open scar was documented withmicrophotographs at time points 0 h and 24 h after wounding. As an inde-pendent method, the Costar Transwell System (8-mm pore size poly-carbonate membrane) was used to evaluate cell migration. BV-2 cells (1 3106 cells in 1.5 ml serum-free medium) were added to the upper well, and2.6 ml serum-free medium was added to the lower chamber. A total of50 ng/ml LPS or ethanol as solvent control were added to the lowerchamber medium. At the end of the 24 h incubation, cells on the top of themembrane were removed by swiping with a damp cotton swab, and cellsthat had migrated to the lower surface were fixed in methanol for 15 min atroom temperature and stained with 1% crystal violet. The migration ac-tivity was quantified by counting the migrated cells on the lower surface ofthe membrane using light microscopy.

Trypsin protease protection assay

Stably AMWAP-GFP and GFP-expressing BV-2 cells were cultured in 24-well plates overnight until 80% confluence was reached. The culture me-dium was removed and 0.25% trypsin in PBS was added to the cells for theindicated time points. Complete medium containing 10% FCS was added tothe cells to stop trypsin activity. The cells were washed three times andstained with 0.2% crystal violet in PBS for 10 min. After additional washingsteps, cell-associated crystal violet was extracted with 10% acetic acid andthe OD was assessed on a microplate reader at 600 nm. OD values werenormalized to untreated control cells.

Ccl2 luciferase reporter assay

The murine Ccl2 promoter (2988/+113) was PCR amplified from genomicDNA using the primers forward 59-CCC CTC GAG CAT GCT ACA GAAAGC CCA AAA-39 and reverse 59-CCC AGATCT GGC CCA GAA GCATGA CAG-39. The PCR product was cut with restriction enzymes XhoI andBglII and cloned into the pGL4.10 vector (Promega,Madison,WI). BV-2 cellswere cotransfected with the luciferase construct and a pTK-Hyg vector(Clontech Laboratories, Palo Alto, CA) for selection of stable clones withHygromycin B (PAA). BV-2 cells stably expressing the Ccl2 luciferase re-porter were transiently transfected with 2 mg of pAMWAP-GFP or pGFPvectors. Transfected cells were harvested after 24 h and luciferase activity wasdetermined with the Luciferase assay system (Promega) on a FLUOstar Op-tima plate reader (BMG Labtech, Offenburg, Germany).

Bacterial expression and purification of recombinant AMWAP

The AMWAP cDNA sequencewas codon-optimized and de novo synthesizedfor recombinant expression in Escherichia coli (Sloning Biotechnology,Puchheim, Germany), resulting in an increased codon adaption index from0.7 to 0.91 (Supplemental Fig. 1) (46). Signal-sequence lacking and codon-optimized AMWAP was PCR amplified from pSlo1.0-AMPWAP with for-ward primer 59-CCC CATATG ACC TAT GTG GTG TCC TGT CC-39 andreverse primer 59-CCC AAG CTT AAA CAC CGG GGT TTT GC-39. ThePCR product was inserted with NdeI and HindIII into the pET21a(+) vector(Novagen, Madison, WI) inframe with a N-terminal His-tag sequence.

E. coli BL21a-DE3 cells transformed with pAMWAP-His were grownin 500 ml LB-medium until an OD600 of 0.7 was reached. Isopropyl-b-D-thiogalactoside was added to a final concentration of 1 mM and culture wascontinued at 37˚C for 4 h. Cells were harvested, the cell pellet was re-suspended in 7 ml lysis buffer (300 mM NaCl, 50 mM sodium phosphate,pH 8.0) containing 7 mg lysozyme, and the sample was incubated for30 min at 4˚C. Thereafter, the cell suspension was sonicated and stirred onice with 5 mg/ml DNase I for 15 min. The crude lysate was centrifuged at10,000 3 g for 30 min at 4˚C and the supernatant was loaded onto Protino

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Ni-TED Columns (Macherey-Nagel, Duren, Germany). Three fractionswere eluted in buffer containing 250 mM imidazole and concentrated inAmicon Ultra-4 Ultracel 5 kDa tubes (Millipore, Bedford, MA) by cen-trifugation. The protein was dialyzed four times against PBS, the con-centration of each fraction was determined by Bradford assay and purifiedrecombinant protein was stored at 280˚C.

Antimicrobial assay

The bacterial strains E.coli (ATCC25922), Pseudomonas aeruginosa(ATCC27853), and Bacillus subtilis (ATCC6633) were grown in LB med-ium at 37˚C to an OD600 of 1.0. Bacteria were then diluted in MT-LB me-dium (16 mM disodium hydrogen phosphate, 5 mM sodium dihydrogen

phosphate, 150 mM sodium chloride, and 1% LB medium) to a concentra-tion of 1 3 104 CFU/ml. The 50 ml bacterial suspensions were incubatedwith PBS as control or 10 mM, 20 mM, or 30 mM AMWAP-His protein for2 h at 37˚C. Subsequently, appropriate serial dilutions were plated on LBagar plates, incubated overnight at 37˚C and CFUs were determined.

Statistical analyses

The Student t test or Mann-Whitney U test were used for the comparison ofexperimental groups as indicated. p , 0.05 was considered significant.

Microarray datasets used in this study

The microarray datasets cited in this study are available at the National Cen-ter for Biotechnology Information Gene Expression Omnibus as series re-cords GSE5581 (Rs1h2/Y versus wt retinas), GSE9011 (PU.1 ChIP-Chip),and GSE13125 (PUER cells) at (www.ncbi.nlm.nih.gov/geo/).

ResultsAMWAP is strongly induced in activated retinal microglia

To identify novel genes involved in the activation ofmicroglia,we havepreviously performed DNA-microarray analysis of isolated retinalmicroglia from degenerating Rs1h2/Yand wt retinas (24, 28, 30). Ninepreviously uncharacterized transcripts showed a significantly differentexpression pattern in activated Rs1h2/Y versus nonactivated wt micro-glia (data not shown). Among these, a more than 7-fold induction ofa predicted gene with the UniGene entry Mm.24097 was detected inactivated retinalmicroglia. In this study,weconfirmed thesemicroarrayfindings by quantitative real-time RT-PCR assays with RNA samplesfrom independently isolated retinal microglia from Rs1h2/Y and wtmice. AMWAP transcript levels were increased in microglia frompostnatal day 14 (P14) Rs1h2/Y retinas and were further substantiallyinduced inP18Rs1h2/Ymicroglia (Fig. 1A).Wehavepreviously shownthat microglia activation starts at P14 in the retina of Rs1h2/Y mice,a time point where neuronal apoptosis and degeneration is not yetevident (24).For a more precise refinement of the temporal expression profile

of AMWAP in relation to the inflammatory process, mRNA levelswere quantified in retinal tissue of postnatal stages (P)11, 12, 14, 18,21, 24, and 28 (Fig. 1B). The highest AMWAP expression was noted

FIGURE 1. Temporal qRT-PCR expression profile of AMWAP during

early retinal microglia activation. A, AMWAP mRNA expression in pri-

mary retinal microglia isolated from postnatal day 14 (P14) and P18 wt

and Rs1h2/Y mice. Temporal expression profiles of AMWAP (B), lyso-

zyme (C), and secreted phosphoprotein 1 (D) in early postnatal retinas

of Rs1h2/Y mice compared with age-matched wt mice. Data repre-

sent means 6 SD of two independent microglia preparations (A) or the

means 6 SD of three independent retina preparations analyzed in tripli-

cates (B–D), respectively.

FIGURE 2. AMWAP is expressed and LPS-inducible in brain microglia

and other tissue macrophages. Brain microglia (A), spleen macrophages

(B), bone marrow-derived macrophages (C), and bone marrow-derived DCs

(D) were cultured in the absence or presence of 100 ng/ml LPS for 24 h.

Total RNAwas isolated and AMWAP transcript levels were determined by

qRT-PCR. Data points represent the means 6 SD of three independent cell

preparations analyzed in triplicates. pp , 0.05; Student t test.

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at P18 and P21 and thereafter declined to intermediate levels (Fig.1B). The AMWAP transcript profile is in good accordance with thetime kinetics of the known early activation markers lysozyme (Lyzs)(Fig. 1C) and secreted phosphoprotein 1 (SPP1, alias osteopontin)(Fig. 1D). This implicates that high AMWAP expression is alreadypresent at the very early stage of retinal microglial activation anddeclines in the chronic and resultion phase of neuroinflammation.The deduced protein sequence contains a WAP domain and togetherwith the data on its specific expression pattern, this gene was namedAMWAP for activated microglia/Macrophage WAP domain protein.

AMWAP is exclusively expressed in activated microglia andmacrophages

We next addressed the question whether AMWAP is also expressedin brain microglia and macrophages outside of the CNS. Primarybrain microglia, spleen macrophages, bone marrow-derived macro-

phages, and bone marrow-derived DCs were isolated and stimulatedwith LPS to induce a proinflammatory state. AMWAP transcriptswere already present in the four different types of macrophages butstimulation with LPS further upregulated AMWAP gene expression(Fig. 2). Examination of AMWAP transcript levels in public domainAffymetrix microarray data (BioGPS, http://biogps.gnf.org andhttp://macrophages.com) further revealed a macrophage-restrictedexpression of probeset 1436530_at, which specifically and exclu-sively targets the AMWAP mRNA (Supplemental Fig. 2). In theBioGPS database, high abundance of AMWAP transcripts wasdetected inmicroglia, bonemarrow-derivedmacrophages, peritonealmacrophages, and osteoclasts, but not in any of the other studiedmouse tissues or cell types (Supplemental Fig. 2). Consistent withour quantitative RT-PCR (qRT-PCR) data, these comprehensive ex-pression profiles indicate that AMWAP is a microglia/macrophage-specific transcript induced by proinflammatory activation.

FIGURE 3. AMWAP is upregulated by multiple

TLR ligands and IFN-g in BV-2 microglia in

a time- and dose-dependent manner. Time-course

(A, C, E, G) and dose-response (B, D, F, H) of BV-2

cells treated with the indicated concentrations for

the given time periods of LPS (A, B), CpG oligo-

nucleotides or control GG oligonucleotides (C, D),

PAM3SCK4 (E, F), or IFN-g (G, H). qRT-PCR was

performed to determine AMWAP levels. Values

represent the means 6 SD of three independent cell

cultures analyzed in triplicates. pp , 0.05; ppp ,0.01; pppp , 0.001; Student t test.

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Multiple TLR ligands and IFN-g rapidly trigger AMWAPtranscription in microglia

We next analyzed AMWAP expression and regulation in the BV-2microglial cell line as an independent invitromodel system.AMWAPmRNAwas present in BV-2 cells, but transcript levels were markedlyelevated after proinflammatory stimulation with TLR ligands andIFN-g (Fig. 3).Specifically, treatment ofBV-2 cellswithLPSasTLR4ligand (Fig. 3A, 3B), CpG oligonucleotides as TLR9 ligand (Fig. 3C,3D), PAM3SCK4 as TLR2 ligand (Fig. 3E, 3F), and IFN-g (Fig. 3G,3H) evenly upregulated AMWAP expression in a time- and dose-dependent manner. The shortest time of treatment (1 h) and the low-est concentration of each compound were sufficient to induce sig-nificant increases inAMWAPlevels.These data suggest thatAMWAPtranscription is very sensitive to proinflammatory stimulation of BV-2microglial cells.We then examined whether AMWAP induction by TLR ligands

depends on the transcription factor NF-kB. BV-2 cells were pre-treated with the specific NF-kB inhibitor CAPE before exposure toLPS, PAM3SCK4, or CpG oligonucleotides. CAPE completely pre-vented TLR-dependent AMWAP upregulation in all three stimulatoryconditions (Fig. 4A), indicating that NF-kB is absolutely required forAMWAP induction. Next, we pretreated BV-2 cells with CHX, aprotein synthesis inhibitor, to studywhether newly synthesized proteinsmay be involved in this process. We found that TLR-dependent ex-pression of AMWAP was diminished by CHX pretreatment (Fig. 4B).This suggests that newprotein synthesis is necessary and that additionalfactors may be relevant for AMWAP induction by TLR ligands. Asa positive control, the LPS-induced expression of IL-1b was criticallydependent on NF-kB, but required no protein synthesis (Fig. 4C).

Cloning and genomic structure of AMWAP

The previous database entry of Mm.24097 (GenBank Accession no.BC089618) was complete at the 39-end including the polyA tail, butcontained only nine nucleotides upstream of the start codon. Wetherefore performed 59-RLM-RACEwith RNA fromLPS-stimulatedRAW264.7 cells (Fig. 5A). Three major 59-RLM-RACE-PCR pro-ducts were amplified and sequencing after cloning of the individualproducts identified four different transcripts with the longest variantextending 68 bp further upstream (Fig. 5A). The 526-bp full-lengthAMWAP cDNA encodes for a 76 aa polypeptide. After predictedcleavage of the 19 aa signal sequence, the mature AMWAP poly-peptide should consist of a 58 aa singleWAPmotif (Fig. 5B). BLASTanalysis against DNA databases resulted in the identification of theAMWAP gene on mouse chromosome 11, consisting of three exonsand two introns (Fig. 5C). The N-terminal signal peptide is encodedby exon 1, whereas exon 2 completely codes for the C-terminalWAP domain with eight cysteines presumably forming four disul-fide bonds (Fig. 5C). The four-disulfide core domain and its genomicstructure as single exon are characteristic features of all WAP pro-teins (47).

Microglia/macrophage-specific AMWAP expression dependson PU.1 binding to its proximal promoter

Becauseofthemicroglia/macrophage-restrictedexpressionofAMWAP,we sought to investigate the relevant cis-regulatory elements. A com-parison of mouse and rat AMWAP promoter sequences and their clo-sest rodent homolog Expi revealed a highly conserved TATA-box, pro-ximal PU.1 sites, a STATmotif and an overlapping IRF/PU.1 sequence(Fig. 6A). Our information from 59-RLM-RACE identified the exis-tence of four alternative transcription start sites (Fig. 6A, arrows). Be-cause the longest AMWAP transcript includes the TATA-motif andextends 40 bp upstream, a nearby PU.1 site could function in the for-mation of the transcription preinitiation complex and thus regulateAMWAP gene activity (Fig. 6A).

To determinewhether PU.1 is directly involved in themacrophage-specific expression of the AMWAP gene, recent PU.1-related largescale genomic data from our group were used as resource for furtheranalyses. In the first dataset, a genome-wide discovery of PU.1 targetgenes inRAW264.7macrophageswas performedusingChIPcoupledto microarrays (ChIP-Chip) (44). In the second dataset, PU.1(2/2)progenitors and PUER cells with restored PU.1 activity were ana-lyzedwith exon-specificmicroarrays to identify PU.1 regulated genes(42). Results from the ChIP-Chip experiments revealed that theproximal AMWAP promoter strongly bound macrophage PU.1 invivo, indicated by the specific enrichment of probes in the immediateupstream region (Fig. 6B, black bars). In full agreement with thesedata, restored PU.1 activity in PUER cells and differentiation alongthe macrophage lineage caused a significant increase of AMWAPtranscript levels, as shownby specific hybridization signals to all threeAMWAP exons (Fig. 6B, red bars).To recapitulate the findings from PU.1 ChIP-Chip experiments

with RAW264.7 macrophages also in microglial cells, ChIP-PCR as-says were performed with BV-2 microglia. The proximal2114/+68

FIGURE 4. AMWAP upregulation by TLR ligands requires NF-kB and

new protein synthesis. To block NF-kB, BV-2 cells were pretreated with

15 mg/ml CAPE for 2 h and cells were thereafter stimulated with 50 ng/ml

LPS, 1mg/ml CpG oligonucleotides, or 1mg/ml PAM3SCK4 for 4 h (A,C). To

block new protein synthesis, BV-2 cells were pretreatedwith 5mg/ml CHX for

1 h and were thereafter stimulated with 50 ng/ml LPS, 1 mg/ml CpG oligo-

nucleotides, or 1 mg/ml PAM3SCK4 for 4 h (B, C). qRT-PCR was performed

to determinemRNA levels of AMWAP (A,B) and IL-1b asNF-kB–dependent

control gene (C). Values represent the means 6 SD of three independent cell

cultures analyzed in triplicates. pp , 0.05; pppp , 0.001; Student t test.

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AMWAP promoter region harboring the conserved PU.1 site wasspecifically enriched in PU.1-precipitated DNA compared with theIgG control (Fig. 6C, upper panel). Moreover, ChIP-PCR productswere obtained with precipitated DNA from Abs directed against thetranscriptional coactivators p300 and Cbp (Fig. 6C, lower panel),factors known to be involved in chromatin relaxation via histoneacetyltransferase activity and RNA polymerase II recruitment totranscription start sites. The2114/+68 AMWAP promoter DNAwasalso enriched in anti-AcH3 Ab precipitated DNA (Fig. 6C, lowerpanel), indicating open chromatin and active AMWAP gene ex-pression in BV-2 cells.We then aimed to validate the microarray findings of increased

AMWAP mRNA expression in differentiated PUER cells by real-time qRT-PCR. In this tuneable system the PU.1-estrogen receptorbinding domain fusion protein PUER is conditionally activated inmyeloid progenitors upon addition of 4-hydroxy-tamoxifen (OHT),leading to rapid cell cycle arrest and differentiation into macro-phages (42, 48). qRT-PCR analysis of independent RNAs froma time series of OHT-treated PUER cells showed very low AMWAPmRNA levels in PU.12/2 progenitors and a steep increase ofAMWAP transcripts in OHT-differentiated cells (Fig. 6D). Thesedata strongly support the hypothesis that AMWAP expression inmacrophages as well as in microglia is critically dependent on PU.1binding to its proximal promoter region.

AMWAP reduces proinflammatory gene expression andtriggers alternative activation markers in microglia

To further investigate the immunological functions of AMWAP inmicroglia cells, the full-length open reading framewas overexpressedin BV-2 cells. A C-terminally 1D4-Rho-epitope-tagged variant ofAMWAPwas transiently expressed in BV-2 cells. The Rho epitope isnot endogenously expressed in BV-2 cells, as shown by the absence ofa Western blot band using a monoclonal anti-1D4 Ab (Fig. 7A). Aspecific band of 10 kDa was detected in cells transfected with thepAMWAP-1D4 construct, demonstrating successful overexpressionof AMWAP in BV-2 cells (Fig. 7A). A milk fat globule protein-1D4fusion protein was used as positive control and produced the expectedband size of 15 kDa (Fig. 7A). To verify these findings and to visualizeAMWAP within cells, BV-2 transformants expressing AMWAP-GFPor only GFP as control were generated. GFP-immunofluorescence

FIGURE 5. AMWAP full-length cDNA, genomic structure, and protein

domains. A, Distinct 59-RLM-RACE-PCR products generated with tem-

plate cDNA from LPS-activated RAW264.7 cells and a reverse primer

located in the second exon. Specific bands are indicated by arrows and the

asterisk denotes a double band. B, Full-length AMWAP cDNA sequence

deposited in GenBank under Accession no. FJ007372. The open reading

frame and translated amino acids are given in bold letters. The potential

signal sequence is shaded in light gray and the WAP domain is marked in

dark gray. The position of the stop codon is indicated by an asterisk and the

polyadenylation signal is underlined. C, Genomic organization of the

AMWAP gene (top), predicted protein domains (middle panel), and eight

cysteine-residues forming four putative disulfide bonds (bottom).

FIGURE 6. PU.1 regulates the macrophage/microglia-specific expres-

sion of AMWAP. A, Alignment of proximal promoter sequences of rodent

AMWAP and Expi. Predicted transcription factor binding matrices are

shaded. Arrows indicate transcription start sites experimentally verified by

59-RLM-RACE. B, Genomic structure of the AMWAP gene and log2 en-

richment/expression values of tiled probes spanning the promoter and all

three exons. Significant probes of PU.1 versus IgG ChIP-Chip analysis of

RAW264.7 cells are shown in black and differentially expressed probes in

exon array analyses of 24 h OHT-treated PUER cells versus untreated

PUER cells are depicted in red. C, ChIP analysis of PU.1 and coactivator

binding to the AMWAP promoter. Agarose gel analysis of AMWAP-specific

PCR products generated from BV-2 DNA after ChIp with anti-PU.1, anti-

Cbp, anti-p300, anti-AcH3, or anti-IgG–control Abs. Input DNAwas used

as positive control in the PCR reaction. D, Time kinetics of AMWAP

transcript expression before and after OHT treatment of PUER cells to

induce macrophage differentiation. Error bars indicate the SD of the mean

from two independent experiments performed in duplicates.

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confirmed that recombinant AMWAP was expressed in BV-2 cells(Fig. 7B). AMWAP-GFP was localized in perinuclear structures andin some cells in dome-shaped structures with a polarity that resem-bled the Golgi apparatus (Fig. 7B, arrows in left panel). In constrast,GFP alone was broadly expressed in the cytoplasmic region (Fig. 7B,right panel). This finding is consistent with proteins that are pro-cessed through the endoplasmic reticulum and Golgi for extracellulartransport.Because AMWAP transcription was regulated by proinflammatory

activation and the related WAP protein SLPI modifies inflammatoryresponses (49, 50), the effect of AMWAP on macrophage/microgliaactivation marker expression was studied. The immune-regulatorypotential of AMWAP was addressed under basal and LPS-treatedconditions. Enhanced AMWAP-GFP expression in nonstimulatedand LPS-treated BV-2 cells reduced transcripts for IL1-b, IL-6, andCcl2, three typical markers for proinflammatory activation of macro-phages (Fig. 7C–E). In the same cells, the alternative macrophageactivation markers arginase 1 and Cd206 were substantially increased(Fig. 7F–G). These effects were not due to different growth conditionsof the cells or varying transfection efficiencies (data not shown). Toinvestigate if AMWAP is also a direct suppressor of proinflammatorygene transcription, we determined its effect on Ccl2 promoter activity.A stable BV-2 cell line was generated with a luciferase reporter underthe control of the murine Ccl2 promoter region. In these cells, tran-sient cotransfection of AMWAP strongly decreased Ccl2 promoteractivity compared with mock transfected cells (Fig. 7H).

We next studied the role of physiological AMWAP levels in themodulation of microglial activation. Knockdown experiments withspecific AMWAP siRNA or control siRNA were performed andmarker transcripts were determined in transiently transfected BV-2 cells. A 30% knockdown of AMWAP expression was achieved inboth the basal and LPS-activated state (Fig. 8A). AMWAP silencingresulted in significantly derepressed transcript levels of IL1-b,IL-6, and Ccl2 (Fig. 8B–D) and markedly reduced arginase 1 andCd206 expression (Fig. 8E, 8F). These data suggest that AMWAPacts as negative regulator of proinflammatory gene expression andmay support alternative activation of microglial cells.

AMWAP blocks microglial migration and acts as serine-protease inhibitor

To assess whether AMWAP influences the motility of microglia asa functional parameter, in vitro wound-healing assays and transwellmigration experiments were performed. A cell-free zone was cre-ated in confluent monolayers of stably transfected BV-2 cells byscraping cells off with a pipette tip. The repopulation of the cell-free zone was then monitored by microscopy. In GFP control cells,migration into the wounded area was completed after 24 h (Fig. 9A,left panels). In contrast, AMWAP-GFP expressing cells showeda clearly reduced capacity to repopulate the cleared area, indi-cating diminished migratory potential (Fig. 9A, right panels).To role out the possibility that the reduced microglial migration

in the scratch assays was due to different proliferation rates of

FIGURE 7. AMWAP overexpression reduces proin-

flammatory gene expression and upregulates alternative

activationmarkers in BV-2microglia.A, BV-2 cells were

transiently transfected with an AMWAP-Rho 1D4 tag

fusion protein, empty 1D4 mock vector, or a milk fat

globule (MFG)-1D4 control protein, respectively. Cells

were then analyzed by Western blot with anti–1D4-Ab.

B, BV-2 cells were transiently transfected with an

AMWAP-GFP fusion protein or GFP vector alone. In-

tracellular GFP was detected by immunofluorescence

microscopy. Dome-shaped Golgi regions are indicated

by arrows.C–G, qRT-PCR analysis of IL-6-, IL-1b-, and

Ccl2-transcripts as proinflammatory markers (C–E) and

arginase 1 and Cd206 levels as alternative activation

markers (F, G) in AMWAP-GFP versus GFP over-

expressing BV-2 cells under basal and LPS-stimulated

conditions. H, Luciferase assays of BV-2 microglia

containing a stable Ccl2-promoter–luciferase-construct.

BV-2 cells stably expressing the Ccl2 reporter were

transiently transfectedwith either AMWAP-GFP or GFP

control vector. Twenty-four hours after transfection, lu-

ciferase acitvity was determined. Bar graphs represent

means6 SD of three independent cell cultures analyzed

in triplicates. pp , 0.05; ppp , 0.01; Student t test.

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stably transfected BV-2 cells, we tested their LPS-induced che-motactic migration in transwell chambers. In this system, stableAMWAP overexpression reduced the LPS-induced transwell mi-

gration of BV-2 cells to 50% compared with GFP expressing cells(Fig. 9B). In contrast, transient siRNA knockdown of AMWAPsignificantly promoted chemotactic migration of BV-2 microglial

FIGURE 8. AMWAP knockdown leads to increased

proinflammatory gene expression and impairs expression

of alternative activation markers in BV-2 microglia. BV-

2 cells were transiently transfected with 33 nM of each

AMWAP-specific siRNA or nontargeting (scrambled)

siRNA for 24 h. Thereafter, cells were left untreated or

were further stimulated with 5 ng/ml LPS for 24 h.

mRNA levels for AMWAP (A), IL1-b (B), IL6 (C), Ccl2

(D), arginase 1 (E), and Cd206 (F) were determined by

qRT-PCR. Bar graphs represent means 6 SD of three

independent cell cultures analyzed in triplicates. pp ,0.05; ppp , 0.01; pppp, 0.001; Student t test.

FIGURE 9. AMWAP attenuates microglial migration and exerts antiserine protease activity. A, Wound-healing assay in BV-2 microglia stably expressing

AMWAP-GFP or GFP as control. AMWAP-GFP and GFP expressing BV-2 microglia were grown to comparable cell density, wounded with a pipette tip

and microphotographs from wounded areas were taken immediately and after 24 h. B, Relative LPS-induced transwell chamber migration of stably

transfected AMWAP-GFP versus GFP expressing BV-2 microglia. C, BV-2 microglia were transiently transfected with AMWAP-specific siRNA or

nontargeting (scrambled) siRNA for 24 h. Thereafter, the lower wells of transwell chambers were treated with LPS for further 24 h and the absolute

numbers of migrating cells were counted. D, Trypsin protease protection assay. Stable AMWAP-GFP and GFP expressing BV-2 microglia were treated with

0.25% trypsin for the indicated time points. Residing attached cells were stained with crystal violet, washed with PBS and photomicrographs were taken. E,

Quantitative analysis of anti-protease activity in AMWAP-GFP and GFP expressing BV-2 microglia. Optical densities were normalized to untreated cells.

Bars represent the percentage of adherent cells after different time points of trypsin incubation. Data are expressed as mean 6 SD of duplicate meas-

urements from three independent biological replicates. pp , 0.05; ppp , 0.01; pppp , 0.001; Student t test.

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cells compared with cells transfected with nontargeting controlsiRNA (Fig. 9C). These findings implicate that modulation ofAMWAP expression levels in microglia directly influences theirmigration capacity.Because SLPI and other WAP proteins are potent serine protease

inhibitors (51), we investigated a similar function of AMWAP ina trypsin-protection assay. In this test system, the adhesive ca-pacity of cells is monitored in the presence of trypsin in a timecourse (52). The rate of adherent cells after trypsinization for 5min was significantly higher in AMWAP-expressing cultures thanGFP control cells (Fig. 9D, 9E). This difference persisted at alltime points analyzed up to 25 min trypsin incubation, indicatingthat AMWAP confers protease resistance and increases microglialadhesion. In agreement with the wound-healing assays, the trans-well chemotaxis tests and the Ccl2 expression data, these resultsindicate an immunomodulatory role of AMWAP by limiting mi-croglial migration.

Recombinant AMWAP has antibacterial activity

As antibacterial activity is a major feature of WAP domain pro-teins, we studied the effect of recombinant AMWAP on differentbacterial strains. Initially, we only achieved low recombinant ex-pression of AMWAP in E. coli (data not shown). Therefore, codon-optimized AMWAP (Supplemental Fig. 1) was synthesized, ex-pressed in E. coli, and purified via an N-terminal His-tag sequenceby affinity chromatography. Western blot analysis of the recom-binant protein via an anti-His Ab detected a single specific bandat the expected molecular mass of ∼10 kDa (Fig. 10A). To studythe antibacterial potential of AMWAP, the target bacteria E. coli(ATCC25922,Gram-negative rod),P. aeruginosa (ATCC27853,Gram-negative rod), and B. subtilis (ATCC6633, Gram-positive rod) wereincubated in the presence of recombinant AMWAP for 2 h. After over-night growth on plates, CFUs were counted to assess the number ofviable bacteria. AMWAP showed a relatively weak (IC50 of 30 mM)but significant bactericidal activity against E. coli (Fig. 10B). Growthof P. aeruginosa (Fig. 10C) and B. subtilis (Fig. 10D) was significantlyinhibitedat lowermicromolar concentrations (IC50of16mM).Thus, theantibacterial potential of AMWAP increased dose dependently andreached similar IC50 values as those of other WAP motif proteins, in-cluding SLPI (34) and elafin (53).

DiscussionThis study is part of an ongoing effort to characterize known andnovel transcripts involved in the activation and regulation of micro-glia during retinal degeneration. We identified the novel mouseproteinAMWAPand investigated its specific expression and functionin activated microglia and macrophages. AMWAP attenuates pro-inflammatory cytokine and chemokine expression, whereas pro-moting expression of alternative activation markers. AMWAP alsoregulates microglial migration and chemotaxis and it directly exertsantibacterial activity.Although initially identified in early activated microglia from

Rs1h2/Y retinas, induced AMWAP transcript levels were also de-tected in activated microglia stimulated with multiple TLR ligands,including LPS, CpG oligonucleotides, PAM3SCK4 as well asINF-g. Upregulation of AMWAP by these proinflammatory stimuliwas critically dependent on NF-kB but also involves new proteinsynthesis. AMWAP mRNA expression was highly enriched inmicroglia and other tissue macrophages, but transcripts were absentin all other mouse tissues studied so far. These findings on TLR-dependent and microglia/macrophage-restricted expression corre-late well with the major influence of NF-kB and PU.1 in the tran-scriptional control of the AMWAP gene. PU.1 is a key transcriptionfactor in myeloid cells, with high expression levels in activated

retinal microglia (28, 54). Although the precise mechanisms un-derlying the activation-dependent induction of AMWAP are notfully understood, we postulate that PU.1 plays a critical role inAMWAP expression by direct binding to the proximal promoter.Immune-related genes, including cytokines and chemokines, as

well as their receptors are oftenorganized in chromosomal clusters orminiclusters (55–57). In agreement with this concept, the murineAMWAP gene is located in close proximity to a cluster of thechemokine genes Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9. In addition, theAMWAP gene is flanked by theWAP protein extracellular peptidaseinhibitor precursor (Expi), which shares 83% sequence homologypossibly suggesting a gene duplication event. Our ChIP-Chip andChIP-PCR assays have revealed that the AMWAP promoter, but notthe Expi region are bound by PU.1 and the coactivators Cbp andp300. These findings are consistent with microglia/macrophage-restricted expression of AMWAP and the ubiquitous expression ofExpi (58). Likely, the different expression patterns and regulation ofboth genes coincides with different cellular functions. In line withthis, Expi was identified as a protein overexpressed in nonmetastaticmammary cancer cells (59, 60) and ectopic expression of Expiresulted in induced apoptosis of mammary epithelial cells (58).Together with SLPI (61), the best studied WAP protein so far,

AMWAP is the only other WAP family member expressed and

FIGURE 10. Expression, purification and anti-microbial activity of

recombinant AMWAP. A, Schematic presentation of the pET21a-AMWAP

expression construct. Codon-optimized AMWAP cDNA sequence lacking

the signal peptide was cloned into pET21a(+) vector in frame with a His-

tag. Expression in E. coli BL21(DE3) was induced by 1 mM isopropyl-

b-D-thiogalactoside and AMWAP-His was purified from the soluble frac-

tion of the bacterial lysate using Ni-TED columns. Purified recombinant

AMWAP-His was separated by SDS-PAGE and detected with anti-His Ab.

B–D, Growth inhibitory activity of different concentrations of recombinant

AMWAP-His on E. coli (B), P. aeruginosa (C), and B. subtilis (D). Log-

phase bacterial cultures were incubated with the indicated concentrations

of AMWAP-His in MT-LB medium for 2 h at 37˚C. Bacteria were then

plated overnight and the number of CFUs was determined by counting

colonies. Cells incubated with PBS were set as 100%. Data are expressed

as mean 6 SD of duplicate measurements from three independent bio-

logical replicates. pppp , 0.001; Mann-Whitney U test.

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regulated in microglia. SLPI transcript levels were markedly in-creased in experimental autoimmune encephalomyelitis and a rolefor SLPI in the promotion of tissue repair was discussed (61). SLPIcan be secreted by activated macrophages and acts as a suppressorof LPS response (62). The inhibition of LPS-induced NO and TNFproduction in macrophage cell lines by SLPI is independent of itsantiprotease function (39). Intracellular SLPI also competes withNF-kB for binding sites in the promoters of proinflammatorycytokines (50) and blocks LPS-induced IkBa-degradation (63).Both mechanisms may also be relevant for AMWAP function.Support for the hypothesis that AMWAP similar to SLPI interfereswith NF-kB signaling comes from our finding that Ccl2 promoteractivity was directly repressed by AMWAP.Two major questions relate to why and how AMWAP expression

is induced during microglial activation in retinal degeneration.Because no obvious infection is present in the gene mutation-basedmodel of Rs1h2/Y retinas, we speculate that AMWAP is induced bydamage-associated molecular patterns (DAMPs) (64) and/or alar-mins (65). Our data on the rapid induction of AMWAP in earlyretinal degeneration and the TLR-dependent upregulation in BV-2 cells support the assumption that AMWAP may respond verysensitive to DAMPs. Innate immune cells are often recruited toinjured areas by matrix degradation products (66) or actively se-creted alarminmolecules like HMGB1 (67), consequently initiatingtissue remodeling and repair. Related to this, a secreted and a non-secreted intracellular form of SLPI were both induced in mousemacrophages after exposure to apoptotic cells (68), indicating thatSLPI may promote clearance of dying cells in the absence of in-flammation. AMWAPmay have a similar “alarmin-like” function inthe degenerating retina and thereby may limit microglial activation.A hallmark of retinal degeneration in the retinoschisin-deficient

mouse model is the rapid migration of activated microglia into thephotoreceptor and ganglion cell layer (30). Ccl2, an important che-moattractant is strongly expressed in Rs1h2/Y retinas (24) and wedemonstrated that AMWAP overexpression downregulated Ccl2transcripts in microglia. Furthermore, AMWAP diminished BV-2 cell migration and inhibited the serine protease trypsin. Recentdata indicated that Ccl2 is proteolytically activated by the serineprotease plasmin and that plasmin-deficient mice were resistant toexcitotoxic neurodegeneration (69). Thus, in addition to its effecton Ccl2 gene expression, AMWAP may impair Ccl2-mediatedmigration of microglia by limiting proteolytic processing of Ccl2.Our study also suggests that recombinant AMWAP has a potent

antimicrobial activity. Most bactericidal proteins have a cationicsurface that bind negatively charged phospholipids and thereby dis-turb the integrity of bacterial membranes (70). Although AMWAPhas a lower predicted positive charge than SLPI and elafin (37), wedetected significant antimicrobial activity against E. coli, P. aeru-ginosa, and B. subtilis at micromolar concentrations. The domainsignature of AMWAP is related to the antimicrobial single-WAP-domain proteins SWAM1 and SWAM2 (71).We therefore speculatethat local positive charges on the three-dimensional protein surfaceof AMWAP could be important for antibacterial activity (72).Although the precise mechanisms remain to be determined, the

data described in this study suggest that AMWAP induction byneuronal degeneration and inflammatory signals is an importantcounter-regulatory response in microglial and macrophage acti-vation. Moreover, AMWAP seems to actively support alternativeactivation of microglia and macrophages. AMWAP could be a po-tential candidate for modulating the homeostasis of microglia andthereby limit neurotoxicity and apoptotic degeneration. AMWAP-deficient mice will be a valuable tool to further characterize thein vivo role of this novel WAP protein in microglia and macro-phage function.

AcknowledgmentsWe thank Bernhard Weber and Joost Oppenheim for critical reading of the

manuscript and David Hume for helpful discussions. We also thank Johann

Rohrl for providing the bacterial strains Pseudomonas aeruginosa and

Bacillus subtilis.

DisclosuresThe authors have no financial conflicts of interest.

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