The Transcriptional Activation Program of Human Neutrophils in Skin Lesions Supports Their Important Role in Wound Healing

This information is current as Kim Theilgaard-Mönch, Steen Knudsen, Per Follin and Niels of September 27, 2021. Borregaard J Immunol 2004; 172:7684-7693; ; doi: 10.4049/jimmunol.172.12.7684 http://www.jimmunol.org/content/172/12/7684 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Transcriptional Activation Program of Human Neutrophils in Skin Lesions Supports Their Important Role in Wound Healing1

Kim Theilgaard-Mo¨nch,2* Steen Knudsen,† Per Follin,‡ and Niels Borregaard*

To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed in PMNs from pe- ripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation Downloaded from of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/ were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of , the migration and proliferation of keratinocytes and fibroblasts, the adhesion of

keratinocytes to the dermal layer, and finally, the induction of anti-microbial expression in keratinocytes. Notably, the up- http://www.jimmunol.org/ regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing. The Journal of Immunology, 2004, 172: 7684–7693.

kin wounding elicits a cascade of repair processes involving mediators released by thrombocytes and microorganisms. Upon several types of cells. First, thrombocytes generate a clot, migration to sites of infection such as skin wounds, PMNs get S which stops the bleeding, and serves as a temporary barrier activated by microorganisms and their products, and by cytokines and a source of chemotactic substances. Subsequently, attracted leu- generated by other leukocytes (monocytes and PMNs) and the kocytes initiate an inflammatory response before fibroblasts and en- stromal environment (fibroblasts, endothelial and epidermal cells). by guest on September 27, 2021 dothelial cells migrate to the wound to regenerate tissue that contracts Following activation, PMNs immediately initiate a first line of the wound margins. Finally, epithelial cells complete the repair pro- defense using a number of distinct mechanisms (2, 3). These de- cess by covering the denuded wound surface (1). fense mechanisms include the release of anti-microbial peptides, 3 Polymorphonuclear neutrophilic granulocytes (PMNs) are at- phagocytosis, and the generation of reactive oxygen intermediates tracted to skin wounds within minutes of injury by chemotactic for killing and degradation of microorganisms. De novo synthesis of chemokines and cytokines, which are essential for the regulation of the cellular immune response and the recruitment of additional *The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, effector cells to the wound, constitutes another defense mechanism University of Copenhagen, Copenhagen, Denmark; †Center for Biological Sequence Analysis, BioCentrum-Technical University of Denmark, Lyngby, Denmark; and ‡Di- of PMNs. vision of Infectious Diseases, Department of Health and Environment, University of More recently, studies using genomic and proteomic approaches Linko¬ping, Linko¬ping, Sweden have demonstrated a significant transcriptional response of human Received for publication February 3, 2004. Accepted for publication April 9, 2004. PMNs upon in vitro activation by single agents such as bacteria, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance LPS, and by phagocytosis of IgG- and complement-coated latex with 18 U.S.C. Section 1734 solely to indicate this fact. beads (4Ð7). However, at present, no genomic approaches have 1 This work was supported in part by the Novo Nordisk Foundation, the Amalie been applied to investigate how PMNs respond in vivo to inflam- J¿rgensens Memorial Foundation, the Danish Cancer Research Foundation, the Dan- matory mediators in skin wounds and whether their response con- ish Medical Research Council, the Gangsted Foundation, the Danish National Re- search Foundation, and by the Lundbeck Foundation. K.T.-M. is the recipient of a tributes to healing of wounds. scholarship from the IMK Foundation and Rigshospitalet. To gain more insight into this complex process, we applied gene 2 Address correspondence and reprint requests to Dr. Kim Theilgaard-Mo¬nch, The array technology to compare changes of of highly Granulocyte Research Laboratory, Department of Hematology-9322, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK 2100 Copenhagen, Denmark. E-mail purified PMNs from peripheral blood (PB) and PMNs that had address: [email protected] transmigrated to inflammatory skin lesions in vivo. For the col- 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophilic granulo- lection of PMNs, we used a model of skin wounding called skin cyte; PB, peripheral blood; pb-PMN, PB PMN; sl-PMN, skin lesions; MIP, macroph- chamber technique. With this model, small areas of denuded der- age-inflammatory ; uPA, urokinase plasminogen activator; MCP, monocyte chemoacttractant protein; GRO, growth-related oncogene; GPR, -coupled mis, termed “skin windows”, are generated and covered with skin ; IER3, immediate early response 3; BCL2A1, BCL2-related protein A1; chambers containing a medium that attracts PMNs (8). CASP8, caspase 8, -related cystein protease 8; CXCL, CXC chemokine ligand; TLR, Toll-like receptor; LAMB3, laminin 5 ␤3; VEGF, vascular endothelial Our study demonstrates that migration of PMNs into skin le- growth factor. sions is associated with an extensive change in gene expression,

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 7685 implicating that the cellular fate and function of PMNs attracted to Total RNA and for gene expression analysis and Western blot skin wounds is partially regulated at the transcriptional level. analysis, respectively, were isolated from purified PMN preparations using TRIzol (Invitrogen, Paisley, U.K.) according to the guidelines of the manufacturer. Materials and Methods Collection and purification of PMNs from PB and skin lesions Gene expression analysis For gene expression analysis, total RNA was biotinylated and hybridized to PB and skin chamber samples were collected in parallel from four healthy Hu95A GeneChips (Affymetrix, Santa Clara, CA) according to instructions individuals. All samples were obtained following informed consent accord- of the manufacturer (www.affymetrix.com/pdf/expression_manual.pdf/). ing to the guidelines established by the Ethics Committee of the Cities of Briefly, first-strand cDNA was generated by reverse transcription of 2Ð5 Copenhagen and Fredricksberg. ␮g of total RNA at 42¡C for 1 h using a T7-oligo(dT)24 primer and Su- PMNs were isolated from PB by density centrifugation and subsequent perscript II (Invitrogen). DNA second-strand synthesis was accomplished immunomagnetic depletion of nongranulocytic cells. Briefly, 60Ð80 ml of using DNA polymerase I and RNase H (Invitrogen) at 16¡Cfor2h.Bi- anti-coagulated venous blood were mixed 1 ϩ 1 with chilled saline/2% otinylated cRNA was subsequently generated by in vitro transcription of dextran (Dextran 500; Amersham Bioscience, Uppsala, Sweden) and kept dsDNA using T7 RNA polymerase at 37¡Cfor6hinthepresence of on ice for 30Ð40 min to sediment erythrocytes. The resultant leukocyte- biotinylated nucleotides (BioArray High Yield RNA transcript labeling kit; rich supernatant was centrifuged (200 ϫ g,4¡C, 6 min), and the pellet was Enzo Diagnostics, Farmingdale, NY). Finally, biotinylated cRNA was gently resuspended in chilled saline. The leukocyte suspension was then fragmented and the quality was confirmed on a test GeneChip before hy- layered on 15 ml of Lymphoprep (1.077 g/ml; Nycomed, Oslo, Norway) bridization to Hu95A GeneChips (Affymetrix). and centrifuged (400 ϫ g,4¡C, 30 min). The supernatant including the The expression index for each gene was calculated using the Li-Wong interphase containing mononuclear cells was discarded and the pellet weighted average difference (10). Array signals on individual gene arrays highly enriched for PMNs subjected to hypotonic lysis of erythrocytes

were normalized by the Qspline method developed by Workman et al. (11). Downloaded from (resuspension in 5 ml of chilled H O, gentle mixing for 30 s, and termi- 2 Qspline is a robust nonlinear method for normalization using array signal nation of lysis by addition 5 ml 1.8% NaCl). After lysis, cells were pelleted distribution analysis and cubic splines. Qspline fits cubic splines to the (200 ϫ g,4¡C, 6 min) and resuspended in PBS/0.5% BSA/2 mM EDTA quantiles of the array signal distribution, and uses those splines to normal- buffer. Subsequently, the PMN preparations were depleted of nongranulo- ize signals dependent on their intensity. cytic cells by immunomagnetic sorting using the MACS system according The increase/decrease of gene expression in PMNs from skin lesions to the instructions of the manufacturer (MACS; Miltenyi Biotec, Bergisch relative to PMNs from PB was calculated as a log -fold change to obtain Gladbach, Germany). The mAbs used for depletion were raised against 2 a symmetric distribution around zero (up-regulated genes have positive Ags expressed by the following cell types: monocytes (anti-CD14), B cells log values and down-regulated genes have negative log values). For func- http://www.jimmunol.org/ (anti-CD19), T cells (anti-CD3), platelets and megakaryocytes (anti- 2 2 tional clustering, genes were annotated with Gene Ontologies (www. CD61), NK cells (anti-CD56), erythroid cells (anti-glycophorin A), and geneontology.org/), which provides a unique identifier for genes having a eosinophils (anti-CD49d; all mAbs were provided by BD Biosciences, San characterized biological function. Diego, CA). To minimize the activation of PMNs, all isolation procedures Յ were performed immediately after cell collection at 4¡C, i.e., on ice, in Western blot analysis a cold room, or a cooled centrifuge, using nonpyrogenic reagents and plasticware. TRIzol (Invitrogen) purified cell lysates corresponding to 5 ϫ 105 PB- PMNs that had transmigrated from the blood circulation to skin lesions PMNs (pb-PMNs) and skin lesion-PMNs (sl-PMNs) were electrophoresed generated by epidermal detachment and blister formation were collected by on 10Ð14% SDS polyacrylamide gels (BDH Laboratory Supplies, Poole, the skin chamber technique as described previously (8, 9). Briefly, a cy- U.K.) and transferred to nitrocellulose membranes (Amersham Bioscience) lindrical acrylic suction device containing 3 holes of 5 mm in diameter, was by electroblotting. Subsequently, the membranes were incubated with pri- by guest on September 27, 2021 placed on the volar surface of the nondominant forearm and negative pres- mary Abs raised against IL-8, macrophage inflammatory protein (MIP)-1␣ sure of 200 mm Hg was applied for2hbyavacuum pump resulting in the (both from R&D Systems, Minneapolis, MN), and urokinase plasminogen formation of suction blisters. After detachment of the vacuum pump, the activator (uPA; a gift from Dr. J. Pass, The Finsen Laboratory, Rigshos- blister roofs were removed with sterile tweezers and scissors, resulting in pitalet, Copenhagen, Denmark) followed by a secondary HRP-conjugated 3 uniform skin lesions, termed “skin windows”,ofϳ0.2 cm2. A collection swine anti-rabbit Ab or rabbit anti-mouse Ab (DAKO, Glostrup, Den- chamber with 3 collection compartments of 7 mm in diameter, was placed mark). Binding of Abs was visualized by ECL (Amersham Bioscience, on top of the skin window, filled with 0.5 ml of autologous serum, and Uppsala, Sweden). Loading of equal amounts of protein was assessed by sealed with tightly fitting lids. The collection chamber was then fixed to the probing membranes with a monoclonal primary anti-␤- Ab (12). forearm by tape and an elastic bandage. After 18 h, the collection com- partments were emptied, washed, and refilled with autologous plasma. Af- Statistics ter an additional 6 h, exudated cells were collected from skin chambers and The statistical analysis was performed using the R statistics program en- depleted of nongranulocytic cells by immunomagnetic sorting as described vironment available from www.r-project.org/. The variability between sub- above. Notably, serum contains biological active components of the coag- jects was low as estimated by the correlation coefficient, which ranged ulation and complement system. When used in skin chambers, serum is a from 0.96 to 0.97 for pb-PMN genechips and 0.91 to 0.98 for sl-PMN documented strong chemoattractant, induces exocytosis of gelatinase gran- genechips. In contrast, the correlation coefficient between pb-PMN gene- ules and secretory vesicles by PMNs, and up-regulates Mac-1/CD11b on chips and sl-PMN genechips ranged from 0.78 to 0.80. exudated PMNs, whereas plasma has no such effects (9). Thus, serum itself A Student t test was applied to identify differentially expressed genes in might activate PMNs. With the applied skin chamber protocol, PMNs were pb-PMNs and sl-PMNs. In the t test, the variance among individuals in the collected in plasma rather than serum to minimize the influence of skin two categories of pb-PMNs and sl-PMNs was calculated for each gene, and chamber fluid on PMN activation. However, plasma alone is a poor che- differences between the two categories were only considered significant if moattractant and does not allow the collection of sufficient PMNs for gene they far exceeded the variance. The p values calculated by the t test were expression analysis. Moreover, with plasma, it is technically not possible to corrected for multiple testing (Benjamini-Hochberg, 13) to estimate the extend the exudation period beyond6htoobtain more cells, since this will false discovery rate for differentially regulated genes. The false discovery activate coagulation and trap exudated cells in a clot (P. Follin, unpub- rate for differentially regulated genes in the present study was 0.032. lished observations). Based on these findings the exudation process was first initiated with serum for 18 h, followed by washing and refilling the skin chambers with plasma, before collecting freshly exudated PMNs after Results an additional 6 h (9). Since the applied skin chamber protocol is repro- Purification of PMNs ducible and uses an aseptic inflammation to attract cells to skin lesions, it is in our opinion currently one of the most suitable techniques that mimics Most critical for the comparison of gene expression in cell popu- the in vivo activation and resultant transcriptional response of PMNs in lations collected in vivo is the application of highly purified cell skin lesions. However, the protocol did not discriminate to what extent the preparations to minimize the false positive rate of differentially migration process or the various stimuli (cytokines etc.) in the skin window regulated genes due to contamination with other cell types. Thus, exudates contributed to the observed transcriptional response of PMNs. The purity of PMN preparations was assessed by microscopy of Wright- we reasoned to use a purification strategy based on density cen- Giemsa-stained cytospins before and after immunomagnetic sorting. Cells trifugation and immunomagnetic depletion of nongranulocytic were enumerated using a Neubauer hemocytometer. cells to obtain highly purified PMN preparations from PB and skin 7686 TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS lesions for array analysis. In alignment with previous studies, den- sity centrifugation of PB cells resulted in PMN preparations con- taining 95Ð97% PMNs, 2Ð4% eosinophils, and Ͻ1% mononuclear cells (5, 7). Additional lineage-depletion increased the purity of PMN preparations from PB to Ͼ99.5% (n ϭ 4, mean purity 99.7%). Cells collected from skin chambers contained 85Ð95% PMNs and 5Ð15% contaminating monocytes/macrophages. After depletion of nongranulocytic cells, the purity of PMN preparations increased to Ͼ99% (n ϭ 4, mean purity 99.4%). Subsequent array analysis revealed no detectable levels of lineage-specific tran- scripts for other relevant cell types such as eosinophils, basophils, monocytes, T cells, endothelial cells, fibroblasts, and epidermal cells in any of the purified PMN preparations (Table I; Affymetrix absent call). These findings demonstrate that the applied purifica- tion protocol resulted in highly purified PMN preparations from FIGURE 1. Differentially regulated genes in PMNs collected from PB PB and skin lesions, and thus, minimized the false positive rate of and skin lesions were assigned to gene categories according to their bio- differentially expressed genes due to contamination of nongranu- logical functions using the database. The numbers of up- locytic cells. and down-regulated genes in PMNs from skin lesions compared with

PMNs from PB are shown for each gene category. Downloaded from Differentially expressed genes are assigned to distinct functional gene categories Microarray analysis was applied to compare differentially the ex- protects cells from Fas-induced apoptosis) (14, 15), BCL2-related pression of Ϸ12,500 genes in highly purified PMNs from PB and protein A1 (BCL2A1; blocks mitochondrial release of cytochrome PMNs, which had transmigrated to skin lesions. Genes were de- c) (16), and FLIP (cFLAR; inhibits Fas-associated death domain

fined as differentially expressed if they were among the 1000 most protein-mediated activation of CASP8) (17). Among the down- http://www.jimmunol.org/ significant differentially expressed genes (range of p values: 2.6 ϫ regulated proapoptotic genes were Fas-associated death domain 10Ϫ3Ð3.6 ϫ 10Ϫ9, estimated false discovery rate 0.032 (Ben- protein (activates CASP8), CASP8 (activates downstream jamini-Hochberg)), and if they changed gene expression by Ն0.5 caspases affecting apoptosis), APAF1 (activates CASP9 in a com- plex with cytochrome c), death-associated protein 2 (18), log2-fold. By these criteria, 314 differentially expressed genes as- signed to various functional gene categories of the Gene Ontology and TNFR (activates apoptosis pathway by ligand binding). This database were detected in PMNs upon migration to skin lesions change of gene expression among members of the apoptotic path- (Fig. 1). Almost no up- or down-regulated genes were detected in way suggests a transient anti-apoptotic priming of PMNs imme- categories critical for defense, cellular movement/transport or cell diately after migration to skin lesions, regulated at the transcrip- structure indicating that functions such as the migration to sites of tional level. by guest on September 27, 2021 infection, changes of cellular structure, and immediate anti- Genes involved in wound healing microbial defense are not regulated at the transcriptional level. In contrast, the high numbers of differentially expressed genes in cat- The complex process of wound healing is orchestrated by signal egories such as apoptosis regulators, signal transducers, and en- transduction through chemokines, cytokines, and their respective zymes, indicated that PMN activity in skin lesions is partially reg- receptors. Upon migration to skin lesions, PMNs up-regulated 26 ulated at the transcriptional level. and down-regulated 68 mediators of (Tables II and III). Up-regulated chemokines and cytokines were critical for Genes involved in apoptosis the recruitment of additional macrophages, T cells, and PMNs, and Detailed analysis of apoptosis regulators demonstrated the up-reg- for the modulation of inflammatory responses (IL-8, monocyte ulation of anti-apoptotic genes concomitant with the down-regu- chemoattractant protein (MCP)-1 (CC chemokine ligand 2), lation of proapoptotic genes (Tables II and III). Up-regulated anti- MIP-1␣ (CC chemokine ligand 3), growth-related oncogene apoptotic genes included IEX1 (immediate early response (IER)3; (GRO)-␤ (CXCL2), GRO-␥ (CXCL3), IL-1␤ (IL1B), and TNF-␣ (TNF)) (19). Of interest, receptors mediating chemotaxis and cel- lular activation (IL-8RA (CXCR1), IL-8RB (CXCR2), G-CSFR, Table I. Detection of lineage-specific transcript in purified PMN Toll-like receptor (TLR)1, and TLR6) (19, 20) were down-regu- a preparations lated concomitant with the up-regulation of other receptors mod- ulating inflammatory responses (IL-1R1, TGF-BR1, G protein- Detectable Transcripts coupled receptors (GPR) 65, GPR18, and HM74). Hence, PMNs Cell Type Expressing in Purified PMN Gene Name Lineage-Specific Gene Preparations (n ϭ 8) might change their responsiveness to chemotactic and immuno- regulatory mediators once activated in skin wounds. G-CSFR PMN Yes Other effects of up-regulated chemokines/cytokines included the IL-5RA Eosinophil/basophil No M-CSFR (CSF1R) Monocyte/macrophage No promotion of angiogenesis (vascular endothelial growth factor TCR T cell No (VEGF), IL-8, GRO-␥, and MCP-1) (21), proliferation of - FGFR1 Fibroblast No ocytes and fibroblasts (IL-8, IL-1␤, and MCP-1) (19), and the in- FGFR2 Fibroblast/epidermal cell No duction of anti-microbial gene expression in keratinocytes (IL-1␤ EGFR Epidermal cell No and TNF-␣) (22). Additional up-regulated genes potentially in- VEGFR Endothelial cell No ␤ VECAM1 Endothelial cell No volved in wound healing were laminin 5 3 (LAMB3) (23), which promotes adhesion of keratinocytes to the dermal layer, and uPA a The table demonstrates the absence of lineage-specific transcripts for nongranulo- cytic cells in highly purified PMN preparations collected from pb-PMNs (n ϭ 4) and (PLAU), which supports tissue remodelling by breakdown of fibrin sl-PMNs (n ϭ 4) as detected by microarray analysis (Affymetrix; present/absent call). clots and degradation of extracellular matrix (24, 25). Other wound The Journal of Immunology 7687

Table II. Genes up-regulated in PMNs upon migration to skin lesionsa

Log2-Fold Gene Category Gene Name Gene Symbol Affymetrix ID Change

Apoptosis regulator Immediate early response 3 IER3 1237_at 2.2 BCL2-related protein A1 BCL2A1 2002_s_at 1.8 Caspase 9 CASP9 486_at 1.5 CASP8 and FADD-like apoptosis regulator CFLAR 1867_at 0.9 Binding Aryl hydrocarbon receptor AHR 40516_at 0.5 Calumenin CALU 37345_at 0.5 Killer cell lectin-like receptor subfamily G, member 1 KLRG1 34975_at 0.5 molecule Laminin, ␤3 LAMB3 36929_at 1.9 CD44 Ag CD44 40493_at 1.7 Plasminogen activator, urokinase PLAU 37310_at 4.5 Omithine decarboxylase antizyme inhibitor OAZIN 33368_at 2.3 Uridine phosphorylase UP 37351_at 2.1 Ceroid-lipofuscinosis, neuronal 2 (tripeptidyl-peptidase I) CLN2 32824_at 1 Legumain (asparaginyl endopeptidase) LGMN 317_at 0.9 Phosphoprotein C8FW 35597_at 0.8 Isopentenyl-diphosphate ␦ IDI1 36985_at 0.8 Oxidative-stress responsive 1 OSR1 39136_at 0.8 specific protease 14 USP14 36982_at 0.5 Downloaded from Enzyme regulator Protease inhibitor 3 PI3 41469_at 3.2 Omithine decarboxylase antizyme inhibitor OAZIN 1959_at 2.2 Protein 2, regulatory subunit B, ␣ isoform PPP2R2 41167_at 1.2 Signal transducer MIP-1␣ (chemokine (CC motif) ligand 3) CCL3 36103_at 4.7 IL-8 IL-8 1369_s_at 3.4 Chemokine (CXC motif) ligand 2 (GRO␤)* CXCL2 37187_at 3.4

Vascular endothenal growth factor VEGF 36100_at 3.3 http://www.jimmunol.org/ IL-1, ␤ IL-1 39402_at 3.2 Pleckstrin PLEK 37328_at 1.8 G protein coupled receptor 65 GPR65 34930_at 1.6 Putative chemokine receptor; GTP-binding protein HM74 34951_at 1.5 TGF-␤ receptor 1 TGF-R1 32903_at 1.5 Lymphocyte cytosolic protein 2 LCP2 39319_at 1.5 Adaptor protein with pleckstrin and src homology 2 domains APS 37136_at 1.4 IL-1 receptor, type 1 IL-1R1 1368_at 1.4 TNF-␣* TNF 1852_at 1.2 Discs, large (Drosophila) homolog-associated protein 1 DLGAP1 40388_at 1.1 TNFR-associated factor 3 TRAF3 37057_s_at 0.9 by guest on September 27, 2021 SKI-like SKIL 1866_g_at 0.9 -activated kinase 3 MAP2K3 1622_at 0.8 Chemokine (CXC motif) ligand 3 (GRO␥) CXCL3 34022_at 0.8 G protein-coupled receptor 18 GPR18 252_at 0.7 Protein tyrosine phosphatase, receptor type, E PTPRE 32916_at 0.7 Guanine nucleotide binding protein-like 1 GNL1 1162_g_at 0.7 Mannose-6-phosphate receptor (cation dependent) M6PR 32547_at 0.6 Ore-B-cell colony-enhancing factor PBEF 33849_at 0.6 TRAF family member-associated NFkB activator TANK 39742_at 0.6 Chemokine (CC motif) ligand 2 (MCP-1) CCL2 34375_at 0.5 Tyrosine phosphatase, ␧ PTPR 1150_at 0.5 Structural molecule , light polypetide 4, alkali MYL4 31421_at 0.8 Transcription regulator TGF-B-inducible early growth response TIEG 224_at 4.2 Early growth response 3 EGR3 40375_at 2.9 V-ets erythroblastosis virus E26 oncogene homolog 2 ETS2 1519_at 2 NP-␬B1 (p105) NFKB1 1377_at 2 corepressor 2 NCOR2 39358_at 1.7 Nuclear factor (erythroid-derived 2)-like 2 NFE2L2 853_at 1.5 Chromodomain DNA binding protein 1 CHD1 39231_at 1.4

Vitamin D (1,25-dihydroxyvitamin D3) receptor VDR 1388_g_at 1.1 EC TFEC 34470_at 1.1 High mobility group AT-hook 1 HMGA1 39704_s_at 1 Zinc finger protein 36 ZFP36 40448_at 0.9 TAF6-like RNA polymerase II TAF6L 39908_at 0.8 PHD finger protein 1 PHF1 40446_at 0.8 Heterogeneous nuclear ribonucleoprotein C (C1/C2) HNRPC 33666_at 0.7 HIV-1 Tat interactive protein, 60kDa HTATIP 465_at 0.6 Eukaryotic translation initiation factor 4A, isoform 1 EIF4A1 1199_at 1.7 Eukaryotic translation 1 ␣ 1 EEF11 1288_s_at 1.1 Eukaryotic translation initiation factor 5 EIF5 167_at 1 Ribosomal protein S16 RPS16 38061_at 1 Ribosomal protein S27 (metallopanstimulin 1) RPS27 32748_at 0.9 Signal recognition particle 54kDa SRP54 36060_at 0.9 (Table continues) 7688 TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS

Table II. Continued

Log2-Fold Gene Category Gene Name Gene Symbol Affymetrix ID Change

Transporter Solute carrier family 25, member 13 (citrin) SLC25A13 38328_at 2.4 ATPase, Hϩ transporting, V1 subunit C, isoform 1 ATP6V1C1 37948_at 2 ATPase, Naϩ/Kϩ transporting, ␣ 1 polypeptide ATP11 32225_at 1.6 RA81A, member RAS oncogene family RAB1A 1074_at 1.2 Chloride channel 7 CLCN7 38069_at 1.1 Aquaporin 9 AQP9 34435_at 1 Phosphotidylinositol transfer protein PITPN 35251_at 0.8 GABA(A) receptor-associated protein-like 2 GABARAL2 35767_at 0.7 Solute carrier family 25, member 6 SLC25A6 40435_at 0.7 Methylene tetrahydrofolate dehydrogenase MTHFD2 40074_at 0.5

a Genes up-regulated in PMNs upon migration to skin lesions including their gene category, gene name, gene symbol, Affymetrix ID, and the log2-fold change of gene expression (range of p values: 2.6 ϫ 10Ϫ3Ð3.6 ϫ 10Ϫ9, estimated false discovery rate 0.032 (Benjamini-Hochberg)). Values of p for CXCL2 and TNF-␣ marked with an asterisk were 0.025 and 0.026, respectively.

healing activities that are stimulated by uPA include the prolifer- Physiologically, these findings are meaningful, as basic functions Downloaded from ation, migration, and adhesion of keratinocytes, fibroblasts, and such as migration and immediate host defense are inherent to cir- endothelial cells in skin wounds (25). culating PMNs and do not require a prolonged phase of transcrip- The up-regulation of tripeptidyl-peptidase I/ceroid-lipofuscino- tional activation. In contrast, the data demonstrated that PMNs sis, neuronal 2, legumain/asparaginyl endopeptidase, and the man- were capable to transcriptionally activate specific functions such as nose-6-phosphate receptor suggested a priming of lysosomal ac- the promotion of wound healing once they have migrated to sites

tivity once PMNs have migrated to skin lesions (26Ð29). of infection. http://www.jimmunol.org/ The cellular fate of PMNs at sites of infection includes necrotic Transcriptionally highly induced genes are up-regulated at the death, immediate apoptosis, or the prolongation of life span by protein level inhibition of apoptosis. Upon necrotic death, PMNs release toxic To investigate whether the transcriptional up-regulation of genes granule proteins resulting in tissue damage, whereas the phagocy- in sl-PMNs detected by array analysis correlated with increased tosis of apoptotic PMNs by macrophages protects against such protein levels, protein lysates were extracted from the same sam- damage (30, 31). When cultured in vitro, PMNs rapidly undergo ples used for array analysis and subjected to Western blot analysis. apoptosis, a process, which is delayed by addition of G-CSF and a These analyses demonstrated that transcriptionally highly induced variety of inflammatory mediators to the medium (32, 33). Hence, genes in sl-PMNs including IL-8, MIP-1␣, and uPA were up-reg- cytokines and inflammatory mediators present at sites of infection by guest on September 27, 2021 ulated at the protein level (Fig. 2). might augment the inflammatory response of PMNs by prolonga- tion of cellular life span. This statement is supported by the present Discussion study showing that PMNs in skin wounds up-regulate anti- The present study demonstrates that the migration of PMNs to skin apoptotic genes and down-regulate proapoptotic genes, and thus, lesions in man is accompanied with a substantial change in gene might acquire a transient “anti-apoptotic state”. Notably, two of expression. These findings are in line with in vitro studies showing the up-regulated anti-apoptotic genes, i.e., IEX1 (IER3) and that human PMNs are capable of extensive changes in gene ex- BCL2A1, have been defined as target genes of NF-␬B, a transcrip- pression upon in vitro activation by single agents such as bacteria, tion factor that was found to be up-regulated in sl-PMNs and, LPS, and by phagocytosis of IgG- and complement-coated latex which is activated through IL-1␤ and TNF-␣ signaling and binding beads (4Ð7). Not unexpectedly, the changes reported in those stud- of pathogens to TLRs (34). Because macrophages and PMNs both ies differ partially from those observed in the present study. For produce IL-1␤ and TNF-␣ at sites of infection (35, 36), the “anti- example, of the top 5 up-regulated genes in the present study apoptotic state” of PMNs in skin lesions might partially be regu- (MIP-1␣, uPA, IL-8, VEGF, and IL-1␤) only IL-8 and IL-1␤ were lated at the transcriptional level through NF-␬B activation induced by bacteria and LPS (4, 5), only MIP-1␣ and VEGF were pathways. induced by phagocytosis of IgG- and complement-coated latex The present study supports the notion that PMNs are a rich beads (7), and uPA was not induced by any of these agents. These source of cytokines and chemokines in skin wounds (36). More- findings demonstrate that PMNs generate distinct transcriptional over, PMNs clearly outnumbered macrophages at 24 h in our skin- responses depending on the type of stimuli and activated signaling wounding model, suggesting that PMNs are the major source of pathway. The different responses of PMNs stimulated in vitro and inflammatory mediators during the initial phase of wound healing. in vivo further demonstrate that multiple, and not individual, stim- Some of the factors found to be up-regulated in the present study uli and signaling pathways contribute to the transcriptional re- including IL-1␤, TNF-␣, and IL-8, have been described as up- sponse of PMNs in skin lesions. regulated in PMNs 1 day after incisional wounding (36, 37). How- To define how migration of PMNs to skin lesions affects bio- ever, to the best of our knowledge, the up-regulation of VEGF, logical functions on a global level, differentially regulated genes MCP-1, MIP-1␣, and GRO-␥ has not been reported earlier in were assigned to categories according to their biological functions PMNs upon migration to skin wounds. These findings demonstrate using the Gene Ontology database. Functional clustering revealed that up-regulated chemokines not only recruit more PMNs (IL-8), that genes critical for migration, cellular structure, and immediate but also specifically attract macrophages (MCP-1 and MIP-1␣) host defense were not activated, but were partially down-regulated, (38) and T cells (MCP-1), which have been reported to be the most whereas genes involved in apoptosis, wound healing, and other abundant leukocyte populations 2 days after incisional distinct cellular functions were highly differentially regulated. wounding (37). The Journal of Immunology 7689

Table III. Genes downregulated in PMNs upon migration to skin lesionsa

Log2-Fold Gene Category Gene Name Gene Symbol Affy ID Change

Apoptosis regulator Caspase 8, apoptosis-related cysteine protease CASP8 33774_at Ϫ2.1 Ret finger protein RFP 40176_at Ϫ1.4 TIA1 cytotoxic granule-associated RNA binding protein-like 1 TIAL1 41762_at Ϫ1.1 Topoisomerase (DNA) II-binding protein TOPBP1 38834_at Ϫ1.1 HIV-1 Tat interactive protein 2, 30kDa HTATIP2 38824_at Ϫ1 Apoptotic protease activating factor APAF1 37227_at Ϫ0.9 Death-associated protein kinase 2 DAPK2 34912_at Ϫ0.8 Fas (TNFRSF6)-associated via death domain FADD 38755_at Ϫ0.7 CASP2 and RIPK1 domain containing adaptor with death domain CRADD 1211_s_at Ϫ0.6 Binding Grancalcin, EF-hand binding protein GCA 37556_at Ϫ1 single-strand binding proteinretropseudogene 1c MSSP1 31671_at Ϫ0.8 Xeroderma pigmentosum, complementation group C XPC 1873_at Ϫ0.7 Folliststin-like 1 FSTL1 40132_g_at Ϫ0.6 Zinc finger protein 185 (LIM domain) ZNF185 32139_at Ϫ0.6 A11 ANXA11 36637_at Ϫ0.5 Cell adhesion molecule Platelet/endothelial cell adhesion molecule (CD31 Ag) PECAM1 37397_at Ϫ1.5 Flotillin 2 FLOT2 32181_at Ϫ1.2 , desmosome associated protein PNN 33543_s_at Ϫ1.1 Downloaded from Ectonucleoside triphosphate diphosphohydrolase 1 ENTPD1 32826_at Ϫ0.8 Fasciculation and elongation protein ␨ 2 (zygin II) FEZ2 38651_at Ϫ0.6 Retinoblastoma-like 2 (p130) RBL2 32597_at Ϫ1.6 Transducer of ERBB2, 1 TOB1 40631_at Ϫ1.2 -dependent kinase inhibitor 2D (p19, inhibits CDK4) CDKN2D 1797_at Ϫ1.2 SET translocation (myeloid leukemia-associated) SET 40189_at Ϫ0.7 Ϫ -specific chaperone c TBCC 36176_at 1.9 http://www.jimmunol.org/ Defense/immunity Protein leukocyte specific transcript 1 LST1 37967_at Ϫ0.8 Enzyme Transketolase (Wernicke-Korsakoff syndrome) TKT 38789_at Ϫ2 Ribosomal protein S6 kinase, 90 kDa, polypeptide 5 RPS6KA5 41432_at Ϫ1.8 Sialyltransferase 8D (␣-2, 8-polysialyltransferase) SIAT8D 33649_at Ϫ1.5 Serine/threonine kinase 24 (STE20 homolog, yeast) STK24 40473_at Ϫ1.5 Ubiquitin specific protease 1 USP1 34383_at Ϫ1.5 Homo sapiens mRNA; cDNA DKFZp686D0521 38581_at Ϫ1.5 Methyl-CpG binding domain protein 4 MBD4 34386_at Ϫ1.4 GNAS complex GNAS 37448_s_at Ϫ1.3 Adrenergic, ␤, receptor kinase 1 ADRBK1 38447_at Ϫ1.2 Inositol polyphosphate-5-phosphatase, 145 kDa INPP5D 172_at Ϫ1.2 by guest on September 27, 2021 C, ␤ 2 PLCB2 210_at Ϫ1.2 1A, magnesium-dependent, ␣ isoform PPM1A 857_at Ϫ1.2 Protein phosphatase 1, catalytic subunit, ␥ isoform PPP1CC 37725_at Ϫ1.2 Ubiquitin-activating enzyme E1C (UBA3 homolog, yeast) UBE1C 40066_at Ϫ1.2 Protein phosphatase 1 ␣ catalytic subunit 954_s_at Ϫ1.2 UDP-galactosamine N-acetylgalactosaminyltransferase 3 GALNT3 36484_at Ϫ1.1 Aminopeptidase puromycin sensitive NPEPPS 39431_at Ϫ1.1 , ␥ 2 (phosphatidylinositol-specific) PLCG2 37180_at Ϫ1.1 Prolylcarboxypeptidase (angiotensinase C) PRCP 36672_at Ϫ1.1 ␳-associated, coiled-coil containing protein kinase 1 ROCK1 34735_at Ϫ1.1 Zinc metalloproteinase (STE24 homolog, yeast) ZMPSTE24 33912_at Ϫ1.1 , ␤ PHKB 37392_at Ϫ1 Regulator of nonsense transcripts 1 RENT1 39404_s_at Ϫ1 Thiosulfate sulfurtransferase () TST 36123_at Ϫ1 Unc-51-like kinase 1 (Caenorhabditis elegans) ULK1 34827_at Ϫ1 Ubiquitin specific protease 15 USP15 34295_at Ϫ1 Iduronate 2- (Hunter syndrome) IDS 40814_at Ϫ0.9 Phosphorylase kinase, ␣ 2 (liver) PHKA2 36480_at Ϫ0.9 Serine/threonine kinase 38 STK38 36218_g_at Ϫ0.9 Tyrosine kinase 2 TYK2 993_at Ϫ0.9 X-ray complementing defective dsDNA repair in Chin, hamster cells 5 XRCC5 584_s_at Ϫ0.9 PTK9L protein tyrosine kinase 9-like (A6-related protein) PTK9L 35796_at Ϫ0.8 A disintegrin and metalloproteinase domain 10 ADAM10 40797_at Ϫ0.8 MAP/ affinity-regulating kinase 2 MARK2 965_at Ϫ0.8 Tyrosylprotein 2 TPST2 35172_at Ϫ0.8 Copine III CPNE3 39706_at Ϫ0.8 O-linked N-acetylglucosamine (GlcNAc) OGT 38614_s_at Ϫ0.7 ATP citrate ACLY 40881_at Ϫ0.7 Cell division cycle 2-like 5 (-related cell division controller) CDC2L5 41821_at Ϫ0.7 Peroxiredoxin 3 PRDX3 36631_at Ϫ0.7 Glycogen synthase kinase 3 ␤ GSK3B 40645_at Ϫ0.6 Myotubularin-related protein 3 MTMR3 35739_at Ϫ0.6 Phosphatidylinositol glycan, class B PIGB 314_at Ϫ0.6 Peroxisome biogenesis factor 1 PEX1 38365_at Ϫ0.5 Serine palmitoyltransferase, long chain base subunit 1 SPTLC1 38818_at Ϫ0.5 (Table continues) 7690 TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS

Table III. Continued

Log2-Fold Gene Category Gene Name Gene Symbol Affy ID Change

Enzyme regulator Omithine decarboxylase antizyme 1 OAZ1 1315_at Ϫ0.8 Cystatin A (stefin A) CSTA 39581_at Ϫ0.7 Motor 2 60/70 kDa KNS2 39057_at Ϫ1.2 Myosin IXB MYO9B 33816_at Ϫ0.7 SMC1 structural maintenance of 1-like 1 (yeast) SMC1L1 32849_at Ϫ0.5 Signal transducer Regulator of G-protein signalling 2, 24 kDa RGS2 37701_at Ϫ2.5 Selectin P ligand SELPLG 37541_at Ϫ2.2 ␳ GTPase activating protein 1 ARHGAP1 553_g_at Ϫ1.9 RalA binding protein 1 RALBP1 36628_at Ϫ1.8 Calcium/-dependent protein kinase (CaM kinase) II ␥ CAMK2G 32105_f_at Ϫ1.7 condensation 1-like CHC1L 35193_at Ϫ1.7 ␤ ␤ Ϫ Integrin, 2 (CD18 (p95) alias mac-1 subunit) ITGB2 37918_at 1.7 , ␤ 1 PRKCB1 1336_s_at Ϫ1.7 Endothelial differentiation, G protein-coupled receptor 6 EDG6 33602_at Ϫ1.6 G protein, ␣ activating activity polypeptide O GNAO1 34138_at Ϫ1.6 HMT1 hnRNP methyltransferase-like 1 (S. cerevisiae) HRMT1L1 39348_at Ϫ1.6 ␣ Ϫ Integrin, L (CD11A (p180)) ITGAL 38547_at 1.6 Leukocyte immunoglobulin-like receptor subfamily A, member 2 LILRA2 34033_s_at Ϫ1.6 Downloaded from ␳ GDP dissociation inhibitor (GDI) ␤ ARHGDIB 1984_s_at Ϫ1.5 G protein ␣ inhib. activity peptide 2 GNAI2 37307_at Ϫ1.4 V-akt murine thymoma viral oncogene homolog 1 AKT1 1564_at Ϫ1.4 V-raf-1 murine leukemia viral oncogene homolog 1 RAF1 1917_at Ϫ1.4 Intercellular adhesion molecule 3 ICAM3 402_s_at Ϫ1.4 Insulin-like growth factor 2 receptor IGF2R 160027_s_at Ϫ1.4 ␤ Ϫ IL-8R IL-8RB 664_at 1.4 http://www.jimmunol.org/ Calmodulin 2 (phosphorylase kinase, ␦) CALM2 911_s_at Ϫ1.3 Protein kinase, cAMP-dependent, regulatory, type I, ␣ PRKAR1A 226_at Ϫ1.3 IL-8R␣ IL-8RA 1353_g_at Ϫ1.3 Membrane protein (CD46) MCP 38441_s_at Ϫ1.3 Protein phosphatase 1, regulatory (inhibitor) subunit 12A PPP1R12A 40438_at Ϫ1.3 Regulator of G protein signalling 14 RGS14 38290_at Ϫ1.3 Regulator of G protein signalling 19 RGS19 34268_at Ϫ1.3 IQ motif containing GTPase activating protein 1 IQGAP1 1825_at Ϫ1.2 RAS guanyl releasing protein 2 (calcium and DAG-regulated) RASGRP2 38359_at Ϫ1.2 Mitogen-activated protein kinase-activated protein kinase 3 MAPKAPK3 1637_at Ϫ1.2 3B, cGMP-inhibited PDE3B 35872_at Ϫ1.2 by guest on September 27, 2021 Protein phosphatase 1, regulatory (inhibitor) subunit 12B PPP1R12B 41137_at Ϫ1.2 CD97 Ag CD97 35625_at Ϫ1.2 Toll-like receptor 1 TLR1 36243_at Ϫ1.2 Calmodulin 1 (phosphorylase kinase, ␦) CALM1 41143_at Ϫ1.1 STE20-like kinase JIK 41646_at Ϫ1.1 CREB-binding protein (Rubinstein-Taybi syndrome) CREBBP 33831_at Ϫ1.1 Mitogen-activated protein kinase kinase kinase 5 MAP3K5 1327_s_at Ϫ1.1 Calmodulin 3 (phosphorylase delta) 955_at Ϫ1 GDP dissociation inhibitor 2 GDI2 35307_at Ϫ1 LIM and SH3 protein 1 LASP1 36181_at Ϫ1 Mitogen-activated protein kinase kinase kinase 3 MAP3K3 1330_at Ϫ1 Inositol 1,4,5-trisphosphate 3-kinase B ITPKB 37272_at Ϫ0.9 Calcium/calmodulin-dependent protein kinase (CaM kinase) II ␥ CAMK2G 32104_i_at Ϫ0.9 G protein, ␣-transducing activity polypeptide 2 GNAT2 34571_at Ϫ0.8 G protein, ␤ polypeptide 2 GNB2 38831_f_at Ϫ0.8 Janus kinase 1 (a protein tyrosine kinase) JAK1 34877_at Ϫ0.8 ␳ GTPase activating protein 4 ARHGAP4 39649_at Ϫ0.8 G protein-coupled receptor 19 GPR19 156_s_at Ϫ0.8 Mitogen-activated protein kinase 3 MAPK3 1000_at Ϫ0.8 Transforming growth factor, ␤ 1 (Camurati-Engelmann disease) TGF-1 1830_s_at Ϫ0.8 Toll-like receptor 6 TLR6 34144_at Ϫ0.8 Protein tyrosine phosphatase, f polypeptide, interacting protein, ␣ 1 PPFIA 1 41780_at Ϫ0.7 Colony stimulating factor 3 receptor (granulocyte), G-CSFR CSF3R 34223_at Ϫ0.7 FK508 binding protein 1A, 12-kDa FKBP1A 880_at Ϫ0.7 Leukocyte immunoglobulin-like receptor, subfamily B, member 5 LILRB5 36789_f_at Ϫ0.7 Lymphotoxin ␤ (TNF superfamily, member 3) LTB 40729_s_at Ϫ0.7 Pleckstrin homology, Sec7 and coiled/coil domains 1(cytohesin 1) PSCD1 38666_at Ϫ0.7 Transportin-SR TRN-SR 35813_at Ϫ0.7 Wiskott-Aldrich syndrome (eczema-thrombocytopenia) WAS 38964_r_at Ϫ0.7 Mitogen-activated protein kinase kinase 4 MAP2K4 1845_at Ϫ0.6 Dedicator of cyto-kinesis 2 DOCK2 32704_at Ϫ0.6 Transforming growth factor, ␣ TGFA 160025_at Ϫ0.5 Receptor (TNFRSF)-interacting serine-threonine kinase 1 RIPK1 40696_at Ϫ0.5 Activin A receptor, type IB ACVR1B 34056_g_at Ϫ0.5 -interacting factor RABIF 38264_at Ϫ0.5 RAS p21 protein activator (GTPase activating protein) 1 RASA1 1675_at Ϫ0.5 (Table continues) The Journal of Immunology 7691

Table III. Continued

Log2-Fold Gene Category Gene Name Gene Symbol Affy ID Change

Structural molecule Coronin, actin-binding protein, IA CORO1A 38976_at Ϫ2.1 Adducin 3 (␥) ADD3 33102_at Ϫ2 Tubulin, ␣, ubiquitous K-␣-1 32272_at Ϫ1.7 Tubulin, ␣ 2 TUBA2 38350_f_at Ϫ1.7 Tubulin, ␣ 1 (testis specific) TUBA1 36591_at Ϫ1.6 , ␣ 4 ACTN4 41753_at Ϫ1.4 Spastic paraplegia 4 (autosomal dominant; spastin) SPG4 35171_at Ϫ1.4 Flightless I homolog (Drosophila) FLII 33133_at Ϫ1.2 Lymphocyte-specific protein 1 LSP1 36493_at Ϫ0.9 Actin related protein 2/3 complex, subunit 2, 34 kDa ARPC2 38445_at Ϫ0.9 Actinin, ␣ 1 ACTN1 39330_s_at Ϫ0.9 Actin related protein 2/3 complex, subunit 1B, 41 kDa ARPC1B 39043_at Ϫ0.9 Actin related protein 2/3 complex, subunit 3, 21 kDa ARPC3 35810_at Ϫ0.7 Capping protein (actin filament) muscle Z-line, ␤ CAPZB 37012_at Ϫ0.5 -cap (telethonin) TCAP 39002_at Ϫ0.5 Transcription regulator ␦ sleep inducing peptide, immunoreactor DSIPI 36629_at Ϫ2.8 Zinc finger protein 36, C3H type-like 2 ZFP36L2 32587_at Ϫ2.7 Nuclear factor (erythroid-derived 2), 45 kDa NFE2 37179_at Ϫ2.6 Downloaded from General transcription factor II, i GTF2I 35450_s_at Ϫ2.5 Arginine-glutamic acid dipeptide (RE) repeats RERE 32253_at Ϫ2.3 B receptor LBR 288_s_at Ϫ2.1 Friend leukemia virus integration 1 FLI1 41425_at Ϫ2 Ubinuclein 1 UBN1 32858_at Ϫ2 Hematopoietically expressed HHEX 37497_at Ϫ1.9 Ϫ Polyhomeotic-like 2 (Drosophila) PHC2 36960_at 1.8 http://www.jimmunol.org/ BarH-like homeobox 2 BARX2 35425_at Ϫ1.8 Leucine rich repeat (in FLII) interacting protein 1 LRRFIP1 41320_s_at Ϫ1.7 Chromobox homolog 1 (HP1 ␤ homolog Drosophila) CBX1 37304_at Ϫ1.6 Zinc finger protein 217 ZNF217 32034_at Ϫ1.6 Nuclear receptor coactivator 4 NCOA4 39174_at Ϫ1.5 YY1 transcription factor YY1 891_at Ϫ1.3 High-mobility group box 2 HMGB2 38065_at Ϫ1.2 Nuclear receptor coactivator 1 NCOA1 36118_at Ϫ1.1 Signal transducer and activator of transcription 6, IL-4 induced STAT6 41222_at Ϫ1.1 Sjogren syndrome Ag A2 (ribonucleoprotein autoantigen SS-A/Ro) SSA2 35294_at Ϫ1 Cbp/p300-interacting transactivator, carboxyl-terminal domain, 2 CITED2 33113_at Ϫ1 by guest on September 27, 2021 COP9 constitutive photomorphogenic homolog subunit 5 (Arabidopsis) COPS5 1789_at Ϫ1 binding protein 1 HSBP1 31906_at Ϫ1 ␣ thalassemia/mental retardation syndrome X-linked ATRX 39147_g_at Ϫ0.9 Forkhead box O1A (rhabdomyosarcoma) FOXO1A 40570_at Ϫ0.9 SWI/SNF related, matrix associated, regulator of chromatin, member 2 SMARCA2 40962_s_at Ϫ0.8 Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse) MEIS2 41388_at Ϫ0.8 TAF4 RNA polymerase II, TATA box binding protein-associated factor TAF4 142_at Ϫ0.7 Histone deacetylase 5 HDAC5 38810_at Ϫ0.7 Retinoblastoma binding protein 1 RBBP1 1849_s_at Ϫ0.6 Polymerase (RNA) II (DNA directed) polypeptide J, 13.3 kDa POLR2J 38055_at Ϫ0.6 Heat shock transcription factor 1 HSF1 244_at Ϫ0.6 , ␣ RAR 1337_s_at Ϫ0.6 Transcriptional adaptor 3 (NGG1 homolog, yeast)-like TADA3L 35749_at Ϫ0.6 Hematopoietic cell-specific Lyn substrate 1 HCLS1 31820_at Ϫ0.5 Myeloid/lymphoid leukemia (trithorax homol., Dros.); translocated to 7 MLLT7 36238_at Ϫ0.5 TBP-like 1 TBPL1 31797_at Ϫ0.5 Core-binding factor, runt domain, ␣ subunit 2; translocated to, 3 CBFA2T3 41442_at Ϫ0.5 Growth arrest-specific 7 GAS7 33387_at Ϫ0.5 General transcription factor IIB GTF2B 37380_at Ϫ0.5 General transcription factor IIE, polypeptide 1, ␣ 56 kDa GTF2E1 37882_at Ϫ0.5 General transcription factor IIIC, polypeptide 1, ␣ 220 kDa GTF3C1 35671_at Ϫ0.5 C-myc binding protein MYCBP 37250_at Ϫ0.5 Translation SFRS protein kinase 2 SRPK2 1213_at Ϫ1.3 RNA-binding motif protein 5 RBM5 1556_at Ϫ1.2 Ribosomal protein, large P2 RPLP2 34091_s_at Ϫ1 Eukaryotic translation initiation factor 4 ␥, 2 EIF4G2 41785_at Ϫ1 RNA binding protein (hnRNP-associated with lethal yellow) RALY 36125_s_at Ϫ0.8 Splicing factor, arginine/serine-rich 1 SFRS1 36098_at Ϫ0.8 Signal recognition particle 9 kDa SRP9 36981_at Ϫ0.8 Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 RPS6KA1 1127_at Ϫ0.8 CUG triplet repeat, RNA-binding protein 2 CUGBP2 32851_at Ϫ0.6 Ribosomal protein L26 RPL26 32444_at Ϫ0.5 Splicing factor, arginine/serine-rich 4 SFRS4 36991_at Ϫ0.5 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9 (DNA helicase) DDX9 662_at Ϫ0.5 Prp28, U5 snRNP 100 kDa protein U5Ð100K 40465_at Ϫ0.5 (Table continues) 7692 TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS

Table III. Continued

Log2-Fold Gene Category Gene Name Gene Symbol Affy ID Change

Transporter Nucleoporin 214 kDa NUP214 40768_s_at Ϫ1.8 SEC14-like 1 (S. cerevisiae) SEC14L1 36207_at Ϫ1.2 Neutrophil cytosolic factor 4, 40 kDa NCF4 38895_i_at Ϫ1.2 Nucleoporin 153 kDa NUP153 32850_at Ϫ1.2 Synaptosomal-associated protein, 23 kDa SNAP23 32178_r_at Ϫ1.2 ATP-binding cassette sub-family G (WHITE), member 1 ABCG1 41362_at Ϫ1 Malic enzyme 2, NADϩ-dependent, mitochondrial ME2 36599_at Ϫ1 Aminopeptidase-like 1 NPEPL1 41121_at Ϫ0.8 Solute carrier family 31 (copper transporters), member 2 SLC31A2 34749_at Ϫ0.8 Phosphatidylinositol transfer protein, membrane-associated PITPNM 38297_at Ϫ0.7 Solute carrier family 25 (mitochondrial phosphate carrier), member 3 SLC25A3 37675_at Ϫ0.7 Ubiquinol-cytochrome c reductase (6.4kD) subunit UQCR 38451_at Ϫ0.7 CDP-diacylglycerol-inositol 3-phosphatidyltransferase CDIPT 33397_at Ϫ0.6 Cytochrome c oxidase subunit Vb COX5B 39443_s_at Ϫ0.6 Solute carrier family 19 (folate transporter), member 1 SLC19A1 33135_at Ϫ0.6 Solute carrier family 23 (nucleobase transporters), member 1 SLC23A1 38122_at Ϫ0.6 E1B-55 kDa-associated protein 5 E1B-AP5 40106_at Ϫ0.5 Downloaded from a Genes downregualted in PMNs upon migration to skin lesions including their gene category, gene name, gene symbol, Affymetrix ID, and the log2-fold change of gene expression (range of p values: 2.6 ϫ 10Ϫ3Ð3.6 ϫ 10Ϫ9, estimated false discovery rate 0.032 (Benjamini-Hochberg)).

The cellular response to cytokines and chemokines highly de- transcripts suggests a change in responsiveness to chemotactic and pends on the profile of receptors expressed on the cell membrane. immunoregulatory mediators once PMNs have migrated into skin

The up-regulation of the IL-1R1 concomitant with its own ligand, lesions and have been activated. http://www.jimmunol.org/ IL-1␤ in sl-PMNs, suggests an autoregulatory enforcement of their The healing of skin wounds is a multistep process where cyto- inflammatory response through the NF-␬B activation pathway. kines and chemokines orchestrate the collaboration of various cell Up-regulation of the TGF-␤R indicates an increased responsive- types. VEGF, probably the most important angiogenic cytokine, ness to TGF-␤, a cytokine that is secreted by activated macro- stimulates both proliferation and migration of endothelial cells phages and perhaps is the most potent endogenous negative reg- (42). The present study demonstrates for the first time the up- ulator of hemopoietic cells (39, 40). Hence, one might speculate regulation of VEGF by PMNs in a human skin wounding model. that once PMN-chemokines have attracted sufficient macrophages, Moreover, sl-PMNs up-regulated the chemokines IL-8, GRO-␥, the macrophages will decrease the activity of PMNs through and MCP-1, which have been reported to stimulate growth of en- ␤ by guest on September 27, 2021 TGF- signaling, and thus, take over and initiate the next step in dothelial cells, keratinocytes, and fibroblasts (19, 21). Other genes wound healing. Indeed, this hypothesis is supported by in vivo that affect wound healing and were up-regulated by sl-PMNs in- experiments showing that leukocyte infiltrates in skin wounds are cluded uPA and LAMB3. This was somewhat surprising as uPA is initially dominated by PMNs, which decline in numbers concom- not up-regulated by PMNs when activated in vitro by various stim- itant with the increase of macrophage numbers 2 days after uli (4, 5, 7). However, incubation of plasma with PMNs has been injury (37). shown to generate thrombolytic uPA activity (24). Hence, acti- Other up-regulated receptors in sl-PMNs that might modulate vated PMNs in skin wounds might generate uPA resulting in plas- cellular activity in skin lesions included the GPR18/65 and HM74. minogen activation and subsequent breakdown of fibrin clots and Whereas induction of GPR18/65 in PMNs has not been described extracellular matrix (25). Moreover, uPA has been reported to pro- so far, the induction of HM74 has been reported upon stimulation mote the proliferation, migration, and adhesion of keratinocytes, of PMNs by LPS and bacteria in vitro (4, 5). HM74 has been fibroblasts, and endothelial cells in skin wounds (25). LAMB3 is defined as a receptor for nicotinic acid, which mediates decrease in an important adhesion molecule of the basal membrane and the cAMP levels in adipose tissue when binding to its ligand (41). deficiency of LAMB3 results in a blistering skin disease (junc- Down-regulated receptor transcripts included IL-8R␣, IL8-R␤,G- tional epidermolysis bullosa) due to the disruption of epidermal- CSFR, and the TLRs 1 and 6, which mediate chemotaxis and cel- dermal coadhesion (23). The up-regulation of LAMB3 by PMNs in lular activation (19, 20). Overall, the altered expression of receptor skin lesions might therefore support adhesion of keratinocytes at wound margins, and thus, promote epitheliazation. An important function of PMNs at sites of infection is the re- lease of antimicrobial proteins as well as the phagocytosis and subsequent lysosomal degradation of microorganisms and cellular debris. Importantly, PMNs in skin lesions demonstrated no transcrip- tional regulation of antimicrobial peptides and lysosomal enzymes. However, the up-regulation of tripeptidyl-peptidase I/ceroid-lipofus- cinosis, neuronal 2 and legumain/asparaginyl endopeptidase, which activate lysosomal enzymes by cleavage (26Ð28), and the up-regula- tion of the mannose-6-phosphate receptor (29), which target proteins FIGURE 2. Comparison of mRNA an d protein levels in sl-PMNs and to the lysosome, suggested a priming of PMNs for degradation of pb-PMNs. The upper row depicts the protein expression detected by West- phagocytosed material upon migration to skin lesions. ern blotting and the lower row depicts the mean relative mRNA expression We conclude that PMNs generate a substantial transcriptional (n ϭ 4) detected by microarray analysis. response upon migration to skin lesions. 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