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Their Important Role in Wound Healing Supports Human 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 References This article cites 41 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/172/12/7684.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 genes 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/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene 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 (4–7). 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- Jørgensens 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 gene expression 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 protein; uPA, urokinase plasminogen activator; MCP, monocyte chemoacttractant protein; GRO, growth-related oncogene; GPR, G protein-coupled mis, termed “skin windows”, are generated and covered with skin receptor; IER3, immediate early response 3; BCL2A1, BCL2-related protein A1; chambers containing a medium that attracts PMNs (8). CASP8, caspase 8, apoptosis-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 proteins 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 2–5 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, 60–80 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%
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