Chemokines IL-8, GROα, MCP-1, IP-10, and Mig Are Sequentially and Differentially Expressed During Phase-Specific Infiltration of Leukocyte Subsets in Human Wound Healing
1998; Elsevier BV; Volume: 153; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65699-4
ISSN1525-2191
AutoresEva Engelhardt, Atiye Toksoy, Matthias Goebeler, E. Sebastian Debus, Eva‐Bettina Bröcker, R. Gillitzer,
Tópico(s)Antimicrobial Peptides and Activities
ResumoHealing of cutaneous wounds requires a complex integrated network of repair mechanisms, including the action of newly recruited leukocytes. Using a skin repair model in adult humans, we investigated the role chemokines play in sequential infiltration of leukocyte subsets during wound healing. At day 1 after injury, the C-X-C chemokines IL-8 and growth-related oncogene α are maximally expressed in the superficial wound bed and are spatially and temporally associated with neutrophil infiltration. IL-8 and growth-related oncogene α profiles also correlate with keratinocyte migration and subsequently subside after wound closure at day 4. Macrophage infiltration reaches the highest levels at day 2 and is paralleled by monocyte chemoattractant protein-1 mRNA expression in both the basal layer of the proliferative epidermis at the wound margins and mononuclear cells in the wound area. Other monocyte-attracting chemokines such as monocyte chemoattractant protein-3, macrophage inflammatory protein-1α and -1β, RANTES, and I309 are undetectable. At day 4, perivascular focal lymphocyte accumulation correlates with strong focal expression of the C-X-C chemokines Mig and IP-10. Our results suggest that a dynamic set of chemokines contributes to the spatially and temporally different infiltration of leukocyte subsets and thus integrates the inflammatory and reparative processes during wound repair. Healing of cutaneous wounds requires a complex integrated network of repair mechanisms, including the action of newly recruited leukocytes. Using a skin repair model in adult humans, we investigated the role chemokines play in sequential infiltration of leukocyte subsets during wound healing. At day 1 after injury, the C-X-C chemokines IL-8 and growth-related oncogene α are maximally expressed in the superficial wound bed and are spatially and temporally associated with neutrophil infiltration. IL-8 and growth-related oncogene α profiles also correlate with keratinocyte migration and subsequently subside after wound closure at day 4. Macrophage infiltration reaches the highest levels at day 2 and is paralleled by monocyte chemoattractant protein-1 mRNA expression in both the basal layer of the proliferative epidermis at the wound margins and mononuclear cells in the wound area. Other monocyte-attracting chemokines such as monocyte chemoattractant protein-3, macrophage inflammatory protein-1α and -1β, RANTES, and I309 are undetectable. At day 4, perivascular focal lymphocyte accumulation correlates with strong focal expression of the C-X-C chemokines Mig and IP-10. Our results suggest that a dynamic set of chemokines contributes to the spatially and temporally different infiltration of leukocyte subsets and thus integrates the inflammatory and reparative processes during wound repair. Undisturbed wound healing follows a course that typically starts with blood clotting, activation of thrombocytes, and, later, immigration of inflammatory cells into the provisional matrix of the wounded region.1Clark RAF Basics of cutaneous wound repair.J Dermatol Surg Oncol. 1993; 19: 693-706Crossref PubMed Scopus (183) Google Scholar, 2Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York and London1996Google Scholar There is increasing evidence that different subpopulations of inflammatory cells are important during the catabolic phase of healing as well as anabolic phase.3Martin P Wound healing: aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3626) Google Scholar Initially, neutrophils accumulate in the damaged tissue, where they form a first line of defense against local infections and aid initial debridement by synthesizing and secreting hydrolases and reactive oxygen intermediates.2Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York and London1996Google Scholar Afterwards, accumulating macrophages enhance the debridement4Brown EJ Goodwin JL Fibronectin receptors of phagocytes: characterization of the arg-gly-asp binding proteins of human monocytes and polymorphnuclear leukocytes.J Exp Med. 1988; 167: 777-793Crossref PubMed Scopus (104) Google Scholar and present antigens to initiate specific immune responses. In addition, macrophages are important producers for a battery of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor (bFGF).3Martin P Wound healing: aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3626) Google Scholar, 5Bennett NT Schultz GS Growth factors and wound healing.Am J Surg. 1993; 165: -737Abstract Full Text PDF Scopus (415) Google Scholar These factors stimulate the synthesis of extracellular matrix components produced by fibroblasts and the ingrowth of blood vessels from the surrounding tissue. More recently, it has become evident that lymphocytes, besides acting as immunological effector cells, are also capable of producing particular growth factors such as bFGF and leukocyte-derived growth factor.6Blotnick S Peoples GE Freeman MR Eberlein TJ Klagsbrun M T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells.Proc Natl Acad Sci USA. 1994; 91: 2890-2894Crossref PubMed Scopus (273) Google Scholar, 7Iida N Haisa M Igarashi A Pencev D Grotendorst G Leukocyte-derived growth factor links the PDGF and CXC chemokine families of peptides.FASEB J. 1996; 10: 1336-1345PubMed Google Scholar Their later appearance in the wound area may further tissue formation and remodeling of the tissue components. Consequently, analyzing the mechanisms responsible for the phase-specific and spatially differential recruitment of leukocyte subsets is a prerequisite for understanding both normal and pathological (ie, delayed or hypertrophic) wound repair. Besides adhesion molecules,8Frenette PS Wagner DD Adhesion molecules part II: blood vessels and blood clotting.N Engl J Med. 1996; 335: 43-45Crossref PubMed Scopus (272) Google Scholar, 9Subramaniam M Saffaripour S Van De Water L Frenette PS Mayadas TN Hynes RO Wagner DD Role of endothelial selectins in wound repair.Am J Pathol. 1997; 150: 1701-1709PubMed Google Scholar the targeting processes of extravasation and tissue homing depend on chemoattractants such as chemokines.10Oppenheim JJ Zachariae COC Mukaida N Matsushima K Properties of the novel proinflammatory supergene “intercrine” cytokine family.Annu Rev Immunol. 1991; 9: 617-648Crossref PubMed Scopus (1817) Google Scholar By virtue of their target-cell specificity, chemokines selectively mediate the regionally specific recruitment of neutrophils, macrophages, and lymphocytes. C-X-C chemokines such as IL-8,11Schroeder JM Christophers E Identification of C5a des arg and an anionic neutrophil-activating peptide (ANAP) in psoriatic scales.J Invest Dermatol. 1986; 87: 53-58Crossref PubMed Scopus (186) Google Scholar, 12Yoshimura T Matsushima K Oppenheim JJ Leonard EJ Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1).J Immunol. 1987; 139: 788-793PubMed Google Scholar, 13Baggiolini M Dewald B Moser B Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines.Adv Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2252) Google Scholar growth-related oncogene α (GROα),14Anisowicz A Bardwell L Sager R Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells.Proc Natl Acad Sci USA. 1987; 84: 7188-7192Crossref PubMed Scopus (196) Google Scholar, 15Richmond A Balentien E Thomas HG Flaggs G Barton DE Spiess J Bordoni R Francke U Derynck R Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to β-thromboglobulin.EMBO. 1988; 7: 2025-2033PubMed Google Scholar and epithelial derived neutrophil attractant-78 amino acids (ENA-78)16Walz A Burgener R Car B Baggiolini M Kunkel SL Strieter RM Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8.J Exp Med. 1991; 174: 1355-1362Crossref PubMed Scopus (304) Google Scholar containing the glutamin acid-leucine-arginine motif17Clark-Lewis I Schumacher C Baggiolini M Moser B Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs.J Biol Chem. 1991; 266: 23128-23134Abstract Full Text PDF PubMed Google Scholar preferentially attract neutrophils and possibly lymphocytes.18Larsen CG Anderson AO Appella E Oppenheim JJ Matsushima K The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes.Science (Wash DC). 1989; 243: 1464-1466Crossref PubMed Scopus (902) Google Scholar, 19Jinquan T Frydenberg J Mukaida N Bonde J Larsen CG Matsushima K Thestrup-Pedersen K Recombinant human growth-regulated oncogene-α induces T lymphocyte chemotaxis.J Immunol. 1995; 155: 5359-5368PubMed Google Scholar The C-X-C chemokines, such as monokine induced by interferon-γ (Mig)20Farber JM A macrophage mRNA selectively induced by γ-interferon encodes a member of the platelet factor 4 family of cytokines.Proc Natl Acad Sci USA. 1990; 87: 5238-5242Crossref PubMed Scopus (256) Google Scholar, 21Farber JM HuMIG: a new human member of the chemokine family of cytokines.Biochem Biophys Res Commun. 1993; 192: 223-230Crossref PubMed Scopus (166) Google Scholar, 22Liao F Rabin RL Yannelli JR Koniaris LG Vanguri P Farber JM Human Mig chemokine: Biochemical and functional characterization.J Exp Med. 1995; 182: 1301-1314Crossref PubMed Scopus (335) Google Scholar and interferon-γ-inducible protein-10 (IP-10),23Luster AD Ravetch JV Biochemical characterization of a γ-interferon-inducible cytokine (IP-10).J Exp Med. 1987; 166: 1084-1097Crossref PubMed Scopus (529) Google Scholar which lack the glutamin acid-leucine-arginine motif, selectively attract lymphocytes.24Taub DD Lloyd AR Conlon K Wang JM Ortaldo JR Harada A Matsushima K Kelvin DJ Oppenheim JJ Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells.J Exp Med. 1993; 177: 1809-1814Crossref PubMed Scopus (683) Google Scholar, 25Loetscher M Gerber B Loetscher P Jones SA Piali L Clark-Lewis I Baggiolini M Moser B Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes.J Exp Med. 1996; 184: 963-969Crossref PubMed Scopus (1046) Google Scholar, 26Farber JM Mig and IP-10: CXC chemokines that target lymphocytes.J Leukoc Biol. 1997; 61: 246-257Crossref PubMed Scopus (685) Google Scholar The C-C chemokines are the second subfamily of chemokines which include monocyte chemoattractant protein-1 (MCP-1),27Yoshimura T Robinson EA Tanaka S Appella E Kuratsu J-I Leonard EJ Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants.J Exp Med. 1989; 169: 1449-1459Crossref PubMed Scopus (405) Google Scholar, 28Matsushima K Larsen CG DuBois GC Oppenheim JJ Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line.J Exp Med. 1989; 169: 1485-1490Crossref PubMed Scopus (636) Google Scholar macrophage inflammatory protein-1α and β (MIP-1α and β),29Wolpe SD Davatelis G Sherry B Beutler B Hesse DG Nguyen HT Moldawer LL Nathan CF Lowry SF Cerami A Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.J Exp Med. 1988; 167: 570-581Crossref PubMed Scopus (457) Google Scholar, 30Sherry B Tekamp-Olson P Gallegos C Bauer D Davatelis G Wolpe SD Masiarz F Coit D Cerami A Resolution of the two components of macrophage inflammatory protein 1, and cloning and characterization of one of those components, macrophage inflammatory protein 1 β.J Exp Med. 1988; 168: 2251-2259Crossref PubMed Scopus (182) Google Scholar I30931Miller MD Krangel MS The human cytokine I-309 is a monocyte chemoattractant.Proc Natl Acad Sci USA. 1992; 89: 2950-2954Crossref PubMed Scopus (124) Google Scholar and the “regulated on activation normal T cells expressed and secreted” (RANTES).32Schall TJ Jongstra J Dyer B Jorgensen J Clayberger C Davis MM Krensky AM Human T-cell specific molecule is a member of a new gene family.J Immunol. 1988; 141: 1018-1025PubMed Google Scholar Chemokines from this family show a partly overlapping specificity for macrophages, lymphocytes,33Loetscher P Seitz M Clark-Lewis I Baggiolini M Moser B Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes.FASEB J. 1994; 8: 1055-1060Crossref PubMed Scopus (296) Google Scholar, 34Taub DD Proost P Murphy WJ Anver M Longo DL Van Damme J Monocyte chemotactic protein-1 (MCP-1), -2 and -3 are chemotactic for human T lymphocytes.J Clin Invest. 1995; 95: 1370-1376Crossref PubMed Scopus (357) Google Scholar and non-neutrophil granulocytes.35Baggiolini M Dahinden CA CC chemokines in allergic inflammation.Immunol Today. 1994; 15: 127-133Abstract Full Text PDF PubMed Scopus (515) Google Scholar So far, the role of individual chemokines during wound healing has been studied mainly in rodent models.36DiPietro LA Burdick MD Low QE Kunkel SL Strieter RM MIP-1α as a critical macrophage chemoattractant in murine wound repair.J Clin Invest. 1998; 101: 1693-1698Crossref PubMed Scopus (234) Google Scholar, 37Iida N Grotendorst G Cloning and sequencing of a new gro transcript from activated human monocytes: expression in leukocytes and wound tissue.Mol Cell Biol. 1990; 10: 5596-5599Crossref PubMed Scopus (95) Google Scholar, 38DiPietro LA Polverini PJ Rahbe SM Kovacs EJ Modulation of JE/MCP-1 expression in dermal wound repair.Am J Pathol. 1995; 146: 868-875PubMed Google Scholar DiPietro and colleagues found maximum levels of JE mRNA, the murine homologue of MCP-1, at 12–24 hours after wounding, followed by a peak of macrophage infiltration 1–2 days later.38DiPietro LA Polverini PJ Rahbe SM Kovacs EJ Modulation of JE/MCP-1 expression in dermal wound repair.Am J Pathol. 1995; 146: 868-875PubMed Google Scholar More recently, DiPietro also detected MIP-1α as a critical mediator of monocyte recruitment in murine wound repair.36DiPietro LA Burdick MD Low QE Kunkel SL Strieter RM MIP-1α as a critical macrophage chemoattractant in murine wound repair.J Clin Invest. 1998; 101: 1693-1698Crossref PubMed Scopus (234) Google Scholar However, it is not clear whether human and murine chemokine homologues exhibit similar functions in vivo, leaving open the question whether their physiological roles during inflammatory reactions are comparable. In view of these discrepancies and due to the lack of available chemokine reagents for animal models with a comparable morphology to human skin (eg, pigs, rabbits, or guinea pigs), we decided to focus on the role chemokines play in wound healing of adult human skin. Recently, expression of GROα and its receptor were studied in human wounds at days 2–12 after accidental burn injuries.39Nanney LB Mueller SG Bueno R Peiper SC Richmond A Distributions of melanoma growth stimulatory activity or growth-regulated gene and the interleukin-8 receptor in human wound repair.Am J Pathol. 1995; 147: 1248-1260PubMed Google Scholar The authors observed immunoreactivity to GROα in exudates and granulation tissue, which was associated with inflammatory infiltrates from days 3–12 after injury. Moreover, expression of the CXC-receptor 2 (CXCR2), previously designated as the IL-8 receptor B, was detected early after wounding in undifferentiated keratinocytes.39Nanney LB Mueller SG Bueno R Peiper SC Richmond A Distributions of melanoma growth stimulatory activity or growth-regulated gene and the interleukin-8 receptor in human wound repair.Am J Pathol. 1995; 147: 1248-1260PubMed Google Scholar However, with the exception of GRO and CXCR2 immunoreactivity in burn wounds, little is known about the temporal and spatial appearance of chemokines and their influences on re-epithelialization and angiogenesis during normal human wound healing. In this study, we investigated the role of neutrophil-, macrophage- and lymphocyte-specific chemokines during human wound healing in a standardized manner, using incisional skin wounds of constant size, constant localization, and defined time intervals in adult volunteers. As several studies have demonstrated, in vivo data on chemokine mRNA expression are highly representative of the presence of their respective target cells.38DiPietro LA Polverini PJ Rahbe SM Kovacs EJ Modulation of JE/MCP-1 expression in dermal wound repair.Am J Pathol. 1995; 146: 868-875PubMed Google Scholar, 40Ritter U Moll H Bröcker E-B Velazco O Becker I Gillitzer R Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.J Infect Dis. 1996; 173: 699-709Crossref PubMed Scopus (107) Google Scholar, 41Gillitzer R Wolff K Tong D Mueller C Yoshimura T Hartmann AA Stingl G Berger R MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.J Invest Dermatol. 1993; 101: 127-131Abstract Full Text PDF PubMed Google Scholar, 42Gillitzer R Ritter U Spandau U Goebeler M Bröcker E-B Differential expression of GRO-α and IL-8 mRNA in psoriasis: A model for neutrophil migration and accumulation in vivo.J Invest Dermatol. 1996; 107: 778-782Crossref PubMed Scopus (128) Google Scholar, 43Goebeler M Toksoy A Spandau U Engelhardt E Bröcker E-B Gillitzer R The C-X-C chemokine Mig is highly expressed in the papillae of psoriatic lesions.J Pathol. 1998; 184: 89-95Crossref PubMed Scopus (77) Google Scholar, 44Schroeder JM Gregory H Young J Christophers E Neutrophil-activating proteins in psoriasis.J Invest Dermatol. 1992; 98: 241-247Crossref PubMed Scopus (121) Google Scholar In contrast, chemokine labeling by immunohistochemical measures is less reliable, because the attachment of immunoreactive epitopes to the extracellular matrix leads to a high background signal. This problem is of particular relevance in early wound healing lesions with the strong deposition of fibrin and fibrinogen. For this reason we used in situ hybridization to identify and localize chemokine mRNA expression and immunohistochemistry to detect the distribution of leukocyte subsets. We demonstrate in this study that during normal healing of adult skin wounds, distinct repertoires of chemokines are expressed. These repertoires correlate spatially and temporally with the phase-specific recruitment and trafficking of neutrophils, macrophages, and lymphocytes. After obtaining informed consent from each of 14 healthy adult volunteers of Caucasian origin, incisions (n = 4–7) 5 mm deep and 5 mm long were made on the ulnar forearm. The volunteers were 8 women and 6 men ranging in age from 25–58 years (mean, 39.9 years). Pending collection of biopsy specimens after various time periods, wounds were covered with sterile dressing. The study was approved by the Ethics Commission at the University of Würzburg and performed according to the Declarations of Helsinki and Tokyo. At defined time intervals after wounding (1, 2, 4, 7, 10, 14, and 21 days), 5-mm punch biopsies were obtained under local anesthesia. Biopsy specimens from healthy volunteers (n = 6) of nonwounded skin were used as controls. The tissue samples were placed in optimal cutting temperature compound (Tissue-Tek, Miles Scientific, Naperville, IL) immediately after excision, frozen, and stored at −80°C. Cryostat sections measuring 5 μm were prepared on gelatine-coated slides (Merck, Darmstadt, Germany) for immunohistology and on polyL-lysin-coated slides (Sigma, Deisenhofen, Germany) forin situ hybridization, respectively. After air-drying, sections were fixed in acetone (10 minutes at 4°C) for immunohistochemistry or in 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 minutes at room temperature (RT) for in situ hybridization. For immunohistological staining the following mouse mAbs were used at the indicated dilutions: anti-CD3 (1:100; Becton Dickinson, Sunnyvale, CA), reacting with the T cell receptor-associated CD3 antigen; anti-CD68 (1:1000; Dako, Hamburg, Germany), reacting with monocytes and macrophages; anti-neutrophil elastase (1:200; Dako), specific for neutrophils; MiB-1 (1:200; Dianova, Hamburg, Germany), detecting the Ki-67 proliferation antigen, EN4 (1:500; Sera Lab, Crawley Down, UK) specific for endothelial cells, anti-GROα (1:50; R+D Systems, Minneapolis, MN), anti-MCP-1 (1:50; R+D Systems), anti-MIP-1α (1:20; Promega, Madison, WI), and anti-RANTES (1:50; R+D Systems). The following antisera were used: anti-Mig (rabbit antiserum, 1:200), raised against denatured human Mig (kindly provided by J. M. Farber, Laboratory for Clinical Investigation, National Institutes of Health, Bethesda, MD), and anti-IP-10 (goat antiserum, 1:500), raised against denatured human IP-10 (R+D Systems). Biotin-conjugated sheep anti-mouse immunoglobulin (1:200; Amersham, Braunschweig, Germany), sheep anti-rabbit immunoglobulin (1:200; Jackson ImmunoResearch, West Grove, PA) and sheep anti-goat immunoglobulin (1:200; Jackson ImmunoResearch) were used as the secondary antibody. For immunohistochemical staining a three-step streptavidin-biotin-peroxidase procedure was used.40Ritter U Moll H Bröcker E-B Velazco O Becker I Gillitzer R Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.J Infect Dis. 1996; 173: 699-709Crossref PubMed Scopus (107) Google Scholar First, slides were washed in 0.1% Tween 20 (Merck)/PBS (Sigma) and the nonspecific binding sites were blocked with 20% sheep serum (Dianova)/0.1% BSA (Merck)/PBS for 20 minutes at RT. Sections were then incubated with the primary antibody in 0.1% BSA/PBS at 4°C overnight. After intense washing in 0.1% Tween 20, the slides were incubated with the biotinylated sheep anti-mouse immunoglobulin as the secondary antibody for 1 hour at RT and, after further washing, were treated with streptABC-peroxidase (Dako) for 1 hour at RT. Labeling was visualized with 0.2 mg/ml 0.5% 3-amino-9-ethyl-carbazole (AEC) (Sigma) inN,N-dimethylformamide (Merck) and 0.005% H2O2 in acetate buffer (50 mmol/L, pH 5.0) at RT. Slides were finally counterstained with Papanicolaou's solution 1b (Merck). For control purposes, the first mAb was omitted and replaced by an isotype-matched antibody to control for nonspecificity. The cDNA probes used for in situ hybridization were kindly provided by T. Yoshimura (National Cancer Institute, Frederick, MD; MCP-1), C. Müller (University of Bern, Bern, Switzerland; MCP-3 and ENA-78), Genetics Institute (Cambridge, MA; MIP-1α), T. Schall (DNAX, Palo Alto, CA; RANTES, MIP-1β), A. Anisowicz (Dana Farber Cancer Institute, Boston, MA; GROα), C. Weissmann (University of Zürich, Zürich, Switzerland; IL-8), R. Kulke (University of Kiel, Kiel, Germany; IP-10), J. Farber (National Cancer Institute, Bethesda, MD; Mig), and M. Krangel (Duke University Medical Center, Durham, NC; I309). Subcloning of specific DNA fragments was performed in vectors containing SP6/T7 (pGem 02, Promega, Madison, WI) or T3/T7 promoters (Bluescript, Stratagene, La Jolla, CA) according to standard protocols.45Sambrook J Fritsch EF Maniatis T Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar In vitro transcription of sense and antisense probes was performed as previously described.40Ritter U Moll H Bröcker E-B Velazco O Becker I Gillitzer R Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.J Infect Dis. 1996; 173: 699-709Crossref PubMed Scopus (107) Google Scholar Briefly, plasmid DNA was linearized with appropriate restriction enzymes. Then35S-labeled sense and antisense probes were obtained by in vitro transcription using SP6, T3, or T7 polymerases (Boehringer Mannheim, Mannheim, Germany) together with ATP, GTP, CTP (Boehringer) and [35S]uridine triphosphate (Amersham) as substrates. Original linearized template cDNA was eliminated with deoxyribonuclease (Pharmacia, Uppsala, Sweden) and protein was removed by sequential phenol extraction steps. Then, alkaline hydrolysis of the 35S-labeled RNA probes was performed for 30–50 minutes at 60°C in a carbonate buffer (pH 10.2) according to the formula: time (minutes) = (L0 - Lf)/0.11 × L0 × Lf(L0 = initial length in kb pairs, Lf = final length in kb pairs).46Angerer LM Stoler M Angerer RC In situ hybridization with RNA-probes.in: Valentin K Eberwine J Barchas J In In situ hybridization: Application to CNS. Oxford University Press, New York1987: 42-70Google Scholar After several ethanol precipitation steps, the radioactive riboprobe was adjusted to the specific activity of 1 × 106 cpm/μl in 0.01 mol/L Tris-HCl (pH 7.5), supplemented with 1 mmol/L EDTA. In situ hybridization was done as previously described.40Ritter U Moll H Bröcker E-B Velazco O Becker I Gillitzer R Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.J Infect Dis. 1996; 173: 699-709Crossref PubMed Scopus (107) Google Scholar Paraformaldehyde-fixed cryostat sections were treated with proteinase K (Boehringer,) (1 μg/ml) for 30 minutes at 37°C, refixed in 4% paraformaldehyde in PBS (30 minutes, RT), acetylated with acetic anhydride in 0.1 mol/l triethanolamine (pH 8.0, 10 minutes), dehydrated in graded concentrations of alcohol, and air-dried. Afterwards, sections were overlaid with 20 μl of hybridization solution containing 50% formamide, 300 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 8.0), 5 mmol/L EDTA, 1 × Denhardt's solution, 10% dextran sulfate, 100 mmol/L DTT, and 2 × 105 cpm heat-denatured radioactive sense or antisense probes per μl. The slides were mounted with coverslips, sealed, and hybridized at 46°C for 12–16 hours. Antisense and sense (negative control) probes were hybridized with at least two sections of the same biopsy. After hybridization, nonhybridized probes were removed by several high-stringency washing procedures with 50% formamide solution containing 2 × SSC buffer (Sigma) and 5 mmol/L EDTA at 54–57°C. To minimize the background signal, noncomplementary unhybridized single-stranded probe RNA was digested with RNase A (20 μg/ml) and RNase T1 (1 U/μl, Boehringer) for 30 minutes at 37°C. For autoradiography, slides were dipped in NTB-2 Kodak solution (1:2 in 800 mmol/L ammonium acetate), air-dried, and exposed for 1–5 weeks at 4°C. Slides were then counterstained with Papanicolaou's solution 1b. As control tissues for chemokine expression, we used lesions of psoriasis (GROα, IL-8, IP-10, Mig, and MCP-1), leishmaniasis, leprosy (MIP-1α and MIP-1β), and lichen planus (RANTES). A Zeiss Axiophot microscope equipped with dark-field illumination (Carl Zeiss, Oberkochen, Germany) was used for evaluation and documentation. Positive cells were counted with an ocular square grid (Carl Zeiss) in one half of the symmetrical wound bed (magnifications, ×250 and ×400) and related to the total number of cells in the area. We examined 3 to 14 individual wounds for each time point. The average percentage of mRNA-expressing or -stained cells was determined and expressed as mean ± SEM. For evaluation of microvascular endothelium development, we counted the number of vessels in 2–3 random counting fields (magnification, ×200) within the wound bed and calculated the average number of vessels per counting field. In the first series of experiments, biopsies from incisional adult skin wounds 5 mm long and 5 mm deep taken at day 0, 1, 2, 4, 7, 10, 14, and 21 were evaluated for their histological features and time of wound closure, as determined by complete re-epithelialization of the wound gap with keratinocytes. In five of eleven individuals, wound closure was already completed after 48 hours (day 2), and in six patients closure took place between days 2 and 4. No obvious differences in wound healing between adult volunteers aged 20–30 (n = 5) and those aged 40–60 (n = 4) were seen. After day 4, a hypertrophic neo-epidermis developed over the previously denuded wound surface. Immunostaining with anti-leukocyte mAb for CD45+cells showed maximum leukocyte accumulation within the first 24 hours and a constant level until day 4. Afterwards, the number of CD45+ cells slowly and constantly decline in the next 3 weeks (data not shown). However, the total number of cells after a dramatic increase between day 0 and day 1 remained rather constant (Figure 1), indicating that the slow decline of inflammatory cells after day 4 (wound closure in all lesions) is accompanied by the proliferation of resident cells as fibroblasts and endothelial cells. Immunohistological labeling with leukocyte subtype-specific mAbs revealed a strong dominance of NE+ neutrophils during the first days, reaching a maximum level at day 1 (44 ± 3% of total cells; mean ± SEM) (Figure 1). The neutrophils concentrated in a rim of densely packed cells contained within the superficial part of the wound defect (Figure 2E), but their relative percentage decreased rapidly on wound closure (after days 2–4). In contrast to neutrophils, CD68+ macrophages reached their maximum level at day 2 (30 ± 8%) (Figure 1). The macrophages were distributed within and near the wounded area with preferential accumulation around dermal vessels. After day 2, the number of macrophages slowly declined. Lymphocytes were present at relatively constant levels (12–18%) (Figure 1) and, like macrophages, accumulated preferentially around superficial dermal vessels. The local appearance of lymphocytes was sl
Referência(s)