FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W98461984> ?p ?o ?g. }

• W98461984 endingPage "147" @default.
• W98461984 startingPage "132" @default.
• W98461984 abstract "Whether the wound macrophage is a key regulatory inflammatory cell type in skin repair has been a matter of debate. A transgenic mouse model mediating inducible macrophage depletion during skin repair has not been used to date to address this question. Here, we specifically rendered the monocyte/macrophage leukocyte lineage sensitive to diphtheria toxin by expressing the lysozyme M promoter-driven, Cre-mediated excision of a transcriptional STOP cassette from the simian DT receptor gene in mice (lysM-Cre/DTR). Application of diphtheria toxin to lysM-Cre/DTR mice led to a rapid reduction in both skin tissue and wound macrophage numbers at sites of injury. Macrophage-depleted mice revealed a severely impaired wound morphology and delayed healing. In the absence of macrophages, wounds were re-populated by large numbers of neutrophils. Accordingly, macrophage-reduced wound tissues exhibited the increased and prolonged persistence of macrophage inflammatory protein-2, macrophage chemoattractant protein-1, interleukin-1β, and cyclooxygenase-2, paralleled by unaltered levels of bioactive transforming growth factor-β1. Altered expression patterns of vascular endothelial growth factor on macrophage reduction were associated with a disturbed neo-vascularization at the wound site. Impaired wounds revealed a loss of myofibroblast differentiation and wound contraction. Our data in the use of lysM-Cre/DTR mice emphasize the pivotal function of wound macrophages in the integration of inflammation and cellular movements at the wound site to enable efficient skin repair. Whether the wound macrophage is a key regulatory inflammatory cell type in skin repair has been a matter of debate. A transgenic mouse model mediating inducible macrophage depletion during skin repair has not been used to date to address this question. Here, we specifically rendered the monocyte/macrophage leukocyte lineage sensitive to diphtheria toxin by expressing the lysozyme M promoter-driven, Cre-mediated excision of a transcriptional STOP cassette from the simian DT receptor gene in mice (lysM-Cre/DTR). Application of diphtheria toxin to lysM-Cre/DTR mice led to a rapid reduction in both skin tissue and wound macrophage numbers at sites of injury. Macrophage-depleted mice revealed a severely impaired wound morphology and delayed healing. In the absence of macrophages, wounds were re-populated by large numbers of neutrophils. Accordingly, macrophage-reduced wound tissues exhibited the increased and prolonged persistence of macrophage inflammatory protein-2, macrophage chemoattractant protein-1, interleukin-1β, and cyclooxygenase-2, paralleled by unaltered levels of bioactive transforming growth factor-β1. Altered expression patterns of vascular endothelial growth factor on macrophage reduction were associated with a disturbed neo-vascularization at the wound site. Impaired wounds revealed a loss of myofibroblast differentiation and wound contraction. Our data in the use of lysM-Cre/DTR mice emphasize the pivotal function of wound macrophages in the integration of inflammation and cellular movements at the wound site to enable efficient skin repair. A comprehensive set of different strategies has been used during the past three decades to explore the function and impact of inflammatory cell lineages on tissue regeneration processes in skin wounds. The field was opened by two pioneer studies by Simpson and Ross1Simpson DM Ross R The neutrophilic leukocyte in wound repair: a study with antineutrophil serum.J Clin Invest. 1972; 51: 200-223Google Scholar and Leibovich and Ross.2Leibovich SJ Ross R The role of the macrophage in wound repair: a study with hydrocortisone and antimacrophage serum.Am J Pathol. 1975; 78: 71-100PubMed Google Scholar Both classic experiments assessed neutrophil and macrophage functions: neutrophil depletion did not impact on healing, but depletion of macrophages resulted in an impaired clearance of wound tissue and a severe disturbance of the healing process. These early studies provided the insight that the presence of macrophages at the wound site might appear central to skin repair. This notion was supported by more recent studies. Inactivation of the chemokine macrophage inflammatory protein (MIP)-1α,3DiPietro LA Burdick M Low QE Kunkel SL Strieter RM MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.J Clin Invest. 1998; 101: 1693-1698Crossref PubMed Scopus (241) Google Scholar as well as genetic knock-out of the CXC chemokine receptor 24Devalaraja RM Nanney LB Du J Qian Q Yu Y Devalaraja MN Richmond A Delayed wound healing in CXCR2 knockout mice.J Invest Dermatol. 2000; 115: 234-244Crossref PubMed Scopus (323) Google Scholar markedly reduced wound macrophage numbers and delayed wound healing by impairing angiogenesis and collagen deposition. Proper activation of wound macrophages appeared to be also of importance: apoptotic neutrophils induced secretion of transforming growth factor (TGF)-β1 from macrophages, which is necessary to subsequently drive myofibroblast differentiation and wound contraction.5Peters T Sindrilaru A Hinz B Hinrichs R Menke A Al-Azzeh EA Holzwarth K Oreshkova T Wang H Kess D Walzog B Sulyok S Sunderkötter C Friedrich W Wlaschek M Krieg T Scharffetter-Kochanek K Wound healing defect of CD18−/− mice due to a decrease in TGF-β1 and myofibroblast differentiation.EMBO J. 2005; 24: 3400-3410Crossref PubMed Scopus (128) Google Scholar Moreover, macrophage-chemoattractant protein (MCP)-1 deficient mice exhibited unaltered numbers of wound macrophages on wounding, but showed a delayed re-epithelialization, angiogenesis, and collagen synthesis.6Low QE Drugea IA Duffner LA Quinn DG Cook DN Rollins BJ Kovacs EJ DiPietro L Wound healing in MIP-1 alpha (−/−) and MCP-1 (−/−) mice.Am J Pathol. 2001; 159: 457-463Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar At present, the wound macrophage is established as a key player in undisturbed cutaneous wound healing, mainly in its function as a rich source of mediators at the wound site.7Martin P Wound healing - aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3791) Google Scholar, 8Singer AJ Clark RAF Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4740) Google Scholar, 9Werner S Grose R Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2667) Google Scholar, 10Eming SA Krieg T Davidson JM Inflammation in wound repair: molecular and cellular mechanisms.J Invest Dermatol. 2007; 127: 514-525Crossref PubMed Scopus (1482) Google Scholar A set of studies recently came out that might carry the potential to question the fundamental role of the wound macrophages in skin repair. One of those studies demonstrated the ‘paradoxical’ finding of an improved wound healing on interference with TGF-β1 signaling using a SMAD-3 deficiency. Wounds of SMAD-3 knock-out mice were characterized by a reduced local infiltration of monocytes associated with an accelerated re-epithelialization.11Ashcroft GS Yang X Glick AB Weinstein M Letterio JL Mizel DE Anzano M Greenwell-Wild T Wahl SM Deng C Roberts AB Mice lacking Smad3 show accelerated wound healing an impaired local inflammatory response.Nat Cell Biol. 1999; 1: E117-E119Crossref PubMed Scopus (36) Google Scholar In addition, disruption of tumor necrosis factor-α signaling led to a significant reduction in wound macrophages and an accelerated repair.12Mori R Kondo T Ohshima T Ishida Y Mukaida N Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration.FASEB J. 2002; 16: 963-974Crossref PubMed Scopus (224) Google Scholar More important, studies from embryos and the PU.1 transcription factor knock-out mouse support the notion of an ambivalent role of macrophage function in skin repair. Wounds in embryos healed without excessive inflammation and scarring as long as the monocyte lineage has not developed.13Hopkinson-Woolley J Hughes D Gordon S Martin P Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse.J Cell Sci. 1994; 107: 1159-1167PubMed Google Scholar Loss of PU.1 results in the loss of macrophages and functional neutrophils in mice. However, wounds from PU.1 null mice showed a scar-free, ‘embryo-like’ healing without inflammation.14Martin P D'Souza D Martin J Grose R Cooper L Maki R McKercher SR Wound healing in the PU. 1 null mouse – tissue repair is not dependent on inflammatory cells.Curr Biol. 2003; 13: 1122-1128Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar These important studies might suggest that the inflammatory response on wounding might only serve the function to prevent infection for the price of an impaired, fibrotic repair and formation of a scar. However, having a second glance on the above mentioned classic and more recent studies on macrophage function in wound healing, some difficulties in the interpretation of findings become obvious: the anti-inflammatory corticosterone has been used to fully deplete macrophages from wounds in the early studies,2Leibovich SJ Ross R The role of the macrophage in wound repair: a study with hydrocortisone and antimacrophage serum.Am J Pathol. 1975; 78: 71-100PubMed Google Scholar wounds had to be kept sterile to enable normal healing,1Simpson DM Ross R The neutrophilic leukocyte in wound repair: a study with antineutrophil serum.J Clin Invest. 1972; 51: 200-223Google Scholar, 14Martin P D'Souza D Martin J Grose R Cooper L Maki R McKercher SR Wound healing in the PU. 1 null mouse – tissue repair is not dependent on inflammatory cells.Curr Biol. 2003; 13: 1122-1128Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar or transgenic knock-out animals must always be considered to potentially develop a compensatory adaptation. To circumvent these problems, we have used a transgenic mouse (lysM-Cre/DTR) containing diphtheria-toxin (DTox)-sensitive macrophages to assess macrophage functions in wound healing. LysM-Cre/DTR mice express a normal phenotype in the absence of the toxin.15Buch T Heppner FL Tertilt C Heinen TJ Kremer M Wunderlich FT Jung S Waisman A A Cre-inducible diphteria toxin receptor mediates cell lineage ablation after toxin administration.Nat Methods. 2005; 2: 419-426Crossref PubMed Scopus (611) Google Scholar Application of DTox allowed a simple, inducible, and rapid depletion of macrophages from wound tissue, thus circumventing problems that might arise from conventional knock-out strategies. In this study, we show the functionality of the lysM-Cre/DTR mouse model to investigate macrophage functions in skin repair. Our data strengthen the evidence that macrophages indeed represent an essential prerequisite for overall tissue regeneration. Using the loxP system, we established a conditional myeloid cell linage-specific expression of the DTox receptor (DTR) by crossing C57Bl/6-ROSA26STOP*DTR mice15Buch T Heppner FL Tertilt C Heinen TJ Kremer M Wunderlich FT Jung S Waisman A A Cre-inducible diphteria toxin receptor mediates cell lineage ablation after toxin administration.Nat Methods. 2005; 2: 419-426Crossref PubMed Scopus (611) Google Scholar with C57Bl/6-LysM-Cre mice.16Clausen BE Burkhardt C Reith W Renkawitz R Förster I Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1592) Google Scholar Mice were kept in barrier and specific pathogen-free animal facilities according to the German Tierschutzgesetz. DNA for genotyping was prepared from tail biopsies. Presence of different alleles was assessed by PCR using the following primer pairs: for wild-type (wt)ROSA26S (600 bp amplicon) 5′-AAAGTCGCTCTGAGTTGTTAT-3′ and 5′-GGAGCGGGAGAAATGGATATG-3′, for ROSA26S/DTR (242 bp amplicon) 5′-AAAGTCGCTCTGAGTTGTTAT-3′ and 5′-CATCAAGGAAACCCTGGACTACTG-3′, for wild-type (wt) lysM (350 bp amplicon) 5′-CTTGGGCTGCCAGAATTTCTC-3′ and 5′-TTACAGTCGGCCAGGCTGAC-3′, and lysM-Cre (700 bp amplicon) 5′-CTTGGGCTG CCAGAATTTCTC-3′ and 5′-CCCAGAAATGCCAGATTACG-3′. At the age of 12 weeks, mice were caged individually, monitored for body weight and wounded as described below. For cell-type specific cell ablation, age- and sex-matched lysM-Cre/DTR mice were injected intraperitoneally for three consecutive days with 100 ng of recombinant DTox (Calbiochem, Darmstadt, Germany) before wounding. Injection of PBS was used as a control. After wounding, mice were injected with DTox or PBS every second day. Topical treatment of lysM-Cre/DTR mice was performed by application of recombinant DTox (25 ng in 20 μl PBS/5% dimethylsulfoxide) directly onto wound sites at days 0, 2, and 4 after wounding. Mice were injected intraperitoneally with a single injection of 0.5 ml of sterile thioglycollate (Sigma, Taufkirchen, Germany) solution (6% w/v in H2O) to induce peritonitis. At 60 hours after injection, mice were sacrificed to isolate cells from the peritoneal cavity. Wounding of mice was performed as described previously.17Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Frank S The function of nitric oxide in wound repair: inhibition of inducible nitric oxide synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (197) Google Scholar, 18Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (215) Google Scholar Briefly, mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg body weight)/xylazine (10 mg/kg body weight). The hair on the back of each mouse was cut, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (5 mm in diameter, 3 to 4 mm apart) were made on the back of each mouse by excising the skin and the underlying panniculus carnosus. The wounds were allowed to form a scab. Skin biopsy specimens were obtained from the animals 1, 3, 5, 7, and 13 days after injury. At each time point, an area that included the scab, the complete epithelial and dermal compartments of the wound margins, the granulation tissue, and parts of the adjacent muscle and subcutaneous fat tissue was excised from each individual wound. As a control, a similar amount of skin was taken from the backs of nonwounded mice. Wounds (n = 15) isolated from animals (n = 5) were used for RNA analysis. For immunoblot analysis, wounds (n = 10) from individual mice (n = 5) were used. All animal experiments were performed according to the guidelines and approval of the local Ethics Animal Review Board. RNA isolation and RNase protection assays were performed as described previously.18Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (215) Google Scholar, 19Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar If not indicated otherwise, every experimental time point depicts 15 wounds (n = 15) isolated from five individual mice (n = 5) for all RNase protection assays analyzing wound tissues samples. All samples were quantified using PhosphoImager PSL counts per 15 μg of total wound RNA. Glyceraldehyde phosphate dehydrogenase (GAPDH) hybridization is shown as a loading control and 1000 cpm of the hybridization probe were used as a size marker. Hybridization against tRNA was used to show the specificity of the probe. The murine cDNA probes were cloned using reverse transcriptase-polymerase chain reaction. The probes corresponded to nucleotides (nt) 181 to 451 (for MIP-2, GenBank accession number NM009140), nt 63 to 323 (for MCP-1, NM011333), nt 816 to 1481 (for lipocalin, X81627), nt 425 (exon 1) to 170 (exon 2) (for lysozyme M, M21047), nt 481 to 739 (for interleukin [IL]-1β, NM008361), nt 796 to 1063 (for Cox-2, M64291), nt 2561 to 2783 (for epidermal growth factor[EGF]-like module-containing, mucin-like, hormone receptor-like sequence [Emr]−1, X93328), nt 139 to 585 (for vascular endothelial growth factor [VEGF], S38083), nt 912 to 1183 (for α-smooth muscle actin [α-SMA], BC064800.1), nt 1335 to 1594 (for extra type III domain A [ED-A] fibronectin, AF095690), nt 1274 to 1546 (for TGF-β1, NM-011577), or nt 163 to 317 (for murine GAPDH, NM002046) of the published sequences. Quantitative real-time PCR (qRT-PCR) was performed to assess the expression of genes of interest in wound tissue at day 7 post-wounding. Changes in fluorescence are caused by the Taq polymerase degrading the probe that contains a fluorescent dye (6-carboxyfluorescein for the genes of interest, VIC for GAPDH) and a quencher (6-carboxytetramethylrhodamine). Pre-designed qRT-PCR assays were purchased at Applied Biosystems (Darmstadt, Germany): Mm00436450-m1 (for MIP-2), Mm00441242-m1 (for MCP-1), Mm01324470-m1 (for lipocalin), Mm00802529-m1 (for Emr-1), Mm00478374-m1 (for Cox-2), or Mm00437404-m1 (for VEGF). The possibility of amplification of contaminating genomic DNA was eliminated by the fact that amplicons crossed an exon/intron boundary. For GAPDH, pre-developed assay reagents were used (4352339E) (Applied Biosystems, Darmstadt, Germany). 1.0 μg of total RNA from day 7 wound tissue was transcribed using random hexameric primers and Superscript II RT (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. qRT-PCR was performed on 7500 Fast real-time PCR system (Applied Biosystems) as follows: one initial step at 95°C for 20 seconds was followed by 40 cycles at 95°C for 3 seconds and 60°C for 30 seconds. Detection of the dequenched probe, calculation of threshold cycles (Ct values), and further analysis of these data were performed by the Sequence Detector software. All results for gene expression were normalized to that of GAPDH. Reverse transcription (RT) PCR was performed to assess the expression of lysozyme M, Cre recombinase and GAPDH in isolated murine immune cell fractions. 1.0 μg of total RNA from the respectice immune cell fraction was transcribed using random hexameric primers and Superscript II RT (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. 5′-ATTGCAGTGCTCTGCTGC-3′ and 5′-GTGAGAAAGAGACCGAATG-3′ (for lysM), 5′-GTGTCCAATTTACTGACCG-3′ and 5′-GTTTTTACTGCCAGACCGC-3′ (for Cre) or 5′-CTGGCATTGCTCTCAATGAC-3′ and 5′-TCTTACTCCTTGGAGGCC-3′ (for GAPDH) primers were used to amplify the murine sequences. PCR was performed on a T3000-Thermocycler (Biometra, Göttingen, Germany). Skin and wound tissue biopsies were homogenized in lysis buffer (1% Triton X-100, 20 mmol/L Tris/HCl pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1 mmol/L dithiothreitol, 10 mmol/L NaF, 2 mmol/L Na3VaO4, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonylfluoride, 5 ng/ml aprotinin, 5 ng/ml leupeptin). Wound lysates for cell culture experiments were homogenized in PBS. Extracts were cleared by centrifugation. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce Inc., Rockford, IL). Fifty micrograms of total protein from skin lysates were separated using SDS gel electrophoresis. After transfer to a nitrocellulose membrane, specific proteins were detected using antisera raised against Ly-6G (GR-1) (BD Biosciences, Heidelberg, Germany), Cox-2 (Cayman, Ann Arbor, MI), α-SMA (Dako, Glostrup, Denmark), and β-actin (Sigma). A secondary antibody coupled to horseradish peroxidase and the enhanced chemiluminescence detection system was used to visualize the proteins. Phenylmethylsulfonyl fluoride, dithiothreitol, aprotinin, NaF, and Na3VaO4 were from Sigma. Leupeptin and ocadaic acid were from BioTrend (Köln, Germany). The enhanced chemiluminescence detection system was obtained from Amersham (Freiburg, Germany). Quantification of murine MIP-2, MCP-1, IL-1β, or VEGF165 protein was performed using the respective murine quantikine enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Wiesbaden, Germany) according to the instructions of the manufacturer. Human IL-8 ELISA kit was obtained from BD Biosciences. Wounding of mice was performed as described above. Animals were sacrificed following the 3-day DTox pretreatment or at day 5 and 7 after injury. Biopsies from non-wounded skin and skin wounds were isolated from the back, fixed in formalin or in zinc fixative solution (0.05% CaAc.2H2O, 0.5 ZnAc.2H2O, 0.5% ZnCl2 in 0.1 M/L Tris-Cl, pH 7.4) and subsequently embedded in paraffin. Four-micrometer sections were counterstained with H&E to document overall wound morphology. Sections were subsequently incubated over night at 4°C with antisera raised against murine MIP-2 (R&D), Cox-2 (Biomol, Hamburg, Germany), Ly-6G (Gr-1), F4/80 (AbD Serotec, Düsseldorf, Germany), processed active caspase-3 (DCS Inc., Hamburg, Germany), VEGF (Santa Cruz, Heidelberg, Germany), CD31 (Chemicon, Eschborn, Germany), or α-SMA (Sigma). Primary antibodies were detected using a biotinylated secondary antibody. The slides were subsequently stained with the avidin-biotin-peroxidase complex system (Santa Cruz) using 3,3-diaminobenzidine tetrahydrochloride or Fast Red Substrate-Chromogen System (Dako, Hamburg, Germany) as a chromogenic substrates. Finally, sections were counterstained with hematoxylin and mounted. Isolated femur and tibia from mice were flushed out using BMDC medium (Gibco, Invitrogen) to obtain the bone marrow content for cell culture. Cells were collected by centrifugation (250 × g), and 2 × 106 cells per dish (10 cm) were plated. Cells were cultured in the presence of a granulocyte-monocyte colony-stimulating factor containing hybridoma cell culture supernatant (5% v/v) to obtain non-adherent and adherent cell fractions. Isolated cell fractions were analyzed by flow cytometry (see below). Primary keratinocytes were isolated from skin of newborn C57Bl/6J mice (1 to 3 days) by overnight incubation using dispase (2.4 U/ml, 4°C). After dispase (Roche Biochemicals, Mannheim, Germany) digestion, the epidermis could be isolated and was subsequently treated with trypsin (0.04%)/EDTA (0.03%) for additional 7 minutes to disaggregate cells. After filtration (70 μm; Falcon, Becton Dickinson Labware), keratinocytes were collected by centrifugation (210 × g, 2 minutes) and plated into collagen IV-coated dishes. Confluent murine keratinocytes were treated with a combination of IL-1β (40 ng/ml), tumor necrosis factor-α (25 ng/ml), and interferon-γ (20 ng/ml) in the presence or absence of TGF-β1 (5 ng/ml). The human keratinocyte cell line HaCaT20Boukamp P Petrussevska RT Breitkreutz D Hornung J Markham A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar was cultured in Dulbecco’s Modified Eagle Medium containing 10% (v/v) fetal calf serum. Quiescent confluent keratinocytes were subsequently stimulated using freshly prepared 5-day wound tissue homogenates (f.c: 100 μg of total protein per ml) isolated from lysM-Cre/DTR in the presence or absence of a neutralizing anti-TGF-β1-3 antibody (clone 1D11) (100 μg/ml) or IL-1 receptor antagonist (IL-1ra) (500 ng/ml) for 24 hours. Dulbecco’s Modified Eagle Medium and fetal calf serum were from Gibco (Invitrogen), cytokines were purchased from Roche, anti-TGF-β1-3 and IL-1ra were from R&D systems. Polymorphonuclear neutrophils were freshly isolated from mouse blood using a standard protocol. Briefly, 400 μl ACD (3% w/v citric acid, 6% w/v sodium citrate, 4% dextrose) were added to 2 ml of mouse blood; 1.2 ml of 6% dextran in 0.9% NaCl was added subsequently and blood was incubated for 1 hour at room temperature. The supernatant was recovered from the settled blood and centrifuged (280 × g). The cellular pellet was suspended in 1.2 ml ice-cold H2O, and 400 μl of 0.6 M/L KCl was added. Mixture was centrifuged to remove red blood cells. Red blood cell-free cells were again centrifuged through a Ficoll-Histopaque (Sigma) gradient to finally isolate a pure neutrophil cellular pellet. The isolated cell fraction was analyzed for the expression of Ly6G (GR-1) (BD Biosciences). Two hundred microliters of blood from lysM-Cre/DTR mice were collected in 5 ml RPMI medium. Blood cells were harvested by centrifugation and treated with 5 ml lysis buffer (0.83% NH4CL) for 5 minutes to lyse erythrocytes. The remaining leukocytes were washed in 5 ml of fresh RPMI medium. Leukocytes were collected and resuspended in 3 ml staining buffer (PBS supplemented with 1% v/v fetal calf serum and 0.1% Na3N). The resuspended pellet was subsequently incubated in 50 μl of phycoerythin-conjugated rat anti-mouse F4/80 (AbD Serotec) in staining buffer at 4°C for 30 minutes, followed by two additional washing steps in 3 ml of staining buffer. Cells were fixed (fluorescence-activated cell sorting [FACS] buffer containing 1% paraformaldehyde) and analyzed for surface F4/80 expression using a FACSCalibur flow cytometer (BD Biosciences). Monocyte and macrophage cell fractions obtained from cultured bone marrow cells or by peritoneal lavage were analyzed using Cy7-conjugated CD11b (mac1), phycoerythin-conjugated F4/80, and fluorescein isothiocyanate-labeled Ly6C (BD Biosciences). TGF-β bioactivity was determined using an in vitro assay based on mink lung epithelial cells containing a truncated TGF-β sensitive plasminogen activator inhibitor-1 promoter fused to the firefly luciferase reporter gene.21Abe M Harpel JG Metz CN Nunes I Loskutoff DJ Rifkin DB An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.Anal Biochem. 1994; 216: 276-284Crossref PubMed Scopus (676) Google Scholar Wounds were homogenized in PBS and mink cells were stimulated with wound lysates (400 μg lysate per ml) in the presence or absence of a TGF-β neutralizing antibody (clone 1D11) (100 μg/ml) (R&D systems). After 20 hours, cells were washed and lysed in 100 μl lysis buffer (Promega, Mannheim, Germany) to determine the induced luciferase activity using a standard protocol. Data are shown as means ± SD. Data analysis was performed using the unpaired Student’s t-test with raw data. Transgenic expression of the DTR, which is identical to the simian/human heparin-binding epidermal growth factor-like growth factor precursor,22Naglich JG Metherall JE Russell DW Eidels L Expression cloning of a diphteria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.Cell. 1992; 69: 1051-1061Abstract Full Text PDF PubMed Scopus (470) Google Scholar represents a molecule to render naturally DTox-resistant mouse cells23Middlebrook JL Dorland RB Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphteriae: differential cytotoxicity.Can J Microbiol. 1977; 23: 183-189Crossref PubMed Scopus (84) Google Scholar susceptible to DTox.24Saito M Iwawaki T Taya C Yonekawa H Noda M Inui Y Mekada E Kimata Y Tsuru A Kohno K Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.Nature Biotechnol. 2001; 19: 746-750Crossref Scopus (364) Google Scholar Here we crossed the ROSA26STOP*DTR transgenic mouse strain,15Buch T Heppner FL Tertilt C Heinen TJ Kremer M Wunderlich FT Jung S Waisman A A Cre-inducible diphteria toxin receptor mediates cell lineage ablation after toxin administration.Nat Methods. 2005; 2: 419-426Crossref PubMed Scopus (611) Google Scholar harboring the simian DTR (simian HB-EGF), containing a loxP-flanked transcriptional STOP cassette, under control of the ROSA26 locus,25Zambrowicz BP Imamoto A Fiering S Herzenberg LA Kerr WG Soriano P Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells.Proc Natl Acad Sci USA. 1997; 94: 3789-3794Crossref PubMed Scopus (710) Google Scholar into lysM-Cre mice. Cre-mediated excision of the STOP cassette in lysM-Cre/DTR double-transgenic mice subsequently allows the expression of a functional DTR in myeloid cells in the animals16Clausen BE Burkhardt C Reith W Renkawitz R Förster I Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1592) Google Scholar, 26Faust N Varas F Kelly LM Heck S Graf T Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.Blood. 2000; 96: 719-726Crossref PubMed Google Scholar(Figure 1A). Genotyping by PCR revealed a decisive amplicon pattern to allow selection of lysM-Cre/DTR double-transgenic mice (see supplemental Figure S1 available at http://ajp.amjpathol.org). Here it is noteworthy that we selected those lysM-Cre/DTR transgenic mice for subsequent studies, which were characterized by the insertion of only one allele of the lysM-Cre construct to allow functional expression of endogenous lysozyme M in the animals from the second allele. To assess the functionality of the inducible DTR-driven cell ablation sytem in vivo, we analyzed the response of the monocyte/macrophage lineage on DTox injection into transgenic lysM-Cre/DTR mice. As shown in Figure 1B, we could not observe a significant reduction in circulating monocyte numbers on DTox administration, which had been injected for three consecutive days before analysis. A reduced lysozyme M promoter activity in monocytes might, at least partially, contribute to ineffective levels of Cre re" @default.
• W98461984 created "2016-06-24" @default.
• W98461984 creator A5002634222 @default.
• W98461984 creator A5025746046 @default.
• W98461984 creator A5030030681 @default.
• W98461984 creator A5063254755 @default.
• W98461984 creator A5072367499 @default.
• W98461984 creator A5074909982 @default.
• W98461984 creator A5076704114 @default.
• W98461984 creator A5084988203 @default.
• W98461984 creator A5089247997 @default.
• W98461984 date "2009-07-01" @default.
• W98461984 modified "2023-10-05" @default.
• W98461984 title "A Transgenic Mouse Model of Inducible Macrophage Depletion" @default.
• W98461984 cites W1483582136 @default.
• W98461984 cites W1487581481 @default.
• W98461984 cites W1498618829 @default.
• W98461984 cites W1582872317 @default.
• W98461984 cites W1595293335 @default.
• W98461984 cites W175241905 @default.
• W98461984 cites W1931315964 @default.
• W98461984 cites W1934754959 @default.
• W98461984 cites W1965434649 @default.
• W98461984 cites W1966999008 @default.
• W98461984 cites W1971248512 @default.
• W98461984 cites W1977289462 @default.
• W98461984 cites W1977993125 @default.
• W98461984 cites W1983346506 @default.
• W98461984 cites W1989021197 @default.
• W98461984 cites W1991342668 @default.
• W98461984 cites W2001356286 @default.
• W98461984 cites W2009482935 @default.
• W98461984 cites W2013537969 @default.
• W98461984 cites W2016193552 @default.
• W98461984 cites W2031408045 @default.
• W98461984 cites W2033260397 @default.
• W98461984 cites W2041591721 @default.
• W98461984 cites W2051347106 @default.
• W98461984 cites W2062093468 @default.
• W98461984 cites W2065871536 @default.
• W98461984 cites W2071247073 @default.
• W98461984 cites W2074585020 @default.
• W98461984 cites W2077733587 @default.
• W98461984 cites W2079238808 @default.
• W98461984 cites W2079517510 @default.
• W98461984 cites W2101771279 @default.
• W98461984 cites W2103957643 @default.
• W98461984 cites W2120520218 @default.
• W98461984 cites W2126853519 @default.
• W98461984 cites W2142460886 @default.
• W98461984 cites W2143464311 @default.
• W98461984 cites W2158943553 @default.
• W98461984 cites W2163470859 @default.
• W98461984 cites W2325727928 @default.
• W98461984 cites W2403010988 @default.
• W98461984 cites W4250007760 @default.
• W98461984 cites W4294216491 @default.
• W98461984 doi "https://doi.org/10.2353/ajpath.2009.081002" @default.
• W98461984 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2708801" @default.
• W98461984 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19528348" @default.
• W98461984 hasPublicationYear "2009" @default.
• W98461984 type Work @default.
• W98461984 sameAs 98461984 @default.
• W98461984 citedByCount "313" @default.
• W98461984 countsByYear W984619842012 @default.
• W98461984 countsByYear W984619842013 @default.
• W98461984 countsByYear W984619842014 @default.
• W98461984 countsByYear W984619842015 @default.
• W98461984 countsByYear W984619842016 @default.
• W98461984 countsByYear W984619842017 @default.
• W98461984 countsByYear W984619842018 @default.
• W98461984 countsByYear W984619842019 @default.
• W98461984 countsByYear W984619842020 @default.
• W98461984 countsByYear W984619842021 @default.
• W98461984 countsByYear W984619842022 @default.
• W98461984 countsByYear W984619842023 @default.
• W98461984 crossrefType "journal-article" @default.
• W98461984 hasAuthorship W98461984A5002634222 @default.
• W98461984 hasAuthorship W98461984A5025746046 @default.
• W98461984 hasAuthorship W98461984A5030030681 @default.
• W98461984 hasAuthorship W98461984A5063254755 @default.
• W98461984 hasAuthorship W98461984A5072367499 @default.
• W98461984 hasAuthorship W98461984A5074909982 @default.
• W98461984 hasAuthorship W98461984A5076704114 @default.
• W98461984 hasAuthorship W98461984A5084988203 @default.
• W98461984 hasAuthorship W98461984A5089247997 @default.
• W98461984 hasBestOaLocation W984619841 @default.
• W98461984 hasConcept C102230213 @default.
• W98461984 hasConcept C104317684 @default.
• W98461984 hasConcept C141035611 @default.
• W98461984 hasConcept C142724271 @default.
• W98461984 hasConcept C202751555 @default.
• W98461984 hasConcept C203014093 @default.
• W98461984 hasConcept C2779244956 @default.
• W98461984 hasConcept C54355233 @default.
• W98461984 hasConcept C71924100 @default.
• W98461984 hasConcept C86803240 @default.
• W98461984 hasConcept C95444343 @default.

• next