Title: Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor
Abstract: Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this “stop and return” process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (−/−) mice. Even at 90 days, long after wound closure, wounds in the CXCR3−/− mice remained hypercellular and presented immature matrix components. The CXCR3−/− mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair. Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this “stop and return” process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (−/−) mice. Even at 90 days, long after wound closure, wounds in the CXCR3−/− mice remained hypercellular and presented immature matrix components. The CXCR3−/− mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair. Skin wound repair is a complex, highly orchestrated event consisting of an early hypercellular infiltrate that resolves over time, with loss of most of the regenerative-phase dermal fibroblasts and vascular conduits.1Clark RAF Epithelial-mesenchymal networks in wounds: a hierarchical view.J Invest Dermatol. 2003; 120: ix-xiCrossref PubMed Scopus (8) Google Scholar This reversion of the dermal cellularity is necessary for the maturation and strengthening of the matrix, which when lacking, leads to chronic wounds.2Jaffe AT Heymann WR Lawrence N Epidermal maturation arrest.Dermatol Surg. 1999; 25: 900-903Crossref PubMed Scopus (12) Google Scholar This leaves open the question of which signals define both the transition from regeneration to resolution and the cellular involution that accompanies these changes. Wound repair requires the ordered immigration of fibroblasts into the provisional matrix and keratinocytes over this matrix. This immigration and replacement of the tissue appears to be under the influence of both soluble factors secreted first by platelets and then by inflammatory cell infiltrates, and also matrix components produced by these cells and the immigrated fibroblasts and endothelial cells. Among the latter, tenascin-C and thrombospondins seem to play a major role and thereby mark the immature, regenerative phase of wound healing.3Harty M Neff AW King MW Mescher AL Regeneration or scarring: an immunologic perspective.Dev Dyn. 2003; 226: 268-279Crossref PubMed Scopus (207) Google Scholar, 4Midwood KS Schwarzbauer JE Tenascin-C modulates matrix contraction via focal adhesion kinase- and rho-mediated signaling pathways.Mol Biol Cell. 2002; 13: 3601-3613Crossref PubMed Scopus (121) Google Scholar, 5Esemuede N Lee T Pierre-Paul D Sumpio BE Gahtan V The role of thrombospondin-1 in human disease.J Surg Res. 2004; 122: 135-142Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar These influence the functionality of the vasculogenesis by acting, directly or indirectly, through growth factor receptors.6Swindle CS Tran K Johnson TD Banerjee P Mayes AM Griffith LG Wells A Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.J Cell Biol. 2001; 154: 459-468Crossref PubMed Scopus (223) Google Scholar, 7Kellouche S Mourah S Bonnefoy A Schoevaert D Podgorniak MP Calvo F Hoylaerts MF Legrand C Dosquet C Platelets, thrombospondin, and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation.Exp Cell Res. 2007; 313: 486-499Crossref PubMed Scopus (39) Google Scholar These events involve a degree of cellular dedifferentiation to enable migration and proliferation. During the remodeling phase, sufficient cells have migrated into the provisional dermal matrix to mature this structure and across the missing epidermal gap to re-establish a keratinocyte covering. These cells then differentiate into synthetic fibroblasts to produce a mature collagen I-rich dermis or basal keratinocytes primed to differentiate vertically. Interestingly, a fully repaired dermis is paucicellular compared with the regenerative phase, implying a significant involution of the stromal support cells (fibroblasts and endothelial cells) present during the regenerative phase.8Gailit J Clark RAF Wound repair in the context of extracellular matrix.Curr Opin Cell Biol. 1994; 6: 717-725Crossref PubMed Scopus (375) Google Scholar Obviously, late in wound repair, signals must be generated to induce dermal cell differentiation and subsequent cellular loss. The nature of such signals is not known. However, a suggestive family of chemokines appears in wounds during late transition to maturation phase. IP-10 (CXCL10) appears in the dermis, being produced by endothelial cells of the neovasculature,9Luster AD Greenberg SM Leder P The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.J Exp Med. 1995; 182: 219-231Crossref PubMed Scopus (417) Google Scholar, 10Strieter RM Kunkel SL Arenberg DA Burdick MD Polverini PJ Interferon γ-inducible protein 10 (IP-10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis.Biochem Biophys Res Commun. 1995; 210: 51-57Crossref PubMed Scopus (264) Google Scholar and IP-9 (CXCL11 or I-TAC) is expressed from redifferentiating keratinocytes behind the leading edge of the wound (Ref. 11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossref PubMed Scopus (48) Google Scholar and herein). These secreted peptide factors, both CXC chemokines that lack the canonical N-terminal sequence ELR (glutamic acid-leucine-arginine), bind in common to the ubiquitous CXCR3 chemokine receptor.12Romagnani P Maggi L Mazzinghi B Cosmi L Lasagni L Liotta F Lazzeri E Angeli R Rotondi M Fili L Parronchi P Serio M Maggi E Romagnani S Annunziato F CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production.J Allergy Clin Immunol. 2005; 116: 1372-1379Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Signaling through CXCR3 blocks growth factor-induced motility of fibroblasts11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossref PubMed Scopus (48) Google Scholar, 13Shiraha H Gupta K Glading A Wells A IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.J Cell Biol. 1999; 146: 243-253Crossref PubMed Google Scholar and endothelial cells14Bodnar R Yates C Wells A IP-10 blocks VEGF-induced endothelial cell motility and tube formation via inhibition of calpain.Circ Res. 2006; 98: 617-625Crossref PubMed Scopus (154) Google Scholar by suppressing m-calpain activation.15Shiraha H Glading A Chou J Jia Z Wells A Activation of m-calpain (calpain II) by epidermal growth factor is limited by PKA phosphorylation of m-calpain.Mol Cell Biol. 2002; 22: 2716-2727Crossref PubMed Scopus (153) Google Scholar Interestingly, such a blockade of rear release during motility converts fibroblasts to a contractile behavior,16Allen FD Asnes CF Chang P Elson EL Lauffenburger DA Wells A EGF-induced matrix contraction is modulated by calpain.Wound Repair Regen. 2002; 10: 67-76Crossref PubMed Scopus (34) Google Scholar, 17Smith KD Wells A Lauffenburger DA Multiple signaling pathways mediate compaction of the collagen matrices by EGF-stimulated fibroblasts.Exp Cell Res. 2006; 312: 1970-1982Crossref PubMed Scopus (22) Google Scholar reminiscent of the function of these cells during dermal matrix remodeling.18Grinnell F Ho C-H Tamariz E Lee DJ Skuta G Dendritic fibroblasts in three-dimensional collagen matrices.Mol Biol Cell. 2003; 14: 384-395Crossref PubMed Scopus (176) Google Scholar Of importance, these chemokines do not block the motility of dedifferentiated keratinocytes but rather increase their motility via lessened adhesiveness19Satish L Blair HC Glading A Wells A IP-9 (CXCL11) induced cell motility in keratinocytes requires calcium flux-dependent activation of μ-calpain.Mol Cell Biol. 2005; 25: 1922-1941Crossref PubMed Scopus (64) Google Scholar and thus would promote more rapid re-epithelialization.11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossref PubMed Scopus (48) Google Scholar Parenthetically, cell homotypic contact inhibition has been proposed as the most likely signal for keratinocyte redifferentiation.2Jaffe AT Heymann WR Lawrence N Epidermal maturation arrest.Dermatol Surg. 1999; 25: 900-903Crossref PubMed Scopus (12) Google Scholar The timing of the expression of IP-9 and IP-10, along with their cellular effects, suggests that these chemokines are at least part of the key communication between the dermis and epidermis that signals an end to the regenerative phase and initiation of the remodeling phase of wound repair. We have shown earlier that keratinocyte-derived IP-9 may act as a soluble paracrine communicator between these compartments.11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossref PubMed Scopus (48) Google Scholar However, although correlative data were present, interventional in vivo models were required to demonstrate this role for CXCR3 signaling as a master organizer in wound repair. Mice have been generated that lack CXCR3.20Hancock WW Lu B Gao W Csizmadia V Faia K King JA Smiley ST Ling M Gerard NP Gerard C Requirement of the chemokine receptor CXCR3 for acute allograft rejection.J Exp Med. 2000; 192: 1515-1520Crossref PubMed Scopus (564) Google Scholar These mice displayed decreased immune responsiveness and inflammation in the skin.20Hancock WW Lu B Gao W Csizmadia V Faia K King JA Smiley ST Ling M Gerard NP Gerard C Requirement of the chemokine receptor CXCR3 for acute allograft rejection.J Exp Med. 2000; 192: 1515-1520Crossref PubMed Scopus (564) Google Scholar, 21Liu L Callahan MK Huang D Ransohoff RM Chemokine receptor CXCR3: an unexpected enigma.Curr Top Dev Biol. 2005; 68: 149-181Crossref PubMed Scopus (134) Google Scholar However, wound repair has not been studied in this model system. Thus, we used the CXCR3-null mice to test our hypothesis that, in the absence of CXCR3 signaling, the later resolving phase of wound repair will be impaired and will exhibit a hypercellular, disorganized dermis. C57BL/6J mice whose CXCR3 expression was abrogated were generated as previously described.20Hancock WW Lu B Gao W Csizmadia V Faia K King JA Smiley ST Ling M Gerard NP Gerard C Requirement of the chemokine receptor CXCR3 for acute allograft rejection.J Exp Med. 2000; 192: 1515-1520Crossref PubMed Scopus (564) Google Scholar In brief, two targeted cell lines were injected into blastocysts derived from C57BL/6 mice. Chimeric males were bred to BALB/c females to yield germline transmission of the targeted allele. Mice were backcrossed at least 6 generations onto the C57BL/6 strain. For this study, CXCR3−/− female mice were bred with CXCR3−/− males, and all offspring were screened for genotype before use. Wild-type C57BL/6J were obtained from Jackson Laboratory. All studies on these animals were performed in compliance with and after approval by the Institutional Animal Care and Use Committees of the Veteran's Administration and University of Pittsburgh. These animals were housed in a facility of Veteran's Affairs Medical Center, Pittsburgh, PA, accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. Serological analyses did not detect blood-borne pathogens or evidence of infection. Mice were housed in individual cages after wounding and maintained under a 12-hour light/dark cycle in accordance with the guidelines approved by the Institutional Animal Care and Use Committee. To verify that the mice were CXCR3−/−, genomic DNA from tail clippings was screened by polymerase chain reaction using specific primers for wild-type and CXCR3−/− mice. Three oligonucleotide primers were used. Primer “A” is the forward primer common to both wild-type and CXCR3−/− (5′-CAGGCGCCTTGTTCAACATCAACT-3′). Primer “B” is a reverse primer specific to normal CXCR3 sequence (5′-GTTGTACTGGCAATGGGTGGCATT-3′). Primer “C” is a reverse primer specific to the inserted phosphoglycerate kinase/neomycin sequence and is specific for screening CXCR3−/− mice (5′-ACCTTGCTCCTGCCGAGAAAGTAT-3′). A band size of 239 bp is expected in wild-type DNA, and a band size of 1.1 kb is expected in CXCR3−/− mice. Screenings were further confirmed by sequencing the polymerase chain reaction product inserted in the pCRII vector using M13 forward and reverse sequence primers. Male and female mice (7 to 8 weeks of age, weighing approximately 25 g) were anesthetized with an intraperitoneal injection containing ketamine (75 mg/kg) and xylazine (5 mg/kg). The backs were cleaned, shaved, and treated with 5% povidone-iodine (Betadine; Purdue Products, Stamford, CT) solution. For full-thickness wounds, sharp scissors were used to make an approximately 2-cm diameter circular full-thickness wound through the epidermis and dermis on one side of the dorsal midline; for comparison, the dorsal length of a mouse was about 7.5 cm long. The contralateral uninjured skin served as unwounded control skin. The wounds were covered with liquid occlusive dressing (New-Skin; Medtech, Jackson, WY).22Hebda PA Whaley DL Kim H-G Wells A Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.Wound Repair Regen. 2003; 11: 373-379Crossref PubMed Scopus (30) Google Scholar This wounding assay was performed three independent times with at least five animals each time. The animals were lightly anesthetized for several seconds. Wounds were traced onto a transparent sheet, at 2-day intervals until complete closure. Wound size was compared between the wild-type and CXCR3−/− groups. The areas described by tracings were measured using Adobe Photoshop image analysis software (version 7.0; Adobe System Inc., San Jose, CA). Wound bed biopsies surrounded by a margin of nonwounded skin were collected at days 3, 5, 7, 14, 21, 30, 60, and 90 after wounding. Wound biopsies were fixed in 10% buffered formalin, processed, and embedded in paraffin blocks using standard protocols. Tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) and analyzed for general tissue and cellular morphology. Collagen deposits were evaluated by Masson's trichrome staining. Histopathological examination of mouse tissues was performed blinded by a veterinary pathologist. Qualitative assessments were made concerning aspects of dermal and epidermal maturation, inflammation, and granulation tissue. The samples were scored on a scale of 0 to 4 for epidermal healing (0, no migration; 1, partial migration; 2, complete migration with partial keratinization; 3, complete keratinization; and 4, normal epidermis) and dermal healing (0, no healing; 1, inflammatory infiltrate; 2, granulation tissue present–fibroplasias and angiogenesis; 3, collagen deposition replacing granulation tissue >50%; and 4, complete healing). Quantification of fibroblastic hypercellularity was performed by using MetaMorph (Molecular Devices, Downingtown, PA). Cell counting function allowed for the direct counting of dermal nuclei per high-powered field. Full-thickness 6-mm (open) wounds were made in the skin of the hip region of healthy young adult human volunteers and biopsies taken on days 0, 2, 4, 14, and 28 after wounding. Paraffin tissue sections (5 μm) were stained with hematoxylin and eosin for morphological observations. The original wound studies were approved after full review by the Virginia Commonwealth University/Medical College of Virginia Institutional Review Board, and the use of the specimens herein were deemed exempt by the University of Pittsburgh Institutional Review Board because this tissue was received as excess pathological tissue devoid of protected health information. Sections for immunohistochemical analysis were incubated with appropriately diluted primary antibody, after antigen retrieval (BioGenex, San Ramon, CA). Antigen staining was performed using diaminobenzidine (Vector Laboratories, Burlingame, CA), then counterstained with Mayer's hematoxylin and coverslipped. In all cases, secondary antibody alone served as a negative control, with various human tumor tissues serving as positive controls. For mouse tissue, paraffin sections of 4 to 5 μm were prepared for antibody staining. The following antibodies were used for immunohistochemical staining for mouse specimens: CXCR3 (rabbit polyclonal; R&D Systems, Minneapolis, MN), fibronectin (rabbit polyclonal; Rockland, Inc., Gilbertsville, PA), IP-10/CXCL10 (rabbit polyclonal; PeproTech, Rocky Hill, NJ), IP-9/CXCL11 (rabbit polyclonal; PeproTech), tenascin-C (rat polyclonal; R&D Systems), and von Willebrand factor (rabbit polyclonal; Abcam Inc., Cambridge, MA). For murine tissue, antibodies were chosen not to be of murine origin to limit background staining from the secondary antibody. For human specimens, the following primary antibodies were used: CXCR3 (rabbit polyclonal; R&D Systems), IP-9/CXCL11 (goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and IP-10/CXCL10 (goat polyclonal; Santa Cruz). Masson's trichrome staining was used to assess collagen content. Collagen content was assessed using MetaMorph analysis (Molecular Devices). Stained wound biopsies were compared with that of the unwounded controls; at all times the color was maintained to compare the blue- and red-stained areas. The final output was integrated intensity based on total area and staining intensity at individual pixels. All wound biopsies were stained at the same time to eliminate staining variations. Picrosirius red staining was used to assess alignment and organization in intact biopsies. Briefly, picric acid (Sigma-Aldrich, St. Louis, MO) was dissolved in 500 ml of distilled water. To this, 0.1 g of Sirius red F3BA was added per 100 ml (Sigma-Aldrich). Paraffin-embedded tissue sections were rehydrated and stained with picric acid. Collagen fibrils were then evaluated by means of polarized light microscopy for both collagen fibril thickness and coherence alignment. Polarization microscopy reveals closely packed thick fibrils of type I collagen fibers as either red-orange intense birefringence in the hypertropic tissue, with thin short loose fibrils as yellow-green. Distribution of fibrils in terms of thickness (cross-sectional area) and arrangement in terms of length of the collagen scars were quantitatively analyzed using MetaMorph (Molecular Devices). Biopsies of unwounded skin served to set the threshold against which the wound biopsies were measured. Percent staining of mature fibers was determined by comparing the total staining intensity of the birefringence (area of staining summed for intensity of pixel) of wound biopsies compared with the biopsies of the contralateral unwounded skin. Biopsies were wrapped flat in foil, snap-frozen in liquid nitrogen, and then stored at −80°C. For the tensile strength measurements, the frozen specimens were divided into two samples, the cross-sectional area measured with calipers, and then the samples were clamped in a custom-built tensiometer and force exerted until wound disruption occurred as previously described.22Hebda PA Whaley DL Kim H-G Wells A Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.Wound Repair Regen. 2003; 11: 373-379Crossref PubMed Scopus (30) Google Scholar Measurements were recorded and tensile strength calculated using the formula: maximum tensiometer reading (converted to grams) divided by cross-sectional area (mm2) = tensile strength (g/mm2). The results for individual specimens from one wound were combined to determine an average tensile strength per wound. The average tensile strength per wound was tabulated for each group at days 7, 14, 21, 30, 60, and 90 after wounding. Cultures were established from newborn CXCR3−/− and C57BL/6 WT mice. The cells were derived from neonatal mice as previously described.23Devalaraja RM 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 (317) Google Scholar In brief, newborn mice (3 to 4 days old) were euthanized by CO2 inhalation. The skin was sterilized with povidone-iodine and then ethanol (70%), and then removed from the dorsum and abdomen areas and placed in phosphate-buffered saline plus 2× antibiotics and antimycotics for tissue culture solution for 2 hours. To isolate fibroblasts, the explant method was used in which the skin is cut into small pieces and carefully laid down with the epidermis facing upward in the tissue culture plates. Tissue is allowed to “dry to dampness” before adding Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum for 4 days to allow cells to migrate from the tissue onto the tissue culture plastic. The cells were characterized as fibroblasts by morphology and immunohistochemistry for vimentin and lack of cytokeratins and CD31. The cells were then incubated in DMEM to allow outgrowth of the fibroblasts. The fibroblasts were used before passage 4. Cell migration was assessed by the ability of the cells to move into an acellular area in a two-dimensional wound healing assay. At approximately 70 to 80% confluence, cells were detached and then replated at 1.0 × 105 cells/well in 24-well culture plates in complete growth media (DMEM) and incubated for 24 hours at 37°C in 5% CO2. Cells were then washed with PBS, and the media were changed to DMEM containing 0.5% dialyzed fetal bovine serum for 24 hours at 37°C in 5% CO2. A denuded area was generated in the middle of each well with a rubber policeman. The cells were then stimulated with epidermal growth factor (EGF) (10 nmol/L) in the presence or absence of IP-9 (25 ng/ml), adenosine 3′5′-cyclic monophosphorothioate 8-bromo (8-Br-cAMP) (250 nmol/L), adenosine 3′5′-cyclic monophosphorothioate 8-bromo-Rp-isomer (8-Br-Rp-cAMP) (50 nmol/L), or CPT-cAMP (2 μmol/L) and then incubated for 24 hours at 37°C in 5% CO2. These concentrations were determined empirically to provide either maximum motility or inhibition without toxicity.13Shiraha H Gupta K Glading A Wells A IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.J Cell Biol. 1999; 146: 243-253Crossref PubMed Google Scholar, 24Glading A Chang P Lauffenburger DA Wells A Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway.J Biol Chem. 2000; 275: 2390-2398Crossref PubMed Scopus (230) Google Scholar Images were taken at 0 and 24 hours, and the relative distance moved into the wounded area at the acellular front was determined. Calpain activity was determined by using the membrane-permeable substrate t-BOC-Leu-Met-chloromethylaminocoumarin (Boc-LM-CMAC) as described previously.24Glading A Chang P Lauffenburger DA Wells A Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway.J Biol Chem. 2000; 275: 2390-2398Crossref PubMed Scopus (230) Google Scholar In brief, primary cells were plated at 2.0 × 103 cells/chamber on an eight-well chamber slide (Nalge Nunc International, Rochester, NY) and grown in complete medium for 24 hours. The cells were then incubated in serum-reduced media (0.5% dialyzed fetal bovine serum for DMEM) and incubated for 24 hours. The cells were incubated with 1, 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester (5 μmol/L) for 15 minutes before the addition of Boc-LM-CMAC (50 μmol/L). The cells were further incubated for 15 minutes and then incubated with EGF (10 μmol/L), IP-9 (50 ng/ml), and/or 8-bromo-cAMP (250 μmol/L). The cleavage of Boc-LM-CMAC by calpain was visualized using a fluorescence microscope (Olympus BX40; Olympus, Tokyo, Japan) with a UV blue filter (Olympus MNUA), and images were digitally captured using a SPOT camera and SPOT software (Diagnostic Instruments, Sterling Heights, MI). Images were quantitatively analyzed using MetaMorph (Universal Imaging Corp). Paraffin sections (5 μm) from each case and time point under investigation were examined for the presence of fragmented DNA in apoptotic cells by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique using the Roche TUNEL in situ staining kit (Roche Molecular Biochemicals, Basel, Switzerland), according to the manufacturer's instructions. To detect DNA fragmentation associated with apoptosis, we used a fluorescence-based TUNEL (false colored red) followed by counterstaining with 4,6-diamidino-2-phenylindole (blue) (Vector Laboratories, Burlingame, CA). Results are expressed as mean ± SD, and all individual assay measurements were performed in replicate. Statistical differences between groups were determined by the Student's t-test. Paired analyses were performed between all groups. Comparisons over time were performed by analysis of variance. Significance was claimed for P < 0.05. The CXCR3 receptor is present on most formed elements of the skin (Figure 1A). In vitro, fibroblasts,13Shiraha H Gupta K Glading A Wells A IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.J Cell Biol. 1999; 146: 243-253Crossref PubMed Google Scholar keratinocytes,11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossref PubMed Scopus (48) Google Scholar and endothelial cells,14Bodnar R Yates C Wells A IP-10 blocks VEGF-induced endothelial cell motility and tube formation via inhibition of calpain.Circ Res. 2006; 98: 617-625Crossref PubMed Scopus (154) Google Scholar in addition to cells of the hematopoietic lineage,12Romagnani P Maggi L Mazzinghi B Cosmi L Lasagni L Liotta F Lazzeri E Angeli R Rotondi M Fili L Parronchi P Serio M Maggi E Romagnani S Annunziato F CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production.J Allergy Clin Immunol. 2005; 116: 1372-1379Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar respond to CXCR3 ligands. Our foundational model, based on cellular effects in vitro, posits that the signaling through this receptor depends on timed expression of key ligands. We found differential expression of the CXCR3 ligands IP-9 and IP-10 during wound healing using a mouse model of wound repair. IP-9 was expressed by keratinocytes just behind the leading edge of the wound, and IP-10 was expressed deep in the dermis as well as at the wound edge in wild-type mice (Figure 1B for IP-9 and Figure 1C for IP-10). These same results were observed in the CXCR3−/− mice, suggesting that CXCR3 is not required for secretion of its ligands. If anything, there seems to be increased protein levels likely due to lack of receptor-mediated ligand attenuation. To determine whether the IP-9 and IP-10 expression patterns during mouse wound healing process are relevant to human wound healing, we found that during the healing process in human wounds, IP-9 was expressed during the early granulation phase and IP-10 in the late granulation/early resolving phase (Figure 1D). The results demonstrate a good correlation in IP-9 and IP-10 expression between mouse and human during the wound healing process. Once we had a system in which CXCR3 signaling is present or absent in skin, we asked whether this paracrine signaling system, which we have previously shown in vitro is necessary for the inhibition of fibroblast motility and induction of a contractile state,11Satish L Yager D Wells A ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair.J Invest Dermatol. 2003; 120: 1110-1117Crossr