ELR-Negative CXC Chemokine CXCL11 (IP-9/I-TAC) Facilitates Dermal and Epidermal Maturation during Wound Repair

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis. In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis. Skin wound healing is remarkable for a biphasic cellularity process that entails an initial hypercellular phase with a provisional, undifferentiated matrix followed by loss of many of the initial cells and structures concomitant with significant turnover resulting in maturation of the extracellular matrix. A key question remains as to which signal(s) dictates this switch. Recent findings have suggested key factors for both vascular involution1Rosenkilde MM Schwartz TW The chemokine system—a major regulator of angiogenesis in health and disease.APMIS. 2004; 112: 481-495Crossref PubMed Scopus (212) Google Scholar, 2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar and dermal regression/maturation.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar However, a key requirement for any wound maturation signal is that it would coordinate responses between the ontologically distinct epidermal and dermal compartments. Thus, the onset of dermal maturation must coincide with successful re-epithelialization, otherwise the wound will be prone to dehiscence or chronicity. Our recent findings suggest that this paracrine epithelial-mesenchymal loop is mediated at least in part by ligands for the CXCR3 chemokine receptor. This seven transmembrane classical G-protein-coupled receptor has two isoforms, with the b isoform predominant on adherent cells as opposed to the form on the hematopoietic lineages.3Lasagni L Francalanci M Annunziato F Lazzeri E Giannini S Cosmi L Sagrinati C Mazzinghi B Orlando C Maggi E Marra F Romagnani S Serio M Romagnani P An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC and acts as functional receptor for platelet factor 4.J Exp Med. 2003; 197: 1537-1549Crossref PubMed Scopus (594) Google Scholar This CXCR3b binds, seemingly interchangeably, CXC chemokines that lack the ELR (aspartic acid, leucine, and arginine) motif. In addition to functioning in other physiological and pathological processes, all four chemokines appear during wound healing. CXCL4 (PF4) is released by platelets during the initial hemostatic phase,4Yamamoto T Chikugo T Tanaka Y Elevated plasma levels of β-thromboglobulin and platelet factor 4 in patients with rheumatic disorders and cutaneous vasculitis.Clin Rheumatol. 2002; 21: 501-504Crossref PubMed Scopus (12) Google Scholar with CXCL9 (Mig) being produced during the early inflammatory response.5Engelhardt E Toksoy A Goebeler M Debus S Brocker EB Gillitzer R 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.Am J Pathol. 1998; 153: 1849-1860Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar The other two ELR-negative CXC chemokines are more intriguing because they appear in the later regenerative phase seemingly preceding the maturation switch. CXCL10 (IP-10) is produced deep in the dermis by the neovascular endothelial cells and in the epidermis.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6Tensen CP Flier J vanderRaaij-Helmer EM Sampat-Sardjoepersad S vanderSchors RC Leurs R Scheper RJ Boorsma DM Willemze R Human IP-9: a keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).J Invest Dermatol. 1999; 112: 716-722Crossref PubMed Scopus (138) Google Scholar IP-9 derives from the redifferentiating keratinocytes just behind the leading tongue of re-epithelializing keratinocytes.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Satish 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 What directs us to hypothesize that these two later factors, IP-10 and IP-9, are critical to wound maturation is not only the timing of appearance but the effects of CXCR3 signaling. CXCR3 activation limits the motility induced by classical growth factors of fibroblasts and endothelial cells.7Satish 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, 8Shiraha 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, 9Bodnar RJ Yates CC Wells A IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain.Circ Res. 2006; 98: 617-625Crossref PubMed Scopus (156) Google Scholar The effect on fibroblasts is to channel the growth factor-induced transcellular contractility to maturation-related matrix compaction.10Smith 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 Furthermore, not only is CXCR3 signaling angiostatic,9Bodnar RJ Yates CC Wells A IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain.Circ Res. 2006; 98: 617-625Crossref PubMed Scopus (156) Google Scholar, 11Proost P Schutyser E Menten P Struyf S Wuyts A Opdenakker G Detheux M Parmentier M Durinx C Lambeir A-M Neyts J Liekens S Maudgal PC Billiau A Van Damme J Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties.Blood. 2001; 98: 3554-3561Crossref PubMed Scopus (203) Google Scholar but its activation of μ-calpain (calpain I) leads to vascular disintegration through anoikis (R. Bodnar, manuscript submitted). Importantly, CXCR3 signaling is not inhibitory to dedifferentiated keratinocytes but actually stimulates motility, being additive with growth factors.7Satish 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, 12Satish 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 Thus, we hypothesized that IP-9 is a key integrator of epidermal and dermal responses, by driving speedy re-epithelialization of the wound simultaneous with directing maturation and strengthening of the dermal wound bed.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar To test this, we have prevented IP-9 expression in keratinocytes during wound repair, by generating transgenic mice in which an antisense construct is driven from the basal keratinocyte-specific cytokeratin K5 promoter.13Byrne C Fuchs E Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice.Mol Biol Cell. 1993; 13: 3176-3190Google Scholar FVB mice were used throughout. The antisense construct was generated by cloning the IP-9 cDNA into the SnaB1 site of the pBK5 vector, which contains 5.2 kb of the bovine cytokeratin K5 promoter (a kind gift from Dr. Jose Jorcano, CIEMAT, Madrid, Spain).13Byrne C Fuchs E Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice.Mol Biol Cell. 1993; 13: 3176-3190Google Scholar After verification that this construct could significantly blunt the interferon-γ induction of IP-9 in human keratinocytes (Figure 1A) this was used to generate transgenic mice in the University of Pennsylvania (Philadelphia, PA) transgenic core facility. Founders were identified by germline transmission of the transgene using polymerase chain reaction of tail clip DNA (forward: 5′-CATATGAAGTCCTGGAAAAGGG-3′; reverse: 5′-ACAACTACACCCTGGTCATCA-3′). Mice were sib-mated by transgene × wild type. The transgenic mice are referred to as IP-9AS. In the transgene, IP-9 production was not noted during wound repair (Figure 1B). All studies on these animals were performed in compliance with 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 Affair 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 and temperature in accordance with the guidelines approved by the Institutional Animal Care and Use Committee. Male and female mice (7 to 8 weeks of age weighing ∼25 g) were anesthetized with an intraperitoneal injection containing ketamine (75 mg/kg) and xylazine (5 mg/kg). The backs were cleaned, shaved, and sterilized with betadine solution. For full thickness wounds, a 2-cm full-thickness wound through the epidermis and dermis was made on one side of the dorsal midline, using sharp scissors, with the contralateral uninjured skin serving as a control. Partial-thickness wounds were made with a specially-modified dermatome.14Hebda PA Whaley D Kim H-G Wells A Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.Wound Repair Regener. 2003; 11: 373-379Crossref PubMed Scopus (30) Google Scholar The wounds were covered with liquid occlusive dressing (New-Skin; Medtech, Jackson, WY). This experiment was performed in duplicate. To follow the wound progression visually, the animals were lightly anesthetized for several seconds with a halothane cone. A transparent sheet was placed over the wound. Re-epithelialization of the wound was traced at 2-day intervals until complete closure. These parameters were compared between the wild-type and IP-9AS. The tracings were measured using Adobe Photoshop image analysis software (version 7.0; Adobe System Inc., San Jose, CA). Wound bed biopsies, including a margin of nonwounded skin, were collected at days 5, 7, 14, 21, 30, and 60 after wounding. Wound biopsies were fixed in 10% buffered formalin, processed, and embedded in paraffin blocks using standard protocols. Four-μm tissue sections were stained with hematoxylin and eosin (H&E) for assessment of general tissue and cellular morphology. Collagen deposition, content, and alignment were evaluated by Masson's Trichrome and Picrosirius Red staining. Slides were quantitatively analyzed using MetaMorph (Universal Imaging Corp., Molecular Devices, West Chester, PA). The histological scoring system was developed based on the scoring system by Greenhalgh.15Greenhalgh DG FACS: models of wound healing.J Burn Care Rehab. 2005; 26: 293-305Crossref PubMed Scopus (85) Google Scholar Most of the scoring systems use more than one determinant to assess the histological parameters, as briefly described below. Acute inflammation was defined as the presence of neutrophils and chronic inflammation by the presence of plasma and monocytic cells (0, none; 1, slight; 2, moderate; 3, abundant). In both situations the scale was secondary to relative level of cells per high-power field. Histopathological examination of mouse tissues was performed blinded by a trained histopathologist. 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). Masson's trichrome staining was used to assess collagen content as previously described by Yates and colleagues.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Collagen content was assessed using MetaMorph analysis (Molecular Devices, Sunnyvale, CA), Stained wound biopsies were compared with that of the unwounded controls: at all times the colors 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 variation. 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 Meta-Morph (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.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Sections for immunohistochemical analysis were incubated with appropriately diluted primary antibody, after antigen retrieval. 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 tissues serving as positive controls. Paraffin sections of 4 to 5 μm were prepared for antibody staining. The following antibodies were used for immunohistochemical staining for mouse: fibronectin (rabbit polyclonal; Rockland, Inc., Gilbertsville, PA), IP-9 (goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), tenascin-C (rat polyclonal; R&D Systems, Minneapolis, MN), collagen IV (rabbit polyclonal; Abcam Inc., Cambridge, MA), and Ki-67 (rabbit polyclonal, Abcam Inc.). 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 tensiometer and force-exerted until wound disruption, as previously described.14Hebda PA Whaley D Kim H-G Wells A Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.Wound Repair Regener. 2003; 11: 373-379Crossref PubMed Scopus (30) Google Scholar The tensiometer was calibrated after every third sample with a wide range of weights. Measurements were recorded by a customized computer software program and tensile strength calculated using the formula: maximum tensiometer reading (converted to g) 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, and 60 after wounding. Pspt 18 plasmid (Roche Molecular Diagnostics, Indianapolis, IN) harboring the IP-9 sequence6Tensen CP Flier J vanderRaaij-Helmer EM Sampat-Sardjoepersad S vanderSchors RC Leurs R Scheper RJ Boorsma DM Willemze R Human IP-9: a keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).J Invest Dermatol. 1999; 112: 716-722Crossref PubMed Scopus (138) Google Scholar in either orientation were linearized and Digoxigenin DIG labeled. Sense (control) and anti-sense RNA probes were obtained by using the DIG labeling RNA kit (Roche Molecular Diagnostics, Indianapolis, IN) with SP6 RNA polymerase. Labeled probes were purified by the Qiagen (Valencia, CA) spin column kit. In situ hybridization was performed on formalin-fixed, paraffin-embedded wound biopsy sections from different days as described previously. These were overlaid with 100 μl of hybridization buffer containing 100 ng of probe and incubated overnight at 37°C in a humidified chamber. After hybridization, nonhybridized probe were removed using high stringency washes. The sections were incubated with anti-DIG-labeled secondary antibody conjugated with alkaline phosphatase. Staining reaction was performed using BCIP/NBT substrate and photographed. Results are expressed as mean ± SD. Statistical differences between groups were determined by the 2 tailed Student's t-test. Paired analyses were performed between all groups. Comparisons throughout time were performed by analysis of variance. Significance is deemed at P < 0.05. Keratinocytes produce IP-9 in response to wounding both in vitro and in vivo.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Satish 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 IP-9 mRNA is detectable just behind the leading edge of the wound (Figure 1C). Protein is noted in murine wounds as early as day 4 and expression persists as late as day 16.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Because IP-9 expression during wound repair appears mainly limited to the dedifferentiated keratinocytes of the epidermis we sought to specifically ablate such production in mice using a promoter that is highly active in the dedifferentiated keratinocytes, the cytokeratin K5 promoter (Figure 1B).16Fuchs E Keratins and the skin.Annu Rev Cell Dev Biol. 1995; 11: 123-154Crossref PubMed Google Scholar The pathophysiological effects of the suppression of IP-9 during healing were determined by creating full and partial thickness excisional wounds in the dorsal skin on the IP-9AS and FVB (wild type) mice. Grossly in partial thickness wounds, a delay in healing was observed in the IP-9AS mice with the eschar being present at least 14 days longer than in the wild-type mice (Figure 2). In the full thickness wounds, the gross deficits were more subtle (data not shown). This was not unexpected because full thickness skin wounds in rodents close mainly by contraction15Greenhalgh DG FACS: models of wound healing.J Burn Care Rehab. 2005; 26: 293-305Crossref PubMed Scopus (85) Google Scholar; furthermore, deep in the dermis, CXCL10/IP-10 appears to be the predominant CXCR3 ligand.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Boulday G Haskova Z Reinders MEJ Pal S Briscoe DM Vascular endothelial growth factor-induced signaling pathways in endothelial cells that mediate overexpression of the chemokine IFN-{gamma}-inducible protein of 10 kDa in vitro and in vivo.J Immunol. 2006 Mar 1; 176: 3098-3107PubMed Google Scholar Analyzed histologically, the wounds in the IP-9AS mice showed both a delayed re-epithelialization and an immature epidermis. In partial thickness wounds, the progressing keratinocyte tongue was delayed (Figure 3A), and even where the underlying dermis was covered, the epidermal layer was thicker (Figure 3B) with more transit-amplifying cells even as long as 30 days after wounding at which time the wounds in wild-type mice appear fully healed. Even in the wild-type mice, the wounds at 30 days are not similar to unwounded skin, although they do demonstrate significant wound maturation; this level of maturation is significantly less in the wounds in the IP-9AS mice. This correlates with the corresponding increase in the number of cell layers that constitute the epidermis in the IP-9AS wounds (0.0202 mm in IP-9AS versus 0.0080 mm in the FVB mice) (Figure 3C). The late-stage wounds in the wild-type mice resemble that of normal skin with a low number of keratinocytes presenting the proliferation marker Ki-67, whereas a greater number of nuclei stain positive for Ki-67 as late as 30 days after injury in the IP-9AS mice (Figure 3D) suggesting a degree of wound retardation or immaturity in these mice. Epidermal maturation is also deficient in the full-thickness wounds (Figure 3E), showing that this maturation aspect is independent of actual wound edge juxtapositioning The number of cell layers also increased in IP-9AS full-thickness wounds (Figure 3F). Fibroplasia and epithelialization are two distant aspects of the skin healing, both of which appear impacted by CXCR3 signaling.2Yates CC Whaley D Kulasekeran P Hancock WW Lu B Bodnar R Newsome J Hebda PA Wells A Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor.Am J Pathol. 2007; 171: 484-495Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar It has been speculated that keratinocyte-derived soluble factors signal the maturation of the underlying dermis to synchronize the wound repair.18Babu M Wells A Dermal-epidermal communication in wound healing.Wounds. 2001; 13: 183-189Google Scholar Even in full thickness wounds, dermal maturation was delayed up to 60 days after wounding (Figure 4A). Masson trichrome staining for collagen content shows a clear morphological difference in the dermal matrix with the wounds in IP-9AS mice having less deposited collagen and greater fibroblast cellularity in comparison to wounds in FVB control mice (Figure 4, B and C). Quantification of the dermal wounds revealed that IP-9AS mice failed to properly remodel the dermal matrix as late as day 60 after wounding; Picrosirius Red staining showed that the IP-9AS wounds had shorter and less well connected collagen fibers resulting in a less well organized with respect to regain of tensile strength and immature scar (Figure 4, D and E). Consistent with the collagen immaturity, provisional matrix components including tenascin C and fibronectin are not present at day 21 (tenascin C) and day 14 (fibronectin) in the wounds of IP-9AS mice in comparison to wild type (Figure 4F). Because it is well documented that tensile properties of a wound depend not only on the amount of collagen, but also on the organization and crosslinking of the matrix we assessed the biomechanical properties of the wounds. Tensiometry demonstrated that IP-9AS mice wounds lagged behind those of the control mice with respect to regain of tensile strength; even at 60 days after wounding, the IP-9AS mice regained only 60 to 65% of the prewounding strength compared to the 75 to 80% for the control mice (Figure 4G). These results suggest that not only does chemokine IP-9 affect epidermal healing but dermal healing as well by contributing to fibroblast immigration, collagen bundling and alignment, and dermal strength (collagen organization). In the partial thickness wounds, the superficial, papillary dermis was removed by the dermatome.14Hebda PA Whaley D Kim H-G Wells A Absence of inhibition of cutaneous wound healing in mice by oral doxycycline.Wound Repair Regener. 2003; 11: 373-379Crossref PubMed Scopus (30) Google Scholar In this region, dermal repair was deficient in IP-9AS wounds, showing collagen and matrix immaturity. Importantly for skin healing, the delineating basement membrane between the dermal and epidermal compartments was deficient. Most obvious, is the elevated expression of collagen IV (Figure 5) in the late healing phase of the IP-9AS mice suggesting that the epidermis-dermal junction has yet to properly form, resulting in a looser connection between these layers in the healed wounds of the IP-9AS mice (Figure 3B). The first aspect of wound healing after hemostasis involves an inflammatory response with a rapid infiltration of polymorphonuclear leukocytes (acute response) followed by macrophages and lymphocytes (chronic response). This is thought to be crucial to re-establishing the microbial barrier function of the skin. Interestingly, although CXCR3 ligands limit migration of stromal and endothelial cells, they are chemotactic for hemat