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Originally published online as doi:10.1189/jlb.0507306 on September 4, 2007

Published online before print September 4, 2007
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(Journal of Leukocyte Biology. 2007;82:1554-1563.)
© 2007 by Society for Leukocyte Biology

Accelerated wound healing in leukocyte-specific, protein 1-deficient mouse is associated with increased infiltration of leukocytes and fibrocytes

JianFei Wang*, Haiyan Jiao*, Tara L. Stewart*, Megan V. H. Lyons*, Heather A. Shankowsky*, Paul G. Scott{dagger} and Edward E. Tredget*,1

* Wound Healing Research Group Division of Plastic and Reconstructive Surgery and Critical Care, Department of Surgery, and
{dagger} Department of Biochemistry, University of Alberta, Edmonton, Canada

1Correspondence: 2D3.81 WMSHC, 8440-112 St., University of Alberta, Edmonton, Alberta T6G 2B7, Canada. E-mail: etredget{at}ualberta.ca


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ABSTRACT
 
Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and ECM. Recently, a newly identified cell type, the fibrocyte, has been reported to contribute to wound healing and fibrotic conditions such as hypertrophic scarring. We previously established leukocyte-specific protein 1 (LSP1) as a marker for fibrocytes. LSP1 is an F-actin binding protein and substrate of p38 mitogen-activated protein kinase and protein kinase C, and has been reported to be important in leukocyte chemotaxis. We examine the biological roles of LSP1 in skin wound healing using Lsp1–/– null mice. These animals showed accelerated healing of full-thickness skin wounds, with increased re-epithelialization rates, collagen synthesis, and angiogenesis. Healing wounds in Lsp1–/– mice had higher densities of neutrophiles, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression, TGF-β1, and VEGF were all up-regulated. These results demonstrate that the absence of LSP1 promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of chemokines and growth factors. Further analysis of Lsp1–/– mice may suggest ways to improve wound healing and/or treat fibrotic conditions of skin and other tissue.

Key Words: extracellular matrix molecule • LSP1 • fibrotic disease • cytoskeletal protein


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INTRODUCTION
 
Cutaneous wound healing involves several overlapping phases including inflammation, cell proliferation and migration, angiogenesis, and extracellular matrix (ECM) molecule production [1 ]. It is a dynamic, complex process involving the integrated action of numerous cell types, extracellular matrices, and soluble mediators. A hallmark of the inflammatory phase is the recruitment of neutrophils and macrophages into the injury [2 ]. In the proliferative phase, the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells results in re-epithelialization and tissue granulation. During the remodeling phase, excess collagen in the wound is degraded by proteolytic enzymes leading to the completion of tissue repair [1 ]. A newly identified cell type, the fibrocyte, has recently been reported to contribute to wound healing [3 4 5 ].

Fibrocytes are a novel cell population that represents 0.1–0.5% of peripheral blood leukocytes [3 , 5 ]. Fibrocytes are thought to play a role in tissue repair by several mechanisms such as the secretion of ECM, antigen presentation, cytokine production, angiogenesis, and wound closure [6 ]. We recently reported that an increased number of fibrocytes in hypertrophic scar [7 , 8 ] and fibrocytes may contribute to hypertrophic scarring via the regulation of fibroblasts [9 , 10 ]. Fibrocytes have been characterized by the expression of the ECM proteins collagen type I and fibronectin, together with cell surface proteins such as CD11b, CD34, and CD45 [5 ]. Unfortunately, these cell surface proteins are not unique to fibrocytes. For example, CD34 is also found on capillary endothelial cells. Moreover, some of these markers (including CD34 and CD45) are gradually lost in culture [11 ]. To identify a more stable and specific marker for fibrocytes, 2-D gel electrophoresis and mass spectrometry was performed in our laboratory, and leukocyte-specific protein-1 (LSP1) was identified as a potential marker for fibrocytes [8 ]. More important, LSP1 is completely absent from fibroblasts. Therefore, dual immunostaining for procollagen type-I and LSP1 can be used to identify fibrocytes and distinguish them from fibroblasts.

LSP1 is a 52 kDa intracellular F-actin binding protein that accumulates on the cortical cytoskeleton. LSP1 is expressed in mature and immature B and T cells, macrophages, and neutrophils [12 ]. Human and mouse LSP1 share the same expression patterns and their level of amino acid identity is high. LSP1 has two putative Ca2+ binding motifs and distributes in three different subcellular fractions: ~25% on the cytoplasmic face of the plasma membrane, ~15% in the cytoskeleton, and ~60% in the cytosol [13 ]. LSP1 is a substrate for mitogen-activated, protein kinase-activated protein kinase 2 and for protein kinase C [12 ], two enzymes implicated in leukocyte migration and chemotaxis. In addition, LSP1 was also reported to regulate a Ca2+-dependent step in the induction of anti-IgM-mediated apoptosis [14 ]. Therefore, LSP1 appears to be a signaling molecule regulating cytoskeletal architecture and mobility and plays a role in receptor-induced apoptosis.

LSP1-deficient mice grew normally, the development of myeloid cells was normal, and there was no difference in the numbers of lymphocytes, monocytes, or neutrophils in various tissues, with the exception of the peritoneum [15 ]. There is controversy surrounding the effect of LSP1 on chemotaxis of leukocytes. Mice deficient in LSP1 have been reported to exhibit significantly higher levels of resident macrophages in the peritoneum compared with wild-type (WT) mice. In addition, Lsp–/– neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (fMLP) and to the C-X-C chemokine. Therefore, these results show that LSP1 is a negative regulator of neutrophil chemotaxis [15 ]. However, Hannigan et al. found impaired chemotaxis of LSP1-deficient neutrophils compared with their wild-type counterparts [16 ]. In addition, Liu et al. recently reported that LSP1 deficiency in endothelial cells resulted in the inhibition of migration into the cremaster muscle induced by IL-1β [17 ].

Since LSP1 is predominantly expressed by leukocytes and fibrocytes, and inflammation is a critical phase in wound healing, elucidating the role of LSP1 in skin wound healing is of great interest. In this study, we examined the biological relevance of LSP1 to skin wound healing using Lsp1–/– mice [15 ]. Skin wound healing was significantly accelerated in these mice. Recruitment of neutrophils, macrophages, and fibrocytes during the inflammatory phase was significantly increased in the Lsp1–/– mice compared with WT mice. Corresponding to the increased recruitment of inflammatory cells to the injured sites, re-epithelialization, collagen synthesis, and angiogenesis were also enhanced. In accordance with these observations, the expression levels of macrophage-derived chemokines and growth factors were also up-regulated. Our results show that LSP1 regulates skin wound healing by mediating leukocyte recruitment and the subsequent production of chemokines and growth factors.


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MATERIALS AND METHODS
 
Animals
129/SvJ WT mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Lsp1–/– mice on the 129/SvJ background were generated by homologous recombination as described previously [15 ] and transferred from the University of Calgary Health Sciences Centre to the University of Alberta Health Science Laboratory Animal Service. Lsp1–/– mice were bred in the University Animal Centre. Mice between 8 and 10 wk of age were used in these experiments. All animal protocols were approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta and met the standards of the Canadian Council on Animal Care. All animals were kept in specific pathogen-free conditions.

Wound model
Skin wounds were prepared as described previously [18 ]. Briefly, mice were anesthetized with isoflurane (Halocarbon Laboratories, River Edge, NJ, USA). After shaving the dorsal hair and cleaning the exposed skin with 70% ethanol, each animal received, aseptically, two full-thickness excisional skin wounds 5 mm in diameter on one side of the dorsal midline using a 5 mm biopsy punch (Acuderm Inc., Ft. Lauderdale, FL, USA). Each punch biopsy was traced using transparency paper and the areas were processed, scanned, and calculated using the NIH Image J program (http://rsb.info.nih.gov/ij). Wound closure was expressed as: (1 – open wound area/initial wound area) x 100. The wounds were also digitally photographed. At the indicated times, mice were euthanized by CO2 inhalation and the wounds and surrounding tissue, including the epithelial margins and any scab, were harvested with an 8 mm biopsy punch (Acuderm Inc.).

Histology and immunohistochemistry
Harvested wound samples were fixed overnight in 4% paraformaldehyde buffered with phosphate-buffered saline (PBS) (pH 7.2), and embedded in paraffin. Sections (5 µm thick) were subjected to hematoxylin and eosin (H&E) staining. For immunohistochemistry, the tissues were embedded in OCT and cut into 8 µm sections. These sections were then fixed in acetone, treated with hydrogen peroxide to quench endogenous peroxidase, and blocked for 1 h with serum from the species in which the secondary antibody had been raised. Sections were then incubated for 2 h at room temperature with either rabbit anti-myeloperoxidase (MPO) polyclonal antibody (diluted 1:100; Neomarkers, Fremont, CA, USA), rat anti-mouse F4/80 monoclonal antibody (mAb) (5 µg/ml; eBioscience Inc., San Diego, CA, USA), or platelet endothelial cell adhesion molecule rat anti-mouse CD31 mAb (diluted 1:200; PharMingen, San Diego, CA, USA) for neutrophils, macrophages, and angiogenesis, respectively. Sections that had been incubated with anti-MPO antibody were subsequently incubated with a goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (Sigma, St. Louis, MO, USA) for 1 h at room temperature. Sections that had been incubated with anti-F4/80 or anti-CD31 antibodies were incubated with a rabbit anti-rat antibody conjugated with HRP (Sigma). The signals in the tissues were revealed using 3, 3'-diaminobenzidine and hydrogen peroxide. Thereafter, counterstaining was performed with hematoxylin.

Analysis of wound re-epithelialization
The analysis of re-epithelialization was performed according to the procedure of Low and colleagues [19 ]. The width of the wound and the distance between the leading edge of the keratinocyte migration were measured on H&E-stained vertical wound sections. The degree of re-epithelialization was calculated as follows: re-epithelialization (%) = [distance covered by epithelium] x 100/[distance between original wound edges].

Detection of proliferating cells
The effect of LSP1 deficiency on keratinocyte proliferation in vivo was analyzed by 5-bromodeoxyuridine (BrdU) incorporation on day 6 after wounding, as described [18 ]. Mice were injected intraperitoneally with BrdU (Sigma) solution (250 µg/g body weight in 0.9% NaCl) and euthanized 1 h after injection. Wound specimens were frozen in OCT compound and cryosections (8 µm) were incubated with a mouse anti-BrdU antibody (RPN202; Amersham Pharmacia Biotec, Inc., Baie d’Urfé, Quebec, Canada), followed by a biotinylated goat anti-mouse secondary antibody. The immunoreaction was visualized using the Vectastain ABC kit (Vector Laboratories, Burlington, Ontario) and 3, 3'-diaminobenzidine. The numbers of BrdU-labeled keratinocytes in 5 high-power fields of the epidermis around the wound edge were counted by a blinded observer and averaged.

Assessment of neutrophil and macrophage recruitment, and angiogenesis
A blinded observer counted F4/80-positive macrophages in the wound bed (defined as the area surrounded by unwounded skin, fascia, regenerated epidermis, and eschar) in five random high-power (x200) fields of each immunohistochemically stained section [19 ]. The neovascular areas (CD31-positive cells) were measured in the whole wound bed areas using the freehand drawing tool of the NIH ImageJ software, and the degree of vascularization was calculated as described previously, with slight modifications [19 ]: vascularization (%) = [CD31-positive area] x 100/[total wound bed area].

Myeloperoxidase activity
An 8 mm biopsy punch was used to excise full-thickness wounds 6 and 24 h after wounding. These tissues were homogenized at 2600 rpm using a Mikro-Dismembrator (B. Braun Biotech International, Allentown, PA, USA) in 400 µl of lysis buffer (10 mmol/L PBS, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 5 mmol/L ethylenediaminetetraacetic acid) (EDTA) containing complete protease inhibitor mixture (Sigma) to extract the proteins. The homogenates were freeze-thawed three times, and the debris was removed by centrifugation at 14,000 rpm. Myeloperoxidase (MPO) was assayed using tetramethylbenzidine (TMB) [20 ]. Briefly, 150 µl TMB substrate solution was added to 50 µl of sample and incubated (ambient temperature, 30 min) prior to termination of the reaction with 50 µl 1 M H2SO4. Plates were read spectrophotometrically at 450 nm (Molecular Devices, Sunnyvale, CA, USA). fMLP stimulated and unstimulated fresh human neutrophils were used as controls.

Detection and quantification of fibrocytes
To detect fibrocytes in tissue, cryosections (8 µm) were subjected to analysis as described [21 ], with slight modifications. Briefly, the sections were fixed with 4% paraformaldehyde, quenched for auto-fluorescence with 0.1% glycine in PBS, and then blocked. After washing, tissue sections were incubated overnight at 4°C with a rat anti-mouse CD13 antibody (Serotec, Raleigh, NC, USA). After washing, sections were incubated with TRITC-conjugated goat anti-rat antibody (Jackson ImmunoResearch, West Grove, PA, USA). Subsequently, slides were incubated with a rabbit anti-mouse collagen type-I polyclonal antibody (Chemicon, Temecula, CA, USA) overnight at 4°C. After washing, slides were incubated with FITC-conjugated goat anti-rabbit antibody. Cell nuclei were detected by incubating the tissue sections with Draq5 (Alexis Biochemicals Corp., San Diego, CA, USA) for 10 min. After washing, tissue sections were mounted using 50% PBS-50% glycerol containing 4 mg/ml of n-propyl gallate (Sigma Aldrich, Oakville, ON, Canada). Stained sections were examined using an LSM510 laser scanning confocal microscope at 489 nm to assess collagen type-I, at 543 nm to assess CD13, and at 688 nm to assess cell nuclei. To quantitate the number of fibrocytes, an algorithm was designed using MetaMorph Image Software (Metamorph, Molecular Devices) using positive nuclear staining, dual fluorescence, and cell size as criteria for inclusion.

Hydroxyproline analysis
The content of collagen in wound tissue was determined by mass spectrometric analysis for 4-hydroxyproline [18 ]. Wound samples taken from WT and Lsp1–/– mice on days 3 and 6 after wounding were freeze-dried. Internal standard (N-methyl-L-proline) and 6 N HCl solution was added to wound tissue, then each sample was hydrolyzed overnight at 115°C. The O-butyl ester derivatives were prepared with 10% BF2-butanol for 30 min at 120°C after drying the hydrolysate. Liquid chromatography (column: Eclipse XDB-C18)/mass spectrometry analysis was performed on a Hewlett-Packard (series 1100; Atlanta, GA, USA) mass selective detector monitoring the ion at m/z 188.

Real-time reverse transcription-polymerase chain reaction
Total RNA was extracted from uninjured skin and wound samples using RneasyTM spin columns (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s recommendations. To eliminate contamination with genomic DNA, DNase digestion was performed for 60 min. First-strand cDNA was synthesized using an enhanced avian first-strand synthesis kit (Sigma Aldrich) at 42°C using 500 ng total RNA extract. Real-time reverse transcription–polymerase chain reaction (RT-PCR) was conducted using Power SYBR® Green PCR Master Mix (ABI, Foster, CA, USA) in a 25 µl tube with a total reaction volume of 25 µl containing 1 µl of a 1:2 dilution of first-strand reaction product, 0.2 µM gene-specific upstream and downstream primers. Amplification and analysis of cDNA fragments were carried out using a 7300 real-time PCR system (ABI). Cycling conditions were initial denaturation at 95°C for 3 min, followed by 40 cycles consisting of a 15 s denaturation interval at 95°C and a 30 s interval for annealing and primer extension at 60°C. Amplification of the housekeeping gene β-actin mRNA was used as to normalize results. The primers used are listed in Table 1 . Levels of mRNA levels were measured as CT threshold levels and normalized with the individual β-actin control CT values. Altered mRNA levels in wound tissues are indicated as fold change compared with WT uninjured skin.


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  		Table 1. Sequences of Primers for Real Time PCR

Enzyme-linked immunosorbent assay (ELISA)
Levels of TGF-β1 in wound samples were measured using an ELISA as described previously, with slight modifications [18 ]. Briefly, samples of wounded skin were homogenized using a Mikro-Dismembrator (B. Braun Biotech International) in 600 µl of lysis buffer (10 mmol/L PBS, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 5 mmol/L ethylenediaminetetraacetic acid) (EDTA) containing complete protease inhibitor mixture (Sigma Aldrich) to extract the proteins. The homogenates were centrifuged at 15,000 rpm for 15 min at 4°C to remove debris, then the TGF-β1 protein contents in the supernatants were measured with an ELISA kit (Quantikine M; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. The total protein concentration was measured with a BCA protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The data were expressed as TGF-β1/total protein (pg/mg) for each sample.

Statistical analysis
Experiments were carried out in triplicate or quadruplicate. Data are expressed as mean ± SE. Statistical analysis was performed using 1-way ANOVA, with Tukey-Kramer multiple comparison and Excel 7 with significance set at P < 0.05.


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RESULTS
 
Accelerated skin wound closure in Lsp1–/– mice
The healing of full-thickness excisional wounds in the dorsal skin of WT and Lsp1–/– mice was monitored for 14 days (Fig. 1 ). One day after injury the wound sites were similar in size and appearance. At 3, 6, and 9 days, however, wounds in the Lsp1–/– mice closed to a significantly greater extent than in WT mice (Fig. 1B) . By day 14, essentially all the wounds were closed in both groups.


Figure 1
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  		Figure 1. Skin wound healing in WT and Lsp1–/– mice. (A) Macroscopic observation of wounds in WT and Lsp1–/– mice. Wounds were photographed using the same exposure and lighting settings, at the indicated time after wounding. Representative results for the six animals in each group are shown. (B) The ratio of wound closure to total initial wound area at each time point after wounding in WT (open bars) and Lsp1–/– mice (filled bars). Data are expressed as the mean ± SE (n=6) (*, P<0.05, **, P<0.01). The results are representative of three experiments performed.

Increased re-epithelialization in LSP1–/–mice
Figure 2 shows histological sections of the epithelium of skin wounds in WT (Fig. 2A 2B 2C) and Lsp1–/– (2D-2F) mice 6 days after injury. There was no difference in re-epithelialization of wounds in WT and Lsp1–/–mice 1 day after injury, but the extent of re-epithelialization was significantly greater in the Lsp1–/– mice on days 3 and 6 (Fig. 2G) . This stereological result was supported by immunohistochemical analysis of proliferating cells: there were more BrdU-positive cells in the Lsp1–/– mice on day 6 than in the WT mice (Fig. 3A 3B 3C ). This greater rate of epithelial cell proliferation may be a consequence of higher levels of TGF-{alpha} [18 ], since the mRNA for this cytokine was more abundant in the Lsp1–/– mouse wounds (Fig. 3D) .


Figure 2
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  		Figure 2. Histological analysis of re-epithelialization. (A–F) Histological sections showing the re-epithelialization of skin wounds in WT (A–C) and Lsp1–/– (D–F) mice 6 days after injury. (A, C) Sections from the middle of wounds were stained with H&E. Arrows indicate the re-epithelialized edges. Regions indicated by the left and right arrows in panels A and D are magnified in panels B, C and E, F, respectively. (G) Re-epithelialization ratio after the injury was measured in WT (filled diamond) and lsp1–/– (open square) mice. Data are expressed as the mean ± SE (n=6) (*, P<0.05; **, P<0.01). original magnifications: x20 (A, D); x200 (B, C, E, F), bar = 50 µm. Results are representative of three experiments performed.


Figure 3
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  		Figure 3. Proliferation of keratinocytes. (A, B) Analysis of cell proliferation by immunohistochemical staining with the anti-BrdU monoclonal antibody in WT (A) and Lsp1–/– (B) mice 6 days after wounding. (C) The number of anti-BrdU-positive cells per five high-power microscopic fields (HPF) in the epidermis was counted. Data are expressed as the mean ± SE (n=6) (**, P<0.01), 1 HPF = 0.012 mm2. (D) Real-time PCR analysis of the expression of the TGF-{alpha} gene at wound sites in WT and Lsp1–/– mice. Representative results for the six animals in each group are shown. The expression levels of TGF-{alpha} normalized to β-actin mRNA levels in WT (open bars) and Lsp1–/– mice (filled bars) wound sites were determined 3 and 6 days after injury. Data are expressed as mean ± SE (n=6) (**, P<0.01). Original magnifications: x400 (A, B), bar = 20 µm.

Increased neutrophil and macrophage infiltration in wounds in Lsp1–/– mice
During wound healing, leukocytes migrate from blood vessels into the wound. The specific cell type (or types) that migrates depends on the nature of the wound and the time after injury. During the inflammatory phase, neutrophils and macrophages are the main infiltrative cells. A large number of neutrophils were detected immunohistochemically 24 h after the injury in WT mice using the anti-MPO antibody (Fig. 4A 4B ), but many more were seen in wounds in Lsp1–/– mice (Fig. 4C 4D) . This observation was confirmed by quantitative measurements of MPO activity at both 6 and 24 h (Fig. 4E) . This increased neutrophil infiltration may be a consequence of elevated keratinocyte-derived chemokine (KC) levels [22 ], since the mRNA for this chemokine is significantly elevated in both WT and Lsp1–/– mouse wounds (Fig. 4F) .


Figure 4
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  		Figure 4. Neutrophil recruitment into wound sites. (A–D) Neutrophil recruitment into skin wounds of WT (A, B) and Lsp1–/– (C, D) mice was analyzed using an anti-MPO antibody in samples taken 24 h after injury. (E) MPO activity at the wound sites of WT (open bars) and Lsp1–/– mice (filled bars) mice 6 and 24 h after injury. Data are expressed as the mean ± SE (n=6) (**, P<0.01). Original magnifications: x20 (A, C), x200 (B, D), bar = 50 µm. The results are representative of four experiments performed. (F) Expression of KC chemokines in wounds, real-time PCR analysis of the gene expression of KC in WT and Lsp1–/– mice. Representative results of the three animals in each group are shown. The expression levels of KC in WT (open bars) and Lsp1–/– (filled bars) mice were analyzed by real-time PCR 4 h after the wounding. Data are expressed as the mean ± SE (n=3).

To analyze the number of macrophages, tissue sections were stained with anti-F4/80 antibody, which recognizes mature macrophages [23 , 24 ]. Figure 5A 5B 5C 5D shows F4/80 staining of skin wounds in WT (Fig. 5A 5B) and Lsp1–/– (Fig. 5C 5D) mice 6 days after the injury. Infiltration of F4/80-positive macrophages was observed 1 day after injury and reached a maximum 6 days after injury (Fig. 5E) . Although the numbers of F4/80-postive macrophages in WT and Lsp1–/– mice were comparable 1 day after injury (data not shown), the wounds of Lsp1–/– mice had significantly more at 3 and 6 days (Fig. 5E) . These results suggest that LSP1 affects leukocyte recruitment in skin wound healing.


Figure 5
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  		Figure 5. Macrophage recruitment into wound sites. (A–D) Macrophage recruitment into skin wounds of WT (A, B) and Lsp1–/– (C, D) mice was analyzed using an anti-F4/80 antibody in samples taken 6 days after the injury. (E) Macrophage recruitment into skin wounds of WT (open bars) and Lsp1–/– (filled bars) mice 3 and 6 days after the injury. The number of recruited macrophages per high-power microscopic field (HPF) was counted. Data are expressed as the mean ± SE (n=12) (**, P<0.01). Original magnifications: x20 (A, C); x200 (B, D), bar = 50 µm, 1 HPF = 0.04 mm2.

Increased number of fibrocytes in wounds in Lsp1–/– mice
When tissue samples from WT and Lsp1–/– mice were stained for fibrocytes, numerous cells coexpressing CD13 and pro-collagen type I were visualized. The numbers of these cells increased over time, whereas very few were seen in normal skin (Fig. 6A 6B ). Many more fibrocytes were seen in the Lsp1–/– mouse wounds harvested on days 3 and 6 than in those from the WT mice. Although the numbers of fibrocytes had fallen by 9 days after injury, there were still significantly more in the Lsp1–/– mouse wounds.


Figure 6
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  		Figure 6. Examination of fibrocytes in wound sites. (A) Immunofluorescent staining and confocal microscopy analysis of fibrocytes in wounds. Cryosections of wounds from WT and Lsp1–/– mice at 6 days post-wounding were stained with CD13 and procollagen, then subjected to confocal microscopy. (B) Quantification of fibrocytes in wound sites. Cryosections of wounds from WT and Lsp1–/– mice 3 and 6 days post-wounding were stained by CD13 and procollagen with immunofluorescence. The fibrocytes were quantified by an algorithm, which was designed using MetaMorph Image Software as described in Materials and Methods. The data represent mean ± SD (n=6), 1 unit area = 6 mm2.

Increased expression of chemokines in wounds in Lsp1–/– mice
Real-time PCR was performed for MIP-1{alpha}, MIP-2, and MCP-1 to compare levels of expression of these chemokines in the wounds, (Fig. 7 ). All were significantly higher at days 3 and 6 after injury in the Lsp1–/– mouse wounds (Fig. 7) . The up-regulated expression of these chemokine genes in the LSP1-deficient mouse wounds is consistent with the observed increased infiltration of neutrophils and macrophages.


Figure 7
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  		Figure 7. Expression of chemokines in wounds, real-time PCR analysis of the gene expression of MIP-1{alpha}, MIP-2, and MCP-1 in WT and Lsp1–/– mice. Representative results of the six animals in each group are shown. The expression levels of MIP-1{alpha} (A), MIP-2 (B), and MCP-1 (C) in WT (open bars) and Lsp1–/– (filled bars) mice were analyzed by real-time PCR at 3 and 6 days after the wounding. Data are expressed as the mean ± SE (n=6) (*, P<0.05; **, P<0.01).

Increased collagen synthesis in wounds in Lsp1–/– mice
The expression of the collagen type I {alpha}1 (COL1{alpha}1) gene was examined by real-time RT-PCR analysis (Fig. 8B ). The level of COL1{alpha}1 mRNA was significantly higher in the LSP1-deficient wounds 3 and 6 days after injury than in the WT wounds. Wounds harvested from Lsp1–/– mice 3 and 6 days after injury contained significantly more collagen, assessed by analysis of hydroxyproline content, than WT mice (Fig. 8A) . Since TGF-β1 is known to induce the expression of COL1{alpha}1 [25 ], mRNA for this cytokine was analyzed by real-time RT-PCR and TGF-β1 protein was analyzed by ELISA. As expected, both TGF-β1 mRNA (Fig. 8C) and protein levels (Fig. 8D) were significantly higher 3 and 6 days after injury in the LSP1-deficient wounds.


Figure 8
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  		Figure 8. Collagen and TGF-β levels at wound sites. (A) Collagen content was assessed by quantitatively measuring the HP content of the wound sites of WT (open bars) and Lsp1–/– (filled bars) mice at 3 and 6 days after wounding. Real-time PCR analysis of the expression of COL1{alpha}1 and TGF-β genes in the wounds of WT and Lsp1–/–mice. The expression of COL1{alpha}1 (B) and TGF-β (C) in WT (open bars) and Lsp1–/– (filled bars) mice were determined by real-time PCR at 3 and 6 days after the wounding. (D) Protein levels of TGF-β1 in wound samples from WT (open bars) and Lsp1–/– (filled bars) mice analyzed by ELISA. All data expressed as the mean ± SE (n=6) (*, P<0.05; **, P<0.01).

Enhanced angiogenesis in wounds in Lsp1–/– mice
Neovascularization is important for sustaining newly formed granulation tissue. To compare the extent of angiogenesis in the wounds from WT and Lsp1–/– mice, we performed immunohistochemical staining with an anti-CD31 antibody. Figure 9A 9B 9C 9D shows CD31 staining of skin wounds in WT (Fig. 9A 9B) and Lsp1–/– (Fig. 9C 9D) mice 6 days after the injury. There was no difference in vessel density of uninjured skin between the WT and Lsp1–/– mice (Fig. 9E) . In contrast, 6 days after injury the vascular density in the wound bed of Lsp1–/– mice was significantly higher than that of WT mice (5.7% in Lsp1–/– mice vs. 2.1% in WT mice, P< 0.01) (Fig. 9E) . The expression level of the VEGF gene, a major regulator of angiogenesis, was also assessed using real-time RT-PCR (Fig. 9F) . Although VEGF mRNA was strongly induced after injury in both mice, its level 6 days after injury was higher in the Lsp1–/– mice than in the WT mice (Fig. 9F) . These results indicate increased angiogenesis in the Lsp1–/– mice during the proliferative phase of wound healing.


Figure 9
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  		Figure 9. Analysis of angiogenesis. (A–D) Immunohistochemical sections of wounds of WT (A, B) and Lsp1–/– (C, D) mice 6 days after injury. Sections were stained with a monoclonal antibody for endothelium (CD31). Representative results from the six animals in each group are shown. (E) Areas of the wound beds that were CD31 positive in WT (open bars) and Lsp1–/– (filled bars) mice were quantified using NIH Imagine J before and after wounding. (F) Real-time PCR analysis of VEGF gene expression in wounds of WT and Lsp1–/– mice. The expression of VEGF mRNA was analyzed in the wounds from WT (open bar) and Lsp1–/– (filled bar) mice at days 3 and 6 post-wounding. All data are expressed as the mean ± SE (n=6) (*, P<0.05; **, P<0.01). Original magnifications: x20 (A, C); x200 (B, D), bar = 50 µm.


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DISCUSSION
 
Wound healing begins soon after injury, with increased vascular permeability causing the release of blood and plasma. Aggregation of platelets and coagulation of blood then leads to the formation of provisional matrices that facilitate cellular infiltration, at first predominantly of neutrophils and macrophages. This early cellular infiltration must be sufficient to stimulate subsequent normal repair. Here we have demonstrated that wound closure was significantly accelerated in Lsp1–/– mice concomitant with increased numbers of leukocytes and fibrocytes. To our knowledge, the present study is the first to report a potential regulatory role for LSP1 in skin wound healing.

The results of our study are generally consistent with earlier literature. Using two experimental models of inflammation, Jongstra-Bilen et al. injected thioglycollate into the peritoneum and zymosan into knee joints and showed increased leukocyte infiltration in Lsp1–/– mice [15 ]. In hairy cell leukemia (HCL), overexpression of LSP1 in neutrophils inhibits leukocyte migration and increases cell adhesion, leading to recurrent infection [26 ]. Moreover, wound healing in HCL patients is delayed [27 ]. Similarly, the expression of high levels of LSP1 in normally LSP1-deficient melanoma or U937 cells diminished cell motility [28 , 29 ]. These reports all support the notion that high levels of LSP1 on microfilaments down-regulate the motility of leukocytes, affecting both homeostasis and recruitment to inflamed sites. However, Hannigan et al. found impaired chemotaxis of LSP1-deficient neutrophils [16 ] and Liu et al. reported that LSP1 deficiency resulted in the inhibition of migration into the cremaster muscle induced by IL-1β. This last observation is in direct contrast to the negative regulation by LSP1 in IL-1β stimulated peritonitis described in the same study [17 ]. These findings might be reconciled if LSP1 participates in the active cross-talk between leukocytes and endothelial cells during leukocyte transmigration. The combined effects of physiological differences in microvasculature and the integrins involved may dictate organ-specific roles for LSP1 in leukocyte recruitment into inflammatory sites.

The requirement for LSP1 in neutrophil chemotaxis is dependent on the type of integrin engaged [12 ]. Mac-1 (CD11b/CD18; {alpha}Mβ2) is negatively regulated by LSP1 [30 ]. Optimal levels of integrin-mediated adhesion by focal contacts are critical for stabilizing the polarization of leukocytes during chemotaxis. Indeed, on fibrinogen gel, which is a β2-integrin substrate used by leukocytes, in addition to increased adhesion and migration rate, Lsp1–/– neutrophils exhibit more robust fMLP-induced polarization of F-actin and increased focal contacts at the anterior and expanded trailing filopodia at the posterior of the cell. Deficiency of LSP1 may up-regulate expression of Mac-1, which in turn speeds up leukocyte recruitment.

Chemokine levels appear to be critical in leukocyte infiltration [31 ]. Although the level of mRNA for KC, a functional homologue of human IL-8 [22 ], in wound site in Lsp1–/– mice was not significantly elevated compared with WT mice, KC mRNA levels nevertheless were significantly up-regulated in injured sites in both types of mice compared with uninjured sites. In vitro, Lsp1–/– neutrophils demonstrate an enhanced chemotactic response to KC and N-formyl-methionyl-leucyl-phenylalanine (fMLP) [15 ], indicating that their enhanced influx into the injured site may be a result of increased mobility. The present results also showed that MIP-1{alpha}, MIP-2, and MCP-1 were all up-regulated in the wounds of Lsp1–/– mice. The most probable source of these chemokines is the neutrophils and macrophages themselves. Moreover, up-regulation of these chemokines might in turn stimulate further leukocyte infiltration. Re-epithelialization, angiogenesis, and collagen synthesis are all delayed in MCP-1-deficient mice without any change in macrophage recruitment [19 ], suggesting that MCP-1 regulates the effector state of macrophages and other cell types.

It is known that macrophages are important for wound healing [32 , 33 ]. Thus, wounds made devoid of macrophages by hydrocortisone and anti-macrophage serum [34 ] or depleted of macrophages by radiation therapy [35 ] show delayed healing. In IL-6- or ICAM-1-deficient mice, skin wound healing is impaired with decreased macrophage infiltration [36 , 37 ]. Furthermore, in IL-10-deficient mice, increased macrophage infiltration resulted in accelerated wound closure [38 ]. Local transfer of macrophages into the wounds of aged mice accelerates healing [39 ]. Monocytes normally arrive at the wound site after neutrophils [1 ] and can differentiate into macrophages [40 ] or fibrocytes [3 ]. Early in wound healing, macrophages produce enzymes and cytokines that are important for debridement and angiogenesis [32 ]. At later stages, they promote fibroblast proliferation and modulate collagen metabolism.

Growth factors, cytokines, and chemokines produced during the inflammatory phase orchestrate the cell migration and proliferation that are necessary for wound repair [1 , 2 ]. Macrophages produce large amounts of TGF-{alpha}, a key regulator of keratinocyte proliferation [41 ]. The increased expression of TGF-{alpha} mRNA noted in the present study may have led to the improved re-epithelialization of the Lsp1–/– mice. Similarly, since TGF-β1 and VEGF are the principal fibrogenic and angiogenic factors, respectively [42 , 43 ], the elevated levels of these cytokines probably account for the increased collagen synthesis and angiogenesis seen at early stages of healing in Lsp1–/– mice.

Fibrocytes are an emerging cell population thought to play a role in tissue repair by several mechanisms such as the secretion of ECM molecules, antigen presentation, cytokine production, angiogenesis, and wound closure [3 , 5 ]. For example, fibrocytes have been shown to rapidly enter the site of tissue injury and contribute to wound healing by producing ECM macromolecules including collagen type I, collagen type III and fibronectin [3 , 4 ]. Fibrocytes act as potent instigators of the immune response by presenting antigen to both CD4+ and CD8+ T lymphocytes and secreting chemoattractants [44 ]. In addition, fibrocytes have been reported to induce angiogenesis both in vitro and in vivo [45 ]. It has recently been reported that fibrocytes may also contribute to the myofibroblast population [21 ]. In this study, the numbers of fibrocytes in LSP1-deficient wounds were significantly higher that in those of WT mice. This increase in fibrocytes probably resulted from the enhanced infiltration of the monocytes from which they are derived [4 , 7 ]. Therefore, the current study indicates that accelerated wound healing in Lsp1–/– mice is also associated with increased fibrocytes. In our earlier work on the role of fibrocytes in healing human burn wounds, we had used dual staining for procollagen type I and LSP1 to identify fibrocytes [8 ], but the present results suggest that expression of LSP1 is not essential for the differentiation of these cells, at least in mice.

In summary, our data demonstrate that the cytoskeletal protein LSP1 plays a regulatory role in skin wound healing. Further analysis of these Lsp1–/– mice may lead to improved wound healing and novel therapies for fibrotic diseases of the skin and other organs.


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ACKNOWLEDGEMENTS
 
This work was supported by the Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research, and the Firefighters’ Burn Trust Fund of the University of Alberta. The authors thank Drs. Jenny Jongstra-Bilen of the Cell and Molecular Biology Division, Toronto Western Research Institute and Jan Jongstra of the Department of Immunology, University of Toronto for providing the Lsp1–/– mice and for critical reading of this manuscript. We also thank Mr. Takashi Iwashina for hydroxyproline measurement and Dr. Page Lacy for help with the measurement of myeloperoxidase.

Received May 17, 2007; revised August 2, 2007; accepted August 3, 2007.


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