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First published online August 24, 2006
Stem Cells Vol. 24 No. 12 December 2006, pp. 2810 -2816
doi:10.1634/stemcells.2006-0264; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

CTACK/CCL27 Accelerates Skin Regeneration via Accumulation of Bone Marrow-Derived Keratinocytes

Daisuke Inokumaa, Riichiro Abea, Yasuyuki Fujitaa, Mikako Sasakia, Akihiko Shibakia, Hideki Nakamuraa, James R. McMillana, Tadamichi Shimizub, Hiroshi Shimizua

aDepartment of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan;
bDepartment of Dermatology, Faculty of Medicine, University of Toyama, Toyama, Japan

Key Words. Bone marrow-derived stem cell • CTACK/CCL27 • CCR10 • Keratinocyte • Wound healing

Correspondence: Hiroshi Shimizu, M.D., Ph.D., Department of Dermatology, Hokkaido University Graduate School of Medicine, N 15 W 7, Kita-ku, Sapporo 060-8638, Japan. Telephone: +81-11-716-1161, ext. 5962; Fax: +81-11-706-7820; e-mail: Shimizu{at}med.hokudai.ac.jp

Received on April 30, 2006; accepted for publication on August 14, 2006.

First published online in STEM CELLS EXPRESS  August 24, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Recent studies have suggested that bone marrow (BM) cells transdifferentiate to regenerate a variety of cellular lineages. Due to the relatively small population of BM-derived cells in each organ, it is still controversial whether these BM-derived cells are really present in sufficient numbers for effective function. Conversely, it is speculated that chemokine/chemokine receptor interactions mediate this migration of the tissue-specific precursor cells from BM into the target tissue. Here, we show that cutaneous T-cell attracting chemokine (CTACK)/CCL27 is the major regulator involved in the migration of keratinocyte precursor cells from BM into skin. By screening various chemokine expression patterns, we demonstrated that CTACK is constitutively expressed in normal skin and upregulated in wounds and that approximately 20% of CD34+ BM cells expressed CCR10, the ligand for CTACK. Intradermal injection of CTACK/CCL27 into the periphery of skin wounds significantly enhanced BM-derived keratinocyte (BMDK) migration, and CTACK/CCL27 neutralizing antibody inhibited this BMDK migration. Furthermore, increased BMDK migration caused by CTACK/CCL27 significantly accelerated the wound-healing process without any influence over either angiogenesis or keratinocyte proliferation. These results provide direct evidence that recruitment of BM keratinocyte precursor cells to the skin is regulated by specific chemokine/chemokine receptor interactions, making possible the development of new regenerative therapeutic strategies.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Bone marrow (BM)-derived stem cells residing in adult BM possess the unique ability to self-renew and differentiate into multiple cell lineages. A number of recent studies have suggested that BM cells might transdifferentiate and contribute to the regeneration of a variety of nonhematopoietic cell lineages in multiple organs [1–3]. Recent studies have shown that epithelial phenotype BM-derived cells can be found in normal and malignant epithelia without evidence of fusion [4–6] and that cutaneous injury leads to increased engraftment of these BM-derived cells as epidermal cells [7].

Thus far, the concept of BM-derived stem cell plasticity has been cautiously accepted; however, several hurdles remain that have blocked the development of clinical applications. The characteristics of BM-derived stem cells are not fully understood (e.g., the mechanisms causing the tissue-specific migration). Tissue repair and regeneration after injury are thought to involve selective recruitment of circulating or resident stem cell populations [8]. BM-derived, transdifferentiated cells have been detected at the wound site in injured tissues; however, the numbers of these cells are so low that it is impossible to confirm any of their specific biological characteristics or functions.

To increase numbers of BM-derived, transdifferentiated cells, two strategies have been employed. One is to increase the number of BM-derived stem cells present in circulating blood (e.g., using granulocyte colony stimulating factor (G-CSF) to induce mobilization of BM cells [9]) and another is to induce tissue-specific recruitment to increase the final number of BM-derived, transdifferentiated cells in the target tissue (e.g., using stromal cell-derived factor-1{alpha} [SDF-1{alpha}] to induce homing of hematopoietic stem cells to the BM by binding to CXCR4 [10, 11]). Chemokine/chemokine receptor interactions are thus predicted to play important roles in tissue-specific BM-derived stem cell recruitment.

To further our understanding of the chemokine/chemokine receptor interactions involved in tissue-specific stem cell trafficking, we have investigated the mechanism that controls in vivo migration of BM-derived keratinocyte (BMDK) precursor cells into the skin. In addition, to elucidate whether BMDKs have any of the functional roles of keratinocytes, we investigated the contribution of BMDKs to the processes involved in wound healing.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Generation of BM-Chimeric Mice
Whole BM cells (5 x 106) from green fluorescent protein (GFP)-transgenic (under control of ß-actin promoter) mice (The Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were transplanted into lethally irradiated (8.5 Gy) wild-type C57BL/6 recipients. Hematopoietic reconstitution was subsequently evaluated in peripheral blood 4 weeks after transplantation.

Wounded and Normal Skin Tissue Preparation
All animal procedures were conducted according to guidelines provided by the Hokkaido University Institutional Animal Care and Use Committee under an approved protocol. We performed skin injury and examined for GFP-expressing cells at least 10 weeks after BM transplantation. The mice were anesthetized, and 6-mm full-thickness punch biopsy wounds were made by folding the back skin. The wounded tissues were subsequently collected after 24 hours in reverse transcription-polymerase chain reaction (RT-PCR) analysis and Western blot analysis or after 3 days in immunofluorescence staining.

RT-PCR Analysis
Total RNA was isolated from normal or wounded skin. RT-PCR analyses of mRNA from chemokines and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed in a thermocycler (GeneAmp PCR system 9600; PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). Primers were as follow: cutaneous T-cell attracting chemokine (CTACK) (sense: 5'-AGCAGCCTCCCGCTGTTACTGTTG-3', antisense: 5'-TGCTTTATTAGTTTTGCTGTTGGG-3'), mucosae-associated epithelial chemokine (sense: 5'-CATACTTCCCATGGCCTCC-3', antisense: 5'-GAGAGGCTTCGTGCCTGTG-3'), secondary lymphoid tissue chemokine (SLC) (sense: 5'-ATGGCTCAGATGATGACTCT-3', antisense: 5'-TACTGGGCTATCCTCTTGA-3'), SDF-1{alpha} (sense: 5'-AGTGACGGTAAACCAGTCAG-3', antisense: 5'-CTTTCTCCAGGTACTCTTGG-3'), macrophage inflammatory protein (MIP)-1{alpha} (sense: 5'-AAGGTCTCCACCACTGCCCTTG-3', antisense: 5'-CTCAGGCATTCAGTTCCAGGTC-3'), MIP-1ß (sense: 5'-CCAGCTGTGGTATTCCTGACC-3', antisense: 5'-AATAGCAGAGTTTCAGCAATGG-3'), MIP-2 (sense: 5'-AGTGAACTGCGCTGTCAATG-3', antisense: 5'-CTTTGGTTCTTCCGTTGAGG-3'), MIP-3{alpha} (sense: 5'-CAAGCGTCTGCTCTTCCTTG-3', antisense: 5'-TGGATCAGCGCACACAGATT-3'), RANTES (sense: 5'-ATAACGCGTATGCATCACCATATGGCTCGGAC-3', antisense: 5'-CCAGATCTAGCTCATCTCCAAATAG-3'), TARC (thymus and activation-regulated chemokine) (sense: 5'-AGTGGAGTGTTCCAGGGATG-3', antisense: 5'-TTTGTGTTCGCCTGTAGTGC-3'), monocyte chemoattractant protein (MCP)-2 (sense: 5'-AGTGCTTCTTTGCCTGCTGCTCATAG-3', antisense: 5'-ATGAGAAAACACGCAGCCCAGGCACC-3') MCP-5 (sense: 5'-CTATGCCTCCTGCTCATAGC-3', antisense: 5'-CTTAACCCACTTCTCCTTGG-3'), TECK (thymus-expressed chemokine) (sense: 5'-CTGGGTTACCAGCACAGGAT-3', antisense: 5'-CCTCTGGATTCCCACACACT-3'), interferon-{gamma} (IFN-{gamma}) inducible protein-10 (sense: 5'-GGGCCAGTGAGAATGAGGGC-3', antisense: 5'-TGAGCTAGGGAGGACAAGGAG-3'), MIG (monokine induced by IFN-{gamma}) (sense: 5'-GATCAAACCTGCCTAGATCC-3', antisense: 5'-GGCTGTGTAGAACACAGAGT-3'), and GAPDH (sense: 5'-GAGGGGCCATCCACAGTCTTC-3', antisense: 5'-CATCACCATCTTCCAGGAGCG-3'). Aliquots from each amplification reaction were analyzed by electrophoresis in 5% acrylamide-Tris-borate gels.

Western Blot Analysis
Protein lysates from normal and wounded skin tissues were electrophoresed on polyacrylamide gels under reducing conditions and then blotted onto nitrocellulose filters. Filters were blocked with nonfat dried milk and followed by incubation with a primary antibody against SDF-1{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), SLC, CTACK, MIP-1{alpha}, and MIP-1ß (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com). After incubation, the filters were treated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G, and the resultant immune complexes were visualized.

Immunofluorescence Staining
After 28 days, the wounded tissues were removed. Skin sections were stained with primary antibodies to keratin-14 (Chemicon International, Temecula, CA, http://www.chemicon.com), and the chemokines were used in Western blot analysis. Primary antibodies were visualized using secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate. Fluorescence staining was detected using a confocal laser scanning fluorescence microscope (Laser Scanning Confocal Imaging System MRC 1024; Bio-Rad, Hercules, CA, http://www.bio-rad.com). Keratinocytes expressing both keratin-14 and GFP were presumed to be BMDKs. The number of BMDKs was quantified and calculated as a percentage of the total number of keratinocytes in wounded skin.

Chemokine Receptor Expression in CD34+ BM Cells
BM cells were incubated with FITC-conjugated antibody against CD34 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) and antibodies to CXCR4 (BD Pharmingen), CCR7 (Santa Cruz Biotechnology, Inc.), and CCR10 (Calbiochem, San Diego, http://www.emdbiosciences.com) with secondary PE-conjugated antibodies and then analyzed by flow cytometry (FACScalibur; Becton Dickinson Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com).

Migration Assays
Migration assays were performed using Costar Transwell (Corning, Acton, MA, http://www.corning.com) inserts (pore size: 3 µm). Isolated CD34+ BM cells purified by fluorescence-activated cell sorting (FACSVantage; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) based on surface CD34 staining (>99% purity) were suspended at 1 x 106 cells per milliliter in RPMI 1640 medium containing 0.1% fetal bovine serum. Medium alone or medium containing SDF-1{alpha}, SLC, or CTACK (R&D Systems, Inc.) at concentrations of 0, 10, 100, or 500 ng/ml was added to individual lower wells of a 24-well plate. CD34+ BM cells were layered on top of the membrane in the upper chamber of the transwell insert and incubated for 18 hours. For checkerboard analysis, chemokines (100 ng/ml) were added to both the bottom and top chambers. Migration was assessed by counting the cell number in the lower wells. Replicate experiments were performed with separate cultures of cells on separate occasions.

Chemokine Intradermal Injection into the Peripheral Wounded Site
BM-chimeric mice were locally anesthetized and given 4-mm, round skin wounds and received a single intradermal injection of SDF-1{alpha}, SLC, or CTACK (1 µg in 30 µl) or phosphate-buffered saline (PBS) (as control) into the peripheral wound sites. After 28 days, the wounded tissue was removed, and the percentage of BMDKs in the wounded skin was calculated.

Neutralizing Antibody Injection into the Wounded Skin
CTACK-neutralizing antibody (0–16 µg in 120 µl) was injected into the periphery of the 4-mm, round wound site, and the percentage of BMDKs in the wounded skin was analyzed as described above.

Mobilization of BM Cells
BM cells of BM-chimeric mice were mobilized into peripheral blood by three daily injections of recombinant mouse G-CSF (TECNE, R&D Systems, Inc.) (150 µg/kg per day). Control mice received a sterile saline solution without G-CSF. Twenty-four hours after the last injection, the mice were given epidermal wounds and received intradermal injections of SDF-1{alpha}, SLC, or CTACK (1 µg in 30 µl) or PBS (as control) into their peripheral wounds. Twenty-eight days after wounding, the sites were examined to detect BMDKs as described above.

CD34+ BM Cell Adoptive Transfer
CD34+ BM cells from GFP-transgenic mice were purified using the MACS (magnetic cell sorting) technique (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Immediately after tail vein injection of CD34+ BM cells (5 x 105 cells/mouse), the mice were given skin wounds and received SDF-1{alpha}, SLC, or CTACK (1 µg in 30 µl) or PBS (as control) in their peripheral wound sites. After 28 days of wound healing, BMDKs were counted as described above.

Wound-Healing Analysis
Ten-millimeter, round skin wounds were created, and CTACK (total 3 µg in 100 µl), CTACK-neutralizing antibody (total 16 µg in 100 µl), or PBS (100 µl) (as a control) was injected into the peripheral wound sites. Standardized images of wounds were recorded using a digital camera for analysis of daily wound closure rates.

Analysis of Wound-Healing Angiogenesis
BM-chimeric mice were given 4-mm, round skin wounds and received an intradermal injection of CTACK (1 µg in 30 µl) into the periphery of wounds. After 3 days, the wound sites were removed and skin sections were cut and stained with primary antibodies to CD31 (a marker of endothelial cells) (BD Pharmingen) followed by a secondary antibody conjugated to rhodamine isothiocyanate. The number of capillaries in the dermis of the wounded skin was calculated per surface area or volume of tissue.

Proliferation Assay
Keratinocytes were prepared from the skin of a newborn C57BL/6 mouse. After separation of the epidermis from the dermis with dispase and then 0.5% trypsin, the keratinocytes were cultured in 96-well plates at 1,000 cells in 100 µl of keratinocyte growth medium (Cambrex, East Rutherford, NJ, http://www.cambrex.com) per well. After 24 hours of culture, CTACK was added at concentrations of 1–100 ng/ml. After 72 hours of incubation at 37°C, 10 µl of the tetrazolium salt WST-1 (Dojindo Laboratories, Kumamoto, Japan, http://www.dojindo.co.jp) was added to each well [12]. The plates were incubated for 2 hours at 37°C, and viable cells were determined using a microplate reader at 450 nm with a 630 nm reference wavelength.

In Vitro Keratinocyte Migration Assay
Keratinocytes from C57BL6 mice were cultured in six-well uncoated plates with keratinocyte growth medium until they reached 80% confluency. A cell-free area was created by scraping the keratinocyte monolayer with a plastic pipette tip. Keratinocyte migration to the cell-free area was evaluated after 48 hours of culture in medium alone or medium containing CTACK at concentrations of 0, 1, 10, or 100 ng/ml. The number of migrating keratinocytes was counted in four nonoverlapping fields [13].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
BMDKs Were Present in the Basal Layer of the Epidermis
We used a GFP transgenic BM transplantation model. The recipient mice had been lethally and completely irradiated, but to enable their long-term survival, BM was reconstituted from the cells of a donor GFP transgenic mouse. Recruitment of BMDKs to the skin was assessed after the induction of full-thickness wounds. Twenty-eight days after the first incision, the wound was excised and examined for BMDK recruitment. Cells expressing both GFP (a marker of BM origin) and keratin-14 (a marker of basal keratinocyte) were present in the basal layer of the epidermis and were classified as BMDKs (Fig. 1A). Some BM-derived cells are likely to be epidermal Langerhans' cells derived from BM, but the GFP+ keratin-14+ cells did not express CD45 (a marker of hematopoietic cells) and CD11c (a marker of Langerhans' cells) (data not shown). Almost all BMDKs were present in the basal layer, but some BMDKs were also present in the bulge region of hair follicles. The percentage of BMDKs as a ratio of all keratinocytes was assessed on three sections from one mouse (n = 5). BMDKs were calculated to be present as a total of 0.025% ± 0.009% of all keratinocytes and comprised approximately 0.1% in the basal cell layer.


Figure 1
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  		Figure 1. Bone marrow-derived keratinocytes (BMDKs) were identified and CTACK was expressed in wounded skin. (A): Engrafted BMDKs in wounded skin expressed both GFP, as a marker of bone marrow origin (green), and keratin-14, as a marker of basal keratinocyte (red), (arrows). (B–D): Normal (N) or wounded (W) skin tissue samples were collected and analyzed for chemokine expression as shown in Table 1. The expressions of SDF-1{alpha}, SLC, and CTACK were detected by RT-PCR (B) and Western blot analysis (C) in normal skin and also in skin 24 hours after wounding. These experiments of RT-PCR and Western blot analyses were performed in triplicate. In immunofluorescence staining in the wound edge 3 days after wounding, CTACK expression, in particular, was upregulated (green) (D). SDF-1{alpha} and SLC were weakly expressed (data not shown). Nuclei were counterstained with propidium iodide (red). Abbreviations: CTACK, cutaneous T-cell attracting chemokine; d, dermis; e, epidermis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; SDF-1{alpha}, stromal cell-derived factor-1{alpha}; SLC, secondary lymphoid tissue chemokine.

 
SDF-1{alpha}, SLC, and CTACK Were Expressed in Normal Skin, and CTACK Expression Was Upregulated in Wounded Skin
We therefore supposed that optimum BMDK migration required a higher level of cell recruitment signal generation, such as after tissue injury, which could induce a greater accumulation of tissue-specific BM progenitor cells through tissue-specific chemokine/chemokine receptor interactions. To test this possibility in skin, we first examined the expression of a panel of chemokines as possible candidates by RT-PCR, Western blot analysis, and immunofluorescence staining (Table 1). RT-PCR and Western blot analyses were performed with normal skin or with skin 24 hours after wounding, including both epidermis and dermis. Experiments using RT-PCR (Fig. 1B) and Western blot analyses (Fig. 1C) showed that SDF-1{alpha}, SLC, and CTACK/CCL27 were constitutively expressed in normal skin and also wounded skin. Only CTACK expression was upregulated in wounded tissue. We examined the location of CTACK expressed in skin. Immunofluorescence staining demonstrated that CTACK expression was upregulated in the epidermis 3 days after wounding (Fig. 1D), whereas SDF-1{alpha} and SLC remained only weakly expressed (data not shown). Western blot analysis and immunofluorescence staining showed that MIP-1{alpha} and MIP-1ß were not detectable, but RT-PCR analysis demonstrated a low level of expression of these chemokines. CTACK has recently been described in mice and humans as being exclusively expressed by keratinocytes [14]. CTACK selectively attracts cutaneous memory T cells [14] by interacting with a specific receptor, CCR10 [15]. CTACK/CCR10 interactions are directly involved in T-cell recruitment to inflamed skin [16]. However, there are no reports that suggest that this interaction is important for other cell types.


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  		Table 1. Chemokine expression in normal and wounded skin

 
CXCR4, CCR7, and CCR10 Expression Were Detected on CD34+ BM Cells
If chemokine/chemokine receptor interactions contribute to the recruitment of BM-derived tissue-specific precursor cells in damaged tissue, a specific chemokine should be upregulated in the target tissue together with a partner receptor expressed on the BM-derived tissue precursor cells. Although the markers identifying keratinocyte precursor cells in BM are still unknown, we chose CD34 as a stem cell marker; CD34 is a known marker for several BM precursor cell populations, including myocytes and neural cells [17, 18]. Expression of CXCR4, CCR7, and CCR10, which are specific receptors for the skin chemokines expressed in wounds (SDF-1{alpha}, SLC, and CTACK, respectively), were also analyzed together with CD34+ BM cells using flow cytometry.

Approximately 19.1% of CD34+ BM cells expressed CCR10 (Fig. 2A). In addition, CXCR4 and CCR7 were expressed in 97.4% and 12.9% on the CD34+ cells, respectively (Fig. 2A).


Figure 2
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  		Figure 2. CCR10, a receptor for CTACK, was expressed in CD34+ bone marrow (BM) cells and these CD34+ BM cells migrated in response to CTACK in vitro. (A): Expression of CXCR4, a receptor for SDF-1{alpha}; CCR7, a receptor for SLC; and CCR10 on CD34+ BM cells was analyzed by flow cytometry. Shown are the staining with a specific antibody for each chemokine receptor (solid line) and the background staining with the nonspecific immunoglobulin antibody (negative isotype-matched control; shaded profile) by the gated CD34+ population. CD34+ BM cells expressed CXCR4 (97.4%), CCR7 (12.9%), and CCR10 (19.1%). (B, C): Chemotaxis assays were undertaken in vitro. Isolated CD34+ BM cells purified by fluorescence-activated cell sorting were added to the upper well of a 3-µm-pore Transwell. Recombinant SLC, SDF-1{alpha}, or CTACK was added to the upper and/or lower plate. CD34+ BM cell migration rates increased in response to medium containing recombinant SLC, SDF-1{alpha}, or CTACK (100 ng/ml) (* p < .05, ** p < .001) versus medium alone (n = 4) (B). CTACK (0–500 ng/ml) induced CD34+ BM cell migration in dose-dependent manner (n = 4) (* p < .05) (C). SDF-1{alpha} and SLC also induced in dose-dependent manner (data not shown). Abbreviations: CTACK, cutaneous T-cell attracting chemokine; SDF-1{alpha}, stromal cell-derived factor-1{alpha}; SLC, secondary lymphoid tissue chemokine.

 
CD34+ BM Cells Migrated in Response to SDF-1{alpha}, SLC, and CTACK In Vitro
To confirm that these receptors were actually functional in these cells, in vitro chemotaxis assays were undertaken. CTACK induced CD34+ BM cell migration in a dose-dependent manner (Fig. 2B, 2C). SDF-1{alpha} was previously known to induce hematopoietic stem cell migration via CXCR4 interactions [19]. Indeed, SDF-1{alpha} enhanced the migration of CD34+ BM cells in a dose-dependent manner (Fig. 2B and data not shown). In addition, SLC enhanced CD34+ BM cell migration (Fig. 2B and data not shown).

CTACK Treatment Specifically Led to Accumulations of BMDKs in Wounded Skin
To assess the ability of chemokines in keratinocyte precursor cell recruitment in vivo, we injected these chemokines to the periphery of wounded skin in enhanced GFP transgenic transplanted mice. The number of GFP-positive BMDKs in the epidermis was calculated (n = 5 mice in each group). Although SDF-1{alpha} and SLC failed to influence the number of BMDKs compared with the controls, CTACK significantly increased the number of BMDKs in wounded skin (Fig. 3A). Next, to clarify whether this increase of CD34+ BM cells could enhance the overall number of BMDKs, we attempted to increase the levels of CD34+ BM cells in peripheral blood by means of cytokine mobilization using G-CSF (five mice) or CD34+ cell adoptive transfer (five mice). Increased numbers of CD34+ BM cells in peripheral blood significantly enhanced the number of BMDKs in wounded skin in each group. In addition, CTACK treatment in this group significantly increased the number of BMDKs by approximately fivefold compared with the controls in each group (Fig. 3A). Furthermore, intradermal injection of CTACK neutralizing antibody inhibited this BMDK migration in a dose-dependent manner (five mice) (Fig. 3B).


Figure 3
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  		Figure 3. CTACK specifically accumulated bone marrow-derived keratinocytes (BMDKs) in wounded skin. (A): The number of BMDKs was quantified as a percentage of the total number of keratinocytes in wounded skin from untreated mice or those treated with G-CSF for cytokine mobilization or those that received CD34+ BM cell adoptive transfer. SLC, SDF-1{alpha}, or CTACK (1 µg in 30 µl) was intradermally injected into the periphery of wounded skin (five mice in each group). CTACK significantly accumulated large numbers of BMDKs as compared with SLC, SDF-1{alpha}, and PBS (** p < .01). Furthermore, the number of BMDKs increased in mice treated with G-CSF for cytokine mobilization or those that received CD34+ BM cell adoptive transfer (*** p < .005). (B): CTACK neutralizing antibody (0–16 µg in 120 µl) was injected to the periphery of wounded skin. The numbers of BMDKs were decreased by CTACK neutralizing antibody in dose-dependent manner (five mice) (* p < .05, *** p < .005). Abbreviations: BM, bone marrow; CTACK, cutaneous T-cell attracting chemokine; G-CSF, granulocyte colony stimulating factor; PBS, phosphate-buffered saline; SDF-1{alpha}, stromal cell-derived factor-1{alpha}; SLC, secondary lymphoid tissue chemokine.

 
CTACK Treatment Accelerated BMDK-Induced Wound Healing
We determined that CTACK was capable of inducing additional BMDKs that could participate in the host skin wound response. Surprisingly, intradermal injection of CTACK significantly accelerated wound closure (six mice) (Fig. 4A, 4B). We speculated that this effect was mediated by increases in BMDK. However, there are several possible reasons why intradermal injection of CTACK might accelerate wound repair. This could represent increased angiogenesis, keratinocyte proliferation, or keratinocyte migration during wound healing. Therefore, the following additional experiments were performed. We examined whether CTACK induced angiogenesis in wound healing. Three days after skin wounding, when the greatest number of new capillaries were formed [20], the number of dermal capillaries was calculated on two sections from each mouse (five mice per treatment group) and on an identical wound site section with or without CTACK injection (PBS vehicle control). CTACK had no effect on angiogenesis in wounded skin (Fig. 4C). Next, to determine whether CTACK induced keratinocyte proliferation, we measured proliferation rates in vitro. CTACK had no effect on the proliferation of the keratinocytes (six mice) (Fig. 4D). It has previously been reported that CCR10 is expressed on only skin-homing memory T cells. In our study, we confirmed that keratinocytes and fibroblasts in normal and wounded skin did not express CCR10 (data not shown). Finally, to determine whether CTACK induced keratinocyte migration, we undertook a keratinocyte migration assay in vitro. CTACK had no effect on keratinocyte migration. These results indicate that intradermal injection of CTACK accelerated wound healing without influencing angiogenesis, keratinocyte proliferation, or migration.


Figure 4
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  		Figure 4. Increased bone marrow-derived keratinocytes (BMDKs) by CTACK accelerated wound closure without angiogenesis or keratinocyte proliferation. (A, B): Wound size was measured at 10 days after wounding and subsequent CTACK treatment (total 3 µg in 100 µl) or PBS (100 µl) as control (six mice in each group). Full-thickness cutaneous wounds were made and subsequently monitored daily. Intradermal injection of CTACK significantly accelerated wound closure (* p < .05) (A). Representative images at 10 days after wounding of wounds treated with PBS (as control) (mice 1–3) or CTACK (mice 4–6) (B). (C): The numbers of capillaries in the dermis treated by CTACK (1 µg in 30 µl) or PBS (30 µl) (as control vehicle) at 3 days after wounding were quantified. There was no statistical difference between CTACK and control (two sections each of five mice). (D): Keratinocytes were cultured with or without CTACK (0–100 ng/ml) for 72 hours, and viable cells were determined by proliferation assay. There was no statistical difference in proliferation of keratinocytes between the two treatment groups (nine mice). Abbreviations: CTACK, cutaneous T-cell attracting chemokine; PBS, phosphate-buffered saline.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
We have shown that CTACK is a major regulator of keratinocyte precursor cell migration from the BM to the skin. In this study, BM-derived cells were able to transdifferentiate into keratinocytes at the sites of skin wounds. Furthermore, we have shown that CTACK is specifically expressed in wounded skin and that CD34+ BM cells express CCR10, the main CTACK receptor. Although the numbers of BMDKs in untreated skin wounds are extremely low, a combination of treatments to increase the number of peripheral blood CD34+ BM-derived cells, together with intradermal injection of CTACK into the wounded skin periphery, resulted in a 10-fold increase of BMDK. Furthermore, an increase in circulating CD34+ BM cells enhanced the number of BMDKs, suggesting that a significant number of CD34+ cells are included in the BMDK precursor population. Intradermal injection of CTACK significantly accelerated wound closure via increased rates of BMDK accumulation. We concluded that small populations of the progenitor cells coexpressing CD34 and CCR10 exist, that a subset of these cells migrated to the epidermis in response to CTACK, and that these cells play important roles in skin wound healing and repair.

Tissue repair and regeneration after injury is thought to involve the selective recruitment of circulating or resident stem cell populations. Chemokine/chemokine receptor interactions are expected to contribute to these mechanisms of stem cell plasticity. However, only one chemokine/chemokine receptor interaction, the SDF-1{alpha}/CXCR4 interaction, has thus far been reported. The SDF-1{alpha}/CXCR4 interaction has been identified as a factor causing hematopoietic stem cell mobilization [19]; however, other tissue cells are recruited from the BM by this interaction, including myocytes [21] and neural cells [22]. In addition, SDF-1{alpha} and CXCR4 are widely expressed on various cell types, and SDF-1{alpha}/CXCR4 interactions play an important role in several developmental and regenerative phenomena, such as cardiogenesis [21], neovascularization [23], hematopoiesis [19], and hepatic development [24]. Furthermore, wounding stimulated the engraftment of BM cells into the skin and induced BM-derived cells to incorporate into and differentiate into nonhematopoietic skin structures [25]. Given that it is unlikely that this receptor interaction is involved in many cell functions, organ-specific chemokine/chemokine receptor interactions are predicted. Indeed, we have demonstrated that the CTACK/CCR10 interaction is involved in skin wound healing. A strategy to detect further tissue-specific chemokines expressed in injured tissue would further benefit future organ-specific BM stem cell enrichment and recruitment.

Several studies have indicated that BM-derived cells have the ability to effect tissue regeneration in protein-deficient mouse models. Mice with damaged liver function caused by a fumarylacetoacetate hydrolase deficiency recovered after normal mouse-derived hematopoietic stem cell transplantation, in which mouse BM cells transdifferentiated into hepatocytes [26]. BM-derived cells may prove beneficial for protein-deficient disease therapy. In the study, the increase in BM-derived cells might further enhance the rate of damaged tissue recovery in wild-type mice. This suggests that cells derived from circulating stem cells are more effective at enlisting host regenerative mechanisms than resident tissue cells, indicating a promising therapeutic strategy for damaged tissues. Our results provide direct evidence that tissue-specific BM precursor cells are recruited with the help of tissue-specific chemokine/chemokine receptor interactions.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
This work was supported in part by grants-in-aid for Scientific Research (number 133,57008 to H.S. and number 157,90563 to R.A.) and the Project for Realization of Regenerative Medicine (to H.S.) from the Ministry of Education, Science, Sports, and Culture of Japan, and Health and Labor Sciences Research Grants (numbers H13-Measures for Intractable Disease-02 and H16-Measures for Intractable Disease-02 to H.S.) from the Ministry of Health, Labor, and Welfare of Japan. We thank Ayumi Honda for excellent technical assistance.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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