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The Journal of Immunology, 2008, 181, 3974 -3983
Copyright © 2008 by The American Association of Immunologists, Inc.
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Defects in Skin {gamma}{delta} T Cell Function Contribute to Delayed Wound Repair in Rapamycin-Treated Mice1

Robyn E. Mills, Kristen R. Taylor, Katie Podshivalova, Dianne B. McKay and Julie M. Jameson2

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Disruptions in the normal program of tissue repair can result in poor wound healing, which perturbs the integrity of barrier tissues such as the skin. Such defects in wound repair occur in transplant recipients treated with the immunosuppressant drug rapamycin (sirolimus). Intraepithelial lymphocytes, such as {gamma}{delta} T cells in the skin, mediate tissue repair through the production of cytokines and growth factors. The capacity of skin-resident T cells to function during rapamycin treatment was analyzed in a mouse model of wound repair. Rapamycin treatment renders skin {gamma}{delta} T cells unable to proliferate, migrate, and produce normal levels of growth factors. The observed impairment of skin {gamma}{delta} T cell function is directly related to the inhibitory action of rapamycin on mammalian target of rapamycin. Skin {gamma}{delta} T cells treated with rapamycin are refractory to IL-2 stimulation and attempt to survive in the absence of cytokine and growth factor signaling by undergoing autophagy. Normal wound closure can be restored in rapamycin-treated mice by addition of the skin {gamma}{delta} T cell-produced factor, insulin-like growth factor-1. These studies not only reveal that mammalian target of rapamycin is a master regulator of {gamma}{delta} T cell function but also provide a novel mechanism for the increased susceptibility to nonhealing wounds that occurs during rapamycin administration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ability of epithelial tissues to rapidly repair and regenerate in the face of damage is fundamental to barrier maintenance. During normal wound healing, a delicate balance of factors and cell types orchestrates the process in a precisely timed manner. Disruptions can result in the debilitating condition of a chronic, nonhealing wound. Much effort has been directed toward determining which factors affect wound repair processes in chronic wounds, but there is less known about what may regulate the cellular sources of the secreted factors important in proper wound healing (1, 2, 3, 4, 5).

Nonhealing wounds are exhibited by patients administered the immunosuppressant rapamycin (also called sirolimus) (6, 7, 8, 9, 10); however, the mechanisms contributing to this wound healing defect are unclear. Rapamycin is commonly administered for the prophylaxis of acute rejection of transplanted solid organs, used to coat arterial stents, and it is currently being examined in clinical trials for the treatment of hematological cancers (11, 12, 13, 14). Rapamycin is known to be a potent inhibitor of certain effector {alpha}β T lymphocyte populations (15, 16, 17, 18, 19), while other populations such as regulatory T cells exhibit a selective survival (20). In other studies, cytokine-driven responses by peripheral naive T cells are not affected by rapamycin, suggesting a reliance on other signaling molecules such as Pim-1 and Pim-2 (21). However, the effects of rapamycin on intraepithelial lymphocytes (IELs)3 such as skin {gamma}{delta} T cells have not been evaluated.

Closely associated with the epithelia is an interdigitating population of resident lymphocytes expressing the {gamma}{delta} TCR (22). Skin {gamma}{delta} T cells express a canonical V{gamma}3V{delta}1 TCR that recognizes an unidentified self Ag expressed on damaged or stressed keratinocytes (23, 24). Intraepithelial {gamma}{delta} T cells are thought to act as primary responders to damage or disease due to their ability to sense and respond to epithelial damage or disruption (25, 26, 27). Specialized roles have been attributed to skin {gamma}{delta} T cells in the regulation of wound repair, epithelial homeostasis, cutaneous malignancy, and contact hypersensitivity (27, 28, 29, 30).

Skin {gamma}{delta} T cells produce cytokines, chemokines, and growth factors with both autocrine and paracrine functions. Homeostatic production of growth factors such as insulin-like growth factor 1 (IGF-1) by skin {gamma}{delta} T cells maintains skin homeostasis (30). IGF-1 is a peptide hormone known to regulate both keratinocyte and skin {gamma}{delta} T cell migration and survival (30, 31, 32, 33). In the context of wound healing, production of factors such as TNF-{alpha}, IGF-1, and keratinocyte growth factor-1 (KGF-1) by skin {gamma}{delta} T cells promotes wound closure, reepithelialization, and inflammatory cell recruitment to the wound site (27, 30, 34, 35). To perform these functions the normally, dendritic skin {gamma}{delta} T cells retract their dendrites, adopt a rounded morphology, and migrate to the site of trauma where they proliferate locally (27). Unlike naive {alpha}β T lymphocytes, skin {gamma}{delta} T cells reside in the epidermis in a preactivated state. They exhibit constitutive IL-2 promoter activation (36) and expression of activation markers CD25 (IL-2 receptor {alpha}) and CD69, suggesting that they are primed for a rapid response. However, little is known about the signaling pathways that regulate this response.

On the molecular level, rapamycin inhibits the serine/threonine kinase mammalian target of rapamycin (mTOR). mTOR is a central protein in a complex network that regulates amino acid and nutrient sensing in particular cell types through the formation of two separate protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (37, 38, 39). Each multiprotein complex regulates distinct pathways of the signaling network. Rapamycin binds to the cellular protein FKBP12 and subsequently induces the disassociation of mTORC1, which consists of mTOR, raptor, and mLST8 (37, 40). This complex has downstream effects that include the regulation of cell cycle, translation machinery, and autophagy (41). The second complex, mTORC2, is involved in cytoskeletal rearrangement (42). mTORC2 is comprised of mLST8, SIN1, and rictor, and it is not immediately disrupted following rapamycin treatment in many types of cells (42). However, prolonged treatment of some cell lines with rapamycin can result in reduced levels of mTORC2 through reduction of rictor-bound mTOR, as well as decreased phosphorylation of Akt Ser473, the downstream target of mTORC2 (43). It has been controversial whether rapamycin results in anergy or apoptosis of transplant-specific effector T cells (19, 44, 45). Some groups suggest that mTOR regulates certain types of {alpha}β T cells by impairing their ability to receive costimulation and cytokine signals (46, 47).

Herein we establish a murine model for the examination of wound healing defects mediated by rapamycin. Data presented here indicate that rapamycin negatively affects skin {gamma}{delta} T cell function during wound repair. Rapamycin arrests the IEL in G1 phase and blocks proliferative responses to cytokines such as IL-2, rendering them anergic. Our data show that skin {gamma}{delta} T cells do not undergo apoptosis when cytokine signals are suppressed by rapamycin. Instead, skin {gamma}{delta} T cells treated with rapamycin enter autophagy in an attempt to survive in the absence of cytokine signaling. As the skin IEL become anergic and autophagic, they exhibit impaired homeostatic and activation-induced functions. This dysfunction is indicated by a diminished ability to produce soluble factors such as IGF-1 and TNF-{alpha} and delayed activation-induced cell migration. These effects are mediated through mTOR, as we observe reduced phosphorylation of both mTORC1 and mTORC2 downstream targets in skin {gamma}{delta} T cells treated with rapamycin. Finally, the addition of the skin {gamma}{delta} T cell-produced factor IGF-1 is able to successfully restore normal wound closure in rapamycin-treated mice. These studies demonstrate the selective inhibition of {gamma}{delta} T cell function in the skin of rapamycin-treated animals resulting in defective wound repair and they represent a novel pathway in the regulation of skin {gamma}{delta} T lymphocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

The skin {gamma}{delta} T cell line 7-17 was maintained in complete RPMI 1640 (Mediatech) supplemented with 10% heat-inactivated FBS (Omega Scientific) and 20 U/ml recombinant IL-2. The keratinocyte line PAM 2-12 was maintained in complete DMEM (Mediatech) supplemented with 10% heat-inactivated FBS. Epidermal cells and skin {gamma}{delta} T cells were isolated from trypsin-digested epidermal sheets (27, 34) and maintained in complete RPMI 1640 with 10% FBS and 10 U/ml recombinant IL-2. For short-term cell lines, skin {gamma}{delta} T cells were initially cultured with 2 µg/ml Con A (Sigma-Aldrich) and 1 µg/ml indomethacin (Sigma-Aldrich). All cells were maintained at 37°C under 5% CO2.

Animals and wounding procedure

TCR{delta}–/– mice on the C57BL/6 background were purchased from Jackson ImmunoResearch Laboratories. C57BL/6 and TCR{delta}–/– mice were bred at The Scripps Research Institute and housed in specific pathogen-free conditions according to The Scripps Research Institute Institutional Animal Care and Use Guidelines. Mice were used between 10 and 16 wk of age. For rapamycin administration, mice were injected i.p. with 200 µl containing 1% rapamycin (Sigma-Aldrich) in 0.2% carboxymethlycellulose (Sigma-Aldrich) and 0.25% Tween 80 (Sigma-Aldrich) in distilled H2O or with vehicle control daily. Since rapamycin is originally diluted in ethanol, the vehicle control contains equal amounts of diluent. For wound healing experiments, mice were administered rapamycin or vehicle control for 3 days before wounding, and daily administration was continued. Wounding was performed on mice anesthetized with isoflurane as previously described (27, 34). Briefly, the dorsal surface of the mouse was shaved, back skin and panniculus carnosus was pulled up, and one or two sets of sterile full-thickness wounds were generated using a sterile 2-mm punch tool. Wounds were left uncovered, and mice were housed individually with sterile paper bedding. In some experiments 100 ng of recombinant IGF-1 (Sigma-Aldrich) or buffer alone was applied to each wound site immediately postwounding and daily thereafter. Wounds on at least six mice were examined per condition in at least three independent experiments. For wound closure kinetics, images were acquired with a Nikon Coolpix S4 and wound size was monitored using ImageJ software (National Institutes of Health). To examine rounding of skin {gamma}{delta} T cells at the wound site, full-thickness wounds were generated in mouse ears using a 1-mm punch tool and wounded tissue was harvested 2 h later.

Antibodies and flow cytometry

FITC-, PE-, or allophycocyanin-conjugated mAbs specific for {gamma}{delta} TCR (GL3), CD25 (PC61), and Thy1.2 (53-2.1) were purchased from BD Biosciences. Other Abs used for flow cytometry include goat anti-IGF-1 (G-17) (Santa Cruz Biotechnology), rat anti-CD69 (H1.2F3) (eBioscience), BrdU flow kit (BD Biosciences), and an annexin V apoptosis kit (BD Biosciences). Rabbit Abs specific for S6 kinase, p-S6 kinase (Thr389), Akt, and p-Akt (Ser473) were purchased from Cell Signaling Technology. Rat anti-Ki-67 Ag (DakoCytomation) was used for immunohistochemistry with biotin-conjugated mouse anti-rat secondary Ab (Jackson ImmunoResearch Laboratories). Abs specific for CD3{epsilon} (500A2) (1 µg/ml) were used for stimulation of 7-17 cells and skin {gamma}{delta} T cells in epidermal sheets. Other secondary Abs used include HRP-conjugated goat anti-rabbit (SouthernBiotec) and FITC-conjugated donkey anti-goat (Jackson ImmunoResearch Laboratories). For flow cytometry, a Cytofix/Cytoperm kit (BD Biosciences) was used for intracellular cytokine/growth factor staining. Cells were acquired with CellQuestPro on a BD FACSCalibur HTS (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Skin organ culture

Skin organ cultures from C57BL/6 and TCR{delta}–/– mice were established as previously described (27, 34). Briefly, gel foam (Pfizer) was soaked in media. Full-thickness biopsy wounds (2 mm) were generated and placed dermis side down on gel foam in 10% DMEM supplemented with rapamycin or ethanol control in 24-well plates. In some cases 7-17 skin {gamma}{delta} T cells were incubated in the presence of 20 ng/ml rapamycin or ethanol control for 15 h, stimulated for 2 h with anti-CD3{epsilon}, washed thoroughly, and plated at a density of 3 x 105 cells per well. Recombinant IGF-1 was added at a concentration of 100 ng/ml to some wells. Images of wounds were acquired and kinetics of closure quantified using ImageJ software.

Western blot analysis

7-17 cells were incubated in starvation media for 2 h followed by culture in the presence of 20 ng/ml rapamycin or ethanol control for 2 h (p-S6 kinase) or 24 h (p-Akt). Next, cells were stimulated for various time points with 40 U/ml IL-2 and harvested in lysis buffer containing 62.5 mM Tris HCL (pH 6.8), 2% (v/v) SDS, 50 mM DTT, 10% glycerol, and 0.01% bromphenol blue. After lysis, insoluble material was removed by centrifugation at 12,000 x g for 10 min. Samples were separated on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h with 1x TBS, 0.02% sodium azide, 3% BSA, and 10% goat serum. Primary Abs diluted in 1x TBS, 1% BSA, 0.2% Tween 20, 0.02% sodium azide, and 3% goat serum were incubated with the membrane overnight at 4°C. HRP-conjugated secondary Abs and ECL (Pierce Chemical) were used to detect primary Abs.

Epidermal sheet immunofluorescence

Wounded or nonwounded ears from rapamycin or vehicle-treated mice were excised, peeled into halves, and digested in 3% ammonium isothiocyanate (Sigma-Aldrich). Epidermal sheets were peeled and stained with PE-conjugated anti-{gamma}{delta} TCR, LysoTracker Green, and/or DAPI (Sigma-Aldrich). Digital images were acquired (Zeiss AxioCam HRc). To examine migration of skin {gamma}{delta} T cells, ear skin was cultured in complete DMEM supplemented with 10% FBS and 20 ng/ml rapamycin in 24-well plates. For these experiments 1 µg/ml anti-CD3{epsilon} (500A2) Ab or 100 ng/ml IGF-1 was added to culture. After culture, epidermal sheets were washed with PBS and processed as described above. Quantifications of dendrite number and autophagosomes were performed using Photoshop CS2 software (Adobe). More than 200 cells were counted per mouse and at least three mice were examined per condition.

Proliferation assays

Cells were cultured between 5 and 8 h in 10% complete DMEM supplemented with rapamycin or ethanol before addition to wells coated with stimulatory anti-CD3{epsilon} or {gamma}{delta} TCR Abs. In some cases, 100 ng/ml IGF-1, 40 U/ml IL-2, or 5 µg/ml Con A (Sigma-Aldrich) was added. Cells were cultured for 14–16 h before addition of 1 µCi/well [3H]thymidine (MP Biomedicals) for 8–10 h. Samples were harvested and [3H]thymidine incorporation was determined by a β-counter. To examine cell cycle, the keratinocyte line PAM 2-12 or the skin {gamma}{delta} T cell line 7-17 was seeded at 2 x 105 cells/well in a 6-well plate in complete DMEM supplemented with 10% FBS overnight before addition of 20 ng/ml rapamycin (Sigma-Aldrich) or ethanol. Fresh rapamycin or ethanol was supplemented daily. After 24 h, 100 µg/ml BrdU was added and proliferation monitored using flow cytometry.

Immunohistochemistry

Wounded skin was excised from rapamycin or vehicle-treated mice, fixed in ethanol, and embedded in paraffin. Sections were prepared and stained with biotinylated Abs to Ki-67 followed by peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories). The presence of positive cells was revealed by incubation in metal-enhanced diaminobenzidine (Pierce Chemical) and counterstained with hematoxylin (Sigma-Aldrich). Control staining was done without primary Ab. At least two wounds from each of three mice per condition were examined.

Acridine orange staining

7-17 skin {gamma}{delta} T cells were seeded on coverslips in the presence or absence of 20 ng/ml rapamycin for 15 h in complete RPMI 1640 supplemented with 10% FBS. Coverslips were washed in PBS supplemented with 10% FBS and 10 U/ml IL-2, incubated with 1 µg/ml acridine orange (Sigma-Aldrich) for 15 min at 25°C, and washed thoroughly before examination with a fluorescence microscope. Starvation was used as a positive control, while bafilomycin treatment was used as a negative control. Digital images were acquired and the number of autophagic cells was quantified using Photoshop software (Adobe). Cells with more than two positively staining vesicles were defined as autophagic.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Rapamycin impairs the ability of skin {gamma}{delta} T cells to mediate wound closure

To examine how the mTOR inhibitor rapamycin affects wound repair, we established a murine model of wound healing in which mice were administered rapamycin daily. The defect in wound repair has been described in humans (6, 7, 8) and rats (9, 48); however, this has not been investigated in mice, and no mechanism has been ascribed to this defect. Wild-type C57BL/6J mice were administered daily with rapamycin or vehicle control for 3 days before wounding, and treatment was continued as wound closure was monitored. Wound size was measured during a period of 14 days (Fig. 1, A and B). Similar to findings in humans, rapamycin-treated mice exhibited delays in the rate of wound closure as compared with those treated with vehicle control. This defect was especially evident on days 4, 7, and 10 postwounding. Furthermore, significantly fewer wounds had completely closed on day 10 in rapamycin-treated animals as compared with wounds from vehicle control-treated animals (Fig. 1C). A delay in complete wound closure of ~3 days was observed, similar to the delay observed in mice lacking {gamma}{delta} T cells (TCR{delta}–/– mice) (27). These data confirm that rapamycin has a negative impact on wound closure.


Figure 1
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  		FIGURE 1. Rapamycin treatment delays wound closure and impairs skin {gamma}{delta} T cell wound healing functions. Wound closure was monitored on mice administered rapamycin as compared with vehicle control (A). Rapamycin-treated mice exhibited a delay in wound closure as compared with those treated with vehicle. Data are means ± SEM and are representative of three experiments. At least six mice per condition have been examined in each experiment. B, Representative images from wounds acquired 4 days postwounding. C, Percentage of observed wounds remaining open 10 days postwounding. Data are means ± SD. D, Rapamycin impairs wound closure in skin from wild-type mice to a similar degree to skin from untreated TCR{delta}–/– mice using a skin organ culture system. E, Rapamycin inhibits the ability of 7-17 skin {gamma}{delta} T cells to restore normal wound closure to TCR{delta}–/– skin in organ culture. Data in skin organ cultures are means ± SEM and are representative of at least three experiments. *, p < 0.05 and **, p < 0.005 vs vehicle control (two-tailed, unpaired Student’s t test).

 
Given the potent immunosuppressive nature of rapamycin and the similarity of wound closure kinetics to TCR{delta}–/– mice, the contribution of skin {gamma}{delta} T cell dysfunction to impaired wound healing in rapamycin-treated mice was examined. Using a well-established system of skin organ culture (27, 49, 50), the early stages of wound healing were monitored in vitro. In cultures supplemented with rapamycin, wound closure in this skin organ culture model was inhibited, similar to our findings in vivo (Fig. 1D). The kinetics of closure observed in wounded skin treated with rapamycin were similar to those observed in skin from TCR{delta}–/– mice (Fig. 1D). Additionally, there was no further defect in wound repair in TCR{delta}–/– skin upon rapamycin treatment (Fig. 1D). To identify whether rapamycin impairs the ability of skin {gamma}{delta} T cells to mediate wound repair, we utilized a model we have previously established to assess skin {gamma}{delta} T cell wound healing functions. In this model, the addition of activated skin {gamma}{delta} T cells to ex vivo cultures of wounded skin from TCR{delta}–/– mice restores normal wound closure kinetics (27). Here, we examined whether rapamycin treatment impairs the ability of skin {gamma}{delta} T cells to affect early wound closure. When skin from TCR{delta}–/– mice was cultured with activated skin {gamma}{delta} T cells, early wound closure was restored as we previously reported (27) (Fig. 1E). However, rapamycin pretreatment of activated skin {gamma}{delta} T cells before their addition to TCR{delta}–/– skin organ culture impaired the ability of skin {gamma}{delta} T cells to restore early wound closure (Fig. 1E). These results indicate that rapamycin impairs skin {gamma}{delta} T cell function, diminishing their capacity to mediate wound closure. This implicates the mTOR signaling cascade in wound healing functions mediated by skin {gamma}{delta} T cells.

Rapamycin treatment inhibits phosphorylation of the mTOR targets p70 S6 kinase and Akt in skin {gamma}{delta} T cells

Little is known about the signaling cascades activated in intraepithelial {gamma}{delta} T cells. In an effort to determine whether T cell trophic factors such as the cytokine IL-2 signal via mTOR in skin {gamma}{delta} T cells, we examined the molecular targets of the two mTOR complexes. Signaling through mTORC1 induces the phosphorylation of p70 S6 kinase at many sites, including Thr389 (37). To evaluate mTOR activity, the skin {gamma}{delta} T cell line 7-17 was stimulated with IL-2 in the presence and absence of rapamycin. Phosphorylation at Thr389 was evident in IL-2-stimulated skin {gamma}{delta} T cells within 15 min and was observed at higher levels by 30 min. This activation-induced phosphorylation is repressed in the presence of rapamycin (Fig. 2A), demonstrating that IL-2-mediated phosphorylation of S6 kinase is dependent on mTOR.


Figure 2
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  		FIGURE 2. Rapamycin treatment of skin {gamma}{delta} T cells directly inhibits IL-2 signaling through mTOR. A, IL-2 (40 U/ml) stimulates phosphorylation of pS6 kinase at Thr389 (a marker of mTOR activation) within 30 min, while rapamycin inhibits phosphorylation of this site. 7-17 skin {gamma}{delta} T cells were treated with rapamycin or ethanol control for 2 h following 2 h of starvation and were then stimulated with IL-2 for various time points. B, Akt is phosphorylated at Ser473 in skin {gamma}{delta} T cells stimulated with IL-2. Prolonged (24-h) rapamycin treatment inhibits this response, suggesting that activity of mTORC2 is inhibited. 7-17 skin {gamma}{delta} T cells were treated with rapamycin for 24 h. During the last 2 h, cells were also starved before stimulation with IL-2. Lysates were prepared for Western blot analysis.

 
mTOR is a component of another complex, mTORC2, that mediates cytoskeletal rearrangement (42). Signaling through mTORC2 induces phosphorylation of Akt at Ser473 (43, 51). Although the mTORC2 complex was originally reported to be rapamycin resistant (38, 42), the phosphorylation of Akt Ser473 by mTORC2 is diminished in particular cell types after 24 h of rapamycin treatment (43). In an effort to determine whether mTORC2 activity is present in skin {gamma}{delta} T cells and whether this activation is rapamycin-sensitive, we treated the skin {gamma}{delta} T cell line with and without rapamycin, stimulated the cells with IL-2 or anti-CD3{epsilon} Abs, and evaluated Akt phosphorylation. Upon IL-2 treatment or TCR stimulation of skin {gamma}{delta} T cells, Akt is phosphorylated at Ser473 within 15 min. After 24 h of rapamycin treatment this phosphorylation is severely impaired (Fig. 2B). Similar to previous reports, rapamycin was unable to inhibit Akt Ser473 phosphorylation at time points earlier than 24 h of treatment (data not shown).

Skin {gamma}{delta} T cells are unresponsive to IL-2 or mitogen stimulation in the presence of rapamycin

IL-2-stimulated proliferation of certain {alpha}β T lymphocyte subsets induces the phosphorylation of p70 S6 kinase in a rapamycin-sensitive manner (52). However, peripheral regulatory T cells are resistant to rapamycin treatment, suggesting that they rely on alternate pathways for proliferation (20). To examine whether skin {gamma}{delta} T cell proliferation is dependent on mTOR, skin {gamma}{delta} T cells were treated with various concentrations of rapamycin before stimulation with Abs specific for CD3{epsilon} (Fig. 3A), Con A (Fig. 3C), or {gamma}{delta} TCR (data not shown), or addition of IL-2 (Fig. 3B, left panel). Rapamycin treatment inhibited proliferation of the skin {gamma}{delta} T cell line 7-17 to either anti-CD3{epsilon} or cytokine stimulation at rapamycin concentrations as low as 10 ng/ml. Similar results were obtained with {gamma}{delta} T cells isolated from the skin of C57BL/6 mice (Fig. 3B, right panel). This proliferative defect could not be restored in the presence of rapamycin by supplementing the stimulus with IL-2 or IGF-1 (Fig. 3C). Removal of the inhibitor from the culture for several days restores normal skin {gamma}{delta} T cell proliferation (data not shown). To identify at which stage of the cell cycle rapamycin-treated skin {gamma}{delta} T cells arrest, BrdU incorporation and DNA content analyses were performed. Rapamycin treatment blocks skin {gamma}{delta} T cells from exiting the G1 phase of the cell cycle, preventing entry into synthesis (S) phase (Fig. 3D). These results suggest that skin {gamma}{delta} T cells depend on mTOR signaling as a major pathway for exit from G1 phase.


Figure 3
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  		FIGURE 3. Inhibition of mTOR suppresses skin {gamma}{delta} T cell but not keratinocyte proliferation. Skin {gamma}{delta} T cell proliferative responses to anti-CD3{epsilon} (A) and IL-2 (B) stimulation are impaired upon mTOR inhibition by rapamycin. 7-17 skin {gamma}{delta} T cells (A and B, left panel) or dendritic epidermal T cells from murine skin (B, right panel) were incubated for 4 h with 20 ng/ml rapamycin before stimulation with 1 µg/ml anti-CD3{epsilon} (A) or 40 U/ml IL-2 (B) as assessed by [3H]thymidine incorporation. C, Neither IGF-1 nor IL-2 addition can rescue the defect. Either 100 ng/ml IGF-1 or 40 U/ml IL-2 was added to the cultures simultaneously with Con A stimulation. Data represent the means ± SD. D, When mTOR is inhibited, skin {gamma}{delta} T cells are blocked at the G1 stage of the cell cycle. 7-17 skin {gamma}{delta} T cells were treated with 20 ng/ml rapamycin for 17 h and pulsed with 100 µg/ml BrdU for 12 h before staining for BrdU incorporation. Numbers indicate percentage of cells within indicated gates. E, Keratinocyte proliferation is not impaired upon rapamycin treatment. The PAM 2-12 cell line was treated with 20 ng/ml rapamycin or vehicle control for 48 h with 100 µg/ml BrdU and analyzed by flow cytometry. F, Five days postwounding, keratinocytes proliferate at the wound site in mice treated with or without rapamycin. Arrows indicate Ki-67-positive cells in the epidermis.

 
The bulk of the epidermis consists of keratinocytes. To examine whether rapamycin affects the ability of keratinocytes to proliferate, the PAM 2-12 cell line was incubated with BrdU for 48 h in the presence of rapamycin (Fig. 3E). In contrast to skin {gamma}{delta} T cells, keratinocytes retain the ability to proliferate during treatment with rapamycin. Similar results were obtained with keratinocytes pulsed with BrdU for 16 or 24 h (data not shown). Since previous studies have identified defects in keratinocyte stem cell proliferation in the presence of rapamycin (53), we monitored keratinoycte proliferation in vivo. We performed biopsy punch wounds on the skin of rapamycin- or vehicle-treated mice and stained skin sections for Ki-67 expression to identify proliferating cells (Fig. 3F). Keratinocyte proliferation at the wound site was comparable between rapamycin- and vehicle-treated mice. Using either BrdU to monitor keratinocyte turnover or Ki-67 staining to identify proliferating cells, no defect in keratinocyte proliferation was found in the presence of rapamycin. Thus, at clinically relevant doses, rapamycin specifically inhibits skin {gamma}{delta} T cell proliferation in the epidermal compartment. These results demonstrate cell-specific regulation of proliferation via mTOR signaling within the epidermis.

Rapamycin induces autophagy, not apoptosis, of skin {gamma}{delta} T cells

In several T cell populations, such as double-positive thymocytes or effector T cells, rapamycin has been shown to inhibit proliferation and increase the susceptibility to apoptosis (18, 54). In other cases, lymphocyte populations undergo macroautophagy, a process in which the cell catabolizes proteins and organelles as a survival mechanism (55). Having observed the diminished proliferative capacity of rapamycin-treated skin {gamma}{delta} T cells, we examined whether these cells were undergoing increased levels of apoptosis. For these experiments, skin {gamma}{delta} T cells were treated with rapamycin and apoptosis was examined by annexin V binding and propidium iodide staining. No increase in apoptosis was evident in skin {gamma}{delta} T cells treated with rapamycin (Fig. 4A). Furthermore, apoptosis is not exacerbated when rapamycin-treated skin {gamma}{delta} T cells are stimulated to undergo activation-induced cell death or are cytokine-starved (Fig. 4A). Additionally, rapamycin does not induce skin {gamma}{delta} T cells to undergo apoptosis when administered for several days in skin organ culture (data not shown). In fact, rapamycin treatment does not seem to affect the viability of skin {gamma}{delta} T cells either in vitro or in vivo. {gamma}{delta} TCR-bearing cells were quantified in epidermal sheets via immunofluorescent staining, and no measurable decrease in the number of cells per square millimeter was detected after up to 14 days of rapamycin treatment (Fig. 4B). Taken together, these results suggest that rapamycin treatment does not induce or exacerbate apoptosis in skin {gamma}{delta} T cells.


Figure 4
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  		FIGURE 4. Rapamycin promotes autophagy rather than apoptosis in skin {gamma}{delta} T cells. A, Inhibition of mTOR with rapamycin does not induce or augment apoptosis. 7-17 skin {gamma}{delta} T cells were left untreated, stimulated with Abs specific for CD3{epsilon}, or starved for 6 h in the presence of rapamycin or ethanol control. Cells were then stained with annexin V and propidium iodide (PI) to assess apoptosis and analyzed by flow cytometry. B, Similar numbers of skin {gamma}{delta} T cells remain in the epidermis of mice treated with rapamycin for 14 days. Epidermal sheets were prepared from mice administered rapamycin or vehicle control and stained with Abs specific for the {gamma}{delta} TCR. The number of {gamma}{delta} T cells was quantified per mm2. C, Rapamycin induces skin {gamma}{delta} T cells to undergo autophagy. 7-17 skin {gamma}{delta} T cells were incubated in the presence or absence of rapamycin for 15 h before staining with acridine orange to identify autophagosomes. An increased number of cells treated with rapamycin exhibit more than two autophagosomes as compared with ethanol control-treated cells. D, An increased number of skin {gamma}{delta} T cells undergo autophagy in vivo after rapamycin administration. Epidermal sheets were prepared from mice administered rapamycin as compared with vehicle control. Sheets were stained with fluorescent Abs to the {gamma}{delta} TCR to identify skin {gamma}{delta} T cells and LysoTracker Green to identify autophagosomes. The number of autophagosomes was quantified per {gamma}{delta} T cell, and cells with more than two autophagosomes were considered autophagic. Arrows indicate autophagosomes within skin {gamma}{delta} T cells. Data represent means ± SD. *, p < 0.05 vs vehicle control (two-tailed, unpaired Student’s t test).

 
Although inhibiting mTOR does not affect skin {gamma}{delta} T cell survival, it is possible that this inhibition causes the cells to undergo other cellular changes that affect activation-induced functions, such as the lysosomal degradation process called autophagy. mTOR can act as a sensor to regulate the autophagy pathway (56, 57). CD4+ T cells treated with rapamycin undergo autophagy in vitro, implicating the process in T cell homeostasis (55). Autophagy is indicated by the presence of large autophagosomes in the cytoplasm. To determine whether rapamycin-mediated mTOR inhibition induces autophagy in skin {gamma}{delta} T cells, we used several common methods to detect autophagosomes in skin {gamma}{delta} T cells. First, cells were treated with rapamycin or vehicle control and stained with acridine orange to monitor the number of autophagic vesicles. Rapamycin treatment significantly increased the incidence of skin {gamma}{delta} T cells undergoing autophagy as compared with the basal level observed in vehicle control-treated cells (Fig. 4C). To identify whether skin {gamma}{delta} T cells also undergo autophagy during rapamycin treatment in vivo, we used LysoTracker Green staining to identify the number of cells with acidic vesicles in the epidermis. In mice administered rapamycin, there were increased numbers of skin {gamma}{delta} T cells exhibiting large numbers of acidic vesicles (Fig. 4D). These data indicate that mTOR inhibition of skin {gamma}{delta} T cells not only inhibits proliferation, but also induces autophagy.

Activation-induced morphology changes in skin {gamma}{delta} T cells are inhibited by rapamycin

The process of autophagy preserves the cytoskeleton, preventing its degradation while recycling cellular macromolecules in an effort to stave off cell death (57). To identify whether blocking mTORC2 function with rapamycin affects the capacity of skin {gamma}{delta} T cells to alter their cellular morphology upon activation, skin from rapamycin-treated mice was subsequently cultured with stimulating Abs specific for CD3{epsilon}. Skin {gamma}{delta} T cells normally exhibit a highly dendritic morphology. Upon TCR ligation, the skin {gamma}{delta} T cells normally become rounded within several hours and initiate migration (Fig. 5A). However, skin {gamma}{delta} T cells in the epidermis of mice treated with rapamycin did not exhibit this characteristic morphology change (Fig. 5A). These results were replicated using rapamycin treatment in vitro, and once again neither IL-2 nor IGF-1 was able to restore normal cell rounding and migration (data not shown).


Figure 5
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  		FIGURE 5. Rapamycin treatment inhibits activation-induced morphology changes. A, Skin {gamma}{delta} T cells from mice administered rapamycin exhibit a decreased ability to "round-up" in response to stimulation. Epidermal sheets were isolated from mice treated with rapamycin or vehicle control and stimulated with Abs to CD3{epsilon} before immunofluorescent staining. The numbers of dendrites per skin {gamma}{delta} T cell were quantified in at least 300 cells per mouse and are representative of at least six mice per condition. Data represent the means ± SD. *, p < 0.05 vs vehicle control (two-tailed, unpaired Student’s t test). B, Skin {gamma}{delta} T cells exhibit a delay in morphology changes at the wound site in rapamycin-treated mice. Epidermal sheets were prepared 2 h postwounding in mice treated with rapamycin and stained with fluorescent Abs specific for the {gamma}{delta} TCR. The white line indicates the wound edge. At least 150–200 cells were quantified from wounds in mice from three separate experiments.

 
One of the primary responses of skin {gamma}{delta} T cells to wounding is activation-induced rounding of these characteristically dendritic cells before migration to the wound edge and proliferation at the wound site (27). To identify whether rapamycin treatment inhibits this response in vivo, skin {gamma}{delta} T cells were examined from wounds isolated from rapamycin- and vehicle-treated mice. In rapamycin-treated mice, skin {gamma}{delta} T cells adjacent to the wound edge maintained a dendritic morphology, while cells from control-treated mice exhibited a rounded appearance (Fig. 5B). Furthermore, skin {gamma}{delta} T cells isolated from the wound site have a reduced capacity to produce factors such as TNF-{alpha} (data not shown). These results provide further evidence that activation-induced functions of skin {gamma}{delta} T cells are negatively regulated by rapamycin. It is possible that normal homeostatic functions are similarly altered.

Rapamycin negatively affects skin {gamma}{delta} T cell homeostatic functions

Since mTOR has the capacity to regulate transcription and translation through p70 S6 kinase and eIF-4E binding proteins (58) and to regulate skin {gamma}{delta} T cell autophagy, there may be consequences of rapamycin treatment on normal cellular homeostasis. Although long-term treatment with rapamycin does not impair surface expression of activation markers such as CD25 or CD69 (Fig. 6A), skin {gamma}{delta} T cells isolated from mice treated with rapamycin express a reproducible reduction in levels of TCR as compared with mice administered vehicle alone (Fig. 6A). Down-regulation of TCR could have negative implications for T cell activation. Additionally, skin {gamma}{delta} T cells isolated from rapamycin-treated mice have reduced levels of homeostatic IGF-1 production (Fig. 6B). Constitutive IGF-1 production by skin {gamma}{delta} T cells has been shown to play an important role in skin homeostasis (30) by promoting keratinocyte migration and survival of both {gamma}{delta} T cells and keratinocytes.


Figure 6
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  		FIGURE 6. Constitutive TCR and IGF-1 expression are reduced in {gamma}{delta} T cells from rapamycin-treated mice. A, {gamma}{delta} TCR expression is reproducibly reduced in mice treated with rapamycin, while the activation markers CD69 and CD25 are unaffected. Epidermal cells were isolated from mice administered rapamycin or vehicle control for 14 days and stained for various markers with fluorescent Abs. B, Skin {gamma}{delta} T cells isolated from rapamycin-treated mice exhibited decreased levels of IGF-1 production. Epidermal cells were isolated from mice administered rapamycin or vehicle control and stained with fluorescent Abs for intracellular IGF-1 production. Gates are set on live, Thy1.2+ cells. Ovals highlight skin {gamma}{delta} T cells without IGF-1 production. These results are representative of at least three mice per condition performed in three experiments. The mean fluorescence intensity (MFI) of IGF-1 expression by {gamma}{delta} TCR+ cells in these experiments is presented in the right panel.

 
Addition of skin {gamma}{delta} T cell-produced growth factors such as IGF-1 restores wound closure in rapamycin-treated mice

Since our data demonstrated that rapamycin has a negative impact on skin {gamma}{delta} T cell function in vivo, we examined the possibility that the addition of growth factors produced exclusively by skin {gamma}{delta} T cells in the epidermal compartment would restore rapid wound closure in rapamycin-treated mice. To examine this, recombinant IGF-1 was added to wounded skin in organ culture and wound closure was assessed. Addition of IGF-1 to skin treated with rapamycin did indeed restore wound closure (Fig. 7A). To assess whether addition of IGF-1 would have the same effect in vivo, mice were administered rapamycin for 3 days before wounding and daily thereafter. Upon wounding, either recombinant IGF-1 or buffer alone was applied directly to the wound. Treatment of wounds in rapamycin-treated mice with IGF-1 restored normal rates of wound healing (Fig. 7B). Notably at days 4, 7, and 10, when the greatest difference between rapamycin-treated and vehicle-treated mice was evident (Figs. 1A and 7B), addition of IGF-1 to rapamycin-treated wounds clearly restored wound healing kinetics to those of untreated animals (Fig. 7B). These studies indicate that dysfunctional wound repair caused by rapamycin can be rescued by the addition of factors normally produced by skin {gamma}{delta} T cells.


Figure 7
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  		FIGURE 7. Application of IGF-1 to wounds rescues rapamycin-induced wound healing defect. A, IGF-1 supplemented to rapamycin-treated skin in culture rescues wound closure. Skin from wild-type mice was wounded and cultured in the presence or absence of rapamycin. Digital images were acquired to monitor wound closure. IGF-1 (100 ng/ml) was supplemented in various wells. B, IGF-1 rescues wound repair in rapamycin-treated mice. Mice were treated with rapamycin or vehicle control, wounded on the dorsal surface, administered IGF-1 or buffer control, and wound closure was measured over time. Data represent means ± SEM. *, p < 0.05 and **, p < 0.005 vs vehicle control (two-tailed, unpaired Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Rapamycin is a powerful clinical therapeutic administered for the prophylaxis of allograft rejection, coatings on arterial stents, and it is currently used in clinical trials for the treatment of cancer. Several studies have described the potent inhibitory action of rapamycin on various {alpha}β T lymphocyte populations, as the mechanism employed to prevent rejection of transplanted solid organs (59). Herein we show that similar to anti-allograft {alpha}β T lymphocytes, intraepithelial {gamma}{delta} T lymphocytes are suppressed by rapamycin treatment. Due to the inhibitory effects of rapamycin on cell cycle and function, there has been speculation that rapamycin renders effector T cells anergic (19, 45). Anergy has been described as a mechanism by which lymphocytes are functionally inactive following stimulation, but survive in a nonresponsive state (60). Skin {gamma}{delta} T cells are present but with diminished functional capacity in rapamycin-treated mice. Our results show that {gamma}{delta} TCR expression is reduced, cytokine and growth factor production are decreased, and proliferation and cell cycle initiation are inhibited. This may be caused by a block in mTOR-mediated costimulatory responses (signal 2) or a defect in IL-2 signals (signal 3). IL-2 receptor signaling is key for the proliferation and cell cycle progression of skin {gamma}{delta} T lymphocytes (61). We show that IL-2 responsiveness is diminished in rapamycin-treated skin {gamma}{delta} T cells, which may be exacerbated by the reduced levels of TCR expression. Upon removal of rapamycin, normal skin {gamma}{delta} T cell function is restored, suggesting that rapamycin inhibition is reversible.

In the presence of rapamycin, skin {gamma}{delta} T cells survive in the hyporesponsive state for a prolonged time. Instead of undergoing apoptosis, skin {gamma}{delta} T cells appear to undergo autophagy as they attempt to survive with diminished mTOR signaling. This mechanism of survival may be vital for cell types such as skin {gamma}{delta} T cells, which are only seeded in the murine skin from the fetal thymus and cannot be repopulated by the adult thymus (62). Since treatment with rapamycin impairs the ability of skin {gamma}{delta} T cells to proliferate or migrate in response to T cell growth factors such as IL-2 or IGF-1, the skin {gamma}{delta} T cell may become autophagic in an attempt to survive in the absence of cytokine/growth factor signaling. Although most cells undergo a basal level of autophagy, this process is induced during nutrient deprivation in an attempt to break down macromolecules and recycle the components (57, 63). With skin {gamma}{delta} T cells undergoing autophagy and unable to proliferate, the number of skin {gamma}{delta} T cells in the epidermal compartment may eventually diminish. In our studies a decrease was not observed after 2 weeks of rapamycin treatment, but long-term, sustained rapamycin administration, as would occur in transplant recipients, may impact skin T cell numbers. The increased level of autophagy may also impair the cellular machinery involved in activation, leading to inhibition of downstream effector functions. Rapamycin treatment directly affects pathways involved in nutrient sensing and cellular growth through inhibition of mTORC1 and mTORC2, but increased levels of autophagy may also induce degradation of signaling molecules involved in other independent pathways. It is still unclear whether particular proteins are specifically targeted for degradation during autophagy.

Skin-resident {gamma}{delta} T cells are known to play roles in the early stages of wound repair (27, 34). Similar to TCR{delta}–/– mice, the delay in wound healing observed in rapamycin-treated mice occurs within the first 3 days of wound healing. The precise timing of wound repair is critical as key growth factors, cytokines, and chemokines participate in the entry, exit, and function of resident and inflammatory cells at the wound site. Although rapamycin has been shown to potently inhibit {alpha}β T lymphocytes, peripheral {alpha}β T lymphocytes do not infiltrate the wound site until 7 days postwounding, and they play roles in the later stages of wound repair (64). Thus, the early timing of the wound healing defect observed during rapamycin treatment makes it unlikely that {alpha}β T cell inhibition by rapamycin is responsible for the delay in wound closure.

The cell-specific nature of rapamycin inhibition is a testament to the complex and sometimes redundant nature of the mTOR pathway. In contrast to allograft-specific {alpha}β T cells, which undergo anergy or apoptosis (19, 45, 65), rapamycin treatment leads to an accumulation of CD25+ regulatory T lymphocytes in mice (66) and humans (17). Additionally, some tumor cell lines are susceptible to apoptosis upon rapamycin treatment (67, 68). Since keratinocytes compose the bulk of the epidermis, their proliferative capacity is essential to wound repair. However, during wound repair, we show that keratinocytes retain their ability to proliferate in the presence of rapamycin. Although signaling through PI3K is important for keratinocyte proliferation (69), it must not depend on mTOR. Additionally, IGF-1 application rescues normal wound closure rates, suggesting that keratinocytes can respond to growth factors normally in the presence of the inhibitor. In the epidermis, IGF-1 is exclusively produced by skin {gamma}{delta} T cells (30), but its receptor is widely expressed among cells in the epidermal compartment including keratinocytes. IGF-1 plays key roles in both keratinocyte and skin {gamma}{delta} T cell migration and survival (30, 31, 32, 33). Addition of this growth factor restores wound closure upon rapamycin treatment, presumably by overcoming the impaired function of the skin {gamma}{delta} T cells and acting upon neighboring keratinocytes to promote migration. Thus, supplementing the wound environment with exogenous IGF-1 restores the paracrine effects of skin {gamma}{delta} T cell-derived IGF-1 on other epidermal cells, thereby reestablishing wound closure.

Rapamycin acts by binding FKBP12 and inhibiting the serine/threonine kinase mTOR. Although there appears to be an ever-expanding list of downstream targets for mTOR, the best studied readout of mTOR function in mTORC1 is phosphorylation of p70 S6 kinase (70). S6 kinase is involved in proliferative responses of {alpha}β T lymphocytes, and our results indicate that IL-2 signaling induces mTOR phosphorylation of S6 kinase in skin {gamma}{delta} T cells as well and that this phosphorylation is inhibited by rapamycin. The second complex formed with mTOR has been shown to regulate spatial aspects of cell growth. mTORC2 affects cytoskeletal reorganization in yeast and is implicated as an upstream regulator of Rho GTPases in mammalian cells (42). Although originally reported as a rapamycin-resistant complex (42), recent evidence shows that in particular cell types mTORC2 is sensitive to prolonged rapamycin treatment (43). Skin {gamma}{delta} T cells exhibit rapamycin-sensitive phosphorylation of Akt Ser473, suggesting that mTORC2 is not functional during rapamycin treatment in these cells. The defect we observe in the capacity of skin {gamma}{delta} T cells to undergo rapid activation-induced morphology changes, both in vitro and in vivo, in the presence of rapamycin highlights the capacity for the drug to inhibit cytoskeletal functions. To our knowledge, this is the first time that a T cell has been shown to exhibit mTORC2 function and cytoskeletal changes that are rapamycin-sensitive.

Both {alpha}β and {gamma}{delta} TCR-expressing cells are found in human skin. Similar to the mouse, human skin T cells exhibit a restricted TCR repertoire specific to the cutaneous environment (71, 72) and are able to produce TNF-{alpha} and IFN-{gamma} upon stimulation (73). Skin {gamma}{delta} T cells in the human epidermis are also able to produce IGF-1 (our unpublished data). Additionally, human skin {gamma}{delta} T cells have tumoricidal capabilities much like the mouse {gamma}{delta} T cell (73). Our studies suggest that prolonged rapamycin treatment would result in skin {gamma}{delta} T cell dysfunction and an inability to respond to injury. Herein we have identified a key signaling pathway vital for skin {gamma}{delta} T cell function and a novel mechanism that may contribute to wound healing defects observed during treatment with rapamycin (6, 7, 8, 9, 10).


   Acknowledgments
 
We thank Dr. Deborah Witherden for technical advice and manuscript review. We thank Alexandre Webster for technical assistance. We are grateful to Dr. Wendy Havran for generously providing advice and support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from National Institutes of Health (DK073098 and AI07244) and the Leukemia and Lymphoma Society. This is manuscript number 19281. Back

2 Address correspondence and reprint requests Dr. Julie M. Jameson, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: jamesonj{at}scripps.edu Back

3 Abbreviations used in this: IEL, intraepithelial lymphocyte; IGF-1, insulin-like growth factor-1; KGF-1, keratinocyte growth factor-1; mTOR, mammalian target of rapamycin; mTORC, mTOR complex. Back

Received for publication April 2, 2008. Accepted for publication July 14, 2008.


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