science pharmaceutical expo biotech jobs
Originally published online as doi:10.1189/jlb.0307137 on September 28, 2007

Published online before print September 28, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0307137v1
83/1/165    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kagimoto, Y.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kagimoto, Y.
Right arrow Articles by Yoshikai, Y.
(Journal of Leukocyte Biology. 2008;83:165-172.)
© 2008 by Society for Leukocyte Biology

A regulatory role of interleukin 15 in wound healing and mucosal infection in mice

Yoshiko Kagimoto*,{dagger}, Hisakata Yamada*, Takahiro Ishikawa*, Naoyoshi Maeda{ddagger}, Fumi Goshima§, Yukihiro Nishiyama§, Masutaka Furue{dagger} and Yasunobu Yoshikai*,1

* Division of Host Defense, Medical Institute of Bioregulation,
{dagger} Department of Dermatology, Graduate School of Medicine,
{ddagger} Digital Medicine Initiative, Kyushu University, Fukuoka, Japan; and
§ Department of Virology, Nagoya University School of Medicine, Nagoya, Japan

1 Correspondence: Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yoshikai{at}bioreg.kyushu-u.ac.jp


arrow
ABSTRACT
 
IL-15 plays a critical role in the development and maturation of {gamma}{delta} intraepithelial T lymphocytes (IEL), which are known to play important roles in wound healing and resolving inflammation in mice. In this study, we found that IL-15 transgenic (Tg) mice, under the control of a MHC Class I promoter, exhibited accelerated wound healing but were highly susceptible to genital infection with HSV-2. The IEL in the skin and reproductive organs of IL-15 Tg mice produced an aberrantly higher level of TGF-β1 upon TCR triggering than in control mice. In vivo neutralization of TGF-β ameliorated the susceptibility of IL-15 Tg mice to genital HSV-2 infection. Taken together, overexpression of IL-15 may stimulate IEL to produce TGF-β1, promoting wound healing but impeding protection against genital HSV-2 infection.

Key Words: {gamma}{delta} T cells • immunity • immune regulation • cytokines


arrow
INTRODUCTION
 
{gamma}{delta} T cells are present in small numbers in the blood and peripheral lymphoid tissues but are relatively abundant in intraepithelial lymphocytes of skin (s-IEL) and female reproductive organs (r-IEL) [1 ]. s-IEL and r-IEL differentiate in the thymus at an early stage of ontogeny and bear truly invariant V{gamma}5/V{delta}1 and V{gamma}6/V{delta}1 TCR without junctional diversity, respectively [2 3 4 ]. Most of the studies about the roles of {gamma}{delta} T cells in the epithelium revealed their immunoregulatory properties [2 , 5 ]. Furthermore, TCR{delta} knockout (KO) mice, lacking {gamma}{delta} T cells in skin, showed a significant delay in wound healing and impaired epidermal cell proliferation, suggesting that s-IEL play specialized roles in wound healing [6 7 8 9 ].

HSV-2 is an important human pathogen, which causes a variety of diseases, ranging from mild mucosa disorders to life-threatening encephalitis [10 , 11 ]. Animal models of infection with HSV-2 have been established to study mechanisms by which the virus causes disease, and studies have demonstrated that the cellular immune system contributes to the recovery from infection [12 ,]. Protective mechanisms against primary infection with HSV-2 are mainly mediated by Class II-restricted CD4+ Th1 cells secreting IFN-{gamma} and/or CTL activity [13 , 14 ]. It has been reported that TCR{delta} KO mice showed increased susceptibility to an intravaginal infection with HSV-2 [15 ], suggesting that {gamma}{delta} T cells also play a role in host defense mechanisms against HSV-2 infection.

IL-15 uses β- and {gamma}-chains of the IL-2R for signal transduction and thus shares many properties of IL-2 in spite of having no sequence homology with IL-2 [16 17 18 19 ]. Mice lacking IL-15R{alpha} or IL-15 lack NK cells or {gamma}{delta}s-IEL almost completely and have severely reduced numbers of NKT cells and memory phenotype CD8+ T cells [20 21 22 ]. Recently, IL-15 KO mice have been shown to be susceptible to genital infection with HSV-2, and IL-15-dependent innate immunity is suggested to play an important role in protection against intravaginal HSV-2 infection [23 , 24 ]. We previously constructed transgenic (Tg) mice expressing IL-15 cDNA encoding a secretable isoform under the control of a MHC Class I promoter [25 ], which showed strong protection against systemic infection with Salmonella, Listeria monocytogenes, Mycobacterium bovis Bacille Calmette-Guerin, or murine acquired immunodeficiency virus [26 27 28 ]. However, the effects of overexpression of IL-15 in the functions of {gamma}{delta} T cells mediating mucosal immunity and wound healing are still unknown.

In this study, to determine the potential role of IL-15 in mucosal immunity, we examined wound healing and mucosal infection with HSV-2 in IL-15 Tg mice, which showed accelerated wound healing but were highly susceptible to intravaginal infection with HSV-2. The levels of TGF-β production by r-IEL and s-IEL were significantly higher, whereas IFN-{gamma} production by r-IEL was severely impaired in IL-15 Tg mice. The implications of these findings for a novel role of IL-15 for IEL functions are discussed.


arrow
MATERIALS AND METHODS
 
Mice
Female C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). IL-15 Tg mice, which were constructed using originally described IL-15 cDNA under the control of a MHC Class I promoter, have been described previously [25 ]. C57BL/6 background IL-15 KO mice were purchased from Taconic (Germantown, NY, USA). Mice were maintained and bred under specific pathogen-free conditions and offered food and water ad libitum. In each experiment, age-matched female mice were used. In some experiments, siblings from the same mother were used. All mice were used at 6–8 weeks of age. The Ethics Committee on Animal Experiments of the Faculty of Medicine, Kyushu University (Japan), approved this study. Experiments were carried out according to the Guidelines for Animal Experiments.

Wound-healing procedure
Epidermal wounding was performed as described previously by excising skin and panniculus carnosus [6 ]. In brief, the mouse backs were shaved, and a sterile, 6-mm punch tool was used to create two or three sets of full-thickness wounds as described previously. At each time-point, the diameter of the wounds was measured three times by at least two investigators, and the percentage of open wound was calculated. In each experiment, at least three wounds on three individual mice were measured at each time-point per mouse strain.

Antibodies and reagents
PE-conjugated anti-TCR-{gamma}{delta} (UC7-13D5), FITC-conjugated anti-TCR-{alpha}β (H57-597) mAb, biotin-conjugated anti-CD3{epsilon} (145-2C11) mAb, and Cy-Chrome-conjugated streptavidin were obtained from PharMingen (San Diego, CA, USA). Purified anti-MHC Class II (I-A/I-E) mAb (M5/114.15.2) was purchased from eBiosciences (San Diego, CA, USA). Anti-CD3{epsilon} mAb (145-2C11) was a gift from Dr. Ralph Kubo (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO, USA). Neutralizing anti-TGF-β antibody and the control rabbit IgG were purchased from R&D Systems (Minneapolis, MN, USA).

Preparation of epidermal sheets and immunohistochemistry
Epidermal sheets were prepared as described previously [22 ]. Epidermal sheets were labeled with FITC-conjugated anti-Thy1.2 mAb or PE-conjugated anti-V{gamma}5 mAb (F536, PharMingen, La Jolla, CA, USA) at 4°C overnight. After rinsing with PBS, the specimens were mounted in PBS/glycerol, overlayed with a coverslip, and viewed under a fluorescence microscope (Axiovert 100, Zeiss, Oberkochen, Germany) with a CellScan image analyzer system (Scanalytic, Billerica, MA, USA).

Virus and infections
The 186 strain of wild-type HSV-2 was obtained originally from Fred Rapp (Pennsylvania State University College of Medicine, Hershey, PA, USA) [29 ]. The viral stock was grown in monolayer cultures of Vero cells overlaid with MEM supplemented with 5% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The stock was stored frozen at 2–5 x 106 PFU/ml concentrations. The virus was diluted into PBS just before infection. Mice were infected at 6–8 weeks of age. Mice were infected intravaginally or s.c. with various doses of strain 186 in 20 µl PBS. To synchronize their estrous cycle at the progesterone-dominant stage, all mice were injected s.c. with 2 µg/mouse β-estradiol 17-cypionate (Sigma Chemical Co., St. Louis, MO, USA) 5 days before infection. In some experiments, mice were injected i.p. with neutralizing anti-TGF-β antibody, 2 µg/g mouse body weight, or isotype control rabbit IgG 2 h before HSV-2 inoculation on Day 0 and daily until Day 3. Vaginal washes from mice were assayed individually for virus infectivity. After centrifugation at ~1000 g for 5 min, samples were stored at –80°C. The supernatant was titrated for infectivity in Vero cells.

Cell preparation
r-IEL and epithelial cells (EC) in the uterus/vagina were prepared as described previously [30 , 31 ]. Briefly, the uterus/vagina was incubated in EDTA at 5 mM at 37°C, repeating until the supernatant became clear. The uterus/vagina was then washed several times with supplemented PBS and incubated for 20 min in 50 ml HBSS to inactivate any remaining EDTA. The remaining tissues were chopped into small fragments and then transferred to another flask containing 20 ml HBSS, supplemented with collagenase (20 units/ml) and 10% FCS and incubated at 37°C for 1.5 h. The supernatant was collected and centrifuged at 1000 rpm, and then the cell pellet was suspended in HBSS. The highly viable IEL were purified by using a discontinuous Percoll gradient and then harvested from the interface of 40%/75%. Uterine/vaginal EC were obtained from the 25%/40% interface and washed twice with Ca2+/Mg2+-free HBSS solution with 0.2% EDTA. The cell viabilities of freshly isolated IEL and EC were more than 95%, as assessed by trypan blue staining in both cases. s-IEL were isolated from murine skin as described previously [30 ]. After removing the s.c. tissue, skin samples were incubated with 3000U/ml dispase (Nakalai Tesque, Kyoto, Japan) in RPMI for 4 h at 37°C. The epidermis was separated from the dermis using fine forceps and incubated with 90U/ml DNase I (Sigma Chemical Co.) in RPMI for 30 min at 37°C. The highly viable IEL were purified by Percoll gradient from the interface of 33.3%/66.6%. The percentage of {gamma}{delta} T cells in CD3+ s-IEL was 95%.

Flow cytometry
r-IEL or s-IEL were stained with PE-, FITC-, and biotin-conjugated mAb for 30 min at 4°C. To detect biotin-conjugated mAb, cells were stained with Cy-Chrome-conjugated streptavidin. To block FcR-mediated binding of the mAb, 2.4G2 (anti-FcR mAb) was added. The stained cells were analyzed by a FACSCaliber flow cytometer (Becton Dickinson, San Jose, CA, USA). Small lymphocytes were gated by a forward- and side-scatter.

Cytokine ELISA
Vaginal washes were collected by injecting PBS into the vaginal canal and then recovering vaginal washes with a micropipette. The s-IEL and r-IEL were cultured with anti-CD3{epsilon} mAb (100 µg/ml), which had been immobilized on the plates by prior incubation for 1 h. To estimate cytokine production, the supernatants were collected after 48 h culture. Draining lymph nodes (LNs; inguinal and iliac LNs) were removed and depleted of MHC Class II+ cells according to the Dynabiotech protocol (Dynal Biotech, Norway). Anti-MHC Class II was coated on sheep anti-rat IgG Dynal beads. Cells were cultured in 200 ml complete culture medium in 96-well flat-bottom plates (Falcon, Becton Dickinson Ltd., Oxford, UK) at a density of 5 x 105 cells/well with the same number of mitomycin-treated, naïve spleen cells from C57BL/6 mice as antigen-presenting cells (APC) with or without 2.5 x 104 PFU UV-inactivated HSV-2 (1.5 J/cm2) or with anti-CD3{epsilon} mAb (100 µg/ml), which had been immobilized on the plates by prior incubation for 1 h. To estimate cytokine production by ELISA, the supernatants were collected after culturing for 48 h. The cytokine activity in the cell-free culture supernatants or vaginal washes was assayed by ELISA using a mouse IFN-{gamma}, IL-4, IL-10 (Genzyme Diagnostics, Cambridge, MA, USA), or TGF-β1 ELISA system (Promega, Madison, WI, USA).

RT-PCR analysis
Total RNA was extracted by the acid-guanidium phenol-chloroform method from IEL or EC. cDNA synthesis and PCR were performed using a cDNA cycle kit (Invitrogen Corp., San Diego, CA, USA). For the V{gamma} and V{delta} repertoire, RNA was primed with 20 pmol {gamma}-chain C region (C{gamma}) primers (5'-CTTATGGAGATTTGTTTCAGC-3') or 6.7 pmol {delta}-chain C region (C{delta}) primers (5'-CGAATTCCACAATCTTCTTG-3') in 20 µl reaction mixtures for RT. For IL-15 gene expression, RNA was primed with 20 pmol random primer in 21 ml reaction mixtures for RT. PCR was performed on a PCR thermal cycler (Takara Corp., Tokyo, Japan). PCR cycles were run for 30 s at 94°C, 30 s at 54°C, and 30 s at 72°C. Before the first cycle, a denaturation step for 7 min at 94°C was included, and after 35 cycles, the extension was prolonged for 4 min at 72°C. The 5' V primers and murine IL-15 primers were as follows: V{gamma} 1/2, 5'-ACACAGCTATACATTGGTAC-3'; V{gamma}2, 5'-CGGCAAAAAACAAATCAACAG-3'; V{gamma}4, 5'-TGTCCTTGCAACCCCTACCC-3'; V{gamma}5, 5'-TGTGCACTGGTACCAACTGA-3'; V{gamma}6, 5'-GGAATTCAAAAGAAAACATTGTCT-3'; V{gamma}7, 5'-AAGCTAGAGGGGTCCTCTGC-3'; V{delta}1, 5'-ATTCAGAAGGCAACAATGAAAG-3'; V{delta}2, 5'-AGTTCCCTGCAGATCCAAGC-3'; V{delta}3, 5'-TTCCTGGCTATTGCCTCTGAC-3'; V{delta}4, 5'-CCGCTTCTCTGTGAACTTCC-3'; V{delta}5, 5'-CAGATCCTTCCAGTTCATCC-3'; V{delta}6, 5'-TCAAGTCCATCAGCCTTGTC-3'; V{delta}7, 5'-CGCAGAGCTGCAGTGTAACT-3'; V{delta}8, 5'-AAGGAAGATGGACGATTCAC-3'; IL-15 sense (5', GTG ATG TTC ACC CCA GTT GC, 3'), antisense (5', TCA CAT TCT TTG CAT CCA GA, 3'); β-actin sense (5', TGG AAT CCT GTG GCA TCC ATG AAA C, 3'), antisense (5', TAA AAC GCA GCT CAG TAA CAG TCC G, 3'). PCR products (4 µl) were subjected to electrophoresis on a 1.5% agarose gel (Gibco, Grand Island, NY, USA) and transferred to a Gene Screen Plus filter (New England Nuclear, Boston, MA, USA). The Southern blots of PCR products were hybridized with MNG6 cDNA containing the C{gamma}2 gene, J{delta}1 probe (oligonucleotide; 5'-TTGGTTCCACAGTCACTTGG-3'), J{delta}2 probe (oligonucleotide; 5'-CTCCACAAAGAGCTCTATGCCCA-3'), IL-15 probe (oligonucleotide; 5', GCA ATG AAC TGC TTT CTC CT, 3'), or β-actin, (5', TTC TGC ATC CTG TCA GCA AT, 3'). The C{gamma}2 probe was labeled with [{alpha}-32P]dCTP using a Megaprime DNA labeling system (Amersham International, Amersham, UK), according to the manufacturer’s instructions. The oligonucleotide probes were labeled with [{gamma}-32P]ATP using a Megalabel 5'-labeling kit (Takara Shuzo Co. Ltd., Kyoto, Japan), according to the manufacturer’s instructions. Before hybridization, the filters were incubated in 1 M NaCl, 1% SDS, 10% dextran sulfate, and 50 µg/ml heat-denatured salmon sperm DNA for 18 h at 60°C, and then the filters were washed in 2x SSC, 1% SDS, for 15 min at 60°C. The radioactivity of each band of PCR product was analyzed with a Fujix BAS2000 bio-image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Statistical analysis
The statistical significance of the data was determined by a Student’s t-test, except for lethality data, which were analyzed by the generalized Wilcoxon’s test. *, P < 0.05, was taken as significant.


arrow
RESULTS
 
s-IEL in IL-15 Tg mice
We compared the morphology and number of {gamma}{delta} T cells in skin between naïve IL-15 Tg mice and naïve, control mice. As shown in Figure 1A , immunohistochemical analysis of {gamma}{delta}T cells in epidermal sheets revealed that there was no difference in morphology of {gamma}{delta} s-IEL between control and IL-15 Tg mice. FACS analysis showed that the relative numbers of V{gamma}5-positive {gamma}{delta} s-IEL in IL-15 Tg mice appeared to increase, although the numbers of {gamma}{delta} s-IEL in IL-15 Tg mice were almost the same as in the control mice (Fig. 1B) .


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. {gamma}{delta} T cells in s-IEL. (A) Immunohistochemical analysis of {gamma}{delta} T cells in epidermal sheets from naïve C57BL/6 and IL-15 Tg mice. Epidermal sheets prepared from 8-week-old C57BL/6 and IL-15 Tg mice were stained with FITC-conjugated anti-Thy1.2 and PE-conjugated anti-V{gamma}5 mAb. Original magnification, x400. Representative photographs are shown here. (B) Flow cytometric analysis of s-IEL from C57BL/6 and IL-15 Tg mice. The IEL were stained with anti-CD3, anti-V{gamma}5, and anti-TCR {gamma}{delta} mAb. Cells gated on lymphocytes were analyzed for their expression of CD3, V{gamma}5, and TCR {gamma}{delta}. Values in the panels indicate percentages of cells within the quadrants and are representative of three independent experiments.

Functions of s-IEL in IL-15 Tg mice
To compare the in vivo function of s-IEL between IL-15 Tg and control mice, we examined wound repair, in which {gamma}{delta} s-IEL is reported to be involved [6 ]. As shown in Figure 2A , wound healing was significantly faster in IL-15 Tg mice than that in control mice (P<0.05).


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		Figure 2. Function of s-IEL in IL15 Tg mice. (A) Wound-closure kinetics of IL-15 Tg mice. At each time-point, the diameter of the wounds was measured, and percent of wound open was calculated. Statistically significant difference from C57BL/6 mice (*, P<0.05, by Student’s t-test). Representative data from three independent experiments are shown. (B) TGF-β1 production by s-IEL of epithelized IL-15 Tg mice on Day 10 after wounding. The s-IEL was cultured on plates coated with anti-CD3 mAb for 48 h. TGF-β1 levels in the supernatants were determined by ELISA. Statistically significant difference from C57BL/6 mice (*, P<0.05, by Student’s t-test). N.D., Not detected.

We next examined the difference in ability of s-IEL to produce cytokines between IL-15 Tg and control mice, which were epithelized on Day 10 after wounding. As shown in Figure 2B , the level of TGF-β1 production was significantly higher in the culture supernatants of s-IEL stimulated with immobilized anti-CD3mAb in IL-15 Tg mice than control mice (P<0.05). Neither IFN-{gamma} nor IL-10 was detected in the supernatants (data not shown). These results suggest that s-IEL in IL-15 Tg mice exhibit functions different from those seen in normal mice with respect to TGF-β1 production.

Susceptibility of IL-15 Tg mice to s.c. or genital infection with HSV-2
To compare functional differences further in s-IEL between IL-15 Tg mice and control mice, we examined the survival rate of IL-15 Tg mice after s.c. infection with 2 x 104 PFU HSV-2 wild-type strain 186. As shown in Figure 3A , 30% of IL-15 Tg mice died within 15 days after infection, whereas all control mice survived beyond 30 days (*, P<0.05, by the generalized Wilcoxon’s test). These results suggest that IL-15 Tg mice are susceptible to s.c. infection with HSV-2.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Figure 3. Susceptibility of IL-15 Tg mice to s.c. or vaginal HSV-2 infection. Survival rates of IL-15 Tg mice after s.c. infection with 2 x 104 PFU HSV-2 strain 186 (A) or after intravaginal infection with 100 PFU HSV-2 (B). C57BL/6, IL-15 KO, or IL-15 Tg mice (20 mice in each group) were s.c. or intravaginally inoculated with of HSV-2 strain 186 and monitored for survival. Statistically significant difference from C57BL/6 mice (*, P<0.05, by the generalized Wilcoxon’s test). (C) Viral titers in vaginal wash fluids on Day 3 after intravaginal infection with 250 PFU HSV-2 strain 186. Each dot indicates an individual mouse. Bars show mean values for the group. Representative data from three independent experiments are shown. *, Statistically significant difference from control group (P<0.05, 15 mice for C57BL/6 and 13 mice for IL-15 Tg).

We next examined the survival rates of IL-15 Tg mice after an intravaginal infection with 100 PFU HSV-2. All IL-15 Tg mice died within 10 days after infection, whereas 50% of control mice survived beyond 12 days (Fig. 3B , P<0.05 by the generalized Wilcoxon’s test). Virus titers in the vaginal washes were compared between IL-15 Tg and control mice on Day 3 after infection with 250 PFU HSV-2. As shown in Figure 3C , a significantly higher virus titer was detected in the vaginal wash of IL-15 Tg mice than in control mice on Day 3 after infection (P<0.05). The pathological changes in vagina, such as erythema, erosion, and epilation, were more exaggerated in IL-15 Tg mice than in control mice (data not shown). Thus, IL-15 Tg mice are highly susceptible to genital HSV-2 infection as well as to s.c. infection.

{gamma}{delta} T cells of r-IEL in IL-15 Tg mice
We examined IL-15 mRNA expression in uterine EC in IL-15 Tg mice after HSV-2 infection. As shown in Figure 4A , the levels of IL-15 mRNA increased in IL-15 Tg mice compared with control mice. The number of {gamma}{delta} T cells in reproductive organs of IL-15 Tg mice was more than that of control mice on Day 3 after intravaginal infection with HSV-2. As shown in Figure 4B , the relative numbers of {gamma}{delta} T cells in IEL were higher in IL-15 Tg mice than in control mice. RT-PCR analysis showed that the {gamma}{delta} T cells in IL-15 Tg mice preferentially used V{gamma}6 gene and V{delta}1 gene rearranged to the J{delta}2 gene, similar to those used by the {gamma}{delta}T cells in control mice (Fig. 4C) . These results suggest that IL-15 Tg mice have an increased number of {gamma}{delta} T cells with a V repertoire in the uterus/vagina and the same as those seen in normal mice 3 days after genital infection with HSV-2.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Figure 4. {gamma}{delta} T cells in r-IEL of IL-15 Tg mice infected with HSV-2. (A) IL-15 gene expression by uterine/vaginal EC of mice intravaginally infected 3 days previously. Total RNA extracted from the EC was reverse-transcribed into cDNA and amplified by PCR. (B) Flow cytometric analysis of r-IEL from IL-15 Tg mice intravaginally infected 3 days previously. The IEL in uterine vagina were stained with anti-CD3{epsilon} anti-TCR {alpha}β and anti-TCR {gamma}{delta} mAb. Cells gated on CD3+ cells were analyzed for their expression of TCR {alpha}β and TCR {gamma}{delta}. Representative data from three independent experiments are shown. (C) V{gamma} or V{delta} use of r-IEL of IL-15 Tg mice intravaginally infected 3 days previously. Total RNA extracted from the IEL was reverse-transcribed into cDNA and amplified by PCR with primers for C{gamma} or C{delta} and various V{gamma} or V{delta} segments. The Southern blot of {delta} PCR products was hybridized with an oligonucleotide probe for J{delta}1 or J{delta}2. The results are representative of those from three independent experiments.

Cytokine production in r-IEL of IL-15 Tg mice after intravaginal infection with HSV-2
We compared cytokine production in vaginal washes after intravaginal infection with 250 PFU HSV-2. As shown in Figure 5A , a higher level of TGF-β1 was detected in the vaginal washes of IL-15 Tg mice on Day 3 after infection than in control mice (P<0.05). Conversely, IFN-{gamma} production was impaired significantly in IL-15 Tg mice at this stage after HSV-2 infection (P<0.05). The IL-10 level was not detected in vaginal washes (data not shown).


Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		Figure 5. TGF-β1 and IFN-{gamma} production in IL-15 Tg mice after genital HSV-2 infection. (A) TGF-β1 and IFN-{gamma} production in vaginal washes, which were collected from IL-15 Tg mice or C57BL/6 mice on Day 3 after intravaginal challenge with 250 PFU HSV-2. Cytokine levels in the vaginal washes were determined by ELISA. Data are means ± SD of five mice in each group. There was a statistically significant difference from the value for C57BL/6 mice (*, P<0.05, by Student’s t-test). Representative data from three independent experiments are shown. (B) IFN-{gamma} and TGF-β1 production by r-IEL, which were collected from IL-15 Tg mice or C57BL/6 mice on Day 3 after 250 PFU HSV-2 infection. IFN-{gamma} and TGF-β1 levels in the supernatants were determined by ELISA. Data are means ± SD of five mice in each group. In the case of IFN-{gamma} and TGF-β1, there was a statistically significant difference from C57BL/6 mice (*, P<0.05, by Student’s t-test). Representative data from three independent experiments are shown. (C) IFN-{gamma} production by draining LN cells in response to UV-inactivated HSV-2. LN cells were depleted of MHC Class II+ cells on Day 6 after an intravaginal challenge with 150 PFU HSV-2 strain 186. There was a statistically significant difference from the value for C57BL/6 mice (*, P<0.05, by Student’s t-test). Data are means ± SD of five mice in each group. Representative data from three independent experiments are shown.

Next, we examined cytokine production of r-IEL in the uterus/vagina upon TCR triggering in mice on Day 3 after HSV-2 infection. As shown in Figure 5B , the level of IFN-{gamma} production by r-IEL stimulated with immobilized anti-CD3 mAb was significantly lower in IL-15 Tg mice than control mice infected with HSV-2 3 days previously (*, P<0.05). Conversely, a significantly higher level of TGF-β1 production was detected in the supernatants of r-IEL of IL-15 Tg mice than control mice (*, P<0.05). There was no difference in the IL-10 level between the two groups of mice (data not shown). Taken together, the balance of r-IEL functions may be shifted to TGF-β1 production in IL-15 Tg mice.

Finally, we compared the HSV-2-specific Th1 cell responses between control mice and IL-15 Tg mice after intravaginal infection, as Th1 cells are indispensable for protection against genital HSV-2 infection [13 ]. We examined IFN-{gamma} production by T cells of inguinal and iliac LNs in response to UV-inactivated HSV-2 on Day 6 after intravaginal infection with 150 PFU HSV-2. As shown in Figure 5C , IFN-{gamma} production by LN T cells from IL-15 Tg mice was significantly lower than that in control mice (P<0.05). Conversely, neither IL-4 nor TGF-β1 was detected in the culture supernatants (data not shown). Thus, the generation of protective Th1 cells was impaired in IL-15 Tg mice after intravaginal HSV-2 infection.

Effects of neutralization of TGF-β on viral clearance and IFN-{gamma} production in IL-15 Tg mice after intravaginal infection with HSV-2
Our results suggest that increased TGF-β1 production is associated with impaired viral clearance in IL-15 Tg mice. To directly address an involvement of TGF-β in the impaired protection against HSV-2 infection in IL-15 Tg mice, we examined the effects of neutralization of TGF-β by in vivo administration of anti-TGF-β antibodies in IL-15 Tg mice infected with HSV-2. The titer of HSV-2 in vagina washes was significantly lower in anti-TGF-β-treated IL-15 Tg mice than that in control, IgG-treated IL-15 Tg mice. The IFN-{gamma} level in the vaginal washes was significantly higher in anti-TGF-β-treated IL-15 Tg mice than that in control IgG-treated IL-15 Tg mice (Fig. 6 ). These results indicated that the increased TGF-β1 production is involved in the increased susceptibility to genital infection with HSV-2 in IL-15 Tg mice.


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Figure 6. Effect of neutralization of TGF-β on viral clearance and IFN-{gamma} production in vagina washes after genital infection with HSV-2. Mice were treated i.p. with neutralizing anti-TGF-β antibody or rabbit IgG from Days 0 to 3 after infection with HSV-2. Vaginal washes were collected from IL-15 Tg mice on Day 3. Data are means ± SD of five mice in each group. There was a statistically significant difference from the value for control, IgG-treated mice (*, P<0.05, by Student’s t-test).


arrow
DISCUSSION
 
In the present study, we found that IL-15 Tg mice exhibited accelerated wound healing, and s-IEL in IL-15 Tg mice produced a higher level of TGF-β1 upon TCR triggering than in control mice. {gamma}{delta} s-IEL was reported to be important for wound healing through production of keratinocyte growth factors (KGF) and fibroblast growth factors (FGF) [6 7 8 9 ]. It is true that TCR{delta}–/– mice, lacking all {gamma}{delta} T cells in skin, showed a significant delay in wound healing and impaired epidermal cell proliferation [6 ]. Wang et al. [32 ] suggest that TGF-β1 has stage- and dose-specific effects on wound healing. It has been reported recently that human {gamma}{delta} T cells synthesize FGF and KGF in response to IL-15 and TGF-β1 [33 , 34 ]. Taken together, overexpression of IL-15 may stimulate s-IEL to produce these factors, accelerating wound healing. Cutaneous application of IL-15 may be a useful biotherapy for wound healing.

It is also notable that IL-15 Tg mice were susceptible to intravaginal infection as well as s.c. infection with HSV-2. r-IEL in the uterus/vagina of IL-15 Tg mice produced a higher level of TGF-β1 but less IFN-{gamma} upon TCR triggering. In addition, generation of antigen-specific, protective T cells capable of producing IFN-{gamma} was impaired in IL-15 Tg mice infected with HSV-2. In support of these results, TGF-β is known to down-regulate IFN-{gamma} production through inhibition of T-box expressed in T cells (T-bet) and direct interaction of the Sma and MAD-related proteins (SMAD) proteins with the IFN-{gamma} promoter [35 , 36 ]. We also demonstrated that in vivo neutralization of TGF-β ameliorated the susceptibility of IL-15 Tg mice to genital HSV-2 infection, indicating a direct association between increased TGF-β1 production and susceptibility to HSV-2 infection in IL-15 Tg mice. We have reported previously that {gamma}{delta} T cells in the uterus during pregnancy produced TGF-β, which may contribute to protecting against abortion [37 ]. Mukasa et al. [38 ] have suggested that {gamma}{delta} T cells expressing invariant V{gamma}6/V{delta}1 chains, comprising most of the {gamma}{delta} T cells in the female reproductive tract, have a function in controlling influence on the host inflammatory responses. Taken together with our current data, it appears that epithelial T cells in reproductive organs play important roles in immunoregulation. We speculate that overexpression of IL-15 may shift r-IEL to TGF-β1-producing T cells, which may inhibit IFN-{gamma} production by effector cells for host defense against genital HSV-2 infection.

Recent studies with IL-15 KO mice revealed that IL-15-dependent innate protection, dependently or independently of NK and NKT cells, was important for protection against genital HSV-2 infection [23 , 24 ]. These results suggest that IL-15 plays a critical role in protection against mucosal infection with HSV-2. We have reported recently that IL-15 is required for functional maturation of {gamma}{delta} intestinal-IEL in addition to maintenance of their size [39 ]. We speculate that IL-15 is essential in the host defense mechanism against genital HSV-2 infection through development and functional maturation of effector cells, and IL-15 overproduction may shift the function of effector cells to resolution of inflammation. IL-15 protein is produced at a low level, only by a limited number of cells, such as activated macrophages and EC, during periods of immune response and inflammation. There are several lines of evidence indicating the existence of multicomplex mechanisms for regulation of IL-15 expression at the levels of transcription, translation, and intracellular trafficking [19 , 40 ]. Strictly regulated IL-15 production may be important to determine the fate of mucosal immunity to host defense or immunoregulation for resolution of excessive inflammation in the mucosa.

It remains unknown how IL-15 preferentially induces differentiation of TGF-β1-producing T cells. Mucosa is the first line in battling invasion of nonself antigens from commensal microflora and constitutively active in terms of immunological aspect [41 ]. This environment develops a unique immunoregulatory system mediated by IEL, natural occurring regulatory T cells (nTreg), and adaptive Treg cells such as Th3 cells and Treg 1 phenotype cells, which play important roles in homeostasis in the intestine through TGF-β and IL-10 production [42 43 44 45 46 47 ]. Recently, it was reported that IL-15 together with dermal fibroblast induce proliferation of nTreg in human skin [48 ]. Overexpression of IL-15 may support such T cells with regulatory functions in mucosa. TGF-β in the environment is known to be important for differentiation of TGF-β-producing T cells [49 ]. TGF-β is produced not only by T cells but also by other cells such as EC and macrophages, which are known to be stimulated directly by IL-15 [50 ]. Keratinocytes are known as one of the major sources of TGF-β1 after cutaneous injury [51 ]. Therefore, it is also possible that TGF-β production by cells other than IEL may be stimulated by overproduction of IL-15, inducing differentiation of IEL-producing TGF-β1. Further experiments are required to elucidate these possibilities.

In summary, our experiments using IL-15 Tg mice highlight the novel role of IL-15 in a unique immunoregulation for wound healing and resolution of excessive inflammation via induction of TGF-β1-producing cells.


arrow
ACKNOWLEDGEMENTS
 
This work was supported in part by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases and was launched as a project commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank Ms. Kaneda and Kobayashi for their excellent technical support. We also thank Dr. Ralph Kubo for providing mAb.

Received March 5, 2007; revised July 19, 2007; accepted September 5, 2007.


arrow
REFERENCES
 
    1
  1. Haas, W., Pereira, P., Tonegawa, S. (1993) {gamma}/{delta} Cells Annu. Rev. Immunol. 11,637-685[Medline]
    2. 2
  2. Hayday, A., Tigelaar, R. (2003) Immunoregulation in the tissues by T cells Nat. Rev. Immunol. 3,233-242[CrossRef][Medline]
    3. 3
  3. Kabelitz, D., Marischen, L., Oberg, H. H., Holtmeier, W., Wesch, D. (2005) Epithelial defense by {gamma} {delta} T cells Int. Arch. Allergy Immunol. 137,73-81[CrossRef][Medline]
    4. 4
  4. Komori, H. K., Meehan, T. F., Havran, W. L. (2006) Epithelial and mucosal {gamma} {delta} T cells Curr. Opin. Immunol. 18,534-538[CrossRef][Medline]
    5. 5
  5. Born, W., Cady, C., Jones-Carson, J., Mukasa, A., Lahn, M., O’Brien, R. (1999) Immunoregulatory functions of {gamma} {delta} T cells Adv. Immunol. 71,77-144[Medline]
    6. 6
  6. Jameson, J., Ugarte, K., Chen, N., Yachi, P., Fuchs, E., Boismenu, R., Havran, W. L. (2002) A role for skin T cells in wound repair Science 296,747-749[Abstract/Free Full Text]
    7. 7
  7. Sharp, L. L., Jameson, J. M., Cauvi, G., Havran, W. L. (2005) Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1 Nat. Immunol. 6,73-79[CrossRef][Medline]
    8. 8
  8. Girardi, M., Lewis, J., Glusac, E., Filler, R. B., Geng, L., Hayday, A. C., Tigelaar, R. E. (2002) Resident skin-specific T cells provide local, nonredundant regulation of cutaneous inflammation J. Exp. Med. 195,855-867[Abstract/Free Full Text]
    9. 9
  9. Jameson, J. M., Cauvi, G., Sharp, L. L., Witherden, D. A., Havran, W. L. (2005) {gamma}{delta} T cell-induced hyaluronan production by epithelial cells regulates inflammation J. Exp. Med. 201,1269-1279[Abstract/Free Full Text]
    10. 10
  10. Stanberry, L. R. (1992) Pathogenesis of herpes simplex virus infection and animal models for its study Curr. Top. Microbiol. Immunol. 179,15-30[Medline]
    11. 11
  11. Whitley, R. J. (1996) Herpes simplex viruses Fields, B. N. Knipe, D. Howley, P. M. eds. Virology 3rd ed. ,2297-2342 Lippincott-Raven Philadelphia, PA, USA.
    12. 12
  12. Parr, M. B., Parr, E. L. (1998) Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes J. Virol. 72,2677-2685[Abstract/Free Full Text]
    13. 13
  13. Schmid, D. S., Rouse, B. T. (1992) The role of T cell immunity in control of herpes simplex virus Curr. Top. Microbiol. Immunol. 179,57-74[Medline]
    14. 14
  14. Manickan, E., Rouse, R. J., Yu, Z., Wire, W. S., Rouse, B. T. (1995) Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes J. Immunol. 155,259-265[Abstract]
    15. 15
  15. Nishimura, H., Yajima, T., Kagimoto, Y., Ohata, M., Watase, T., Kishihara, K., Goshima, F., Nishiyama, Y., Yoshikai, Y. (2004) Intraepithelial {gamma}/{delta} T cells may bridge a gap between innate immunity and acquired immunity to herpes simplex virus type 2 J. Virol. 78,4927-4930[Abstract/Free Full Text]
    16. 16
  16. Bamford, R. N., Grant, A. J., Burton, J. D., Peters, C., Kurys, G., Goldman, C. K., Brennan, J., Roessler, E., Waldmann, T. A. (1994) The interleukin (IL) 2 receptor β chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells Proc. Natl. Acad. Sci. USA 91,4940-4944[Abstract/Free Full Text]
    17. 17
  17. Giri, J. G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., Anderson, D. (1994) Utilization of the β and {gamma} chains of the IL-2 receptor by the novel cytokine IL-15 EMBO J. 13,2822-2830[Medline]
    18. 18
  18. Grabstein, K. H., Eisenman, J., Shanebeck, K., Rauch, C., Srinivasan, S., Fung, V., Beers, C., Richardson, J., Schoenborn, M. A., Ahdieh, M., Richardson, L., Alderson, M. R., Watson, J. D., Anderson, D. M., Giri, J. G. (1994) Cloning of a T cell growth factor that interacts with the β chain of the interleukin-2 receptor Science 264,965-968[Abstract/Free Full Text]
    19. 19
  19. Tagaya, Y., Bamford, R. N., DeFilippis, A. P., Waldmann, T. A. (1996) IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels Immunity 4,329-336[CrossRef][Medline]
    20. 20
  20. Lodolce, J. P., Boone, D. L., Chai, S., Swain, R. E., Dassopoulos, T., Trettin, S., Ma, A. (1998) IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation Immunity 9,669-676[CrossRef][Medline]
    21. 21
  21. Kennedy, M. K., Glaccum, M., Brown, S. N., Butz, E. A., Viney, J. L., Embers, M., Matsuki, N., Charrier, K., Sedger, L., Willis, C. R., Brasel, K., Morrissey, P. J., Stocking, K., Schuh, J. C., Joyce, S., Peschon, J. J. (2000) Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice J. Exp. Med. 191,771-780[Abstract/Free Full Text]
    22. 22
  22. De Creus, A., Van Beneden, K., Stevenaert, F., Debacker, V., Plum, J., Leclercq, G. (2002) Developmental and functional defects of thymic and epidermal V {gamma} 3 cells in IL-15-deficient and IFN regulatory factor-1-deficient mice J. Immunol. 168,6486-6493[Abstract/Free Full Text]
    23. 23
  23. Ashkar, A. A., Rosenthal, K. L. (2003) Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection J. Virol. 77,10168-10171[Abstract/Free Full Text]
    24. 24
  24. Gill, N., Rosenthal, K. L., Ashkar, A. A. (2005) NK and NKT cell-independent contribution of interleukin-15 to innate protection against mucosal viral infection J. Virol. 79,4470-4478[Abstract/Free Full Text]
    25. 25
  25. Nishimura, H., Yajima, T., Naiki, Y., Tsunobuchi, H., Umemura, M., Itano, K., Matsuguchi, T., Suzuki, M., Ohashi, P. S., Yoshikai, Y. (2000) Differential roles of interleukin-15 mRNA isoforms generated by alternative splicing in immune responses in vivo J. Exp. Med. 191,157-170[Abstract/Free Full Text]
    26. 26
  26. Umemura, M., Nishimura, H., Hirose, K., Matsuguchi, T., Yoshikai, Y. (2001) Over-expression of IL-15 in vivo induces a predominant Tcl response in mice inoculated with Mycobacterium bovis Bacille Calmette-Guerin infection J. Immunol. 167,946-956[Abstract/Free Full Text]
    27. 27
  27. Yajima, T., Nishimura, H., Ishimitsu, R., Yamamura, K., Watase, T., Busch, D. H., Pamer, E. G., Kuwano, H., Yoshikai, Y. (2001) Memory phenotype CD8+T cells increased in IL-15 transgenic mice are involved in early protection a primary infection with Listeria monocytogenes Eur. J. Immunol. 31,757-766[CrossRef][Medline]
    28. 28
  28. Umemura, M., Nishimura, H., Yajima, T., Wajjwalku, W., Matsuguchi, T., Takahashi, M., Nishiyama, Y., Makino, M., Nagai, Y., Yoshikai, Y. (2002) Over-expression of interleukin 15 prevents the development of murine retrovirus-induced acquired immunodeficiency syndrome FASEB J. 16,1755-1763[Abstract/Free Full Text]
    29. 29
  29. Nishiyama, Y., Rapp, F. (1981) Repair replication of viral and cellular DNA in herpes simplex virus type 2-infected human embryonic and xeroderma pigmentosum cells Virology 110,466-475[CrossRef][Medline]
    30. 30
  30. Havran, W. L., Chien, Y. H., Allison, J. P. (1991) Recognition of self antigens by skin-derived T cells with invariant {gamma} {delta} antigen receptors Science 252,1430-1432[Abstract/Free Full Text]
    31. 31
  31. Rakasz, E., Hagen, M., Sandor, M., Lynch, R. G. (1997) {gamma} {delta} T cells of the murine vagina: T cell response in vivo in the absence of the expression of CD2 and CD28 molecules Int. Immunol. 9,161-167[Abstract/Free Full Text]
    32. 32
  32. Wang, X. J., Han, G., Owens, P., Siddiqui, Y., Li, A. G. (2006) Role of TGF β-mediated inflammation in cutaneous wound healing J. Invest. Dermatol. Symp. Proc. 11,112-117[CrossRef]
    33. 33
  33. Workalemahu, G., Foerster, M., Kroegel, C. (2004) Expression and synthesis of fibroblast growth factor-9 in human {gamma}{delta} T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-β1/IL-15 J. Leukoc. Biol. 75,657-663[Abstract/Free Full Text]
    34. 34
  34. Workalemahu, G., Foerster, M., Kroegel, C., Braun, R. K. (2003) Human {gamma} {delta}-T lymphocytes express and synthesize connective tissue growth factor: effect of IL-15 and TGF-β1 and comparison with {alpha} β-T lymphocytes J. Immunol. 170,153-157[Abstract/Free Full Text]
    35. 35
  35. Monteleone, G., Pallone, F., MacDonald, T. T. (2004) Smad7 in TGF-β-mediated negative regulation of gut inflammation Trends Immunol. 25,513-517[CrossRef][Medline]
    36. 36
  36. Young, H. A. (2006) Unraveling the pros and cons of interferon-{gamma} gene regulation Immunity 24,506-507[CrossRef][Medline]
    37. 37
  37. Suzuki, T., Hiromatsu, K., Ando, Y., Okamoto, T., Tomoda, Y., Yoshikai, Y. (1995) Regulatory role of {gamma} {delta} T cells in uterine intraepithelial lymphocytes in maternal antifetal immune responses J. Immunol. 154,4476-4484[Abstract]
    38. 38
  38. Mukasa, A., Born, W. K., O’Brien, R. L. (1999) Inflammation alone evokes the response of a TCR-invariant mouse {gamma} {delta} T cell subset J. Immunol. 162,4910-4913[Abstract/Free Full Text]
    39. 39
  39. Nakazato, K., Yamada, H., Yajima, T., Kagimoto, Y., Kuwano, H., Yoshikai, Y. (2007) Enforced expression of Bcl-2 partially restores cell numbers but not functions of TCR {gamma} {delta} intestinal intraepithelial T lymphocytes in IL-15-deficient mice J. Immunol. 178,757-764[Abstract/Free Full Text]
    40. 40
  40. Nishimura, H., Fujimoto, A., Naoyuki, T., Yajima, T., Wajjwalku, W., Yoshikai, Y. (2005) A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing FASEB J. 19,19-28[Abstract/Free Full Text]
    41. 41
  41. Macdonald, T. T., Monteleone, G. (2005) Immunity, inflammation, and allergy in the gut Science 307,1920-1925[Abstract/Free Full Text]
    42. 42
  42. Weiner, H. L. (2001) Induction and mechanism of action of transforming growth factor-β secreting Th3 regulatory cells Immunol. Rev. 182,207-214[CrossRef][Medline]
    43. 43
  43. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., Weiner, H. L. (1994) Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis Science 265,1237-1240[Abstract/Free Full Text]
    44. 44
  44. Groux, H., O’Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E., Roncarolo, M. G. (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis Nature 389,737-742[CrossRef][Medline]
    45. 45
  45. Kemper, C., Chan, A. C., Green, J. M., Brett, K. A., Murphy, K. M., Atkinson, J. P. (2003) Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype Nature 421,388-392[CrossRef][Medline]
    46. 46
  46. Izcue, A., Coombes, J. L., Powrie, F. (2006) Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation Immunol. Rev. 212,256-271[CrossRef][Medline]
    47. 47
  47. Wing, K., Fehervari, Z., Sakaguchi, S. (2006) Emerging possibilities in the development and function of regulatory T cells Int. Immunol. 18,991-1000[Abstract/Free Full Text]
    48. 48
  48. Clark, R. A., Kupper, T. S. (2007) IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin Blood 109,194-202[Abstract/Free Full Text]
    49. 49
  49. Weaver, C. T., Harrington, L. E., Mangan, P. R., Gavrieli, M., Murphy, K. M. (2006) Th17: an effector CD4 T cell lineage with regulatory T cell ties Immunity 24,677-688[CrossRef][Medline]
    50. 50
  50. Obermeier, F., Hausmann, M., Kellermeier, S., Kiessling, S., Strauch, U. G., Duitman, E., Bulfone-Paus, S., Herfarth, H., Bock, J., Dunger, N., Stoeck, M., Scholmerich, J., Falk, W., Rogler, G. (2006) IL-15 protects intestinal epithelial cells Eur. J. Immunol. 36,2691-2699[CrossRef][Medline]
    51. 51
  51. O’Kane, S., Ferguson, M. W. (1997) Transforming growth factor β s and wound healing Int. J. Biochem. Cell Biol. 29,63-78[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Virol.Home page
N. Gill and A. A. Ashkar
Overexpression of Interleukin-15 Compromises CD4-Dependent Adaptive Immune Responses against Herpes Simplex Virus 2
J. Virol., January 15, 2009; 83(2): 918 - 926.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0307137v1
83/1/165    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kagimoto, Y.
Right arrow Articles by Yoshikai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kagimoto, Y.
Right arrow Articles by Yoshikai, Y.