Abstract

FTY720, a sphingosine-derived immunomodulator, causes immunosuppression via enhancement of lymphocyte sequestration into secondary lymphoid organs, thereby preventing their antigen-activated T cell egress to sites of inflammation. FTY720 is highly effective in inhibiting autoimmunity in various animal models. However, there is little known about how FTY720 controls the migration property of memory T cells. Here, we demonstrated that FTY720 prevents the development of colitis induced by the adoptive transfer of lamina propria (LP) colitogenic effector memory CD4+ T cells (TEM cells; CD45RBlowCD44highCD62L) into severe combined immunodeficiency (SCID) mice and suppresses interferon-γ, interleukin-2, and tumor necrosis factor-α production by LP CD4+ T cells. The numbers of spleen, peripheral blood, mesenteric lymph node, and LP CD4+ T cells in FTY720-treated mice were significantly reduced compared with those in control mice. Notably, LP CD4+ TEM cells as well as splenic CD4+CD45RBhigh T cells expressed several spingosine-1-phosphate receptors that are targets for FTY720. Furthermore, FTY720 also prevented the development of colitis induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells into SCID mice. Collectively, the present data indicate that FTY720 treatment may offer the potential not only to prevent the onset of disease but also to treat memory T cell-mediated autoimmune diseases including inflammatory bowel diseases.

  • therapy
  • migration
  • I cell

2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride (FTY720) is a chemical derivative of myriocin (thermozymocidin), a substance found in the fungi Myriococcum albomyces and Isaria sinclairii (1, 2, 5, 6). FTY720 is a potent immunomodulator that has been shown to prevent graft rejection in various animal allotransplantation models (23, 24), autoimmune disease animal models (11, 16, 20), and viral infection animal models (17, 21). Good synergy with cyclosporine and sirolimus has been reported (3, 28, 30). FTY720 elicits a lymphopenia resulting from a reversible redistribution of naïve lymphocytes from the circulation to secondary lymph nodes (4) without evoking a generalized immunosuppression (31). Another study (21) has suggested that low blood lymphocyte counts may reflect reduction in the emigration of effector cells to the periphery.

After naïve lymphocytes enter a secondary lymphoid organ from the blood, they travel to separate subcompartments, where they survey for antigen. In the absence of an antigen encounter, cells leave the organ via the efferent lymphatics or, in the case of the spleen, via the red pulp. Timely egress ensures that the cells travel rapidly to further lymphoid organs to continue their antigen surveillance process. Recent studies (1, 4, 22) have demonstrated that FTY720 blocks the egress of lymphocyte from the lymph nodes, Peyer's patches, and thymus. In vivo, FTY720 is immediately phosphorylated, and in vitro analysis has established that FTY720-phosphate is an agonist for sphingosine-1-phosphate (S1P) receptors (S1P1–S1P5 except for S1P4) (2, 5, 13). The S1P1 ligand, S1P, is present at high concentrations (100–300 nM) within blood and body fluids (26). It has been suggested that the egress-blocking activity of FTY720 can be explained by its S1P1 downmodulating activity in lymphocytes (8, 15).

In inflammatory bowel diseases (IBDs), it is believed that memory T cells are intermittently reactivated in secondary lymphoid organs and thereafter return to inflammatory tissues, such as, in this case, the gut (22). These memory T cells can survive for a long period, and they provide the basis for long-term immunological memory. However, it is little known how memory T cells are controlled by FTY720 in IBDs. In this study, we evaluated the effect of FTY720 using our recently established CD4+CD44highCD62L effector memory T cell (TEM cell)-mediated chronic colitis model.

MATERIALS AND METHODS

Animals.

Female BALB/c and CB-17 severe combined immunodeficiency (SCID) mice were purchased from Japan Clear (Tokyo, Japan). Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Tokyo Medical and Dental University. The Institutional Committee on Animal Research of Tokyo Medical and Dental University approved the experiments.

Antibodies.

The following monoclonal antibodies were used for the purification of cell populations and flow cytometric analysis: RM4-5, CyChrome- or phycoerythrin (PE)-conjugated anti-mouse CD4 (BD PharMingen; San Diego, CA); 16A, FITC-conjugated anti-mouse CD45RB (BD PharMingen); IM7, PE-conjugated anti-mouse CD44; and MEL-14, PE- or FITC-conjugated anti-mouse CD62L.

T cell reconstitution and FTY720 treatment.

FTY720 (Novartis Pharma; Basel, Switzerland) was dissolved in sterile distilled water (DW). For in vivo treatment, FTY720 was administered via per os gavage. To exclude the possibility that in vivo FTY720 treatment might be cytotoxic for CD4+ T cells, we treated normal BALB/c mice with a single dose of FTY720 (0, 0.3, and 3.0 mg/kg). Seventy-two hours after the treatment, dead and apoptotic cells were detected by annexin V-FITC/propidium iodide (PI) staining (MBL; Nagoya, Japan). As shown in Fig. 1, there were no significant differences in the numbers of dead (PI+ and annexin V+) or apoptotic (PI and annexin V+) cells between mice treated with each dose. Thus we adopted the 0.3 mg/kg dose of FTY720 in a line of in vivo experiments.

Fig. 1.

FTY720 treatment does not induce apoptosis of peripheral blood (PB) monoclonal cells (PBMC) and splenic CD4+ cells. Normal BALB/c mice were administerred with FTY720 at a single dose of 0, 0.3, or 3.0 mg/kg of FTY720. A: 48 h after the administration, the numbers of dead or apoptotic cells were determined by annexin V-FITC/propidium iodide (PI). Data are from 5 mice/group. NS, not significantly different. B: data are means ± SE of the percentages of PI+ and annexin V+ (dead) cells and PI and annexin V+ (apoptotic) cells from 5 mice/group.

Colitis was induced in CB-17 SCID mice by the adoptive transfer of syngeneic CD4+CD45RBhigh T cells as described previously (10). Briefly, CD4+ T cells were isolated from splenocytes from normal BALB/c mice using the anti-CD4 (L3T4) MACS magnetic separation system (Miltenyi Biotec; Auburn, CA). Enriched CD4+ T cells were labeled with PE-conjugated anti-mouse CD4 monoclonal antibodies and FITC-conjugated anti-CD45RB monoclonal antibodies and the isolated CD45RBhigh (highest staining: 30%) fraction on a FACS Vantage (Becton-Dickinson; Sunnyvale, CA). Each SCID mouse was injected intraperitoneally with syngeneic 3 × 105 CD4+CD45RBhigh T cells. Colitic mice were killed at 5–7 wk after the transfer to isolate the colitogenic lamina propria (LP) memory CD4+ T cells (10). The entire length of the colon was opened longitudinally, washed with PBS, and cut into small pieces. The dissected mucosa was incubated with Ca2+-and Mg2+-free HBSS containing 1 mM DTT (Sigma) for 30 min to remove mucus and then treated with 1 mg/ml collagenase (Worthington Biomedical; Freehold, NJ) and 0.01% DNase (Worthington) for 2 h. Cells were pelleted two times through a 40% isotonic Percoll solution and then subjected to Ficoll-Hypaque density gradient centrifugation (40%/75%). Enriched CD4+ LP T cells were obtained by positive selection using anti-CD4 (L3T4) MACS magnetic beads. The resultant cells, when analyzed by FACS Calibur, contained >96% CD4+ cells. To investigate LP memory CD4+ T cell migration properties and the preventive effect by FTY720, we next induced a colitogenic LP memory CD4+ T cell-mediated colitis as previously described (25). In brief, seven SCID mice from each group were injected intraperitoneally with 200 μl PBS containing 3 × 105 colitic LP CD4+ T cells and were treated with DW or FTY720 (0.3 mg/kg) daily starting 1 day before the transfer over a period of 4 wk. In another experiment, seven SCID mice from each group were injected intraperitoneally with 200 μl PBS containing 3 × 105 normal splenic CD4+CD4RBhigh T cells and were treated with DW or FTY720 (0.3 mg/kg) daily starting 1 day before T cell transfer over a period of 5 wk. SCID mice after transfer were weighed initially and then three times per week thereafter. They were observed for the following clinical signs of illness: hunched over appearance, piloerection of the coat, diarrhea, and blood in the stool. The mice were then killed and assessed for a clinical score, that is, the sum of the following four parameters: hunching and wasting, 0 or 1; colon thickening, 0–3 (0, no colon thickening; 1, mild thickening; 2, moderate thickening; and 3, extensive thickening); and stool consistency, 0–3 (0, normal beaded stool; 1, soft stool; and 2, diarrhea; and an additional point was added if gross blood was noted) (29).

Histological examination and immunohistology.

Tissue samples were fixed in PBS containing 6% neutral buffered formalin. Paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin. Three tissue samples from the middle part of the colon were prepared. Sections were analyzed without prior knowledge of the type of T cell reconstitution or treatment. The area most affected was graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of the previously described scoring system (29).

Flow cytometry.

Flow cytometry three-color analysis was performed. Isolated peripheral blood, spleen, mesenteric lymph node (MLN), peripheral lymph node (PLN; inguinal, axillary, and lateral axillary), and LP cells obtained from FTY720- and DW-treated mice were preincubated with Fcγ receptor-blocking monoclonal antibodies for 20 min followed by an incubation with CyChrome-conjugated anti-mouse CD4, PE-conjugated anti-CD44, and FITC-conjugated anti-CD62L monoclonal antibodies for 30 min on ice. After the cells had been stained, flow cytometry and data analysis were performed using FACS Calibour and CELLQUEST software (BD Biosciencesl San Jose, CA).

Cytokine production assay.

To measure cytokine production, 1 × 105 LP CD4+ T cells were cultured in 200 μl culture medium at 37°C in a humidified atmosphere containing 5% CO2 in 96-well plates (Costar; Cambridge, MA) precoated with 5 μg/ml hamster anti-mouse CD3ε monoclonal antibodies (145-2C11, BD PharMingen) and hamster 2 μg/ml anti-mouse CD28 monoclonal antibodies (37.51, BD PharMingen) in PBS overnight at 4°C. Culture supernatants were removed after 48 h and assayed for cytokine production. Cytokine concentrations were determined by a specific ELISA [interleukin (IL)-10] (R&D; Minneapolis, MN) or a mouse T helper (Th)1/Th2 cytokine bead array kit [IL-2, IL-4, IL-5, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] (BD Biosciences) per the manufacturer's recommendation.

RT-PCR.

Total cellular RNA was extracted from 7 × 105 cells using the RNeasy Mini Kit (Qiagen; Valencia, CA). Five micrograms of total RNA were reverse transcribed using SuperScript reverse transcriptase (Invitrogen). S1P receptor (S1P1, S1P2, S1P3, S1P4, and S1P5) levels were measured by a QuantiTect SYBER green PCR kit using an ABI7500 real-time PCR system and 7500 system SDS software (Applied Biosystems; Foster city, CA). The following primers were used: S1P1, forward 5′-GTG TAG ACC CAG AGT CCT GCG-3′ and reverse 5′-AGC TTT TCC TTG GGA GAG-3′; S1P2, forward 5′-GGC CTA GCC AGT GCT CAG C-3′ and reverse 5′-CCT TGG TGT AAT TGT AGT GTT CCA GA-3′; S1P3, forward 5′-GGA GCC CCT AGA CGG GAG T-3′ and reverse 5′-CCG ACT GCG GGA AGA GTG T-3′; S1P4, forward 5′-CCT GGA ACT CAC TTT ATA GAC CAG G-3′ and reverse 5′-AGA AAG CGT GCC ATA GGC AG-3′; S1P5, forward 5′-GAG TGC CGG TTA CAG GAG ACT T-3′ and reverse 5′-CGC TGC TGT GTC CTG CC-3′; and glyceraldehyde-3-phosphate dehydrogenase, forward 5′-CTA CTG GCG CTG CCA AGG CAG T-3′ and reverse 5′-GCC ATG AGG TCC ACC ACC CTG-3′. PCR cycling conditions consisted of 95°C for 15 min followed by 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 40 s. Real-time PCR analysis of S1P receptors was expressed as the relative amount of the indicated mRNA normalized by that of glyceraldehyde-3-phosphate dehydrogenase.

Statistical analysis.

Results are expressed as means ± SD. Groups of data were compared by the Mann-Whitney U-test. Differences were considered to be statistically significant at P < 0.05.

RESULTS

Treatment with FTY720 suppresses the development of colitis induced by adoptive transfer of naïve CD4+CD45RBhigh T cells.

We first tested whether FTY720 suppressed the development of colitis originally induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells from normal BALB/c mice into CB-17 SCID mice (Fig. 2A). Mice were administered daily with FTY720 (0.3 mg/kg) or DW orally starting 1 day before transfer. The control DW-treated mice manifested progressive weight loss from 2 wk after transfer (Fig. 2B) and clinical symptoms of colitis (Fig. 2C) such as diarrhea with increased mucus in the stool, anorectal prolapse, and hunched posture by 3–4 wk. In contrast, FTY720-treated mice appeared healthy with a gradual increase of body weight and without clinical symptoms during the whole period of observation (Fig. 2, B and C). In total, the assessment of colitis by clinical scores showed a clear difference between the control DW-treated mice and FTY720-treated mice (Fig. 2D). Histological examination showed a marked elongation of the villi with a massive infiltration of mononuclear cells in the LP of the colon from control DW-treated mice (Fig. 2E). In contrast, the elongation of the villi was mostly abrogated and only a few mononuclear cells were observed in the LP of the colon from FTY720-treated mice (Fig. 2E). This difference was also comfirmed by the histological scoring of multiple colon sections, which was 0.4 ± 0.8 in FTY720-treated mice versus 4.2 ± 0.8 in control DW-treated mice (P < 0.05; Fig. 2F).

Fig. 2.

Preventive effect of FTY720 on the development of CD4+CD45RBhigh T cell-transferred colitis. A: 7 CB-17 SCID mice from each group were administered with FTY720 at a dose of 0.3 mg/kg or distilled water (DW) daily by a gavage for 5 wk starting from 1 day before CD4+CD45RBhigh T cell transfer. B: change in body weight over time expressed as a percentage of the original weight. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice. C: gross appearance of the colon, spleen, and mesenteric lymph node (MLN) from CD4+CD45RBhigh T cell-transferred severe combine immunodeficiency (SCID) mice treated with DW (top) or FTY720 (bottom) at 5 wk after transfer. D: clinical scores were determined at 24 days after transfer as described in materials and methods. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice. E: histological examination of the colon from DW-treated (left) or FTY720-treated (right) mice at 24 days after transfer. Original magnification: ×40 in a and c and ×200 in b and d. F: histological scoring of colitis in DW-treated mice and FTY720-treated mice at 5 wk after transfer. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice.

A further quantitative evaluation of CD4+ T cell infiltration was made by isolating CD4+ T cells from each tissue or the peripheral blood. Only a few CD4+ T cells were recovered from the colonic tissue of FTY720-treated mice compared with control DW-treated mice (Fig. 3). The number of CD4+ cells recovered from the colon of control DW-treated mice (23.6 ± 18.4 × 105 cells) far exceeded the number of originally injected cells (3 × 105 cells), indicating extensive T cell migration and/or proliferation in the inflamed colon, which was mostly abrogated in FTY720-treated mice. Similarly, the numbers of CD4+ cells in the spleen, MLN, and peripheral blood from FTY720-treated mice was significantly decreased compared with those from control DW-treated mice (Fig. 3). In contrast, the number of CD4+ cells in the PLN from FTY720-treated mice was comparable with that in DW-treated mice (Fig. 3).

Fig. 3.

Mononuclear cells from the spleen, PB, peripheral lymph node (PLN), MLN, and lamina propia (LP; LPMC) were isolated from the colon at 5 wk after transfer. Cells were stained with FITC-anti-CD3 and PE-anti-CD4, and the total number of CD4+ cells was determined by flow cytometry. Data are means ± SE of 7 mice/group. *P < 0.01. Data are indicated as the mean ± SD of seven mice in each group.

We next examined cytokine production by LP CD4+ T cells from control PBS-treated mice and FTY720-treated mice. As shown in Fig. 4, LP CD4+ cells from FTY720-treated mice produced significantly less TNF-α, IFN-γ, and IL-2 compared with those from control DW-treated mice upon in vitro stimulation. In contrast, the production of IL-4, IL-5, or IL-10 was not significantly affected. These results suggested that FYT720 prevented the development of colitis primarily by promoting the sequestration of naïve CD4+ T cells and/or inhibiting the egress of colitogenic CD4+ T cells in the MLN, followed by inhibiting the development of pathogenic Th1 cells producing TNF-α, IFN-γ, and IL-2.

Fig. 4.

Cytokine production by LP CD4+ T cells. LP CD4+ T cells were isolated from PBS- or FTY720-treated mice 5 wk after CD4+CD45RBhigh T cell transfer or normal BALB/c mice (12 wk old) and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 48 h. Interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-10, and tumor necrosis factor (TNF)-α concentrations in culture supernatants were measured by a specific ELISA (IL-10) or a mouse T helper (Th)1/Th2 cytokine bead array kit (IL-2, IL-4, IL-5, TNF-α, and IFN-γ). Data are means ± SD of 6 mice/group. *P < 0.05.

We further evaluated whether FTY720 affected cell differentiation of the transferred CD4+CD45RBhigh T cells in vivo. As shown in Fig. 5, almost all CD4+ T cells in any tissue and the blood from DW- or FTY720-treated SCID mice had a phenotype of TEM cells, which express CD62LCD44high, in contrast to the originally transferred CD4+CD45RBhighCD62L+CD44low T cells (data not shown). The results indicated that cell activation/differentiation from naïve T cells to TEM cells in this model was not impaired by FTY720 treatment.

Fig. 5.

Expression of CD62L and CD44 on CD4+ T cells in various organs and the blood of DW-treated or FTY720-treated mice transferred with CD4+CD45RBhigh T cells. Freshly isolated cells from DW-treated or FTY720-treated mice transferred with CD4+CD45RBhigh T cells at 5 wk after transfer were stained with FITC-labeled anti-CD4 and PE-labeled anti-CD62L or PE-labeled anti-CD44 monoclonal antibodies. Samples were analyzed by flow cytometry. Lymphocytes were identified by characteristic forward angle and sidescatter profiles. LPL, LP lymphocytes. Data are displayed as a dotted plot (4-decade log scale), and quadrant markers were positioned to include >98% of control Ig-stained cells in the bottom left. Percentages in each quadrant are indicated. Results shown are representative of 3 mice/group.

Treatment with FTY720 suppresses the development of colitogenic CD4+ TEM cell-mediated colitis.

Although we found that FTY720 suppressed the development of chronic colitis induced by the adoptive transfer of naïve CD4+CD45RBhigh T cells into SCID mice (Figs. 25), it was still unclear how FTY720 controlled the migration property of colitogenic memory T cells in memory T cell-mediated chronic colitis. Because it was possible that the effect of FTY720 in the CD4+CD45RBhigh T cell transfer model was owing to the promotion of the sequestration of naïve CD4+ T cells and/or to the inhibition of the egress of colitogenic CD4+ memory T cells in the MLN, as previously demonstrated by others (3), we used our recently established colitogenic CD4+ TEM cell-mediated chronic colitis model (29) under the condition without impact of naïve T cells. CB-17 SCID mice were injected intraperitoneally with LP CD4+ TEM (CD44highCD62L; as shown in the inset in Fig. 6A) cells obtained from colitic SCID mice originally induced by CD4+CD45RBhigh T cells and were treated with DW or FTY720 (0.3 mg/kg) daily starting 1 day before transfer over a period of 4 wk (Fig. 6A). As shown in Fig. 6B, control DW-administered mice manifested progressive weight loss from 2–4 wk after the transfer. These mice had diarrhea with increased mucus in the stool, anorectal prolapse, and hunched posture by 4 wk. In contrast, FTY720-treated mice appeared healthy without any clinical signs during the whole period of observation (Fig. 6B). At 4 wk after the transfer, the colon from control DW-treated mice, but not from FTY720-treated mice, was enlarged and had a greatly thickened wall (Fig. 6C). In total, the assessment of clinical scores showed a clear difference between control DW-treated mice and FTY720-treated mice (Fig. 6D). Histological examination showed a marked inflammation in the LP of the colon from control DW-treated mice (Fig. 6E). In contrast, it was mostly abrogated in the LP of the colon from FTY720-treated mice (Fig. 6, E and F).

Fig. 6.

Effect of FTY720 on the development of colitogenic CD4+ effector memory T cell (TEM cell)-mediated colitis. A: colitogenic CD4+ TEM cells (CD44highCD62L; as shown in the inset by fluorescence-activated cell sorting) were isolated from inflamed mucosa of colitic mice transferred with CD4+CD45RBhigh T cells. Seven CB-17 SCID mice from each group were administered with FTY720 at a dose of 0.3 mg/kg or DW daily by a gavage for 4 wk starting from 1 day before colitogenic CD4+ TEM cell transfer. B: changes in body weight over time expressed as a percentage of the original weight. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice. C: gross appearance of the colon, spleen, and MLN from colitogenic CD4+ TEM cell-transferred SCID mice treated with DW (top) or FTY720 (bottom) at 4 wk after transfer. D: clinical scores were determined at 4 wk after transfer as described in materials and methods. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice. E: histological examination of the colon from DW-treated (left) or FTY720-treated (right) mice at 4 wk after transfer. Original magnification: ×40 in a and c and ×200 b and d. F: histological scoring of colitis in DW- and FTY720-treated mice at 4 wk after transfer. Data are means ± SE of 7 mice/group. *P < 0.05 compared with DW-treated mice.

Furthermore, a few CD4+ T cells were recovered from the colonic tissue of FTY720-treated mice compared with control DW-treated mice (Fig. 7). The numbers of CD4+ cells recovered from the colon of control DW-treated mice (91.4 ± 63.7 × 105 cells) far exceeded the number of originally injected memory cells (5 × 105 cells), indicating extensive T cell migration and/or proliferation in the inflamed colon, which was mostly abrogated in FTY720-treated mice. Similarly, the numbers of CD4+ cells in the spleen and peripheral blood from FTY720-treated mice was significantly decreased compared with control DW-treated mice (Fig. 7). Somewhat at odds, the numbers of CD4+ cells in the MLN from FTY720-treated mice was comparable with that from DW-treated mice (Fig. 7). Unlike the CD4+CD45RBhigh T cell-transferred model, transferred colitogenic LP CD4+ TEM cells could not be detected in the PLN.

Fig. 7.

Mononuclear cells from the spleen, peripheral blood, PLN, MLN, and LP were isolated from the colon at 4 wk after transfer. Cells were stained with FITC-anti-CD3 and PE-anti-CD4, and the total number of CD4+ cells was determined by flow cytometry. Data are means ± SE of 7 mice/group. ND, not determined. *P < 0.05.

Cytokine production by LP CD4+ T cells from control PBS- or FTY720-treated mice transferred with colitogenic LP CD4+ TEM cells was assessed. As shown in Fig. 8, LP CD4+ cells from FTY720-treated mice produced significantly less TNF-α, IFN-γ, and IL-2 compared with control DW-treated mice upon in vitro stimulation. In contrast, the production of IL-4, IL-5, or IL-10 was not significantly affected.

Fig. 8.

Cytokine production by LP CD4+ T cells. LP CD4+ T cells were isolated from control PBS- or FTY720-treated mice 4 wk after the transfer of colitogenic LP CD4+ TEM cells and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 48 h. Cytokine production was measured by the same methods as those described in Fig. 4. Data are means ± SD of 6 mice/group. *P < 0.05.

We further determined whether FTY720 affected the cell differentiation of transferred CD4+CD62LCD44high TEM cells in vivo, because we initially speculated that memory T cells residing in lymph nodes preferentially express CD62L, which is termed as central memory T (TCM) cells. As shown in Fig. 9, however, almost all CD4+ T cells in any organ and the blood from DW-treated or FTY720-treated SCID mice retained the characteristics of TEM cells, including the MLN, indicating that FTY720 did not affect the TEM-to-TCM conversion in this model.

Fig. 9.

Expression of CD62L and CD44 on CD4+ T cells in various organs and the blood of DW- or FTY720-treated mice transferred with colitogenic CD4+ TEM cells. Freshly isolated cells from DW- or FTY720-treated mice transferred with colitogenic CD4+ TEM cells at 24 days after transfer were analyzed by flow cytometry. Percentages in each quadrant are indicated. Results shown are representative of 3 mice/group.

Colitogenic CD4+ TEM cells express receptors for S1P.

Because it has been reported FTY720-phosphate acts via S1P receptors (5), we finally evaluated the expression pattern of S1P receptors in sorted normal splenic CD4+, CD4+CD45RBhigh, and colitic LP CD4+ TEM cells using quantitative PCR analysis. As depicted in Fig. 10, colitogenic LP TEM cells contained mRNA encoding S1P1, S1P2, and S1P4 receptors, albeit to a lesser extent compared with naïve CD4+CD45RBhigh T cells, with only a minimal representation of S1P3 and S1P5 receptors.

Fig. 10.

Quantitative PCR analysis of spingonsine-1-phosphate (S1P) receptors in sorted normal splenic CD4+, CD4+CD45RBhigh, and colitic LP CD4+ TEM cells expressed as the relative amounts of indicated mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase. Results shown are representative of 3 experiments.

DISCUSSION

In the present study, we demonstrated that FTY720 suppressed the development of colitis induced by the adoptive transfer of colitogenic LP TEM cells into SCID mice under the condition without the impact of naïve T cell migration. In addition, we also found that FTY720 ameliorates the development of colitis induced by the adoptive transfer of naïve CD4+CD45RBhigh T cells into SCID mice, in which all processes responsible for the development of colitis, such as the initiation of naïve T cells by antigen-bearing dendritic cells and the elicitation to effector or TEM cells in secondary lymphoid organs, are involved. Because we demonstrated that colitogenic LP TEM cells as well as normal splenic CD4+CD45RBhigh T cells expressed several S1P receptors that are targets for FTY720-phosphate, the present study provides the suggestion that FTY720 can directly control memory CD4+ T cell-mediated immune diseases in addition to the impact of naïve T cells.

Because it has been largely thought that the effect of FTY720 treatment on autoimmune diseases in the previous preventive protocols is owing to the sequestration of naïve T cells rather than pathogenic memory T cells, it was unclear whether FTY720 directly affects the migration property of memory T cells in autoimmune established stages. To overcome this issue, we conducted the adoptive transfer of only colitogenic LP CD4+ TEM cells into SCID mice in the present study. As there were no naïve T cells in this transfer system because almost all isolated LP CD4+ T cells from colitic mice had a characteristic of CD44highCD62LCD45RBlow TEM cells, we could directly evaluate the effect of FTY720 on colitogenic LP CD4+ TEM cells. Surprisingly, we found that FTY720 suppressed the development of colitogenic memory TEM cell-mediated colitis, indicating that FTY720 affects the migration of colitogenic LP CD4+ TEM cells. Consistent with this, we demonstrated that colitogenic LP TEM cells as well as normal splenic CD4+CD45RBhigh T cells expressed several S1P receptors (S1P1, S1P2, and S1P4). However, we found that the numbers of CD4+ T cells in the MLN were also significantly decreased in FTY720-treated mice transferred with colitogenic LP TEM cells compared with DW-treated mice. Although the result might indicate that FTY720 could not sequester colitogenic CD4+ TEM cells in FTY720-treated mice, it is more likely that colitogenic CD4+ TEM cells in PBS-treated mice had a greater chance to repeat the migration into the MLN and the egress from the MLN to sites of inflammation and could expand their number, which exceeded the original transferred cell number (3 × 105 cells). However, it is possible that the finding that there were decreased numbers of CD4+ T cells in the various compartments may be a result of the lack of the colitis in FTY720-treated mice and not necessarily a direct effect of FTY720 on egress or sequestration, because we could not detect any differences of cell numbers in the MLN between TEM cell-transferred DW-treated mice and FTY720-treated mice. Nonetheless, we found that FTY720 caused a significant loss of CD4+ TEM cells from the blood (lymphopenia) and spleen, reducing numbers by ∼100- to 1,000-fold compared with DW-treated mice, indicating that FTY720 induced a smaller chance to recirculate into the MLN repeatedly, followed by no development of colitis. Further studies will be needed to address this issue using another short-term in vivo transfer system.

The present study would provide another impact in terms of a characteristic of memory T cell trafficking for the maintenance of autoimmune diseases. Although some investigators have suggested that LP T cells do not migrate out of the gut (12), our results indicated that LP CD4+ TEM cells are needed to constitutively recirculate into the MLN and are restimulated by antigen-bearing dendritic cells in the MLN to maintain colitogenic CD4+ TEM cells for sustaining chronic colitis. An earlier study (32) has demonstrated that the thoracic duct lymph (TDL), which empties into the blood, contains many lymphocytes. In a landmark experiment, Gowans and Knight (7) transferred radiolabeled thoracic duct lymphocytes intravenously into naïve syngeneic recipients and detected donor memory lymphocytes in their TDL again, providing the first formal demonstration that memory lymphocytes recirculate continuously between blood and lymph. Consistent with this notion, we also found that LP CD4+ T cells obtained from FTY720-treated SCID mice transferred with colitogenic CD4+ TEM cells in FTY720-treated mice produced less amounts of Th1 cytokines compared with DW-treated SCID mice, indicating that the recirculation and restimulation of Th1 cells in lymph nodes, in this case, the MLN, are essential for the maintenance of Th1-mediated autoimmune disorders.

In nohuman primates treated with FTY720 for over 100 days, however, ∼10% of peripheral blood CD4+ T cells were refractory to depletion by FTY720 (23). It is likely that these cells represent long-lived TEM cells. Because TEM cells lack the lymph node homing receptors CCR7 and CD62L (28) and express high levels of inflammatory chemokine receptors (28), and they therefore could preferentially reside in nonlymphoid tissues (16) in contrast to naïve and TCM cells. Thus it is possible that TEM cells might not be trapped by FTY720 treatment in the lymph node but be controlled by the unidentified mechanism of action of FTY720 in our model. Conversely, Henning and colleagues (9) recently reported that FTY720 rescues the homing defect in both CCR7−/− mice and plt mice, which lack expression of CCL19 and CCL21-ser, both ligands for CCR7 on high endothelial cells (9), suggesting that FTY720 enables TEM cells to sequester into lymph nodes in a CCR7/CD62L-independent mechanism for lymphocyte homing through endothelial venules, which is strongly augmented in the presence of FTY720. Further studies will be needed to address this issue.

The ultimate goal of any treatment for autoimmune diseases, including IBDs, is antigen-specific suppression of pathology. Autoaggressive antigen-specific lymphocytes need to be eliminated or controlled to prevent tissue damage and halt the progression of clinical disease. Although it is so far poorly understood regarding the self-antigens and commensal bacteria flora responsible for the pathogenesis of IBDs, colitogenic memory T cells rather than naïve T cells should be the targets for the treatment of IBDs, even if the primary, initiating self-antigens/commensal bacteria flora are unknown and inflammation is progressive (19, 25). Although Mizushima and colleagues (18) recently demonstrated that FTY720 treatment of established colitis in IL-10−/− mice ameliorated colitis, it was still unclear whether FTY720 affected the migration of naïve T cells or colitogenic memory T cells. In light of this, the present SCID colitis model induced by the adoptive transfer of colitogenic CD4+ TEM cells is a useful model, because we can assess a character of memory T cells without the impact of naïve T cells. Because we demonstrated that FTY720 could control and suppress colitogenic CD4+ TEM cell-mediated colitis, FTY720 might be beneficial for the treatment of ongoing or established IBDs as well as their prevention.

In conclusion, we demonstrated here that FTY720 was useful to suppress memory T cell-mediated chronic colitis without the impact of naïve T cell activation using memory CD4+ TEM cell adoptive transfer experiments. Although we have to pass many critical checkpoints, this study indicates that FTY720 offers a hope for the treatment of human IBDs.

GRANTS

This work was supported in part by Grants-In-Aid for Scientific Research, Scientific Research on Priority Areas, Exploratory Research, and Creative Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare; the Japan Medical Association; the Foundation for the Advancement of International Science; Terumo Life Science Foundation; Ohyama Health Foundation; Yakult Bio-Science Foundation; and Research Fund of Mitsukoshi Health and Welfare Foundation.

Acknowledgments

The authors express special thanks to Novartis Pharmaceuticals (Basel, Switzerland) for kindly providing FTY720.

Footnotes

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

REFERENCES

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