Ten mg of Tim-1 vector was injected intraperitoneally 4 times at 2 day intervals into BD mice, followed by the observation for 2 weeks. In this study, skin and genital ulcers were improved after Tim-1 vector administration when compared to the control vector injected group (Fig 5A). However, Tim-1 vector was not effective in eye involvement (Table 1).
Additionally, in control vector group, the severity score was 2.83±0.41 before and 2.83±0.75 at one week and 2.67±1.21 at two week after first injection of control vector (p=0.8, n=6). In Tim-1 vector group, severity score was 3.17±0.75 before the injection, 1.50±1.22 at one
week after injection (p=0.004), 1.33±1.51 at two weeks after injection in BD mice (p=0.03) (Fig 5B). After injection of Tim-1 vector, the severity score was decreased, whereas, control vector injection was not different in BD mice.
Two weeks after the first administration of Tim-1 vector to BD mice, isolated lymph node and PBMC were analyzed for Tim-1(+) cells by FACS. In LN, the frequency of Tim-1(+) cells in Tim-1 vector injected group was slightly high compared to control vector injected group [Con vs. Tim-1 (%): 10.3±1.7 (n=4) vs. 11.9±3.2 (n=5), p=0.19]. However, CD4(+)Tim-1(+) and CD8(+)Tim-1(+) cells were similar to control vector injected group (Fig 5C). The frequencies of 4(+) cells were also not different (Fig 5D). In PBMC, Tim-1(+) cells was slightly lower in Tim-1 vector injected group. CD4(+)Tim-Tim-1(+), CD8(+)1(+) and 4(+) cells were not different (Fig 5C-D). Interestingly, CD4(+) T cells in Tim-1 vector injected group were higher than control vector injected group [Con vs. Tim-Tim-1 (%):
21.3±10.7 (n=8) vs. 27.6±13.7 (n=8), p=0.36, Fig 5C]. One week after administration of Tim-1 vector, the frequencies of those marker expressing cells were similar compared with
- 19 -
two weeks after injection (data not shown). Up-regulated Tim-1 expression improved BD-like symptoms in BD mice.
- 20 -
Figure 5. Administration of Tim of Tim-1 expressing cells were not
injected intraperitoneally 4 times at 2 days interval into BD mice, followed by observation for 2 weeks. A. photographs of mice were taken before and
treatment of Tim-1 vector and control vector injected group compared before and at one and two
Tim-1 vector injection, the frequencies of Tim
CD4(+) T cells, and D. Tim-4(+) cells in LN and PBM 0.1) Con: control vector injection to BD mice, Tim
- 21 -
tration of Tim-1 improved the BD-like symptoms, but the frequencies cells were not changed. Ten mg per mouse of Tim-1
injected intraperitoneally 4 times at 2 days interval into BD mice, followed by observation for 2 weeks. A. photographs of mice were taken before and at one and two weeks
vector and control vector injected group. B. The severity score was at one and two weeks after treatment of them. C. Two weeks after
he frequencies of Tim-1(+), CD4(+)Tim-1(+), CD8(+)Tim 4(+) cells in LN and PBMC by FACS analysis. (n=5~6) vector injection to BD mice, Tim-1: Tim-1 vector injection to BD mice
frequencies vector was injected intraperitoneally 4 times at 2 days interval into BD mice, followed by observation weeks after first
- 22 -
Abdominal skin inflammation + +
Tim-1
- 23 -
No. BD-like symptoms Before 1 wk 2 wks
Tim-1
vector
4 Genital ulcer + - -
Back skin ulcer + - -
5
Neck skin ulcer + + -
Back skin crust + - -
Right eyeball inflammation + + +
6
Left eyeball inflammation + + +
Right eye vision loss + + +
Back skin crust + - -
Left earlobe erythema + + -
BD symptom: +, deteriorated: ++, improved: -
- 24 -
E. Tim-1 vector administration affects the regulatory cellular phenotypes
After Tim-1 vector administration, several cellular phenotypes in LN and PBMC were analyzed in BD mice. The frequencies of CD4(+)CD25(+), CD4(+)Foxp3(+) and CD4(+)CD25(+)Foxp3(+) (regulatory T, Treg) cells in LN were not significantly changed in Tim-1 vector compared to control vector injected group [Con (n=5) vs. Tim-1 (n=6) (%):
CD4(+)CD25(+), 6.5±1.6 vs. 6.1±2.3 p=0.35; CD4(+)Foxp3(+), 5.3±1.5 vs. 4.7±1.7, p=0.29;
Treg, 5.2±1.4 vs 4.7±1.7, p=0.3] (Fig 6A). But, in PBMC, CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg cells in Tim-1 vector injected group were significantly higher than control vector injected group [Con (n=8) vs. Tim-1 (n=8) (%): CD4(+)CD25(+), 0.31±0.18 vs. 0.61±0.21 p=0.01; CD4(+)Foxp3(+), 0.10±0.10 vs. 0.31± 0.12, p=0.004 ; Treg, 0.04±0.08 vs 0.16±
0.10, p=0.03] (Fig 6A).
CD8(+)CD122(+) T cells are newly identified and regarded as Treg cells (Saitoh et al., 2007) and reported the effect as an anti-inflammatory responses (Rifa’i et al., 2008).
Another type of Treg cells, CD8(+)CD122(+) T cells were also analyzed in LN of Tim-1 vector injected group. [Con (n=5) vs. Tim-1 (n=6) (%): 1.3±0.3 vs. 1.6±0.7 p=0.25] (Fig 6B).
In PBMC, CD122(+) and CD8(+)CD122(+)T cells in Tim-1 vector injected group were also higher than control vector injected group [Con (n=8) vs. Tim-1 (n=8) (%): CD122(+), 1.2±1.0 vs. 9.8±7.9 p=0.04; CD8(+)CD122(+), 1.9±1.5 vs. 2.4±2.1 p=0.58).
Our study indicated that Tim-1 vector up-regulated Treg cells and CD8(+)CD122(+) Treg cells in BD mice. Up-regulation of these cellular phenotypes may be involved in the improvement of BD-like symptoms after injection of Tim-1 vector.
Figure 6. The frequencies of Treg cells in PBMC were affected in
BD mice. Two types of Treg cells and apoptotic cell phenotypes were confirmed in LN and PBMC after Tim-1 administration to BD mice. A. The frequencies of CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg cells were compared between control and
group. B. CD8(+)CD122(+) T cells were up
significant. CD122(+) cells was significantly increased in (n=5~8) (**p < 0.05, ***p < 0.01)
vector injection to BD mice
- 25 -
Figure 6. The frequencies of Treg cells in PBMC were affected in Tim-1 vector injected Two types of Treg cells and apoptotic cell phenotypes were confirmed in LN and administration to BD mice. A. The frequencies of CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg cells were compared between control and Tim-1 vector injected group. B. CD8(+)CD122(+) T cells were up-regulated compared to control group but not significant. CD122(+) cells was significantly increased in Tim-1 vector injected grou
**p < 0.05, ***p < 0.01) Con: control vector injection to BD mice, Tim
vector injected Two types of Treg cells and apoptotic cell phenotypes were confirmed in LN and administration to BD mice. A. The frequencies of CD4(+)CD25(+), vector injected regulated compared to control group but not vector injected group.
vector injection to BD mice, Tim-1: Tim-1
- 26 -
F. Pro-inflammatory cytokines were down-regulated by Tim-1 vector administration in BD mice
To determine the level of cytokines, two weeks after Tim-1 vector injection into BD mice, sera were analyzed by ELISA. The IL-17 level in Tim-1 vector injected group was significantly lower than control group [Con (n=7) vs. Tim-1 (n=7) (pg/ml): 11.53±4.45 vs.
6.84±2.17, p=0.03]. TNF-a was also significantly decreased in the Tim-1 vector injected group compared to control group [Con (n=11) vs. Tim-1 (n=11) (pg/ml):16.5±13.8 vs.
6.9±5.1, p=0.04]. The IL-6 level in Tim-1 vector injected group was decreased [Con (n=8) vs.
Tim-1 (n=8) (pg/ml):145.7±176.9 vs.72.2±38.8, p=0.27]. In contrast, IL-4 was slightly increased in Tim-1 vector injected group [Con (n=8) vs. Tim-1 (n=9) (pg/ml):9.3±5.4 vs.
10.8±5.0, p=0.53]. But IFN-g did not differ between the Tim-1 and control vector injected group (Fig 7). Consequently, these results indicated that Tim-1 vector might be served as down-regulator for pro-inflammatory cytokines in BD mice.
Figure 7. Tim-1 vector administration decreased pro mice. Two weeks after first injection
17, TNF-a, IL-6, IFN-g and IL injected BD mice by ELISA. (n=7~11
mice, Tim-1: Tim-1 vector injection to BD mice
- 27 -
vector administration decreased pro-inflammatory cytokines in BD injection of Tim-1 vector isolated sera in blood. The levels of IL and IL-4 were analyzed in the sera of Tim-1 and control vector
(n=7~11) (**p < 0.05) Con: control vector injection to BD vector injection to BD mice
inflammatory cytokines in BD levels of IL-and control vector vector injection to BD
- 28 -
G. Tim-4 siRNA treatment down-regulated the expression of Tim-4 in normal healthy mice
In BD mice, the frequencies of Tim-4(+) cells were higher than Nor and BDN mice in LN cells and peritoneal macorphages (Fig 2). For down-regulation of Tim-4(+) cells, siTim-4 was injected into Nor mice intraperitoneally and the frequencies of Tim-4(+) cells were analyzed in peritoneal macrophages by FACS. Tim-4 siRNA (siTim-4) at 2, 5 or 10 μg per mouse or negative control (N.C.) scramble siRNA (2 and 5 μg per mouse) were injected [N.C - 5 μg vs. siTim-4 - 2, 5 and 10 μg (n=3~4): 8.9±1.4% vs. 6.6±3.4% (p=0.42), 3.7±0.9%
(p=0.013), 2.8±2.2% (p=0.04)] (Fig 8A). siTim-4 down-regulated Tim-4(+) macrophages by dose dependent manner and showed statistical significance at 5 μg and 10 μg administered groups. Therefore, 5 μg injection was used for the following experiment. To see the time dependent efficacy of Tim-4 siRNA, 5 μg of siTim-4 was injected, and after 24, 48, and 72 hours, the frequencies of Tim-4(+) macrophages were analyzed by FACS. Until 72 hours, siTim-4 significantly down-regulated Tim-4(+) macrophages compared to N.C. The frequencies of Tim-4(+) at 48 hours were lowest [N.C vs. siTim-4 (n=2): 24h, 7.9±0.6% vs.
4.9±0.2%, p=0.02; 48h, 8.9±1.4% vs. 2.9±0.4%, p=0.03; 72h, 15.4±0.1% vs. 4.2±3.0%, p=0.03] (Fig 8B).
Figure 8. The expression of Tim
Tim-4 siRNA was applied intraperitoneally and the frequencies of Tim analyzed in peritoneal macrophages
mouse or negative control (N.C.) siRNA (2 intraperitoneally into Nor mice. B. Five μg of siTim
hours, the frequencies of Tim-4(+) peritoneal macrophages were ana was used as negative control (n=3~4)
mice, siTim-4: siRNA Tim-4 injection to normal mice
- 29 -
Figure 8. The expression of Tim-4(+) cells was down-regulated after siTim-4
4 siRNA was applied intraperitoneally and the frequencies of Tim-4(+) cells were analyzed in peritoneal macrophages in Nor mice by FACS. A. siTim-4 2, 5 and 10 mouse or negative control (N.C.) siRNA (2 and 5 μg per mouse) were injected
mice. B. Five μg of siTim-4 was injected, and after 24, 48, and 72 4(+) peritoneal macrophages were analyzed. Scramble siRNA egative control (n=3~4) (**p < 0.05). N.C: negative control injection to normal
4 injection to normal mice, Mac: macrophage
treatment.
- 30 -
H. Administration of siTim-4 changed the BD-like symptoms
Five mg of siTim-4 was injected intraperitoneally 3 times at 2 days interval into BD mice and followed by observation for 2 weeks (Fig 9A). After administration of siTim-4, BD-like symptoms, such as skin ulcer and genital ulcer were compared to the control group.
However, siTim-4 was not effective in eye involvement (Table 2). In negative control group, the severity score was 2.25±0.46 before and 2.00±1.07 at one week (p=0.35), and 1.88±1.13 at two weeks after first injection of BD mice (p=0.28, n=8). In siTim-4 treated group, score was 2.63±0.52 before and 1.25±0.89 at one week (p=0.001), 1.25±0.89 at two weeks after injection into BD mice (p=0.001, n=8) (Fig 9B). At one and two weeks after first administration of siTim-4 into BD mice, macrophages were isolated from peritoneal cavity and analyzed for Tim-4 by FACS. The frequencies of Tim-4(+) cells at one and two weeks after siTim-4 treated group were lower than negative control group even though not significant (1 week: 18.6±4.8% vs. 15.7±3.7%, p=0.31, 2 weeks: 20.7±6.5%vs. 18.8±3.3%, p=0.42) (Fig 9B). However in LN, 4(+), CD11b(+)4(+), CD11c(+)4(+), Tim-1(+), CD4(+)Tim-1(+) and CD8(+)Tim-1(+) cells of siTim-4 treated group were similar to control treated group (Fig 9C~D). At one week after first injection of siTim-4, those markers assessed but not different to two weeks (date not shown). In these results, BD-like symptoms were improved with si4 treatment and decreased severity score but the changes of Tim-4 related cellular phenotypes could not be found in this study.
- 31 -
- 32 -
Figure 9. BD-like symptoms were changed after siTim-4 administration. Five mg per mouse of Tim-4 siRNA was injected intraperitoneally 3 times at 2 days interval into BD mice and observed for 2 weeks. A. Photographs of mice taken before and at one and two weeks after treatment with siTim-4 and N.C treated group. B. The severity score was compared before and at one and two weeks after treatment between the siTim-4 and N.C treated group. The frequencies of Tim-4(+) cells in peritoneal cavity were compared between siTim-4 and N.C treated groups. C~D. The frequencies of Tim-4(+), CD11b(+)Tim-4(+), CD11c(+)Tim-4(+), Tim-1(+), CD4(+)Tim-1(+) and CD8(+)Tim-1(+) cells in LN were compared to siTim-4 and negative control treated group. (n=7~8) (**p < 0.05) N.C: negative control injection to BD mice, siTim-4: siRNA Tim-4 injection to BD mice, LN: lymph node
- 33 -
Table 2. The change of BD-like symptoms in siTim-4 administered BD mice
No. BD symptoms Before 1 wk 2 wks
- 34 -
BD symptom: +, deteriorated: ++, improved: -
- 35 -
I. Treg cells were up-regulated in siTim-4 treated BD mice
The frequencies of CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg [CD4(+)CD25(+) Foxp3(+)] cells were also analyzed by FACS at one and two weeks after first treatment with siTim-4 into BD mice. After one week, Treg cells were slightly increased in siTim-4 treated BD mice compared with N.C treated group [N.C vs. siTim-4: CD4(+)CD25(+), 5.82±2.01%
vs. 6.84±2.03%, p=0.45, n=5; CD4(+)Foxp3(+), 4,64±1.9% vs. 5.82±2.4%, p=0.41, n=5;
Treg, 3.28±1.57% vs. 4.08±1.73%, p=0.47, n=5]. After two weeks, Treg cells were significantly higher in siTim-4 treated BD mice than N.C treated group [N.C vs. siTim-4:
CD4(+)CD25(+), 4.8±0.6% vs. 5.9±1.2%, p=0.03, n=6; CD4(+)Foxp3(+), 4.1±0.7% vs.
5.2±1.2%, p=0.03, n=7; Treg, 2.7±0.5% vs. 3.5±0.9%, p=0.04, n=7] From this result, the increases of Treg cells are associated with knock down of Tim-4 in BD mice.
Figure 10. The frequencies of Treg cells were up
in BD mice. Treg cells and granzyme B(+) cells were confirmed in LN of BD mice after treatment with siTim-4. At One and two weeks after
frequency of CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg were compared between
siTim-negative control injection to BD mice, siTim lymph node
- 36 -
Figure 10. The frequencies of Treg cells were up-regulated after treatment with siTim Treg cells and granzyme B(+) cells were confirmed in LN of BD mice after
One and two weeks after first treatment with siTim frequency of CD4(+)CD25(+), CD4(+)Foxp3(+) and Treg [CD4(+)CD25(+)Foxp3(+)]
-4 and control treated group. (n=5~7) (**p < 0.05) negative control injection to BD mice, siTim-4: siRNA Tim-4 injection to BD mice, LN:
regulated after treatment with siTim-4 Treg cells and granzyme B(+) cells were confirmed in LN of BD mice after ment with siTim-4, the [CD4(+)CD25(+)Foxp3(+)] cells (**p < 0.05) N.C:
4 injection to BD mice, LN:
- 37 -
J. Treatment with siTim-4 decreased the serum level of IL-17 in BD mice
After administration of siTim-4 into BD mice, serum level of IL-17 was analyzed by ELISA and compared with N.C siRNA treated BD mice. The level of IL-17 was decreased in siTim-4 treated group compared to N.C treated group, but not significant [N.C vs. siTim-4 (n=8): 19.4±11.5 pg/ml vs. 15.6±8.1 pg/ml, p=0.25] (Fig 11).
Figure 11. Administration of siTim Serum obtained at two weeks after of IL-17 was analyzed by ELISA.
siRNA Tim-4 injection to BD mice
- 38 -
siTim-4 decreased the serum level of IL-17 in BD mice erum obtained at two weeks after first injection of siTim-4 and N.C in BD mice.
17 was analyzed by ELISA. (n=8) N.C: negative control injection to BD mice, 4 injection to BD mice
17 in BD mice.
in BD mice. The level N.C: negative control injection to BD mice, siTim-4:
- 39 -
K. Gal-9 treatment up-regulated the expression of Gal-9 in vitro and in vivo
We checked the expression of Gal-9 after Gal-9 treatment in in vitro and in vivo normal healthy mice Lymph node cells and splenocytes from Nor mice were cultured with or without Gal-9, including anti-CD3 and anti-CD28 antibodies, for 48 h in vitro. In LN cells, the frequencies of Gal-9(+) cells were significantly higher in the Gal-9-treated group than in the non-treated or IgG1-treated control group (non-treated 1.4±0.4% vs. Gal-9-treated 46.7±1.5, p=0.000001; IgG1-treated 1.5±1.2 vs. Gal-9-treated 46.7±1.5, p=0.000002). The frequencies of Gal-9(+) cells in splenocytes were significantly higher in the Gal-9 treated group than in the non-treated group or IgG1-treated group (non-treated 1.3±0.4% vs. Gal-9-treated 64.6±6.2%, p=0.00003; IgG1-Gal-9-treated 1.7±0.8% vs. Gal-9-Gal-9-treated 64.6±6.2%, p=0.00006] (Fig 12A). For in vivo administration, 100 μg per mouse of Gal-9 was injected intraperitoneally once a day for 3 days. One day after the last injection, the frequency of 9(+) cells was compared to those of the PBS-injected control group. The expression of Gal-9(+) cells was higher in lymph node cells of Gal-9 treated mice (45.5±7.4%) compared to control mice (35.5±7.6%) (p= 0.069) (Fig12B). We confirmed that treated with Gal-9 can increase Gal-9 expression in in vitro and in vivo Nor mice.
Figure 12. Gal-9 treatment increased the expression of Gal node cells and splenocytes isolated from
CD28 antibodies with or without Gal
higher in the Gal-9-treated group than in the non
vivo treatment, 100 μg of Gal-9 was injected 3 times once a day. One day after the last
injection, the frequencies of Gal-(PBS) group. (n=5) (*p < 0.1, ***p
- 40 -
9 treatment increased the expression of Gal-9 in vitro and in vivo node cells and splenocytes isolated from Nor mice were cultured with anti-CD3 and anti CD28 antibodies with or without Gal-9 for 48 h. A. The frequencies of Gal-9(+) cells were
treated group than in the non-treated or IgG1-treated group. B. For 9 was injected 3 times once a day. One day after the last
-9 were higher in the Gal-9 treated group than in the control (*p < 0.1, ***p < 0.01)
in vivo. Lymph
CD3 and anti-9(+) cells were treated group. B. For in 9 was injected 3 times once a day. One day after the last group than in the control
- 41 - L. Gal-9 administration improved BD-like symptoms
Gal-9 (100 μg per mouse) was injected intraperitoneally into BD mice 5 times at 3 days interval, followed by observation for 2 weeks. Changes in symptoms are shown in Figure 13A. The BD-like symptoms, such as skin ulcers and arthritis, improved after Gal-9 administration as compared to the control group. The severity score was significantly different between the control (2.4±0.6 to 2.7±0.8) and Gal-9 treated groups (2.8±0.8 to 1.2±0.5) (p=0.016, n=5) (Fig 13B). Two weeks after administration of Gal-9 into BD mice, lymph node cells were isolated and analyzed for 3 by FACS. The frequencies of Tim-3(+) cells were 3.7±0.4% in the Gal-9 treated group and 5.9±1.1% in the control group (p=0.003, n=5) (Fig 13C). In this study, administration of Gal-9 was significantly decreased the frequencies of Tim-3(+) cells in the lymph nodes. These results suggest that the down-regulation of Tim-3(+) cells may be related to the improvement of BD-like symptoms.
Figure 13. Gal-9 treatment improved BD
9 was injected intraperitoneally five times at 3 days interval, followed by observation for 2 weeks. A. Photographs of mice were taken before and after treatment. B. The severity score was compared before and after treatment b
C.The frequency of Tim-3(+) cells in Gal in LN by FACS analysis. (n=5)
Gal-9: Gal-9 injection to BD mice
- 42 -
9 treatment improved BD-like symptoms. For each mouse, 100 μg of Gal 9 was injected intraperitoneally five times at 3 days interval, followed by observation for 2 weeks. A. Photographs of mice were taken before and after treatment. B. The severity score was compared before and after treatment between the Gal-9 and control treated groups.
3(+) cells in Gal-9 treated group was compared with control group (**p < 0.05, ***p < 0.01) Con: PBS injection to BD mice, 9 injection to BD mice
For each mouse, 100 μg of Gal-9 was injected intraperitoneally five times at 3 days interval, followed by observation for 2 weeks. A. Photographs of mice were taken before and after treatment. B. The severity score treated groups.
9 treated group was compared with control group on: PBS injection to BD mice,
- 43 -
M. Gal-9 induced the expression of cell death-related molecules in BD mice
To confirm the apoptotic cell death in Gal-9 administered BD mice, lymph node cells were analyzed by flow cytometry after staining with Annexin V and propidium iodide (PI). The frequencies of Annexin V(+) and PI(-) apoptotic cells were 12.0±2.9% in the Gal-9-treated group (n=6) and 6.9±2.4% in the PBS treated control group (n=7) (p=0.006) (Fig 14A). In this study, Gal-9 administration induced apoptosis in lymph node cells in BD mice.
Granzyme B induces apoptosis (Saito et al., 2008) and is expressed in resting and activated plasmacytoid dendritic cells,monocytes, resting T-cells, B-cells, activated granulocytes, and activated monocyte-derived dendriticcells (Rissoan et al., 2002). Granzyme B(+) cells were also significantly higher in the Gal-9 treated group (11.0±6.3%) compared to the control group (5.6±3.6%) (p=0.04, n=9) (Fig 14B). CD107a is a marker of functional NK cells (Alter G et al., 2004) and CD8 T cells (Betts et al., 2003). CD107a is a transmembrane glycoprotein found in lysosomal and cell membranes and a marker of degranulation on lymphocytes (Betts et al., 2003). The frequencies of CD107a(+) cells were significantly increased after Gal-9 administration (10.9±4.9%) when compared to the PBS-administered control BD mice (0.4±0.2%) (p=0.001, n=5) (Fig 14C).
For confirmation of apoptosis induced by Gal-9, lymph node cells were cultured for 24 h with Gal-9, after which the frequencies of Tim-3(+) and Gal-9(+) among the apoptotic cells were analyzed by FACS. In apoptotic cells, the Gal-9 treated group showed 42.5±3.5%
frequencies of Gal-9(+) cells compared to 17.9±0.9% in the control group (Fig 14D). In the Gal-9-treated group, the frequencies of Tim-3(+) cells were also up-regulated to 1.7±1.1%
compared to the control group (0.6±0.5%) (Fig 14D). Further, Gal-9 treatment for 48 h also
- 44 -
up-regulated the frequencies of CD11b(+)Gal-9(+)CD107a(+) cells lymph node (1.80±0.3%) compared to the control group (0.20±0.1%) (Fig 14E). The frequencies of CD11c(+)Gal-9(+)CD107a(+) cells were also higher in the Gal-9 treated group (1.2±0.1%) compared to the control group (0.2±0.1%). 9 treatment increased the expression of CD107a(+) in Gal-9(+) macrophages (CD11b) and Gal-Gal-9(+) dendritic cells (CD11c). Furthermore, we applied CD11b(+) or Gr-1(+) staining as a neutrophil marker to lymph node cells and splenocytes for FACS analysis (Fig 14F). The lymph node cells and splenocytes isolated from BD mice were treated with Gal-9 (10 μg/ml) for 16 h. Then, anti-CD11b and anti-Gr-1 staining were performed on Annexin V(+) and PI(-) cells in order to measure the frequencies of apoptotic neutrophils. The frequencies of CD11b(+) cells among apoptotic lymph node cells were 11.5±1.2% in the Gal-9 treated group compared to the control group (8.4±2.4%). In splenocytes, the frequencies of CD11b(+) cells were 44.5±20.7% in the Gal-9 treated group compared to the control group (23.3±0.8%). The frequencies of Gr-1(+) cells among the apoptotic cells were also higher in the Gal-9 treated group (7.7%±2.1%) than in the control group (5.9±0.4%) among lymph node cells. In splenocytes, the frequencies of Gr-1(+) cells among the apoptotic cells were 29.3±22.1% in the Gal-9 treated group compared to the control group (23.1±1.3%) (Fig 14F). The frequencies of Gr-1(+) apoptotic cells were also higher in the Gal-9 treated group than in the control group.
After Gal-9 treatment to BD mice, to determine which cell populations were regulated, additionally, PBMC were isolated from Gal-9-treated BD mice and subjected to transmission electron microscopy (TEM). Gal-9-treated BD mice showed rare neutrophils compared to the control group under TEM (Fig. 14G).
- 45 -
Figure 14. Gal-9 treatment increased the expression of cell death BD mice. A. Annexin V(+) and PI(−
B Frequencies of granzyme B(+) cell
control group. C. CD107a(+) cells were increased in the Gal
control group. (A-C, n=7~9) D–F. Lymph nodes and spleen were isolated from BD mice, and isolated cells were incubated with 10 μg/mL of Gal
Annexin V(+) and PI(−) apoptotic cells were analyzed after staining with anti
Annexin V(+) and PI(−) apoptotic cells were analyzed after staining with anti