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Many human diseases are well known to be caused by uncontrolled immune responses. Although nowadays a lot of drugs have been discovered to suppress the unexpected immunity potently and have been used for the treatments, they are still not as good as we expected. In other words, they would bring some side effects and further worsen a patient’s condition. PTX and TPT, the two drugs we focused here, are used as clinical drugs for a long time. PTX is used for the treatment of peripheral vascular disease; however, it’s immune regulatory functions are being focused in recent years.
TPT is a Chinese herb commonly used to treat some inflammatory diseases and autoimmune diseases despite the exact mechanism are still unclear. In this study, we provided some explanation on their immune effects on CD4 T cells and DCs.
Part I. Pentoxifylline (PTX)
Firstly, we tested the cell toxicity of PTX and found that PTX has very low toxicity to cells. Even under high concentration of this drug, the target cells are still alive.
To explore the effect of PTX on the activation of T cells, we analyzed the effect of this drug on the secretion of IL-2 and cell proliferation. Our results showed that PTX inhibited the IL-2 secretion and proliferation of CD4 T cells upon stimulation. It is well known that IL-2 can induce the expression of CD25, which is a high affinity IL-2 receptor (Depper et al., 1985). We found that when the T cells were cultured with PTX
44
and exogenous IL-2, the up-regulation of CD25 was inhibited. Previous studies showed that the signals delivered by the TCR/CD3 complex and accessory molecules induce the expression of IL-2 receptor and the secretion of IL-2. Furthermore, the signaling through CD25 in response to IL-2 is required for the T cell proliferation (Cantrell and Smith, 1984). Here, we proposed that PTX is a strong inhibitor for CD4 T cell activation.
To further investigate the immunomodulatory function of PTX, we tested the potency of this drug on the induction of regulatory T cells, which have been mentioned to be important in controlling immune responses. However, we found that the expression of Foxp3 was not induced by PTX, and this drug had no synergistic effect in conjunction with TGF-β, a Treg inducing cytokine, on Treg induction. A previous study demonstrated that the mRNA level of Foxp3 in total spleen cells was significantly increased after i.p injection of PTX in to the mice (Aricha et al., 2006). But in our data, we could not see the expression of Foxp3 increased by PTX treatment. It is possible that the inducible effect of PTX on Foxp3 is at the mRNA level, but not at protein level.
Additionally, PTX seemed to inhibit the generation of Treg cells by TGF-β. It has been reported that IL-2 is essential for TGF-β to induce CD25+Foxp3+ regulatory T cells (Zheng et al., 2007). In our data, we can see that PTX could inhibit the CD25 expression on CD4 T cells. Therefore, we propose that PTX might interfere with the
45
generation of Treg cells by TGF-β due to decreasing IL-2 signaling. Apart from Foxp3 expression, IL-10 secretion is one important function of regulatory T cells. So we also detected the IL-10 secretion level from CD4 T cells and found that the IL-10 level was not increased by PTX. Therefore, in our study, we could not identify the ability of PTX on induction of regulatory T cells.
In addition to CD4 T cells, DCs also play an important role in modulating immune responses. The ways of DCs to influence T cells are through the ligation of costimulatory molecules and cytokine secretion. In our data, we found that the expression level of CD86 was significantly increased by PTX, but the levels of MHC class II and CD80 were not affected. On the other hand, PTX increased IL-12 secretion by DCs, and this effect existed in both PTX only or combined with the LPS treatment.
We also identified that the induction of IL-12 secretion of DCs by PTX is through NF-κB pathway, since the induction phenomenon was disappeared when the NF-κB pathway was blocked. It is well known that IL-12 is involved in Th1 differentiation (Jacobson et al., 1996). It is possible that PTX promotes Th1 differentiation by inducing IL-12 secretion from DCs, but this hypothesis requires further examination. However, there is a confliction between our data and previous reports. They showed that in the presence of PTX and LPS, the expression of CD86 on the human DCs were significantly decreased; and moreover, IL-12 secretion from DCs was also inhibited by
46
this drug (Vukanic et al., 2007). The time point of PTX treatment was different between our experimental designs and theirs. They treated DCs with PTX on the day when they isolated CD14+ cells from PBMCs and also maintained these cells in the presence of PTX during the whole culture period. On the contrary, we treated DCs with PTX after six days of culture in the medium containing IL-4 and GM-CSF, therefore, these treated DCs were already differentiated. In the same paper they indicated that PTX impaired the differentiation of human DCs. It is possible that PTX has different effects on DCs at different differentiation stages.
Part II Triptolide (TPT)
TPT is extracted from Tripterygium wilfordii Hook. f, which is a toxic plant. After tested the cell toxicity, we found that the safety dose of this drug is at about ng/ml. This drug has been mentioned to be a special drug with narrow therapeutic window, Therefore, this drug requires more attention on the safety usage.
We explored the effect of TPT on the activation of T cells and analyzed the secretion of IL-2 and cell proliferation. Our data showed that under non-toxic concentration, TPT inhibited CD4 T cell activation including lower IL-2 secretion and cell proliferation. In our study, we could not demonstrate the relationship between its immunosuppressive function of TPT and regulatory T cell induction. We found that the
47
TPT did not induce Foxp3 expression; in addition, it has no synergy between TPT and TGF-β, which has been proved to have the potential for Foxp3 induction on CD4 T cells.
Additionally, we not only detected the molecule specialized for regulatory T cells but also examined whether the TPT was able to induce IL-10-secreting cell, which were one important kind of inducible regulatory T cells. Our result showed that TPT did not induce the IL-10-secreting CD4 T cell production. In this part, we suggested that the immunosuppressive function of TPT on CD4 T cells is to inhibit the activation but not to induce regulatory T cells production.
We further explored the effects of TPT on DCs and analyzed the expression of costimulatory molecules and the cytokine secretion. Our data showed us that in the present of TPT or combined with 100ng/ml of LPS, the expression of MHC class II on DCs was not affected by this drug. Also, the expression levels of costimulatory molecules including CD80 and CD86 were not influenced by TPT treatment either.
Although a published paper suggested that the percentages of CD80 and CD86 expressing cells in total DC population were decreased by TPT under 10ng/ml LPS stimulation (Liu et al., 2007b). There was another paper showed that when DCs were treated with TPT and stimulated by 1μg/ml of LPS, the expression level of CD80 and CD86 were not affected (Liu et al., 2004). LPS can activate DCs through Toll-like receptor 4 and induce costimulatory molecules expression. It is possible that the effects
48
of TPT on costimulatory molecules expression might be diminished under high LPS stimulation.
The cytokine secretion is also an important function of DCs. We showed that TPT significantly inhibited the secretion of IL-12 by DCs, both cultured in drug treatment only or combined with LPS stimulation, and the suppressive effect might through ERK and p38 MAPK pathway. As described before, IL-12 is involved in the differentiation of Th1 cells. In our data, we identified that the effect of TPT on DCs is through the cytokine secretion instead of the expression of costimulatory molecules, which may further regulating the differentiation of CD4 T cells.
Conclusion
PTX and TPT have been widely used in clinical treatment of autoimmune diseases and other immune disorders, but the mechanisms are still unclear. In our study, we showed that PTX is an inhibitor of CD4 T cell activation, and it could promote DCs to secrete IL-12. As for TPT, we identified that this drug could inhibit the activation of CD4 T cells and the secretion of IL-12 by DCs. The different effects of these two drugs on DCs suggested that they can be used to treat different diseases.
49
FIGURES
50
Fig1. Cell toxicity test of PTX
The EL-4 cell line was cultured in the concentration of 2 × 10 5 cells/ml and treated with two-fold serial dilution of PTX and. After 48 hours, cells were counted by Trypan blue. The number of live cells was counted in a minimum of 500 cells per slide. The cell viability was shown in percentage. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test.
* indicated P < 0.05.
51
(A)
(B)
Fig2. The effect of PTX on CD4 T cell activation
CD4 T cells were stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml and cultured with five-fold serial dilution of PTX. After 48 hours, the IL-2 levels in the supernatant were determined by ELISA. After 72 hours, cells were pulsed with 3H-thymidine for 16~18 hours and harvested. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05.
medium cell only BSA10 αCD3/28 2 10 50 250
CPM
αCD3/CD28 2 10 50 250
IL-2 (pg/ml)
52
(A)
(B) (C)
Fig3. The ability of PTX on the induction of CD4+ CD25+ Foxp3+ regulatory T cells CD4 T cells were cultured with IL-2 100U/ml, stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml, and then treated with PTX (250, 25μg/ml) with or without the presence of TGF-β (10ng/ml). After 72 hour of culture, the percentage of CD25+ Foxp3+ T cells was analyzed by flow cytometry.
Control TGF-β
Foxp3
CD25
42.46 2.81
PTX 25 + TGF-β PTX 250 +TGF-β
PTX 25 PTX 250
30.50 12.25
3.36 9.88
53
Fig4. The effect of PTX on the IL-10 secretion of CD4 T cells upon stimulation CD4 T cells were stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml and treated with ten-fold serial dilution of PTX (250, 25μg/ml). After 72 hour of culture, IL-10 levels in the supernatant were determined by ELISA. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’
t-test. * indicated P < 0.05.
0 1000 2000 3000 4000
Control 25 250
IL-10 (pg/ml)
(μg/ml) PTX
*
54
Fig5. The effect of PTX on the cell marker expression of BMDCs
0
Control LPS L-P10 L-P50 L-P250
MFI
Control LPS L-P10 L-P50 L-P250
MFI
Control LPS L-P10 L-P50 L-P250
MFI
*
55
BMDCs were treated with five-fold serial dilution of PTX (250, 50, 10μg/ml) together with or without LPS (100 ng/ml) for 48 hours. The mean fluorescence intensity (MFI) of MHC class II, CD80, CD86 on CD11c+ cells were analyzed by flow cytometry. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05.
56
(A)
(B)
(C)
Fig6. The effect of PTX on the cytokine secretion of BMDCs
0
Control LPS L-P10 L-P50 L-P250
IL-12p40 (pg/ml)
Control LPS L-P10 L-P50 L-P250
IL-12p70 (pg/ml)
Control LPS L-P10 L-P50 L-P250
IL-10 (pg/ml)
57
BMDCs were treated with five-fold serial dilution of PTX (250, 50, 10μg/ml) together with or without LPS (100 ng/ml) for 48 hours. After 48 hours, the IL-12p40, IL-12p70, and IL-10 levels in the supernatant were determined by ELISA. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05. N.D indicated not detectable.
58
Fig7. The effecs of inhibiting the NF-κB, p38 MAPK, ERK1/2, or JNK pathways on the PTX-induced of IL-12p40 up-regulation on BMDCs
BMDCs were pretreated with 0.1% DMSO, 10μM Helenalin (NF-κB inhibitor), 20μM JNK inhibitor II (JNK pathway inhibitor), 50μM PD98059 (ERK pathway inhibitor), or 20μM SB203580 (p38 MAPK inhibitor) for 1 hour, and then incubated in the presence of PTX (250μg/ml). After 24 hours, IL-12p40 levels in the supernatant were determined by ELISA. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P
< 0.05.
0 100 200 300 400
Control 0.1% DMSO PTX 0.1%DMSO Helenalin JNK inhibitor II PD98059 SB203580
IL-12p40 (pg/ml)
PTX
*
*
59
Fig8. Cell toxicity test of TPT
The EL-4 cells were cultured in the concentration of 2 × 10 5 cells/ml and treated with two-fold serial dilution of TPT. After 48 hours, cells were counted by trypan blue. The number of live cells was counted in a minimum of 500 cells per slide.
The cell viability was shown in percentage and expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05.
0%
20%
40%
60%
80%
100%
0 156.25 312.5 625 1250 2500 5000 10000 20000
viability
TPT concentration (pg/ml)
*
*
*
60
(A)
(B)
Fig9. The effect of TPT on CD4 T cell activation
CD4 T cells were stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml and cultured with five-fold serial dilution of TPT. After 48 hours, the IL-2 levels in the supernatant were determined by ELISA. After 72 hours, cells were pulsed with 3H-thymidine for 16~18 hours. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05.
0
medium cell only BSA10 αCD3/28 20 100 500 2500
CPM
αCD3/CD28 20 100 500 2500
IL-2 (pg/ml)
61
(A)
(B) (C)
Fig10. The ability of TPT on the induction of CD4+ CD25+ Foxp3+ regulatory T cells
CD4 T cells were cultured with IL-2 100U/ml and stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml, and then treated with TPT (2500, 250pg/ml) in combination with or without TGF-β (10ng/ml). After 72 hour of culture, the percentage of CD25+ Foxp3+ T cells was analyzed by flow cytometry.
TGF-β Control
CD25
Focp3
42.46 2.81
TPT250 + TGF-β TPT2500 + TGF-β
TPT2500 TPT250
37.13 49.31
5.99 3.54
62
Fig11. The effect of TPT on the IL-10 secretion of CD4 T cell upon stimulation CD4 T cell were stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml and treated with ten-fold serial dilution of TPT (2500, 250pg/ml). After 72 hour of culture, the IL-10 levels in the supernatant were determined by ELISA. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05.
0 1000 2000 3000
Control 250 2500
IL-10 (pg/ml)
(pg/ml) TPT
63
(A) MHC class II
(B) CD80
(C) CD86
Fig12. The effect of TPT on the cell marker expression of BMDCs
0
Control LPS L-T100 L-T500 L-T2500
MFI
Control LPS L-T100 L-T500 L-T2500
MFI
Control LPS L-T100 L-T500 L-T2500
MFI
64
BMDCs were treated with five-fold serial dilution of TPT (2500, 500, 100pg/ml) in combination with or without LPS (100 ng/ml) for 48 hours. The mean fluorescence intensity (MFI) of MHC class II, CD80, and CD86 on CD11c+ cells were analyzed by flow cytometry. The results were expressed as the mean ± SEM (N=3) The P value was analyzed by Students’ t-test. * indicated P < 0.05.
65
(A)
(B)
(C)
Fig13. The effect of TPT on the cytokine secretion of BMDCs
0
Control LPS L-T100 L-T500 L-T2500
IL-12p40 (pg/ml)
0 500 1000 1500
Control T100 T500 T2500
IL-12p70 (pg/ml)
0 100 200 300
Control LPS L-T100 L-T500 L-T2500
IL-12p70 (pg/ml)
Control LPS L-T100 L-T500 L-T2500
IL-10 (pg/ml)
66
BMDCs were treated with five-fold serial dilution of TPT (2500, 500, 100pg/ml) in combination with or without LPS (100 ng/ml) for 48 hours. After 48 hours, the IL-12p40, IL-12p70, and IL-10 levels in the supernatant were determined by ELISA. The results were expressed as the mean ± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P < 0.05. N.D indicated not detectable.
67
Fig14. The effect of inhibiting the NF-κB, p38 MAPK, ERK1/2, or JNK pathways on the TPT-suppressed the IL-12p40 production on BMDCs BMDCs were pretreated with 0.1% DMSO, 10μM Helenalin (NF-κB inhibitor), 20μM JNK inhibitor II (JNK pathway inhibitor), 50μM PD98059 (ERK pathway inhibitor), or 20μM SB203580 (p38 MAPK inhibitor) for 1 hour, and then incubated with TPT (2500pg/ml). After 24 hours, the IL-12p40 levels in the supernatant were determined by ELISA. The results were expressed as the mean
± SEM (N=3). The P value was analyzed by Students’ t-test. * indicated P <
0.05.
0 10 20 30 40 50 60
Control 0.1%DMSO TPT 0.1%DMSO Helenalin JNK inhibitor II PD98059 SB203580
IL-12p40 (pg/ml)
*
*
TPT
68
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