Data were presented as mean±SD. One way analysis of variance (ANOVA) was conducted on every time point to compare differences between experimental groups. A chi-square analysis was conducted using SPSS 10.0 (SPSS Inc., IL, USA) to investigate the relationship between a dichotomous variable and experimental groups. P <0.05 was considered statistically significant.
Proliferative response of recipient PBMCs to donor-specific stimulation
Proliferative responses of recipient PBMCs in MLR to donor alloantigens incubated with MHC-II MAb, MAb-188Re, or medium were examined. It was revealed that 10 μg/ml MHC-II MAb treated group had the lowest SI (Figure 1A), and was considered to be the best inhibiting concentration. However, 10 μg/ml MAb-188Re treated group only had half the SI (3.08±1.64) as compared to the MHC-II MAb group (6.64±1.28, P <0.05) and were much lower than the index of positive group (20.36±5.21, P <0.01), which confirmed MAb-188Re treated group had more thorough inhibiting effect of recipient PBMCs in MLR (Figure 1B).
Morphology and immunostaining
The morphology of cells was shown in Figure 2. The normal dormant lymphocytes had small round volume and nucleus, diaphanous and meager cytoplasm (Figure 2A). When activated, they turned into double or triple volume, copious cancellous or impellucidus cytoplasm and trachychromatic versiform nucleus (Figure 2B). In MHC-II MAb group (Figure 2C), partial activated lymphocytes could be found, while in MAb-188Re group (Figure 2D), characteristic activated lymphocytes could not be found, the majority of cells maintained inactivated phase and some presented signs of apoptosis.
Fluorescence was detected after incubation with MHC-II MAb and fluorescein isothiocyanate (FITC) goat anti mouse antibody (Figure 3), which indicated that MHC-II MAb could effectively block the recipient's high MHC-II expression cells, especially the APCs.
PBMC cytokine mRNA expression in response to donor alloantigen stimulation
The cytokine mRNA expression by recipient PBMCs in response to donor alloantigen stimulation was analyzed (Figure 4). All the cytokines produced in MHC-II MAb and MAb-188Re group were significantly decreased as compared to the untreated group.
In the first 24 hours, the lymphocyte production of IL-2, IFN-γ and TNF-α (type 1 cytokines) was significantly lower in MAb-188Re group than the MHC-II MAb group; in contrast, the production of IL-10 (type 2 cytokines) was higher in MAb-188Re group than the MHC-II MAb group (Figure 5). At 48 hours point, the production levels of IL-2 and IFN-γ were also significantly lower in MAb-188Re group as compared to the MHC-II MAb group, but no difference was noted in the TNF-α level. Besides, the production of IL-10 was no longer elevated in MAb-188Re group, but decreased to a level lower than the MHC-II MAb group.
The results in this study demonstrated that isotope 188Re marked MHC-II antibody could help the graft acceptance and had advantages over normal MHC-II antibody by more thorough inhibiting of T cell proliferation, decreasing type 1 cytokines expression, and elevating type 2 cytokines. Although up to now, the presence or absence of graft rejection is determined by the biopsy and histology, serum cytokines still play an important role in the acute rejection. Their interactions construct a complicated cytokine network which appears to be crucial in directing host immune reactions toward allograft rejection or graft acceptance.10-12 It was revealed that type 1 or type 2 cytokine-producing lymphocyte populations can be elicited by donor antigen stimulation, and that these patterns correlate with the allograft acceptance.13-15 Though the paradigm remains controversial, it was supported by more and more studies which revealed that a predominant type 2 cytokine response (IL-4, IL-10) exists in these orthotopic liver transplant recipients with immunologically stable liver grafts. Significant increase of type 1 cytokines (IL-2, IFN-γ, and TNF-α) mostly exists in immunologically unstable grafts whose acute rejection developed within short interval.16 It was also revealed that IL-2 or IFN-γ downregulation would inhibit the occurrence and advancement of acute rejection, such as neonatal tolerance,17 total lymphoid irradiation- induced tolerance,18 blockade of co-stimulatory pathway,19 or graft acceptance induced by donor-specific transfusion,20 the activation-induced elimination or anergy by anti-CD3 treatment,21 and neutralization by IFN-specific natural antibodies in intravenous immunoglobulins.22
In this study, IL-2, IL-10, IFN-γ, and TNF-α produced by PBMCs were all detected significantly lower in MHC-II MAb group than the untreated group. Consistent with the research above, it confirmed the effect of MHC-II MAb in blocking MHC-II antigen on donor APCs and therefore promoted the reduction of the overall cytokine production. Whereas, in the first 24 hours, type 1 cytokines (IL-2, IFN-γ, and TNF-α) in MAb-188Re group were found significantly lower than the normal MHC-II MAb group, while type 2 cytokine IL-10 was higher expressed. After the first 24 hours, the expression level of IL-10 had no difference between the two groups. Thus, we assume that MAb-188Re could impair the type 1 cytokine-producing lymphocyte proliferation and consequently hampered the acute alloantigen stimulation as compared to MHC-II MAb. But the mechanism could not be explained by the radioactivity of isotope 188Re alone and needs further study. Besides, the time course of cytokine expression following lymph node stimulation could be varied by using different stimulus (mitogens or antigens), and the time points of porcine cytokines responses could peaked very early (0-24 hours) or very late (more than 72 hours).23 But in this research, the differential expression of cytokines between MAb-188Re and MHC-II MAb group were only evaluated within 48 hours, and the responses over 48 hours still needs to be evaluated.
Furthermore, we found the overall lymphocyte proliferation was dramatically inhibited in MAb-188Re group, which was only half of the MHC-II MAb group and one seventh of the untreated group. It was presumed that though MHC-II MAb had partial inhibiting effect, the function was considered non-persistent, because only mature DCs (mDCs) which express high levels of MHC-II on cell membrane could be blocked by MHC-II MAb but not immature DCs (imDCs). Lack of co-stimulated molecules such as CD86, intercellular adhesion molecule-1 (ICAM-1), lymphocyte function- associated antigen 1 (LFA-1), imDCs could induce the recipient alloreactive T cells to devitalize when they were connected. In certain conditions, imDCs could turn to mDCs and express MHC-II as well and it could no longer be blocked by MHC-II MAb.24 MAb-188Re had advantages of more thorough proliferation inhibition over normal MHC-II MAb due to its suitable radiation range and long-lasting radioactivity.
In summary, we introduced in this study isotope 188Re marked MHC-II antibody as a new way which could effectively inhibit the lymphocyte proliferation and benefit the allograft acceptance as compared to normal MHC-II antibody. The promising results above provided another way to reduce the dose and time of immunosuppressant usage in the early period after organ transplantation in the future.
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Keywords:© 2011 Chinese Medical Association
MHC class II antibody; dendritic cells; cytokine; 188Re; acute rejection