DING, Guo-ping; CAO, Li-ping; LIU, Jie; LIU, Da-ren; QUE, Ri-sheng; ZHU, Lin-hua; ZHOU, Yi-ming; MAO, Ke-jie; HU, Jun-an

Since the invention of immunosuppressant from the 1980's, considerable improvements of long- and short-term outcomes of grafts in clinical transplantations have been obtained. However, currently available immunosuppressants possess lots of inborn disadvantages such as higher incidence of opportunistic infection, tumor initiation and other metabolic complications. Besides, the application of these drugs mainly relies on empirical usage, and precise immunologic monitoring to determine the appropriate dose of drugs is not yet available. Thus, various methods were investigated in order to reduce or even replace the usage of existing immunosuppressant, such as transplantation of microencapsulated cells¹ or tissues and combined usage of antibodies.² The application of MHC-II antibodies was also involved. Donor-located “Passenger” leukocytes especially antigen presenting cells (APCs) express high level MHC-II and co-stimulated molecules which could bind T cell receptor (TCR) on the surface of recipient T cells as alloantigen and lead to the beginning of acute rejection through direct pathway. Thus the blockage of MHC-II on donor APCs helps the long-term acceptance of the graft. However, previous studies showed that anti MHC-II monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The reason is considered the MHC-II MAb could only block the mature DCs (mDCs) which expressed more MHC-II and co-stimulated molecules on cell membrane but not immature DCs (imDCs) which could turn to mDCs gradually in certain conditions.

The present study investigated a new way for thorough down regulation of donor MHC II and type 1 cytokines produced by porcine peripheral blood mononuclear cells (PBMCs) in vitro. ¹⁸⁸Re, a new isotope belongs to group 7 (VIIB) family in periodic chart, has advantages of shorter demiperiod, more suitable radiation range and rapid metabolism³ over isotope ⁹⁹Tc, which is also from the same family and has been extensively used in clinic. ¹⁸⁸Re-radiopharmaceuticals have already been used for diagnosis and treatment of various tumors such as liver cancer and melanoma,^4-7 coronary artery restenosis,⁸ rheumatoid arthritis⁹ and so on. In this study, the radioactive function of isotope ¹⁸⁸Re was incorporated with donor MHC class II specific antibody and was expected to exert relative long-lasting radioactivity to block the donor DCs, both mature and immature. The PBMCs tested were either unstimulated or stimulated with donor alloantigen incorporated with MHC-II MAb or ¹⁸⁸Re marked MHC-II MAb (MAb-¹⁸⁸Re). The findings were prospectively correlated with the immune tolerance status in allograft transplantation.

METHODS

Cell culture

Blood samples were obtained from healthy, conventionally reared, 2-4 month old fragrant pigs. Pigs were anaesthetized with Nembutal (Gibco BRL, Life Technologies, USA) and 40 ml of fresh blood was aseptically collected directly from the trachelo-peripheral vein into tubes containing Alsever's solution (50% v/v) (Gibco). PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque. The PBMCs from each animal were suspended at a concentration of 1×10⁷ cells/ml in RPMI-1640 with 10% fetal bovine serum (FBS), 1 mmol/L sodium pyruvate (Gibco), 2 mmol/L L-glutamine (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin and 5×10^-5 mol/L 2-mercapto-ethanol. Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂.

Monoclone antibodies and isotope ¹⁸⁸Re

2E9/13F(ab')₂ which is a fragment hydrolyzed from 2E9/13 monoclone antibody (a murine immuno-globulin G2b antibody against SLA-II DR/DQ antigen complex) by proteolytic enzyme, was used in this experiment. Isotope ¹⁸⁸Re, gain from ¹⁸⁸W-¹⁸⁸Re deviser, was incorporated with 2E9/13F(ab')₂ in isotope laboratory via a series of reaction and got ¹⁸⁸Re-2E9/13F(ab')₂.

MLR

Allogenic stimulator cells were treated with mitomycin-C and incubated with MHC-II MAb (group 2), MAb-¹⁸⁸Re (group 3) and culture medium (group 4). After an incubation in 5% CO₂ at 37°C for one hour, equal numbers (5×10⁵) of stimulator and responder cells from each group were suspended in RPMI-1640 and added in quadruplicate in a 96-well round bottom plate and incubated for six days. The pH of the MAb was adjusted to 7.4 with 1 N sodium hydroxide prior to performance of all experiments. Four hours before termination, the cultures were pulsed with 74 kBq/well [3H]-TdR (ICN Canada Ltd., Montreal, Quebec, Canada).

At the end of culture, 4°C cold normal sodium was added to stop the infiltration. Cells were harvested and the thymidine uptake was measured by liquid scintillation counting. Controls included responder cells only (group 1) and results were expressed as the mean±SD. The stimulation index (SI) was calculated as: SI = (cpm responder cells + cpm control)/(cpm control).

Immunostaining

Porcine PBMCs were incubated with MHC-II MAb at 37°C in 5% CO₂ for half an hour; a FITC goat anti mouse antibody was employed as second antibody. After washing, cells were observed by fluorescence microscope to confirm the incorporation of MHC-II MAb on cell membrane.

Reverse transcription-polymerase chain reaction (RT-PCR)

Cells were harvested at 24 hours or 48 hours for total RNA extraction, RNAgents^®, total RNA isolation system (Promega, USA) were used following the manufacturer's instructions. The oligonucleotide primers for porcine interleukin 2 (IL-2), IL-10, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) were designed from the published nucleic acid sequences available from GenBank/EMBL databases (Table 1). β-actin was used as a control. The conditions used in RT-PCR consisted of denaturing at 94°C for 45 seconds, annealing for 60 seconds and extension at 72°C for 45 seconds with a final extension at 72°C for 10 minutes. The annealing temperature and the numbers of cycles used were shown in Table 2. Gels were scanned using the ImageMaster VDS (GE Healthcare, USA) and the density of bands was assessed using the ImageMaster 1D prime software.

Table 1

Table 2

Statistical analysis

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.

RESULTS

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).

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

DISCUSSION

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.

REFERENCES