Effect of interleukin-10 on human anti-porcine xenogeneic cellular response in vitro

Pigs are being used as an alternative source of tissues for humans and we are interested in the xenotransplantation of fetal pig islet-like cell clusters (ICC) into type 1 diabetic patients. Interleukin-(IL) 10 is a Th2 cytokine with immunosuppressive properties that down-regulate the cell-mediated response. In this study, we evaluated the effects of recombinant human IL-10 on human anti-pig xenogeneic cellular response in mixed lymphocyte culture (MLC) and in mixed islet lymphocyte culture (MILC).

Human peripheral blood mononuclear cells as responder cells were cultured in one-way MLC with pig and human peripheral blood mononuclear cells as stimulant cells in xeno and allo-MLC, respectively, and also with fetal pig ICCs in MILC. IL-10 was added at the time of culture.

The addition of IL-10 significantly inhibited the xeno-MLC (human anti-pig) in a dose-dependent manner, the percentage inhibition being 36, 60, and 73% at 1, 10, and 50 ng/ml, respectively. Inhibition in xeno-MLC was significantly lower than that of the allo-MLC (human anti-human) at all concentrations used, the percentage inhibition of the latter being 58, 84, and 92% at 1, 10, and 50 ng/ml, respectively. Further, the addition of IL-10 also significantly inhibited the proliferation of human peripheral blood mononuclear cells when they were cocultured with fetal pig ICCs, the inhibition being 59, 72, and 80% at 1, 10, and 50 ng/ml, respectively. IL-10 was not toxic to ICCs as determined by 3H-thymidine incorporation over 5 days culture. Preincubation of IL-10 with the pig stimulant cells or the human responder cells did not confer additional benefit in the inhibition of xeno-MLC. IL-10 needs to be present at the start or at an early stage (within 4 hr) in the xeno-MLC because if the addition of IL-10 was delayed by 4 hr, the effect was lost. Next, the production of cytokines was examined in MLC and MILC. In xeno-MLC, levels (pg/ml) of tumor necrosis factor-alpha (TNF-alpha) (163+/-17), interferon-gamma (IFN-gamma) (278+/-60), IL-5 (24+/-10), IL-6 (2959+/-923), and IL-10 (17+/-2) were produced in greater amounts than autologous controls (P<0.05). The levels of TNF-alpha, IFN-gamma, IL-6, and IL-10 but not IL-5 were significantly (P<0.05) lower in xeno-MLC than those produced in allo-MLC. All of these cytokines were also produced in MILC when human peripheral blood mononuclear cells (PBMC) were cocultured with ICCs, levels (pg/ml) being TNF-alpha (308+/-47), IFN-gamma (93+/-17), IL-5 (6.2+/-3), IL-6 (5649+/-421), and IL-10 (122+/-18). No detectable levels of IL-2 and IL-4 were produced in the MLC and in MILC. Addition of IL-10 significantly inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 76, 96, 100, and 93%, respectively, in xeno-MLC. Addition of IL-10 also significantly (P<0.05) inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 88, 91, 100, and 96%, respectively, in MILC. Exogenous addition of IL-2 was partially able to reverse the effect of IL-10 although addition of TNF-alpha had no effect on xeno and allo-MLC. Synergism was seen between IL-10 and cyclosporine in the inhibition of xeno and allo-MLC.

Taken together, the results demonstrated that IL-10 has an immunomodulatory role to play in the inhibition of cellular immune responses associated with the xenotransplantation of fetal pig ICCs.