Objective: Macrophages have been suggested to be involved in xenograft rejection, but the effects of immunosuppressants at different stages of macrophage differentiation have not been fully elucidated. In this study, we investigated the suppressive effect of mycophenolic acid (MPA), which mainly targets lymphocytes, on the cytotoxic activity of human macrophages against swine endothelial cells (SECs).
Method: CD14-positive monocytes were isolated from peripheral blood mononuclear cells of healthy donors using magnetic activated cell sorting (MACS). The isolated monocytes were cultured with 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days to induce differentiation into macrophages. Macrophages were then co-cultured with SECs in the presence of MPA at concentrations of 2.5 µg/mL or 5.0 µg/mL to evaluate its effect on cytotoxicity against SECs. 
To assess the effect of MPA on the differentiation of monocytes into macrophages, 2.5 µg/mL MPA was added simultaneously with GM-CSF and its effect on cytotoxic activity was evaluated. Cytotoxicity was measured using a water-soluble tetrazolium salt-8 (WST-8) assay. In addition, flow cytometry was used to assess changes in the expression of immunoglobulin-like transcript 4 (ILT4) and CD14, as well as cell viability.
Results: Addition of MPA after macrophage differentiation significantly reduced cytotoxicity compared to the control group, although a dose-dependent reduction was not observed (control 82.9%, MPA 2.5 µg/mL group 60.3%, MPA 5.0 µg/mL group 55.7%; control group vs. MPA 2.5 µg/mL, p=0.02). Next, we examined the effect of MPA on macrophage differentiation. When MPA was added concurrently with GM-CSF and cultured for 7 days, cytotoxicity was markedly suppressed (control 94.2%, MPA 2.5 µg/mL 0.2%; p<0.001). Flow cytometry analysis revealed that the expression of ILT4 and CD14 in the MPA-treated group gradually decreased over time compared to the control group, and that these ILT4/CD14-negative cells were not detectable, suggesting loss of cell viability during the differentiation process.
Conclusion: While MPA exhibited a mild inhibitory effect on cytotoxicity in fully differentiated macrophages with GM-CSF, it demonstrated a strong suppressive effect on cytotoxicity by inducing cell death during the macrophage differentiation stage.