The Hybrid Molecule CL-SP-D Has the Potential to Regulate Xenogeneic Rejection by Human Neutrophils

Objective: In this study, we studied the effect of expression of human CD47 and CL-SP-D on swine endothelial cells (SECs) for the inhibition of xenogeneic rejection by neutrophils. Methods: The cDNA for the carbohydrate recognition domain (CRD) of human surfactant protein D (SP-D) was swiched to that of a membrane-type protein, collectin placenta 1 (CL-P1). The cDNA of CD47 was also prepared as a control. We first examined the expression of SIRPα on HL-60, a neutrophil-like cell line, and on neutrophils isolated from periferal blood. The hybrid molecule, CL-SP-D-expressing SECs or CD47-expressing SECs were prepared by transfection of the plasmids containing the genes for human CL-SP-D or CD47 into SECs by electroporation. The suppressive function of the CL-SP-D to neutrophil was then studied and the results compared with that for CD47. Results: The expression of SIRPα on HL-60 and neutrophils from peroferal blood was confirmed. The CL-SP-D-expressing SECs or CD47-expressing SECs were co-cultured with HL-60 or neutrophils using naive SECs as controls. After the co-culture, the degree of cytotoxicity was calculated by WST-8 assay, and the ROS assay was next performed. CL-SP-D significantly suppressed the cytotoxicity of HL-60 and neutrophils, while CD47 showed a suppressive effect on neutrophil from periferal blood but HL-60. Corroborating the results of the cytotoxicity, the results of ROS assay indicated significant downregulation on SEC with CL-SP-D. In addition, IL-10 was also upregulated in the SEC/CL-SP-D. Conclusion: These results indicate that CL-SP-D is very effective on neutrophils in xenogeneic rejection. Moreover, CL-SP-D was significantly more effective than CD47 as a molecule to inhibit the neutrophil-mediated xenogeneic rejection. References: 1. Patmika Jiaravuthisan, Akira Maeda, Chihiro Takakura, Han-Tang Wang, Rieko Sakai, Afifah Mohd Shabri, Pei-Chi Lo, Rei Matsuura, Tasuku Kodama, Hiroshi Eguchi, Hiroomi Okuyama, Shuji Miyagawa A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells. Transplant Immunol 2018; 47:44-48. doi: 10.1016/j.trim.2018.02.003.