The hybrid molecule CL-SP-D has the potential to regulate xenogeneic rejection by human neutrophils
Shuji Miyagawa1, Keigo Iemitsu1, Akira Maeda1, Rieko Sakai1, Daiki Yasufuku1, Makiko Tani1, Fitri Hasnaulia 1, Katarzyna Gadomska1, Kazunori Masahata1, Masafumi Kamiyama1, Hiroshi Eguchi1, Hiroshi Nagashima1, Hiroomi Okuyama1.
1Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Japan
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.
[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.
