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.