Study On Porcine Alveolar Macrophages Removing Opsonized GFP-E.coli From Erythrocytes

OBJECTIVE:To investigate CR1-like(like complement receptor type 1,CR1)by porcine alveolar macrophages(PAMs)for the clearance of porcine erythrocyte immune-adhering onopsonized genetically engineered bacteria(GFP-E.coli).CR1-like(like complement receptor type 1),CR1-like,Fc R(Fc receptor,Fc R)the biological role of PAMs in the removal of onopsonized immune complexes from porcine erythrocytes.Methods: 1)The porcine erythrocytes of the immune-adhering onopsonized GFP-E.coli were flow-passed through the flow chambers covered with PAMs,and the peristaltic pump was determined according to the shear force requirements of the speed of circulation system to maintain stability during the cycle.2)Flow cytometry was used to detect changes the surface fluorescence intensity of porcine erythrocytes and PAMs,and the efficiency of PAMs removal of onopsonized GFP-E.coli was calculated.3)CR1-like,Fc R-mediated PAMs removal of onopsonized GFP-E.coli in vitro: The CR1-like surface of PAMs was isolated and identified by staining immunocytochemistryt echnique,immunoprecipitation technique and Western-blot technique.PAMs were incubated with CR1-like Mc Ab and Fc R Mc Ab alone and co-incubated with immune-adhering onopsonized GFP-E.coli The porcine erythrocytes interaction was used to detect the changes of surface fluorescence intensity of porcine erythrocytes and PAMs by flow cytometry.The mediation of CR1-like and Fc R on PAMs clearance onopsonized immune complexes was studied.RESULTS: 1)The flow rate of the flow cell peristaltic pump was 4 rpm to meet the test requirements of the cycle detection system;2)By flow cytometry,there was no significant difference in the average fluorescence intensity of porcine erythrocytes within 60 min and the system was stable.3)The fluorescence intensity of the porcine erythrocytes immune-adhering onopsonized GFP-E.coli after interaction withPAMs,the reduction rate was 8.0%.The fluorescence intensity of PAM surface increased 6.5%.It can be seen that onopsonized GFP-E.coli is transferred from the surface of porcine erythrocytes to the surface of PAMs;4)On the surface of PAMs has positive yellow fluorescence after incubation with CR1-like Mc Ab;magnetic beads pair coated with CR1-like Mc Ab the total protein of PAMs membrane was immunoprecipitated,and the precipitated protein showed a positive protein band at 55 k Da by reducing electrophoresis.5)Loss of onopsonized GFP-E.coli on porcine erythrocyte surface after treatment of PAMs by CR1-like Mc Ab,The rate decreased;meanwhile,the growth rate of PAMs combined with GFP-E.coli decreased.After Fc R Mc Ab single-blocking treatment of PAMs,the loss rate of porcine erythrocyte surface onopsonized GFP-E.coli decreased;meanwhile,the growth rate of PAMs combined with GFP-E.coli was also decreased.When PAMs was blocked by CR1-like Mc Ab and Fc R Mc Ab,the loss rate of erythrocyte surface onopsonized GFP-E.coli decreased;Meanwhile,the growth rate of PAMs combined with GFP-E.coli decreased.Conclusion: In this study,we found that CR1-like molecules are distributed on the surface of PAMs.Under the circulatory flow detection system,it is found that under the Fc R and CR1-like mediate,PAMs can remove immune complexes of porcine erythrocyte immune adhesion;these are identified the porcine erythrocyte and PAMs interaction in the process of removing IC in the body.