Crucial Role Of Dendritic Cells In The Neuro-immune Cross-talk Mechanism

Objective: To investigate the'handwired'neural pathways of the neuro-immune cross-talk mechanism.Methods:FR antergrade tracingExperimental group: 3 SD rats, male and weight 260g±20g. After anesthesia, rats were cut into a 3 cm long incision in the median line of cervical part in supine position. Rats was slowly injected 1μl 3%FR and retained for 15 min into right superior cervical ganglion exposed in the crotch of internal carotid artery and external carotid artery. Samples were collected after rats which survived nine days from operation had perfusion with formalin.Control group: 2 SD rats, male and weight 260g±20g. After superior cervical ganglion was resected, 1μl of 3% FR was injected into the point of sympathectomy. Samples were collected after rats which survived nine days from operation had perfusion with formalin.FR labeling and plasma membrane double-labelingSections from antrogradely FR‐labeled were rinsed three times in buffer, and then incubated in the DiOC18(3) diluted in DMSO(1:80) for 30 min. After rinsed thoroughly in buffer, counterstain with DAPI for 30 min and rinsed three times in buffer. Sections were mounted and photographed under a confocal microscope.FR labeling and Immunocytochemical double-labelingSections from antrogradely FR-labeled were rinsed thoroughly in buffer, and incubated at room temperature for 30 min in 10% normal goat serum and then incubated at 4°C overnight with primary antibody against CD3, CD14, CD22, S100, NPY and VIP diluted in 0.01M PBS (1:500). On the next day, sections were rinsed three times in buffer, and then incubated in the secondary antibody diluted in buffer (1:100) for 30 min. Then sections were rinsed three times in buffer, and incubated SABC-FITC diluted in buffer (1:100) for 30 min. After rinsed thoroughly in buffer, counterstain with DAPI for 30 min and rinsed three times in buffer, sections were mounted and photographed under a confocal microscope .Results:1. FR antergrade tracing of superior cervical ganglionAfter injecting Fluoro-Ruby into the superior cervical ganglion labeled anterogradely, sympathetic nerve endings labeled by FR were found in the cervical lymph node. The result showed sympathetic nerve endings from superior cervical ganglion were labeled by FR, but sympathetic nerve endings labeled by FR only located surrounding some nuclei of cells. It reaveled cells around by sympathetic nerve endings paly a crucial role in the neuro-immune cross-talk.2. The resulet of FR labeling and plasma membrane double-labelingIt is known that sympathetic nerve endings labeled by FR only located surrounding some cells. For defining the relative positions between these cells and sympathetic nerve endings labeled by FR, we used DiOC18(3) to stain membrane. Then we observed that sympathetic nerve endings labeled by Fluoro-Ruby was located at cytomembrane.3. The resulet of FR labeling and immunocytochemical double-labelingIn order to definine the kind of cells around by the labeled sympathetic nerve endings, double immunofluorescence method was adopted, we used separately specific antibody against T cells(CD3), B cells(CD22), macrophages(CD22) and dedritic cells(S100) . The results showed that these cells around by sympathetic nerve endings labeled by FR expressed S100 and not expressed CD3, CD14 and CD22. It revealed sympathetic nerve endings labeled by FR only innervated dedritic cells but not T cells, B cells, macrophages. In addition, nerve fiber types such as VIP and NPY were co-expressed in sympathetic nerve endings labeled by FR.Conclusion:1. In this study, we provide compelling morphological evidence that sympathetic nerve endings only innervated dendritic cells (DCs) in lymph organs a and it did not innervate directly other lympocytes (such as T cells, B cells, macrophages). Therefore, it is suggested that DCs may be the neuro-immune cross-talker in lymph organs and has implications in the neuro-immune cross-talk mechanism. 2. Our experimentis also confirmed that sympathetic nerve endings labeled by FR in lymph nodes co‐expressed VIP and NPY neuropeptides. They may play a key role in neuro‐immune regulation.3. Dedritic cell in lymphoid organs is impotant for the neuro‐immune path. It may be a neuro‐immune cross‐talk, but the role of dedritic cell in neuro‐immune regulation is need to further explored.