The Architecture Of Blood-spleen Barrier And The Mechanism Of Lymphocyte Homing In The Chicken Spleen

The spleen is the specialized peripheral lymphoid organs which can capture the blood-borne antigens to induce immune response. The white and red pulp compartments of the spleen can effectively remove the aging red blood cells and the circulating antigen. As the largest lymphoid organ, the spleen contains abundant immunocompetent cells- T and B lymphocytes, which can directly mediated the cellular and humoral immunity protecting the body against bacterial and viral infections.The structure and composition of the avian spleen is special, which is different from that mammals, but is similar to that of the reptiles. In avian spleen, there is absent from the marginal zone. The area of PALS of white pulp decreased, but it increased the PELS area. T lymphocytes are mainly distributed in the PALS, while B lymphocytes mainly distributed in the PELS. Although the study on the morphology and function of the spleen in mammals has been described, the detailed immune function and its immunological significance of the spleen in poultry have yet to be revealed. So far, the report on the BSB has been limited to mammals and reptiles. BSB is a new concept of the immunology barrier, which is closely related to the maintenance of the immune function of the spleen. Since the lack of the marginal zone, whether the BSB existed in the chicken spleen and what is the structural composition of the BSB? There has been reported the high endothelial cells in the reptiles spleen, which is the site of the lymphocyte homing. Unlike mammals, whether the high endothelial cells of lymphocyte homing existed in the chicken spleen? In view of these problems, our study first identified the location and structural composition of the BSB in chickens; investigated the chicken BSB during postnatal development. The endothelial cells of sheathed capillary were revealed as the site of lymphocyte homing. And also we further investigate the mechanism of the molecular basis of lymphocytes homing in the chicken spleen. These researches demonstrates the immune mechanism and immunological characteristics of the chicken spleen in cell and molecular level, which would contribute to enrich the content of comparative immunology and provide a scientific basis for the pathogenesis and treatment of avian infectious diseases.Experiment I Identification and structural composition of the blood-spleen barrier in chickens. To identify the existence and composition of the blood-spleen barrier(BSB) in chickens, the microanatomical features of the spleen were investigated by light microscopy, transmission electron microscopy and intravenous injection of ink. The results showed that the white pulp in chicken spleen consists of periarteriolar lymphatic sheaths(PALS), periellipsoidal lymphatic sheaths (PELS) and lymphoid nodules. There was no evidence for the presence of a marginal zone. The endothelial cells of the sheathed capillary is high and cuboidal in appearance. The sheathed capillary continued as the sinus of the red pulp. It suggested the closed circulation of the chicken spleen. After 30min, 1h, 6h, 12h,24h, 2d, 3d, 5d, 7d of ink injection, uptake of carbon particles was limited to the ellipsoid and PELS without in the red pulp and lymph nodule. Carbon particles were strictly resisted in the ellipsoid and PELS within 2d. With the time course, carbon particles diffused to the PALS and lymph nodule 7d after ink injection. BSB functioned as a mechanical and biological barrier, which prevented and phagocytized the carbon particles. Carbon particles migrated to PALS and lymph nodules, playing an important role in adaptive immune response. Silver impregnation revealed the reticular fibres of the supporting framework of the BSB. Two layers reticular fibres arranged on the ellipsoid as a concentric circle.Ellipsoid-associated cells (EACs), reticular cells and macrophages were acid phosphatase positive. The immunohistochemistry results showed MHC II expressed on the EACs, which located in the outside of the ellipsoid of BSB. These findings suggest that BSB of chickens is present in the ellipsoid and PELS. The BSB was a reticular framework, between the arterial and venous vessels, which included cuboidal-shaped endothelial cells, supporting cells, EACs, macrophages, reticular cells and fibres.Experiment II Study on the blood-spleen barrier in the chicken spleen during postnatal development. As the primary site for immunocyte proliferation and differentiation, the spleen is the largest secondary lymphoid organ in chickens. This study identified the location of the resisted carbon particle in different ages 1, 7,14, 21,35 and 60 days after hatched by intravenous injection of ink. The immunohistochemistry of CD3,Bu-1 and MHC ? mAbs were used to detected the distributions of T, B lymphocytes and antigen presenting cells in different ages of chicken spleen. Immune-related gene expressions of TLRs (TLR2 and TLR4) and cytokines (IL-2, IFN-y and TNF-a) were investigated in different ages of chicken spleen. Results showed that BSB formed one day after hatched, but there is also carbon unresisted in the red pulp. With the increase of age,barrier function gradually improved from 14 days to the adult chicken. The carbon particles was limited in the ellipsoid and PELS of BSB. CD3+T cells distributed in the PALS and red pulp. With the growth of age, the T cells of PALS and red pulp has increased. Bu+ B cells distributed in the PELS and lymph nodule. With the growth of age, the B cells of PELS and lymph nodule has increased. MHC 11+ antigen presenting cells mainly distributed in the spleen and red pulp surrounding the ellipsoid and red pulp, and the increasing trend was not significant from one day old to adulthood after hatching. In gene level, TLR2, TLR4, IL-2,IFN-y and TNF-a mRNA expressed higher in the beginning after hatched, while decreased two weeks later. The results suggested that the development of the chicken BSB did not reach the maturity level at the beginning after hatched. During the age of 1 to 14 days old,the resistance ability of chicken spleen is weak. The expression of immune-related factors in the chicken spleen is relatively stable since the age of 21 days old.Experiment III The ultrastructure characteristics and the lymphocyte homing-related adhesion expression on the sheathed capillary in the chicken spleen.The sheathed capillary of chicken spleen is a specialized capillary with its high endothelial cells which was different from that of mammals. The ultrastructure of the high endothelial cells of sheathed capillaries in the chicken spleen was investigated by light and electron microscopy. The lymphocyte homing related addressin- VCAM-1 and MADCAM-1 were detected by the immunohistochemistry method. Results showed that the endothelial cells of the sheathed capillary were high and cuboidal in appearance. Lymphocytes presented in the ellipsoid, which located near the endothelial cells and the vascular channels. The vascular channels extended to the ellipsoid, making the basement membrane discontinuous.Lymphocytes were found in the extended vascular channels in the chicken spleen. VCAM-1 and MADCAM-1 were immunopositive expressed on the sheathed capillary, which suggested that the high and cuboidal endothelial cells of the sheathed capillary were the site of lymphocyte homing to the chicken spleen. These research findings contribute to better understanding of the mechanism of lymphocyte homing to the chicken spleen.Experiment IV The comparison of cytological evidence of lymphocyte homing in the spleen. During the process of the spleen evolution, the spleen of birds is similar to that of reptiles, which lack marginal zone and the white pulp is composed of PALS and PELS.The difference between the spleen of birds and reptiles is the germinal center, which existed in the avian spleen but absent in the reptiles. The Chinese soft-shelled turtle, P. sinensis belongs to the Reptilia Chelonia. The endothelial cells of the sheathed capillary in turtle spleen is similar to that of chicken spleen, which is equivalent to the mammalian high endothelial venule. It also can be used as an important model to investigate the lymphocyte homing to the spleen. In this study, the structural characteristics of the splenic sheathed capillary in Chinese soft-shelled turtle and chicken were investigated by light microscopy and transmission electron microscopy. Results showed that the sheathed capillaries in the turtle and chicken spleen both were high endothelial venule (HEV)-like vessels. The sheathed capillaries in turtle spleen consist of micro-channels that facilitate lymphocyte migration to the splenic white pulp. The pattern of lymphocyte migration in the micro-channel of the turtle spleen includes the following steps: (i) lymphocyte first adheres to the endothelium of the sheathed capillary, passes through the endothelial cells, and traverses through the basement membrane of the sheathed capillary; (ii) it then enters into the ellipsoid combined with supporting cells and EACs; and (iii) lymphocyte migrates from the ellipsoid to the PELS. This study depicted the morphological evidence for lymphocyte migration in the turtle spleens and also provides cytological basis of lymphocyte homing to the spleen of birds and reptiles.Experiment V (1) Prokaryotic expression and polyclonal antibody preparation of integrin linked kinase. Integrin linked kinase (ILK) located in the focal adhesion, is the threonine / serine protein kinase. It functions as the combination of integrin extracellular and intracellular signal transduction. To investigate the lymphocyte homing related factors expression in the chicken spleen, the polyclonal antibody of chicken ILK was performed in our study. In the present study, the gene sequence of ILK coding region was designed as a template, which was added the enzyme digestion site to design primers. The ILK antigen region was amplified. The products were characterized by restriction endonuclease KpnI and XhoI, which constructed into the prokaryotic expression vector pET-3 0(a)+. The recombination plasmid pET-3 0(a)+- ILK was transformed and the recombination protein was expressed in BL21 (DE3) by optimizing the expression conditions with the use of the IPTG induction. SDS-PAGE results showed that the recombinant protein expressed in the form of inclusion body with the molecular weight of 27kD. The recombinant protein was purified by Ni2+ affinity chromatography column. The polyclonal antibody were prepared by immunized rabbits with purified ILK protein. The fourth immune sera were collected and purified. ELISA measurement showed the titer was more than 1:32000. Western blot showed the specificity of the polyclonal antibody ILK is good, which could be used for the next experiment of the molecular mechanism of lymphocyte homing in the chicken spleen.(?) Mechanism of lymphocyte homing in the chicken spleen during LPS stimulation. Lipopolysaccharide (LPS) is an acceptable administration for studying systemic inflammation. This study demonstrated CFSE labeled lymphocytes homing to the chicken ellipsoid of the splenic white pulp via the sheathed capillary, and with the time increase, lymphocytes gradually migrated to the red pulp. An immunohistochemical analysis revealed that the number of lymphocytes in the PELS decreased, but the number of lymphocytes in the red pulp increased after LPS stimulation. Electron microscope observation showed lymphocytes in the blood circulation transmigrated to the spleen ellipsoid via the endothelial cells of the sheathed capillary under the stimulation of LPS.Furthermore, the qPCR and Western blot results showed the mRNA and protein levels of integrin ?1 and VCAM-1 are up-regulated after LPS stimulation. The integrin ?1 and VCAM-1 pathways regulating lymphocyte homing during LPS stimulation might be associated with the ILK-dependent regulation of protein kinase B (PKB/AKT). This is the first study of lymphocyte homing to the chicken spleen, which would contribute to the avian immunology and provide a better means for investigating the strategy of prevention and therapy of lymphocyte homing in inflammation and immunological reactions in infectious disease.