A Comparative Study Of Blood Spleen Barrier And Lymphocyte Homing Between Chicken And Duck

The spleen combines the innate and adaptive immune system in a uniquely organized way.The structure of the spleen enables it to remove older erythrocytes from the circulation and leads to the efficient removal of blood-borne microorganisms and cellular debris.This function,in combination with a highly organized lymphoid compartment,makes the spleen the most important organ for antibacterial and antifungal immune reactivity.Poultry lack lymph nodes on the immune system,but has bursa of fabricius which distincts from mammalian.Spleen is the largest peripheral lymphoid organ in poultry and mammal,and there are differences in its structure and cell composition.Unlike mammals,poultry spleen lacks marginal zone,and white pulp has increased the periellipsoidal lymphocytic sheath.The white pulp of birds consists of the splenic nodules(SN),the T cells region from the thymus origin(PALS),the B cells region from the bursa of fabricius(PELS)and the ellipsoid structure.At present,lots of literatures have been reported on the morphological characteristics and functions of the spleen of mammals,but the histomorphological characteristics and immunol functions of the spleen have yet to be further studied in avian species.At present,the BSB has been reported only in mammals and reptiles.The BSB plays an important role in maintaining the homeostasis of the body,regulating the balance of the body,protecting the normal physiological function and blocking the intruding foreign antigen in the blood.In recent years,Zhang Qian established the concept of BSB on the chicken and described its function,she found that the sheathed capillary endothelial cells of spleen are high endothelium.The high endothelial structure is the basis for the lymphocyte homing.But whether BSB exists in duck and what’s the cellular composition of BSB?Chicken and duck as the most commercial value of birds differ in their immunol system.Like most poultry,chicken lacks the lymph nodes,but the duck as a waterfowl has two lymph nodes,the cervicothoracic lymph nodes and the lumbar lymph nodes.There are also difference in avian influenza virus tolerance and host immune response between chicken and duck.Whether the difference in the immunol system of poultry and the immunol reactivity in the disease can cause the difference in morphology of BSB and the expression of lymphocyte homing related molecules,and what’s the molecular mechanism.In view of these questions,we first explored the cellular and structural characteristics of BSB in duck and compared the structural and cellular composition of BSB between chicken and duck.Then the cytological characteristics of chicken and duck BSB and the differences in the expression of lymphocyte homing related molecules were studied after LPS stimulation.The results demonstrate the immunological differences between chicken and Duck in cellular and molecular level,which will provide theoretical support for prevention and control avian diseases.Experiment Ⅰ Microstructural comparative study of the blood-spleen barrier between duck and chicken.To identify the difference of composition of the blood-spleen barrier(BSB)between chicken and duck,the microanatomical structures of the spleen were compared by light microscope and transmission electron microscopy,using microtomy and enzymatic histochemistry technique.The results of light microscopy showed that both chicken and duck lacked marginal zone and contained special ellipsoidal structures of birds.Compared with chicken,the interface of red and white pulp in duck was obscure and lymphatic sheath was undeveloped;Chicken ACP positive reaction was mainly distributed at ellipsoid and PALS,while mainly distributed at PELS and red pulp in duck;ANAE positive reaction was mainly distributed at the outer surface of the chicken ellipsoid,around PALS and SN,while that in duck is mainly distributed in the red pulp and PELS.The ultrastructure showed that the capillary endothelium in duck is cubic,belonging to the high endothelial.There were interlaced fibroblasts and small amounts of macrophages in PELS,and a small number of granulocytes and lymphocytes were distributed in the red pulp.while a few EAC were visible at red pulp of chicken spleen,and a number of dendritic cells were seen encircling the lymphocytes.The results indicated that there were subtle differences in ellipsoid structure between chicken and duck spleen.In addition to reticular cells,fibers,macrophages,and dendritic cells,EACs were also involved in the composition of chicken BSB,while in the spleen of duck,macrophages and dendritic cells have been observed,but not typical EAC.Experiment Ⅱ A functional comparative study of blood spleen barrier between chicken and duck.the microanatomical structures were investigated by light and electron microscopy technique.we compared the phagocytosis of BSB between chicken and duck by the method of intravenous injection of ink to represet the strength of immunal function of BSB.After different time point of ink injection(30min,3h,6h,15h,ld,3d,5d),we found the carbon particles were mainly concentrated around the ellipsoid of chicken spleen,subsequently migrated to outer PELS and the sorroundings of PALS and splenic nodules,while absent in splenic nodules.carbon particles in duck within 3h distributed a large quantity at the surroundings of basement menmbrence of capillaries,a small amount at PELS,then migrated to PELS and red pulp.The total number of carbon particles decreased gradually under microscope.The results showed the mechanical force of BSB in chicken blocking carbon particles were stronger than that in duck.The mechanical barrier of chicken spleen is mainly located at the ellipsoid,while that in duck spleen is mainly located around the basement membrane of capillaries and at PELS.The biological barrier of chicken spleen is mainly located on the surface of ellipsoid,while that in duck spleen is mainly located at PELS.The cells capturing foreign antigen in chicken spleen are mainly EAC,while in duck spleen are mainly macrophages and dendritic cells.This provides a morphological basis for the existence of different cellular and molecular mechanisms in the immunol response of chicken and duck spleen.Experiment Ⅲ Comparative study on the lymphocyte homing related molecules of chicken and duck after LPS stimulation.The aim of this study was to compare the different levels of expression of lymphocyte homing related molecules in chicken and duck after LPS stimulation.intraperitoneal injection of LPS 3h,under the light microscope and electron microscope,a large number of lymphocytes and granulocytes were found in the red pulp of chicken and duck spleen.The intercellular space of endothelial cells and ellipsoid structure in duck spleen were relatively loose,also the ellipsoid related cells migrated to PELS were visible.Western blot result showed that the expression of intercellular connexin Claudin-10 protein was significantly increased in chicken and significantly decreased in ducks(P<0.05),The adhesion molecule VCAM-1 increased significantly in chicken spleen(P<0.05),and decreased in duck spleen.The expression of lymphocyte homing gene,like MAdCAM-1,VCAM-1 and Integrina4 of treatment group increased significantly in the spleen of the chickens(P<0.05),while in duck spleen expression significantly decreased(P<0.05);after the injection of LPS,the expression of cell movement related gene,like RHO-A and FAK in chickens decreased significantly(P<0.05)and increased significantly in ducks;the gene expression of Cdc42 in chickens decreased not significantly,while increased significantly in duck(P<0.05).After LPS stimulation,chicken and duck spleen all initiated a host immune response,and recruited a large number of lymphocytes and neutrophils involved in inflammatory reaction.The lymphocyte homing in chicken is mainly attributed to the adhesion between the endothelial cells and lymphocytes,and in duck may be attributed to two aspects:one is that the decrease of tight junction proteins induces enhancement of the permeability between endothelia cells,which indirectly promotes the migration of lymphocyte.and the second is to upregulate the migration and movement related genes which causes enhancement of the ability of lymphocyte migration.This provides a basis for the comparison of mechanism of lymphocyte homing molecular between two avian species after inflammation.