Comparison Of Histological Characteristics And Expression Of Immune Factors Among Yak Hemal Nodes,Lymph Nodes And Spleens

ObjectsHemal nodes,lymph nodes and spleens are important secondary immune organs in the body.They constitute the main components of mammalian peripheral lymphoid tissues and play an important role in local and systemic immune responses.Knowledge on immune organs development is mainly based on the morphology,and studies combining morphology and function of immune organs are the most fundamental and informative ways of understanding the immune system.Morphological methods were used to observe the structure and ultrastructure of the yak hemal nodes,lymph nodes and spleen,so as to elucidate the developmental characteristics of the three secondary immune organs.Immunohistochemical methods were used to analyze the variations of immune cells in hemal nodes,lymph nodes and spleens.Real-time PCR(qRT-PCR)and Western bloting techniques were used to detect the expression patterns of major immune molecules in the hemal nodes,lymph nodes and spleens of yaks at different age groups.The comparison of the structure,immune cells,and immune factors variation of the yak hemal nodes,lymph nodes and spleens,will provide new data for further studies on local and systemic immune system in yak,and provide theoretical basis for immunopathology and comparative immunology.Methods1.Morphological method was used to observe the age-related morphological changes of health yak hemal nodes,viz newborn(2-5 days old,n=5,the same below),juvenile(5-7 months old,n=5,the same below),adult(3-4years old,n=5,the same below)and aged(8-12years old,n=5).2.Light and transmission electron microscopy methods were used to observe the microstructure and ultra-structure of the hemal nodes,lymph nodes and spleens of newborn,juvenile and adult yak.Their similarities and differences were also compared.3.The histological and immunohistochemical methods were used to analyze the distribution of CD3,CD79a,IgA,IgG,SIRP?and CD68 positive immunoreactive cells of the hemal nodes,mesenteric lymph nodes and spleen of newborn,juvenile and adult yak.4.Real-time PCR and Western bloting methods were used to quantitatively analyze the expression of CD3,CD79a,IgA,IgG,SIRP?,and CD68 mRNA as well as proteins in the hemal nodes,mesenteric lymph nodes and spleens of newborn,juvenile and adult yak.Results1.The hemal nodes of yak were dark-red,spherical or oval with variable size and enclosed with a thin capsule.The parenchyma consisted of lymphoid tissues and a lot of blood sinuses.The cortical and medullary layer could not be clearly distinguished.There were many reticular fibers extending into the sinus.A large number of eosinophils,neutrophils,and a small number of megakaryocytes,platelets and mast cells,and hemosiderin were observed in the yak hemal node.The hemal node of newborn yak was hypoplasia,lymphoid tissues located peripherally were very loose,and mostly resembled lymphocyte infiltration,and no lymphoid follicles were observed.In addition,there were only a few eosinophils in the newborn yak hemal node and no mast cells were observed.In the juvenile,the yak hemal node was well developed,the lymphoid tissue was tight,and the primary and secondary lymphoid follicle had appeared.The histology structure of the adult yak hemal node was similar to that of the juvenile.Both of them contained a large number of eosinophils.In the aged,the connective tissue had increased in the parenchyma,some blood sinuses were substituted by the connective tissue and the lymphoid tissue was sparse.In some hemal node,the lymphoid follicles had disappeared.2.Being different from lymph nodes which was divided into cortex and medulla or the spleen which was divided into white pulp and red pulp,the hemal node consisted of blood sinus and lymphoid tissue.The hemal node was an independent secondary lymphoid organ and appeared to share the histological characteristics of the spleen and lymph nodes:the lymphoid follicles of the hemal nodes were much like those of the lymph nodes,which were not surrounded by the central artery and periarterial lymphoid sheath.The lymphoid cords of the hemal node had a tendon-like feature with those in the lymph node,but contained many erythrocytes,which were much like the splenic cords.The blood sinus wall of hemal node had obvious endothelial cells like the lymph sinus in lymph nodes,but it contained a lot of blood like the spleen sinus.Interestingly,there were two different splenic sinuses of yak:the sinuses with obvious endothelial cells or those consisting of reticular cells.In newborn yaks,there were no lymphoid follicle formation in the hemal nodes and the spleens,but it had appeared in the lymph nodes.And there were three different types of splenic follicles:primary,secondary and transitional splenic follicles.3.CD3~+cells were mainly located in the paracortex area of the mesenteric lymph node and the T-cell dependent area in the hemal node and spleen.Many CD79a~+cells were detected in the mantle zone of all examined lymphoid organs.Both the expression of CD3 and CD79a mRNA as well as protein increased from newborn to juvenile and then decreased in the adults.CD3 expression was always higher in the spleen than in the mesenteric lymph node and hemal node.CD79a was always highest in the mesenteric lymph node.IgG~+and IgA~+cells were found in all examined samples,except in the newborn yak hemal node.IgG and IgA mRNA and protein were up-regulated with age,and the highest expression was observed in the lymph node,followed by the hemal node,with the lowest in the spleen.The SIRP?~+cells and CD68~+cells were widely expressed in all examined organs,and especially in the lymphatic cords along the sinus and the lymphoid follicles.A significant feature was that the expression of SIRP?in the spleen was lower than that in the mesenteric lymph nodes and hemal node in newborns,but higher than that in juvenile and adult yaks.The expression of CD68 in the hemal node was always higher than that in the lymph node and spleen.Moreover,the age-related expression patterns of SIRP?and CD68 were consistent with IgG and IgA,which were increased in all examined organs from newborn to adult.Conclusions1.This study described the age-related morphological changes of yak hemal node for the first time.The function of the yak hemal node,such as in immunity,blood storage,blood filtration and platelet formation were proposed.The yak hemal node was not fully developed in newborns.The lymphoid tissue in the parenchyma was similar to lymphocyte infiltration,and no lymphoid follicles were formed.In juvenile,the hemal node had fully developed.The structure and function were still stable in the adult but then degraded in the aged.2.The yak hemal node appeared to share the histological characteristics of the spleen and lymph nodes.In newborn yaks,there were no lymphoid follicle formed in the hemal nodes and the spleen,but it had appeared in the lymph nodes.And there were three different splenic follicles:primary,secondary and transitional splenic follicles.There were two different splenic sinuses in yak spleen:the sinuses with obvious endothelial cells or those consisting of reticular cells.The histology structure may be related to its adaptability to the altitude environment.3.The detection of the histological structure,immune cells and immune factors of yak hemal nodes,lymph nodes and spleens indicated that the yaks were born with a relatively incompetent immune system,and the structure and function of the immune system gradually develop by juvenile age.The yak hemal nodes were as immunologically active as the lymph nodes and spleens.