Role Of Pineal Gland And Melatonin In The Differentiation And Development Of T Cells In Rat Thymus

Since Berman proposed the concept that pineal gland is related to the immune system in 1926, there have been a lot of studies indicating the obvious functional relationship between them. Most studies so far, however, are in vitro, and the reported methods limited. Therefore the structural and functional relationship between pineal gland and thymus still remains unclear. By utilizing pinealectomized model of rats, this study was to investigate the role of pineal gland and melatonin(MLT) of rats in the differentiation and development of T cells in rat thymocytes, and the possible mechanism as it is going to discuss in the following four sections.1. Contribution of pineal gland and melatonin to the differentiation and development of rats thymocytesUp to now, there are few reports about studies to the contribution of pineal body and its melatonin to the thymocytes of rats in their development and the possible mechanism. By utilizing immunocytochemistry, computer morphometric analyzing technology and Immunofluorescent dual staining of CD4/CD8, this section approached the variation of the proportion of thymic and hematic T cell subsets after pinealectomy and melatonin administration, explored the effects of pineal gland and the MLT on the thymocytes differentiation and development of rats.The results showed that the decrease of the rat body weight was statistically significant 4 weeks after pinealectomy (Px), and the decrease of the rat thymic weight was significant 8 weeks later, which was partly restored by exogenous MLT, especially by the high dose. The decreasing area of the CD4~+ cells in cortexhas statistical significance 8 weeks after Px? which was restored by exogenous MLT in dose-dependent manner. The area of CD8+ cells in medulla increased apparently 4 weeks after Px, which was also statistically significant in the group dealt with Px and exogenous melatonin in spite of the less increase compared with Px group. Flow cytometry analysis indicated no significant variation of the number of CD4+ cells and the proportion of CD4+CD8+(double-positive, DP) or CD4"CD8" (double-negative, DN) T cells 4 weeks after Px- The number of CD8+ cells, on the contrary, reduced significantly. Then the proportion of CD8+cells increased 8 weeks after Px, whereas that of DN cells decreased instead. Exogenous melatonin gradually raised the proportion of CD8+cells in dose-dependent manner and statistically significant in the group of high dose either 4 or 8 weeks after Px, and the proportion of DP cells decreased instead. Meanwhile, the variation of CD4+cells in blood is not obvious after Px, while the proportion of CD8+cells and the sum of CD4+ /CD8+cells decreased whereas the proportion of DP or DN increased. Although the proportion of CD8+cells in blood was restored gradually with exogenous MLT, the proportion of both low and high dose group was still lower than that of control group 8 week after Px, and the proportion of DN cells increased obviously and simultaneously. All these indicated that thymus atrophied obviously by Px, which decresed the proportion of blood CD8+ cells and had no strong effect on CD4+ cells. The proportion of thymic CD8+ cells decreased obviously 4 weeks after Px, but increased 8 weeks after Px as a compensation. MLT administration can restore the effects of Px on blood and thymic T cells significantly, suggesting that MLT may play the key role in the process.2. Effect of pineal gland and melatonin on the cell cycle of rats thymocytes The continuous release of thymocytes into the blood and mass apoptosis need immediate supplement and renewal, so thymus is an organ in which cell division is active. This section investigated the different proportion of thymocytes in different phases of the development with PI one step method, observed the effects of Px and MLT on variation of Cyclin A/E in rat thymocytes with immunocytochemistry, and then explored the effects of Px and MLT on differentiation and development of rat thymocytes and its mechanism.The results showed that the proportion of thymocytes in Gl phase decreased significantly 8 weeks after Px, but the parameters in S phase and G2+M phase increased accordingly, which had been statistically significant especially in G2+M phase 4 weeks after Px. Exogenous MLT administration down-regulated the proportion of G1/G0 phase and S phase, but they kept increasing in G2+M phase. The area of CyclinA positive cells tended to decrease after Px, which was more significantly in cortex than in medulla and could be restored with exogenous MLT, but not in dose-dependent manner. The area of Cyclin E positive cells, however, enlarged and demonstrated statistical significance in medulla 8 weeks after Px, which could be reversed by exogenous MLT in medulla rather than in cortex. These indicated that Px has an obvious impact on the process of thymocyte cycle. It reduced the expression of cyclin A in cortex, but promoted cycline E expression in medulla; it then decreased the proportion of G1/G0 phase thymocytes but increased S and G2+M phase thymocytes. MLT administration promoted the production of thymocytes and restored the thymic atrophy caused by Px finally, by increasing expression of cyclin A and cyclin E, promoting the transformation of thymocytes from G1/G0 phase to S and G2+M phase, decreasing the proportion of G1/G0, and maintaining the high proportion of S and G2+M phase thymocytes,.3. Regulation of thymocytes apoptosis by pineal gland and melatoninApoptosis is a normal phenomenon in the development of immune system and the immune response. Most thymocytes are eliminated by apoptosis during positive and negative selection. By utilizing TUNEL technique and ABC immunocytochemistry staining, this section detected the expression of Fas/FasL, caspase-3, caspase-8, caspase-12 in thymocytes, and then explored the effects of pineal gland and MLT on thymocytes apoptosis and the mechanism.The results showed that the apoptosis of thymocytes was on the rise after Px, and compared with those of the control group, the number of apoptosis cells in both cortex and medulla increased statistically significantly 8 weeks after Px, and when MLT administration 4 weeks after Px, the number of apoptosis thymocytes in cortex of two dose groups kept increasing, and then restored 8 weeks after Px, which showed no statistical significance as compared with that of control group. The grayscale of Fas positive thymocytes tended to decrease significantly as compared with that of the control 8 weeks after Px, whereas the area of the positive cells enlarged with statistical significance in cortex 4 weeks after Px, furthermore, the discrimination continued to enlarge 8 weeks after Px and even demonstrated differences in medulla as compared with that of the control group. The result of FasL staining was similar to the former, but it raised first in medulla 4 weeks after Px and then became significant both in cortex and in medulla 8 weeks after Px. With MLT administration; the effect caused by Px attenuated. The expression of Fas in the cortex cells of low dose group became larger 8 weeks after Px than that of the control group; FasL expression in medulla increased and was significant in low dose group 4 weeks after Px ,and it increased significantly in cortex and medulla of low dose group as well as in cortex of high dose group 8 weeks after Px.The area of Caspase-3 positive cells in cortex raised significantly, which was down-regulated by exogenous MLT and demonstrated statistical significance both 4 and 8 weeks after Px; the number of Caspase-8 positive thymic T cells increased significantly in both cortex and medulla after Px, while exogenous MLT had no strong effect on the gray scale of Caspase-8, positive staining was only enhanced in the cortex and medulla of low dose group 8 weeks after Px , and the positive area of medulla in low dose enlarged 4 weeks after Px, high dose MLT partly reversed the increasing of Caspase-8 by Px, but the parameters were still statistically significant compared with the control group; Caspase-12 expressed evenly in cortex and medulla, the gray scale of caspase-12 positive cells in thymic cortex was on the rise 8 weeks after Px, and the positive area of cortex enlarged but that of medulla reduced 4 weeks after Px; then it reduced both in cortex and medulla 8 weeks after Px but showed no significance. MLT administration enhanced rather than reversed the decrease of Caspase-12 caused by Px, and the reduction of Caspase-12 postive area in the two dose groups was significant 8 weeks after Px.The results showed Px could induce the apoptosis of thymocytes, and it may be one of the main mechanisms of the thymic atrophy. The mechanisms in which Px induced apoptosis of thymocytes included activating death signal path and causing cascade reaction of Caspase: increase the Fas/FasL expression and up-regulate thelevels of Caspase-3, -8 protein. That is, with MLT administration the apoptosis of thymocytes was markedly attenuated, and the signal expression of death signal path and the expression of Fas/FasL, Caspase-3, -8,-12 decreased.4. Effect of melatonin on gene expression profiles of rat thymic cells Gene expression profiles are widely used now, through which mRNA or cDNA in cells of different individuals can be detected. This section is to detect primary thymocyte culture by MTT and explore thymocyte gene expressing changes in primary cultured thymocytes after MLT intervention by high-flux gene expression profiles. The detected 1176 genes fell into 42 categories: cell surface antigen, transcription, cell cycle, oncogene/tumor suppressor, membrane channel/ transporter, extracellular matrix protein, post-tranlational modification/protein folding, translation, apoptosis associated protein, cell receptor/ligand, cell signal /extracellular communication protein, intracellular transducer/effector/ modulator, protein turnover, cytoskeleton/motility protein, DNA synthesis/ recombination/ repair etc. They were checked by RT-PCR technology.The results showed that the expression of 40 genes of thymocytes was either up-regulated or down-regulated after MLT administration. 15 genes were up-regulated, including CDK inhibitor IB, integrin-Pl, CSF factor 1 receptor, phospholipase A2, protein tyrosine phosphatase, villin etc, while 25 genes were down-regulated, including Annexinl,cytochrome b5, carbonic anhydrase 4, Heat shock protein 70-1, TNF-2, IL-15 etc. The results of RT-PCR were coincident with that of gene expression profiles. This indicated that MLT could either up-regulate or down-regulate gene expression levels of related factors, then finally protect the thymocytes by influencing the growth and metabolism of thymocytes, by inhibiting apoptosis of thymocytes , and by sympathic nervous system. Px induced mass thymocytes apoptosis and resulted in thymic atrophy because of losing MLT protection.