Effect of FasL High Expression on Immune Regulative Function of Sertoli Cell in Transgenic Mouse

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1 Journal of Reproduction and Contraception (2004) 15 (4): Effect of FasL High Expression on Immune Regulative Function of Sertoli Cell in Transgenic Mouse Wei-yi LI 1, Zhao-ping LIU 1, Ye-bin XI 1, Bo LIN 2, Ying-hua MA 2, Jia-hua HU 2, Guang-jie CHEN 1, Bao-guo WANG 1, Shi-san BAO 3, Li-he GUO 2 1. Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai , China 2. Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai , China 3. Department of Veterinary Anatomy & Pathology B14, University of Sydney, NSW 2006, Australia Objective To study the immune regulative function of Sertoli cell on testis local infection Methods Ureaplasma urealyticum (UU) was directly injected into bladders of FasL transgenic mice and wild-type mice, which mimicked an ascending infectious way. At week 1, 2 and 3 after injection respectively, the mice were killed to observe the pathological alterations in testis section. And at the same time cytokines was tested by immunohistochemistry. Comparison of levels of FasL, TGF-β, IL-1α, and IL-6 between UU-infected and control groups of wild mice and FasL transgenic mice was made respectively. Then the capability of Sertoli cell (FasL + ) to mediate apoptosis of Fas + cells between wild control and wild UU-infected groups was analyzed. Results The pathological changes of testis in FasL transgenic mice were more seriously compared with wild counterpart and the changing mode of cytokines secreted by Sertoli cells were different between the two kinds of mice. The UU-infected Sertoli cells increased Fas + Jurkat cell apoptosis. Conclusions High expression of FasL in FasL transgenic mice can influence the cytokines secretion during anti-infection, thus affecting the testis immune response to infection and immune balance. The high expression of FasL is not beneficial for body s anti-inflection immune response. Key words: Sertoli cell; immune regulative function; FasL; TGF-β; IL-1α; IL-6 This study was supported by National Natural Science Foundation of China (No ). Corresponding author: Wei-yi LI; Tel: ext ; liweiyi@shsmu.edu.cn 209

2 Sertoli cell, the main stromal cell in testis, provides the microenvironment for the differentiation and maturation of spermatogenic cell [1]. It can also form a blood-testis barrier. In addition, Sertoli cell attracts great attention for its immune regulation because it can secrete many cytokines, such as proinflammatory factors IL-1α [2], IL-6 [3] and immunosuppressive molecule TGF-β [4]. In 1995, Bellgrau et al. [5] reported that FasL expressed by Sertoli cell was the key factor to maintain the testis immune privilege. However, later researches found that transgenic FasL can induce conflicting results: immune tolerance [6] and inflammation [7]. It is possible that the consequences of enforced FasL expression are controlled by FasL expression level [8] or tissue microenvironment factors [9]. It has been demonstrated that IL-1 [10] or IL-6 [11] can be often tested when FasL induced inflammation. While TGF-β is a great negative regulative cytokine in immune system, which can counteract the proinflammatory factors function. And it can shut down the signal of immune response and induce the cell apoptosis during executing inflammation and anti-tumor cells [12]. It has been shown that coexpression of TGF-β and FasL promoted immune tolerance and protection, preventing inflammation of allografts [12]. Thus it has great significance to study the immune functions of Sertoli cell and high-expression of FasL in anti-infection and maintainance of immune privilege by investigating the influence of enforced high expression of FasL on Sertoli cell secreting TGF-β, IL-1α, and IL-6 when testes is infected by ureaplasma urealyticum (UU). Materials & Methods Mice Twenty four male DBA/C57BL FasL transgenic mice (6 weeks, g) were kindly provided by the laboratory of Prof. Li-he GUO, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences. The mice were divided into 2 groups randomly: transgenic control group (Tg-C) and UU infected group (Tg-U). Another 24 male DBA/C57BL wild-type mice (6 weeks, g) were obtained from Shanghai Animal Research Center, Chinese Academy of Sciences. These mice were also divided into 2 groups randomly: wild-type control group (W-C) and UU-infected group (W-U). There were 12 mice in each group. In vitro Incubation of UU and test of UU titer [13] UU (10 6 CCU/mL, CCU for color change unit) was of log phase. Sodium pentobarbital, 40 mg/kg, was used to anesthetize mice. Then 0.1 ml UU suspension (10 6 CCU/mL) was injected into mice s bladders after laparotomy. And at the same time 0.1 ml culture medium was injected into the bladder of each mouse in the control groups. Then their abdomen was stitched. All the procedures were operated in an aseptic environment. Histopathology The testes were obtained from the infected mice after 1, 2 and 3 week(s) respectively. 210

3 Part of the testicular tissues were fixed in 10% neutral formalin, embedded in paraffin, sliced into 4 µm thick piece and stained by hematoxylin and eosin for histopathological examination; whereas other part were fixed in 2% glutaraldehyde solution and underwent routine electron microscope (EM) examination by transmission EM. Immunohistochemistry Immunohistochemistry staining and Sertoli cell labeling index (LI) calculating were performed as was described previously [13]. All data was statistically analysed by Student s t test. Separation of Sertoli cells The mice were killed after being infected for 1, 2 and 3 week(s), respectively. Testis tissues were isolated and digested by collagenase type II and hyaluronidase (both digestive enzyme in half quantity comparing with rat experiment). One point five ml of both kinds of enzyme was used every time to digest a rat testis, while we used 0.75 ml to replace 1.5 ml in the isolation of mice Sertoli cell twice, then filtered and centrifuged. Sertoli cells were in high purity and survival rate. The number of cells was adjusted to /ml in serum free DMEM/F-12 culture medium. In 6 well plates, each well contained 6 ml [14]. Jurkat cells (Fas + ) preparation Jurkat cells used in this study were incubated in RPMI1640 medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 at 37, suspending growth. Co-culture of Sertoli and Jurkat cell After 24 h of culture and Sertoli cells tight adhesion to the plate wall, the culture medium was removed. Jurkat cells were then added according to the effector/target ratio (3: 2). The cells were incubated in an atmosphere of 5% CO2 at 37. Apoptosis of Jurkat cells Suspending Jurkat cells were harvested and labeled according to the procedures of Annexin V-FITC apoptosis detection kit. Samples were analyzed within 1 h on a FAC Scan flow cytometer. Data from 10 4 cells were collected and analyzed with Cellquest software (Becton Dickinson). Then the capability of Sertoli cell (FasL + ) was compared to mediateapoptosis of Fas + cells between wild control and wild UU-infected groups. Results Histopathology Testicular tissues of wild-type control mice The spermatogenic cells lined up in order, and had no shedding (Figure 1A). At the 1 st week after UU-infection, there were significant morphological changes, including exfoliation of the germ cells, irregular location of the epithelial cell and many acidophilic aggregates in the seminiferous (Figure 1B). There was obvious restoration at the 2 nd week after infection, 211

4 but there were still some abnormalities, including formation of the multinucleated giant cells (Figure 1C). There was another better pathological changes in the testis at the 3 rd week, compared with the 2 nd week post-infection (Figure 1D). Testicular tissues of transgenic control mice There were few epithelial cell damages in the seminiferous tubule, orderly-lined-up spermatogenic cells, few shedding and lymphocytes invading in the stroma; whereas some local parts of the testis exhibited extensive exfoliation of the germ cells and vacuolated spermatogenic epithelium within the seminiferous tubules (Figure 1E). There was serious damage, focal lesions in the testicular tissues 1 week after infection, including irregular location of the epithelial cell (Figure 1F). And there were no recovery at the 2 nd and 3 rd week (Figure 1G, 1H). Lots of lymphocytes were seen in the interstitial (Figure 1I). A: W-C group B-D: W-U groups (B: 1 week; C: 2 weeks; D: 3 weeks) E: Tg-C group F-I: Tg-U groups (F: 1 week; G: 2 weeks; H, I: 3 weeks) W: wild-type Tg: transgenic C: control U: UU infected Figure 1 Mouse testis tissues under light microscope (HE staining; A-H 66; I 132 ; : lymphocyte) 212

5 A: W-C group B-D: W-U groups (B: 1 week ; C: 2 weeks 3 000; D: 3 weeks 4 000) E: Tg-C group F-I: Tg-U groups (F: 2 weeks G: 2 weeks 5 000; H: 3 weeks ) W: wild-type Tg: transgenic C: control U: UU infected Figure 2 Observation of mouse testis tissues under electron microscope P S1 S2 M A: negative control B: positive cells in FasL immunochemical staining ( : Sertoli cell) Figure 3 Immunohistochemical staining ( 132) 213

6 mice of week 3, vacuoles appeared in the spermatid acrosome, and in the middle of the vacuole there was an abnormal black dot (Figure 2D). Most Sertoli cells and germ cells of the FasL transgenic control groups (Tg-C) showed no abnormality under EM, but very few spermatocytes showed nuclear cavitation (Figure 2E). In the 1st week, Tg-U cell was very dispersing; in the 2 nd week, some Tg-U spermatocytes were damaged seriously, remarkable emptiness of n uc lear and swollen of mitochondria can be observed (Figure 2F). In the 3 rd week, some Tg-U spermatogonia exhibited abnormal morphology, including mitochondria swollen, mitochondrial cristae and membrane damage, endoplasmic reticulum expanded to be vacuoles (Figure 2G). Furthermore, macrophages were seen in the interstitial (Figure 2H). Cytokines and FasL expression Immunochemistry showed that FasL, TGF-β, IL-1α, IL-6 in Sertoli cells were positively stained in all samples. The negative control was unstained (Figure 3A, B). Cytokines and FasL expression were compared through Sertoli cell labeling index (LI) calculation (Figure 4). Cytokines and FasL expression in wild-type mice When W-U group is compared with W-C group, FasL expression in Sertoli cells increased significantly at the 1 st, 2 nd and 3 rd week after UU infection (P<0.05, P<0.01, P<0.05, respectively). In Tg-U mice, TGF-β secretion had an obvious rise at the 1 st and 2 nd weeks (both P<0.01), IL-1α production showed an elevation at week 1 (P<0.05) and IL-6 expression increased at week 1 (P<0.05) (Figure 4A). Cytokines and FasL expression in transgenic mice FasL had a significant rise at week 2 in Sertoli cells of UU-infected group compared with control group (P<0.01). TGF-β had no obvious change at week 1, but increased evidently at week 2 and 3 after UU infection (P<0.01, P<0.05, respectively). IL-1α for UU-infected increased at 1 st to 3 rd weeks after UU infection (P<0.05 for all subjects). IL-6 increased at week 1 (P<0.05) and reduced at week 3 (P<0.01) (Figure 4B). Apoptosis of Jurkat cells After wild-type mice were infected by UU, the Sertoli cells were separated at week 1, 2 and 3 and co-cultured with Jurkat cells for 14 hours. When UU-infected Sertoli cells were compared with wild-type control groups, apoptosis rates of Jurkat cells mediated increased (Figure 5). The apoptosis rate of Jurkat cells was 42% induced by in W-C Sertoli cells at the 1 st week, while 45% was induced by W-U at the 1 st week; while in W-C at the 2 nd week it was 52% and 56% in W-U at the 2 nd week, 47% in W-C and 61% in W-U at the 3 rd week. 214

7 Sertoli cell labelling index(li Wild-type group W-C W-U W-C W-U W-C W-U W-C W-U FasL TGF-β IL-1α IL-6 Week 1 Week 2 Week 3 Sertoli cell labeling index (LI) :P <0.05,:P <0.01, vs W-C group at the same time Transgenic group Week 1 Week 2 Week 3 0 Tg-C Tg-U Tg-C Tg-U Tg-C Tg-U Tg-C Tg-U FasL TGF-β IL-1α IL-6 :P <0.05, :P <0.01, vs Tg-C group at the same time Figure 4 Expression levels of FasL and TGF-β, IL-1α, IL-6 of Sertoli cells at the 1 st, 2 nd, 3 rd weeks after infection Figure 5 Comparison of apoptosis of Jurkat cells induced by Sertoli cells in vivo at the 1 st, 2 nd and 3 rd week after injection 215

8 Discussion Testis is an immune privileged site. Normally testis does not induce inflammation. Sometimes it can also be infiltrated by various microorganisms. To protect the individual, immune system will have immune response to microorganism (Ag). At the same time, inflammatory response process will inevitably produce pathological injury by immune response and inflammatory response. Through histopathology analysis, we found that the spermatogenic cells in the wild-type control groups lined up in order without Shedding. While in the transgenic control groups, testes had slightly morphological abnormalities. Abnormal spermatocytes were also found through transmission EM examination. The result was coincide with those of other discoveries. FasL transgenic mice can develop a natural local morphological changes in testis, kidneys and some other tissues [15]. These changes may have some relation with the over-expression of FasL. For human FasL integrated in mice chromosome could mediate Fas positive cells of mice apoptosis [16]. But there were no severe tissue destruction in other examples of FasLdirected proinflammation [17]. The data also suggested that the other tissue-specific microenvironment factors can modulate the proinflammatory consequences of FasL. Serious damage was observed in the testicular tissues in week 1 after infection in wildtype mice, but these tissues obviously recovered 3 weeks after infection. While in the UUinfected mice of transgenic groups, from week 1 to 3, we found that the epithelial cells in the testicular tissue were irregularly located. No recovery was seen at the 3 rd week. These light microscopic findings were further confirmed by EM examination. All those suggested that FasL will not certainly supply an immune protection. It may be attributable to down-regulation of body s immune response mediated by high expression of FasL through apoptosis of Fas + lymphocytes. We found that FasL expressed by Sertoli cell can induced Fas + cells apoptosis. Further more, FasL expression in Sertoli cells increased especially at the 3 rd week. Meanwhile the Sertoli cell-induced apoptosis of Jurkat cells were also most apparent. It is suggested that infected Sertoli cells could stimulate the production of Fas + lymphocytes and induce apoptosis of germ cells through up-regulating of FasL expression. As a result, body can maintain the balance of immune privilege and microenvironment, thus protecting reproductive and physiological functions of testis. But over high expression of FasL could be unfavorable for anti-inflammation immune response. Results demonstrated that in FasL-transgenic mice, the level of FasL did not rise obviously in week 1, but it increased greatly at week 2, then recovered at week 3. This suggested that suitable increase of FasL expression might have positive impression on body antiinflammation. While in transgenic mice, constant FasL expression rose after UU infection could have negative effect in balancing and sustaining immune privilege and anti-inflammation (Figure 4). 216

9 At the same time, we compared the levels of TGF-β, IL-1α, and IL-6 between UUinfected and control groups of wild mice and transgenic mice respectively. The results showed: after UU infection, testis tissue in the wild mice was damaged most seriously at the 1 st week after infection, and all the 4 cytokines showed most obvious changes at that time all upregulated. IL-1α and IL-6, as proinflammatory factors, could strengthen the local inflammation reaction and were good for the control of inflammation. While up-regulation of FasL and TGF-β expression might increase Sertoli cell s capacity to induce apoptosis of other cells. It was kind for maintaining testis immune privilege and thus beneficial to protect testis functions. At week 2 and week 3, IL-1α and IL-6 restored to normal level, while FasL and TGF-β continued to increase. This might be caused by that pathogen had almost been cleared. However it still needed Sertoli cells increasing FasL expression to facilitate the elimination of the damaged cells and remained lymphocytes that express Fas. So it could provide a good microenvironment for tissue recovery. While in the transgenic mice, the cytokines change in another pattern. From Figure 4B we could find almost all the 3 cytokines up-regulate expression from week 1 to 3 after infection. It was consistent with the pathologic examination results that testes tissues showed continue inflammation at the 3 rd week after infection. IL-1α and TGF-β expression of UUinfected group increased significantly after infection from week 1 to 3. For one reason, IL- 1α might rise as for FasL [18]. On the other hand, as an proinflammatory factor, IL-1α could strengthen the local inflammation reaction and was good to control the inflammation [19] ; meanwhile IL-1α could strengthen the testis local immunity through affecting some immune cells immune response [20]. Whereas TGF-β was an important negative regulative factor in immune system. Its role was to suppress the local immune response and inflammatory reaction, counteract the function of IL-α, resist inflammation and sustain testis immune privilege to protect the testis reproductive function. In conclusion, Sertoli cell had great regulative function on immunology; high expression of FasL was not always helpful for anti-infection, and too high expression could influence the cytokines secretion during anti-infection, thus affecting the testis immune response to antiinfection and immune balance. References 1. Pierantoni R, Cobellis G, Meccariello R, et al. The amphibian testis as model to study germ cell progression during spermatogenesis. Comp Biochem Physiol B Biochem Mol Biol, 2002, 132(1): Huleihel M, Zeyse D, Lunenfeld E, et al. Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of Sertoli cells. Am J Reprod Immunol, 2001, 45(3): Stephan JP, Syed V & Jegou B. Regulation of Sertoli cell IL-1 and IL-6 production in vitro. Mol Cell Endocrinol, 1997, 134(2): Avallet O, Gomez E, Vigier M, et al. Sertoli cell-germ cell interactions and TGF beta 1 expression and secretion in vitro. Biochem Biophys Res Commun, 1997, 238(3):

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