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Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells
Biochemical and Biophysical Research Communications 404 (2011) 829–833
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Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells Ping Fan ⇑, Lan He, Dan Pu, Xiaohong Lv, Wenxu Zhou, Yining Sun, Nan Hu Department of Rheumatism and Immunity, The First Affiliated Hospital Xi’an Jiaotong University School of Medicine, Xi’an, Shaanxi 710061, PR China
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Article history: Received 25 November 2010 Available online 21 December 2010 Keywords: Endothelial cells Sertoli cells Proliferation Immunogenicity Co-culture
a b s t r a c t The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 103, 1 104 or 1 105 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-c and TNF-a were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 104 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 104 cell/ ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-c-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-a induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In cell transplantation, inadequate and delayed angiogenesis after transplantation result in shortage in the supply of oxygen and nutrients, leading to apoptosis or necrosis of the graft [1]. Endothelial cells (ECs) facilitate angiogenesis, and the loss of these cells is one of the important reasons for the delay in the reconstruction of microcirculation of transplanted cells [2–4]. In recent years, ECs have been used for islet transplantation [5]. But Cheng et al. showed that, although a certain number of ECs are transplanted, these ECs are in a resting state, and there is no extension to the inside of the islet to form endothelial growth sinusoid. Therefore, these cells are unable to establish microvascular system within the islet in a short term [6]. In addition, ECs themselves are strongly immunogenic. ECs do not express major histocompatibility complex (MHC) class II antigens at resting state, ⇑ Corresponding author: Fax: +86 29 85323976. E-mail address: [email protected] (P. Fan). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.068
but express MHC class II antigens upon stimulations such as virus infections or INF-c induction [7,8]. In addition, through expression of different inflammatory genes, such as interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM), promoted by the activation of tumor necrosis factor (TNF)-a [9,10], ECs recruit white blood cells and thus play an important role in the inflammatory process [11,12]. These reactions are able to induce the occurrence of rejection, resulting in the loss of grafts [13,14]. Thus, the key to solve the problem is to find a way to promote the proliferation while reduce the immunogenicity of ECs. Testis Sertoli cells (SC) has been confirmed to induce immune tolerance in organ transplantation [15]. SC secretes a variety of cytokines such as transforming growth factor (TGF)-b1 and basic fibroblast growth factor (bFGF) [16]. TGF-b1 is a potent immunosuppressive factor that suppresses the secretion of INF-c and TNF-a by immune cells [17], thus reduce graft rejection. In addition, TGF-b1 has a role in regulating cell proliferation; it promotes proliferation of ECs at certain concentrations [18]. bFGF also promotes angiogenesis activity [19]. However, the specific mechanism
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of TGF-b1 in promoting EC proliferation is not clear. Additionally, there has been no report on whether TGF-b1 reduces the upregulation of the expressions of MHC class II antigens and inflammatory cytokines induced by INF-c and TNF-a. Therefore, we cocultured the Sertoli cells with ECs and examined the impact of the Sertoli cells on EC proliferation and immunogenicity in order to find better ways to use ECs in cell transplantation. 2. Materials and methods
respectively. At 24, 48 and 72 h, five wells were sampled at each time point. Culture media were discarded and the culture insert containing the Sertoli cells were taken out. Trypsin at 2.5 g/L was added to each well and the cells were dispersed by trituration and then mixed with RPMI 1640 medium with 10% FCS. Cell density was determined with a hematocytometer, and the total number of cells per well was calculated. The proliferation rates of ECs in two groups were compared at different time points. Proliferation rate = cells harvested at different time point/initial seeding cells (1 104) 100%.
2.1. Cell line and main reagents The human umbilical vein endothelial cell (HUVEC) line ECV304 was obtained from the Cell Bank of Chinese Academy of Medicine. The cells were seeded into 25 ml culture flask at 1 107 cells per flask in 5 ml RPMI 1640 culture medium (Sigma, USA) containing 100 ml/L fetal calf serum (FCS, Gibco, USA). The cells were then incubated in a humidified incubator at 37 °C with 5% CO2. Culture media were changed every day. 2.2. Isolation of Sertoli cells Adult testes were retrieved in sterile conditions from deceased male multi-organ donors. Briefly, seminiferous tubules separated from testes were subjected to a two-step sequential enzymatic treatments at 37 °C with trypsin (Sigma, USA) and DNase (Sigma, USA) for 30 min in the first step and collagenase P and hyaluronidase (Sigma, USA) for 20 min in the second step, followed by RPMI-1640 culture medium containing 100 ml/L fetal bovine serum to terminate the digestion at 4 °C. After 5–10 min incubation at room temperature, digested tissue was filtered through a 75 lm mesh stainless steel filter. Sertoli aggregates were plated and incubated at 39 °C under 5% CO2 for 48 h. Cells were then subjected to hypotonic treatment with 20 mM Tris–HCl buffer (Sigma, USA). Sertoli cells were replenished with RPMI-1640 medium supplemented with 100 ml/L FCS and incubated at 37 °C under 5% CO2. Culture media were changed every other day and the resulting pretreated Sertoli-enriched monocultures contained greater than 95% Sertoli cells. 2.3. Preparation of spleen lymphocytes Human spleen lymphocytes were isolated as previously described with minor modification [20]. The human spleen placed in 200 mesh aseptic milling steel line was repeatedly milled. After filtered twice, the cell suspension was then centrifuged at 1500 r/min at 4 °C for 10 min. The cell pellet was washed twice with PBS, and then mixed with the human lymphocyte separating medium (LYMPHOLYTE, USA), and centrifuged at 1500 r/min for 20 min to absorb the mononuclear cell layer. 2.4. Observation of the effect of Sertoli cells on the proliferation of ECs When confluent, ECs were digested with trypsin at 2.5 g/L and prepared into a cell suspension of 1 104 cell/ml. The cells were divided into two groups, and each group had 10 wells, with 1 104 cell in 1 ml medium in each well. In the control group, the cells were seeded on normal 24-well plates. The plates were incubated at 37 °C under 5% CO2 for 6 h to allow cell adherence, and the culture media were then discarded and replaced with 2 ml of RPMI 1640 medium. In the experimental group, the cells were seeded on a special 24-well cell culture plate (HTS transwell, Corning, USA) for co-culturing. Three sub-groups were set up with 2 103, 2 104 and 2 105 Sertoli cells in the cell culture inserts (HTS transwell, Corning, USA), and RPMI 1640 medium with 10% fetal calf serum was added to reach 2 ml for co-culturing. The densities of Sertoli cells were 1 103, 1 104 and 1 105 cell/ml,
2.5. Western blot analysis of vascular endothelial growth factor (VEGF) receptor-2 (KDR) of ECs ECs in each group were collected after culturing for 24 and 48 h respectively. Cells were homogenized in SDS-1 solubilization buffer (Sigma, USA). Twenty milligram of protein were loaded and separated on a 12% SDS–PAGE followed by transferring to a polyvinyldifluoride membrane (Immobilon-P transfer membrane; Millipore, Billerica, MA, USA). After blocking non-specific binding sites, the membrane was incubated for 2 h at room temperature with a mouse polyclonal antibody against KDR (1:1000 dilution, Cell Signaling Technology, USA) followed by incubation with a secondary peroxidase-linked anti-mouse antibody (Tropix, PE Applied Biosystems, Germany; 1:10000 dilution). Protein expression was visualized by enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech, Germany) and digitized with ChemiDocTM XRS System (Quantity One, Bio-Rad Laboratories GmbH, Munich, Germany). Signals were densitometrically assessed (Quantity One) and normalized to the b-actin signals as loading controls (mouse monoclonal anti-b-actin antibody, 1:10000; Sigma). 2.6. Western blot analysis of MHC class II proteins of ECs ECs in each group were collected after culturing for 24 h. Except the mouse polyclonal antibodies against human nonpolymorphic MHC class II molecules (1:100 dilution) were used, other western blot steps were the same as described above. Protein expression was visualized by enhanced chemiluminescence and digitized with ChemiDocTM XRS System. Signals were densitometrically assessed and normalized to the b-actin signals as loading controls. 2.7. Detection of IL-6, IL-8 and sICAM by ELISA The optimum concentration of Sertoli cells was selected to promote ECs proliferation in the co-culture experiments described above. ECs were divided into three groups, seeded on the glass coverslips in a 24-well plate with each group consisted of five wells with 1 104 cell/well. In the negative control group, 2 ml of RPMI 1640 medium containing 10% FCS was added to each well; in the positive control group, 2 ml of 1640 medium containing 10% FCS and 0.5 lg/L TNF-a (Sigma, USA) were added; in the experimental group, ECs were co-cultured with Sertoli cells, 1640 medium containing 10% FCS and 0.5 lg/L TNF-a. Culture supernatants in each group were collected after culturing for 24 h and kept frozen at 80 °C. Commercially available ELISA kits were used to determine the concentrations of IL-6, IL-8 and sICAM (Bender, Austria). 2.8. 3H TdR incorporation in one-way mixed lymphocyte culture (MLC) ECs were set as stimulating cells and spleen lymphocytes as response cells. The experimental group was divided into two subgroups, EC single culture group and co-culture group. Each group had five wells. In the single culture group, 1 104 cell/well ECs were seeded in each well in ordinary 24-well plate. In the coculture group, 1 104 cell/well ECs were seeded in each well in
P. Fan et al. / Biochemical and Biophysical Research Communications 404 (2011) 829–833
the 24-well plate with inserts containing 1 104 of the Sertoli cells, cultured in a final volume of 800 ll 1640 medium containing 50 mg/L of mitomycin C. Pure spleen lymphocyte group was used as the control group, and wells with medium alone were used as blank control. The cells were cultured for 96, and 16 h before the end of culturing, 3H TdR (2 lCi/well) was added. The radioactivity (CPM) of ECs was measured with an automatic liquid scintillation counter. Stimulation index (SI) was calculated as measured CPM/ control CPM. 2.9. Statistical analysis The data were expressed as mean ± SEM. Inter-group statistical comparisons were made by one-way analysis of variance (ANOVA) using SPSS statistical software (version 13.0, SPSS Inc., USA). A P value less than 0.05 was considered statistically significant. 3. Results 3.1. The effect of Sertoli cells on the proliferation of co-cultured ECs The counting of ECs showed (Fig. 1) that the proliferation rates at 24, 48 and 72 h when co-cultured with 1 103 cell/ml Sertoli
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cells were 135.8 ± 21.0, 208.4 ± 29.0 and 295.1 ± 37.0, respectively, significantly higher than those at the same time point in the control group (24 h, 107.1 ± 15.9; 48 h, 167.2 ± 23.8; 72 h, 227.3 ± 30.5; P < 0.05). When co-cultured with 1 104 cell/ml Sertoli cells, the proliferation rates at 24, 48 and 72 h were as high as 175.7 ± 26.0, 237.6 ± 49.0 and 363.5 ± 55.0 respectively, not only significantly higher than the control group, but also significantly higher than the co-culture group with 1 103 cell/ml Sertoli cells (P < 0.05). When co-cultured with 1 105 cell/ml Sertoli cells, the proliferation rates at 24, 48 and 72 h were 105.8 ± 14.3, 130.2 ± 19.4 and 180.3 ± 20.4, respectively. While there was no significant difference compared with the control group at 24 h, the rates at 48 and 72 h were significantly lower than the control group (P < 0.05). 3.2. The effect of Sertoli cells on ECs VEGF receptor-2 (KDR) expression Western blotting results showed that ECs in the control group expressed KDR (Fig. 2A), and the relative expression intensities at 24 and 48 h were 0.59 ± 0.13 and 0.65 ± 0.15, respectively (Fig. 2B). When co-cultured with 1 103 and 1 104 cell/ml Sertoli cells for 24 and 48 h, KDR expression was significantly increased, and increased continually with extended incubation time (Fig. 2A). In co-culture with 1 103 cell/ml Sertoli cells, the relative expression intensities of KDR at 24 h and 48 h were 0.89 ± 0.19 and 1.13 ± 0.22, respectively, significantly higher than that in the control group (P < 0.05). In co-culture with 1 104 cell/ml Sertoli cells, the relative expression intensities of KDR at 24 and 48 h were 1.45 ± 0.27 and 1.73 ± 0.30, respectively, significantly higher than those in the control group and co-culture group with 1 103 cell/ml Sertoli cells (P < 0.05). In co-culture group with 1 105 cell/ml Sertoli cells, KDR expression in ECs was decreased (Fig. 2A) and the relative expression level at 24 h was 0.55 ± 0.12, not significantly different from the control group; the relative expression level decreased to 0.37 ± 0.08 at 48 h, significantly lower than that in the control group (Fig. 2B, P < 0.05). 3.3. The effect of Sertoli cells on INF-c -induced MHC II class protein expression in co-cultured ECs
Fig. 1. Microscopic observation of ECs in different groups. ECs were cultured for 24 h and examined under an inverted phase contrast microscope. Co-culture group with 1 104 cell/ml of Sertoli cells had the largest number of ECs and smallest gaps between cells. Co-culture group with 1 105 cell/ml of Sertoli cells had the least number of ECs and the largest gaps between cells. The proliferation rate of ECs in the co-cultured group with 1 104 cell/ml Sertoli cells was significantly higher than the other three groups.
Western blotting results showed that the expression of MHC II-like protein in the control group was very low without adding INF-c (Fig. 3A), and the relative expression intensity was 0.13 ± 0.08 (Fig. 3B). In the presence of INF-c, MHC II-like protein expression in pure ECs culture and co-culture group was increased (Fig. 3A), and the relative expression intensity of the two groups
Fig. 2. KDR expressions in ECs detected by Western blotting after 24 and 48 h culture. Co-culture group with 1 104 cell/ml of Sertoli cells had the highest expression level of KDR on ECs. Co-culture group with 1 105 cell/ml of Sertoli cells had the lowest expression level of KDR on ECs. The KDR expression level of ECs in co-culture group with 1 103 cell/ml of Sertoli cells was higher than that in 1 105 but lower than that in 1 104 cell/ml of Sertoli cells co-culture group.
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Fig. 3. MHC II class protein expression by Western Blotting. ECs had low expression of MHC II-like protein and significantly increased in the presence of IFN-g added into culture media. But the expression of MHC II-like protein level in ECs induced by in the IFN-g decreased significantly after co-cultured with Sertoli cells, though was still higher significantly than control group.
were 1.02 ± 0.21 and 0.52 ± 0.15 respectively, significantly higher (P < 0.05) than the control group. The INF-c-induced MHC II-like protein expression in pure ECs culture was significantly higher than in the co-culture group (P < 0.05) (Fig. 3B).
that in the pure ECs culture group (P < 0.05), and not significantly different from that in the control group (P > 0.05).
3.4. The effect of Sertoli cells on TNF-a-induced secretions of IL-6, IL-8 and sICAM in co-cultured ECs
In this study, two-chamber co-culture system was adopted [21], so that the two types of cells in co-culture had no direct contact with each other and thus avoided interference. Additionally, the two-chamber system was easy for assays and suitable for examination of the influence of Sertoli cells on ECs via the secretion of cytokines. In this study, three concentrations of Sertoli cells were co-cultured with a certain number of ECs, and their effects on the proliferation of ECs were a little different. The different numbers of Sertoli cells led to differences in the concentrations of cytokines that affect the proliferation of ECs, especially TGFb1, which affects EC proliferation dose-dependently [18], TGF-b1 promotes cell proliferation at low concentrations (0.1–5 lg/L), but inhibits cell proliferation at higher concentrations (>10 lg/L) [22,23]. The effect of co-culturing on KDR expression was similar to that on cell proliferation. In islet transplantation, islet cells express angiogenesis factor VEGF at high levels due to the hypoxic stimulation received during the isolation process [19,24]. Up-regulation of KDR expression enhances the responsiveness of ECs for VEGF. Therefore, Sertoli cells cannot only directly affect the proliferation of co-cultured ECs, but also indirectly affect EC proliferation by influencing cell VEGF receptor expression, both facilitate faster angiogenesis to complete the microvascular reconstruction. Because 1 104 cell/ml of Sertoli cells had the greatest effect on the proliferation of co-cultured ECs, we selected this concentration
Without TNF-a induction, ECs in the control group secreted IL-6, IL-8 and sICAM. Their concentrations as measured by ELISA were 89.9 ± 12.3 pg/ml, 105.5 ± 15.6 pg/ml and 55.4 ± 11.5 ng/ml respectively. With TNF-a induction, secretions of IL-6, IL-8 and sICAM by ECs in the EC single culture group increased to 254.2 ± 26.0 pg/ml, 386.6 ± 36.7 pg/ml and 98.7 ± 20.9 ng/ml respectively, significantly higher than in the control group (P < 0.05). With TNF-a induction, secretions of IL-6, IL-8 and sICAM by ECs in the co-culture group with Sertoli cells were 101.6 ± 15.3 pg/ml, 120.1 ± 19.4 pg/ml and 67.5 ± 15.2 ng/ml, respectively, significantly lower than in the single culture group plus TNF-a (P < 0.05), and not significantly different from the control group (P > 0.05, Fig. 4). 3.5. The effect of Sertoli cells on MLC of co-cultured ECs MLC results showed that the stimulation index of the control group on pure cultured spleen lymphocyte was 1.74 ± 0.05; the stimulation index of pure ECs culture group on spleen lymphocytes was 2.40 ± 0.12, significantly higher than that of the control group (P < 0.05); the stimulation index of the ECs in the co-culture group on spleen lymphocytes was 1.67 ± 0.05, significantly lower than
4. Discussion
Fig. 4. Cytokine concentrations in ECs medium by ELISA detection. IL-6, IL-8 (A) and sICAM (B) in the single culture group plus TNF-a were significantly higher than the other two groups.
P. Fan et al. / Biochemical and Biophysical Research Communications 404 (2011) 829–833
of Sertoli cells in the subsequent experiments to examine the immunogenicity of co-cultured ECs. MHC II antigen-induced lymphocyte activation is one of the important factors leading to rejection after transplantation [25]. ECs are antigenic because of their expression of MHC antigens. In the resting state, ECs do not express MHC II antigen [7]. Upon induction by INF-c produced by the lymphocytes in the receiver of transplantation, ECs up-regulate MHC II antigen expression [8], and thus further enhance the immunogenicity. Sertoli cells secrete TGF-b1, which inhibits the production of INF-c by lymphocytes [18]. Sertoli cells can inhibit INF-c-induced MHC II antigen protein expression, although not to the level in the control group. The reason may be that Sertoli cells secrete TGF-b1, which inhibits MHC II protein expression. This inhibition is able to reduce the antigenicity of ECs substantially. Together with the suppression on INF-c secretion by lymphocyte, Sertoli cells are able to maximally inhibit MHC II antigen expression on the surface of ECs. Cytokine is also one of the important factors contributing to transplantation rejection [26]. Another important display of EC immunogenicity is the increased expression of inflammatory cytokines secreted by macrophages after induction by TNF-a including IL-6, IL-8 and sICAM, which can recruit leukocytes and cause damage to the graft [9,10]. There was no report that Sertoli cells can inhibit macrophage secretion of TNF-a, thus it is particularly important to inhibit TNF-a-induced up-regulation of inflammatory cytokine expression in ECs. Sertoli cells exert strong inhibition on the expressions of inflammatory factors such as IL-6, IL-8 and sICAM in co-cultured ECs. Since TNF-a induces increased expression of inflammatory factors in ECs by NF-jB pathway [9,10], we speculate that the effect of Sertoli cells on EC secretion of inflammatory factors may also be achieved by inhibiting NF-jB. This is one of the directions for our future research. MLC is the best detection method for immunogenicity of ECs co-cultured with Sertoli cells. This study showed that cultured ECs alone can stimulate spleen lymphocytes leading to their rapid proliferation. The reason is that ECs are strongly immunogenic. This result may indicate that Sertoli cells reduced the immunogenicity of co-cultured ECs. At the same time, TGF-b1 secreted by Sertoli cells has a broad inhibitory effect on lymphocytes [27]. This may also be the reason that the lymphocyte stimulation index showed no obvious increase in the co-cultured group. In summary, we found that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate posttransplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes. However, the effect of Sertoli cells on ECs still needs to be validated in vivo through co-transplantation. Furthermore, it is not clear what effect Sertoli cells have on ECs when the cells are in direct contact, and this needs to be investigated in future experiment. Nevertheless, our research provides a new approach for the application of ECs in cell transplantation. References [1] A. Pileggi, C. Ricordi, M. Alessiani, L. Inverardi, Factors influencing islet of Langerhans graft function and monitoring, Clin. Chim. Acta. 310 (2001) 3–16. [2] C. Beger, V. Cirulli, P. Vajkoczy, P.A. Halban, M.D. Menger, Vascularization of purified pancreatic islet-like cell aggregates (pseudoislets) after syngeneic transplantation, Diabetes 47 (1998) 559–565. [3] F.A. Merchant, K.R. Diller, S.J. Aggarwal, A.C. Bovik, Angiogenesis in cultured and cryopreserved pancreatic islet grafts, Transplantation 63 (1997) 1652–1660.
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Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells Ping Fan ⇑, Lan He, Dan Pu, Xiaohong Lv, Wenxu Zhou, Yining Sun, Nan Hu Department of Rheumatism and Immunity, The First Affiliated Hospital Xi’an Jiaotong University School of Medicine, Xi’an, Shaanxi 710061, PR China
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Article history: Received 25 November 2010 Available online 21 December 2010 Keywords: Endothelial cells Sertoli cells Proliferation Immunogenicity Co-culture
a b s t r a c t The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 103, 1 104 or 1 105 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-c and TNF-a were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 104 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 104 cell/ ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-c-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-a induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction In cell transplantation, inadequate and delayed angiogenesis after transplantation result in shortage in the supply of oxygen and nutrients, leading to apoptosis or necrosis of the graft [1]. Endothelial cells (ECs) facilitate angiogenesis, and the loss of these cells is one of the important reasons for the delay in the reconstruction of microcirculation of transplanted cells [2–4]. In recent years, ECs have been used for islet transplantation [5]. But Cheng et al. showed that, although a certain number of ECs are transplanted, these ECs are in a resting state, and there is no extension to the inside of the islet to form endothelial growth sinusoid. Therefore, these cells are unable to establish microvascular system within the islet in a short term [6]. In addition, ECs themselves are strongly immunogenic. ECs do not express major histocompatibility complex (MHC) class II antigens at resting state, ⇑ Corresponding author: Fax: +86 29 85323976. E-mail address: [email protected] (P. Fan). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.068
but express MHC class II antigens upon stimulations such as virus infections or INF-c induction [7,8]. In addition, through expression of different inflammatory genes, such as interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM), promoted by the activation of tumor necrosis factor (TNF)-a [9,10], ECs recruit white blood cells and thus play an important role in the inflammatory process [11,12]. These reactions are able to induce the occurrence of rejection, resulting in the loss of grafts [13,14]. Thus, the key to solve the problem is to find a way to promote the proliferation while reduce the immunogenicity of ECs. Testis Sertoli cells (SC) has been confirmed to induce immune tolerance in organ transplantation [15]. SC secretes a variety of cytokines such as transforming growth factor (TGF)-b1 and basic fibroblast growth factor (bFGF) [16]. TGF-b1 is a potent immunosuppressive factor that suppresses the secretion of INF-c and TNF-a by immune cells [17], thus reduce graft rejection. In addition, TGF-b1 has a role in regulating cell proliferation; it promotes proliferation of ECs at certain concentrations [18]. bFGF also promotes angiogenesis activity [19]. However, the specific mechanism
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of TGF-b1 in promoting EC proliferation is not clear. Additionally, there has been no report on whether TGF-b1 reduces the upregulation of the expressions of MHC class II antigens and inflammatory cytokines induced by INF-c and TNF-a. Therefore, we cocultured the Sertoli cells with ECs and examined the impact of the Sertoli cells on EC proliferation and immunogenicity in order to find better ways to use ECs in cell transplantation. 2. Materials and methods
respectively. At 24, 48 and 72 h, five wells were sampled at each time point. Culture media were discarded and the culture insert containing the Sertoli cells were taken out. Trypsin at 2.5 g/L was added to each well and the cells were dispersed by trituration and then mixed with RPMI 1640 medium with 10% FCS. Cell density was determined with a hematocytometer, and the total number of cells per well was calculated. The proliferation rates of ECs in two groups were compared at different time points. Proliferation rate = cells harvested at different time point/initial seeding cells (1 104) 100%.
2.1. Cell line and main reagents The human umbilical vein endothelial cell (HUVEC) line ECV304 was obtained from the Cell Bank of Chinese Academy of Medicine. The cells were seeded into 25 ml culture flask at 1 107 cells per flask in 5 ml RPMI 1640 culture medium (Sigma, USA) containing 100 ml/L fetal calf serum (FCS, Gibco, USA). The cells were then incubated in a humidified incubator at 37 °C with 5% CO2. Culture media were changed every day. 2.2. Isolation of Sertoli cells Adult testes were retrieved in sterile conditions from deceased male multi-organ donors. Briefly, seminiferous tubules separated from testes were subjected to a two-step sequential enzymatic treatments at 37 °C with trypsin (Sigma, USA) and DNase (Sigma, USA) for 30 min in the first step and collagenase P and hyaluronidase (Sigma, USA) for 20 min in the second step, followed by RPMI-1640 culture medium containing 100 ml/L fetal bovine serum to terminate the digestion at 4 °C. After 5–10 min incubation at room temperature, digested tissue was filtered through a 75 lm mesh stainless steel filter. Sertoli aggregates were plated and incubated at 39 °C under 5% CO2 for 48 h. Cells were then subjected to hypotonic treatment with 20 mM Tris–HCl buffer (Sigma, USA). Sertoli cells were replenished with RPMI-1640 medium supplemented with 100 ml/L FCS and incubated at 37 °C under 5% CO2. Culture media were changed every other day and the resulting pretreated Sertoli-enriched monocultures contained greater than 95% Sertoli cells. 2.3. Preparation of spleen lymphocytes Human spleen lymphocytes were isolated as previously described with minor modification [20]. The human spleen placed in 200 mesh aseptic milling steel line was repeatedly milled. After filtered twice, the cell suspension was then centrifuged at 1500 r/min at 4 °C for 10 min. The cell pellet was washed twice with PBS, and then mixed with the human lymphocyte separating medium (LYMPHOLYTE, USA), and centrifuged at 1500 r/min for 20 min to absorb the mononuclear cell layer. 2.4. Observation of the effect of Sertoli cells on the proliferation of ECs When confluent, ECs were digested with trypsin at 2.5 g/L and prepared into a cell suspension of 1 104 cell/ml. The cells were divided into two groups, and each group had 10 wells, with 1 104 cell in 1 ml medium in each well. In the control group, the cells were seeded on normal 24-well plates. The plates were incubated at 37 °C under 5% CO2 for 6 h to allow cell adherence, and the culture media were then discarded and replaced with 2 ml of RPMI 1640 medium. In the experimental group, the cells were seeded on a special 24-well cell culture plate (HTS transwell, Corning, USA) for co-culturing. Three sub-groups were set up with 2 103, 2 104 and 2 105 Sertoli cells in the cell culture inserts (HTS transwell, Corning, USA), and RPMI 1640 medium with 10% fetal calf serum was added to reach 2 ml for co-culturing. The densities of Sertoli cells were 1 103, 1 104 and 1 105 cell/ml,
2.5. Western blot analysis of vascular endothelial growth factor (VEGF) receptor-2 (KDR) of ECs ECs in each group were collected after culturing for 24 and 48 h respectively. Cells were homogenized in SDS-1 solubilization buffer (Sigma, USA). Twenty milligram of protein were loaded and separated on a 12% SDS–PAGE followed by transferring to a polyvinyldifluoride membrane (Immobilon-P transfer membrane; Millipore, Billerica, MA, USA). After blocking non-specific binding sites, the membrane was incubated for 2 h at room temperature with a mouse polyclonal antibody against KDR (1:1000 dilution, Cell Signaling Technology, USA) followed by incubation with a secondary peroxidase-linked anti-mouse antibody (Tropix, PE Applied Biosystems, Germany; 1:10000 dilution). Protein expression was visualized by enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech, Germany) and digitized with ChemiDocTM XRS System (Quantity One, Bio-Rad Laboratories GmbH, Munich, Germany). Signals were densitometrically assessed (Quantity One) and normalized to the b-actin signals as loading controls (mouse monoclonal anti-b-actin antibody, 1:10000; Sigma). 2.6. Western blot analysis of MHC class II proteins of ECs ECs in each group were collected after culturing for 24 h. Except the mouse polyclonal antibodies against human nonpolymorphic MHC class II molecules (1:100 dilution) were used, other western blot steps were the same as described above. Protein expression was visualized by enhanced chemiluminescence and digitized with ChemiDocTM XRS System. Signals were densitometrically assessed and normalized to the b-actin signals as loading controls. 2.7. Detection of IL-6, IL-8 and sICAM by ELISA The optimum concentration of Sertoli cells was selected to promote ECs proliferation in the co-culture experiments described above. ECs were divided into three groups, seeded on the glass coverslips in a 24-well plate with each group consisted of five wells with 1 104 cell/well. In the negative control group, 2 ml of RPMI 1640 medium containing 10% FCS was added to each well; in the positive control group, 2 ml of 1640 medium containing 10% FCS and 0.5 lg/L TNF-a (Sigma, USA) were added; in the experimental group, ECs were co-cultured with Sertoli cells, 1640 medium containing 10% FCS and 0.5 lg/L TNF-a. Culture supernatants in each group were collected after culturing for 24 h and kept frozen at 80 °C. Commercially available ELISA kits were used to determine the concentrations of IL-6, IL-8 and sICAM (Bender, Austria). 2.8. 3H TdR incorporation in one-way mixed lymphocyte culture (MLC) ECs were set as stimulating cells and spleen lymphocytes as response cells. The experimental group was divided into two subgroups, EC single culture group and co-culture group. Each group had five wells. In the single culture group, 1 104 cell/well ECs were seeded in each well in ordinary 24-well plate. In the coculture group, 1 104 cell/well ECs were seeded in each well in
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the 24-well plate with inserts containing 1 104 of the Sertoli cells, cultured in a final volume of 800 ll 1640 medium containing 50 mg/L of mitomycin C. Pure spleen lymphocyte group was used as the control group, and wells with medium alone were used as blank control. The cells were cultured for 96, and 16 h before the end of culturing, 3H TdR (2 lCi/well) was added. The radioactivity (CPM) of ECs was measured with an automatic liquid scintillation counter. Stimulation index (SI) was calculated as measured CPM/ control CPM. 2.9. Statistical analysis The data were expressed as mean ± SEM. Inter-group statistical comparisons were made by one-way analysis of variance (ANOVA) using SPSS statistical software (version 13.0, SPSS Inc., USA). A P value less than 0.05 was considered statistically significant. 3. Results 3.1. The effect of Sertoli cells on the proliferation of co-cultured ECs The counting of ECs showed (Fig. 1) that the proliferation rates at 24, 48 and 72 h when co-cultured with 1 103 cell/ml Sertoli
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cells were 135.8 ± 21.0, 208.4 ± 29.0 and 295.1 ± 37.0, respectively, significantly higher than those at the same time point in the control group (24 h, 107.1 ± 15.9; 48 h, 167.2 ± 23.8; 72 h, 227.3 ± 30.5; P < 0.05). When co-cultured with 1 104 cell/ml Sertoli cells, the proliferation rates at 24, 48 and 72 h were as high as 175.7 ± 26.0, 237.6 ± 49.0 and 363.5 ± 55.0 respectively, not only significantly higher than the control group, but also significantly higher than the co-culture group with 1 103 cell/ml Sertoli cells (P < 0.05). When co-cultured with 1 105 cell/ml Sertoli cells, the proliferation rates at 24, 48 and 72 h were 105.8 ± 14.3, 130.2 ± 19.4 and 180.3 ± 20.4, respectively. While there was no significant difference compared with the control group at 24 h, the rates at 48 and 72 h were significantly lower than the control group (P < 0.05). 3.2. The effect of Sertoli cells on ECs VEGF receptor-2 (KDR) expression Western blotting results showed that ECs in the control group expressed KDR (Fig. 2A), and the relative expression intensities at 24 and 48 h were 0.59 ± 0.13 and 0.65 ± 0.15, respectively (Fig. 2B). When co-cultured with 1 103 and 1 104 cell/ml Sertoli cells for 24 and 48 h, KDR expression was significantly increased, and increased continually with extended incubation time (Fig. 2A). In co-culture with 1 103 cell/ml Sertoli cells, the relative expression intensities of KDR at 24 h and 48 h were 0.89 ± 0.19 and 1.13 ± 0.22, respectively, significantly higher than that in the control group (P < 0.05). In co-culture with 1 104 cell/ml Sertoli cells, the relative expression intensities of KDR at 24 and 48 h were 1.45 ± 0.27 and 1.73 ± 0.30, respectively, significantly higher than those in the control group and co-culture group with 1 103 cell/ml Sertoli cells (P < 0.05). In co-culture group with 1 105 cell/ml Sertoli cells, KDR expression in ECs was decreased (Fig. 2A) and the relative expression level at 24 h was 0.55 ± 0.12, not significantly different from the control group; the relative expression level decreased to 0.37 ± 0.08 at 48 h, significantly lower than that in the control group (Fig. 2B, P < 0.05). 3.3. The effect of Sertoli cells on INF-c -induced MHC II class protein expression in co-cultured ECs
Fig. 1. Microscopic observation of ECs in different groups. ECs were cultured for 24 h and examined under an inverted phase contrast microscope. Co-culture group with 1 104 cell/ml of Sertoli cells had the largest number of ECs and smallest gaps between cells. Co-culture group with 1 105 cell/ml of Sertoli cells had the least number of ECs and the largest gaps between cells. The proliferation rate of ECs in the co-cultured group with 1 104 cell/ml Sertoli cells was significantly higher than the other three groups.
Western blotting results showed that the expression of MHC II-like protein in the control group was very low without adding INF-c (Fig. 3A), and the relative expression intensity was 0.13 ± 0.08 (Fig. 3B). In the presence of INF-c, MHC II-like protein expression in pure ECs culture and co-culture group was increased (Fig. 3A), and the relative expression intensity of the two groups
Fig. 2. KDR expressions in ECs detected by Western blotting after 24 and 48 h culture. Co-culture group with 1 104 cell/ml of Sertoli cells had the highest expression level of KDR on ECs. Co-culture group with 1 105 cell/ml of Sertoli cells had the lowest expression level of KDR on ECs. The KDR expression level of ECs in co-culture group with 1 103 cell/ml of Sertoli cells was higher than that in 1 105 but lower than that in 1 104 cell/ml of Sertoli cells co-culture group.
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Fig. 3. MHC II class protein expression by Western Blotting. ECs had low expression of MHC II-like protein and significantly increased in the presence of IFN-g added into culture media. But the expression of MHC II-like protein level in ECs induced by in the IFN-g decreased significantly after co-cultured with Sertoli cells, though was still higher significantly than control group.
were 1.02 ± 0.21 and 0.52 ± 0.15 respectively, significantly higher (P < 0.05) than the control group. The INF-c-induced MHC II-like protein expression in pure ECs culture was significantly higher than in the co-culture group (P < 0.05) (Fig. 3B).
that in the pure ECs culture group (P < 0.05), and not significantly different from that in the control group (P > 0.05).
3.4. The effect of Sertoli cells on TNF-a-induced secretions of IL-6, IL-8 and sICAM in co-cultured ECs
In this study, two-chamber co-culture system was adopted [21], so that the two types of cells in co-culture had no direct contact with each other and thus avoided interference. Additionally, the two-chamber system was easy for assays and suitable for examination of the influence of Sertoli cells on ECs via the secretion of cytokines. In this study, three concentrations of Sertoli cells were co-cultured with a certain number of ECs, and their effects on the proliferation of ECs were a little different. The different numbers of Sertoli cells led to differences in the concentrations of cytokines that affect the proliferation of ECs, especially TGFb1, which affects EC proliferation dose-dependently [18], TGF-b1 promotes cell proliferation at low concentrations (0.1–5 lg/L), but inhibits cell proliferation at higher concentrations (>10 lg/L) [22,23]. The effect of co-culturing on KDR expression was similar to that on cell proliferation. In islet transplantation, islet cells express angiogenesis factor VEGF at high levels due to the hypoxic stimulation received during the isolation process [19,24]. Up-regulation of KDR expression enhances the responsiveness of ECs for VEGF. Therefore, Sertoli cells cannot only directly affect the proliferation of co-cultured ECs, but also indirectly affect EC proliferation by influencing cell VEGF receptor expression, both facilitate faster angiogenesis to complete the microvascular reconstruction. Because 1 104 cell/ml of Sertoli cells had the greatest effect on the proliferation of co-cultured ECs, we selected this concentration
Without TNF-a induction, ECs in the control group secreted IL-6, IL-8 and sICAM. Their concentrations as measured by ELISA were 89.9 ± 12.3 pg/ml, 105.5 ± 15.6 pg/ml and 55.4 ± 11.5 ng/ml respectively. With TNF-a induction, secretions of IL-6, IL-8 and sICAM by ECs in the EC single culture group increased to 254.2 ± 26.0 pg/ml, 386.6 ± 36.7 pg/ml and 98.7 ± 20.9 ng/ml respectively, significantly higher than in the control group (P < 0.05). With TNF-a induction, secretions of IL-6, IL-8 and sICAM by ECs in the co-culture group with Sertoli cells were 101.6 ± 15.3 pg/ml, 120.1 ± 19.4 pg/ml and 67.5 ± 15.2 ng/ml, respectively, significantly lower than in the single culture group plus TNF-a (P < 0.05), and not significantly different from the control group (P > 0.05, Fig. 4). 3.5. The effect of Sertoli cells on MLC of co-cultured ECs MLC results showed that the stimulation index of the control group on pure cultured spleen lymphocyte was 1.74 ± 0.05; the stimulation index of pure ECs culture group on spleen lymphocytes was 2.40 ± 0.12, significantly higher than that of the control group (P < 0.05); the stimulation index of the ECs in the co-culture group on spleen lymphocytes was 1.67 ± 0.05, significantly lower than
4. Discussion
Fig. 4. Cytokine concentrations in ECs medium by ELISA detection. IL-6, IL-8 (A) and sICAM (B) in the single culture group plus TNF-a were significantly higher than the other two groups.
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of Sertoli cells in the subsequent experiments to examine the immunogenicity of co-cultured ECs. MHC II antigen-induced lymphocyte activation is one of the important factors leading to rejection after transplantation [25]. ECs are antigenic because of their expression of MHC antigens. In the resting state, ECs do not express MHC II antigen [7]. Upon induction by INF-c produced by the lymphocytes in the receiver of transplantation, ECs up-regulate MHC II antigen expression [8], and thus further enhance the immunogenicity. Sertoli cells secrete TGF-b1, which inhibits the production of INF-c by lymphocytes [18]. Sertoli cells can inhibit INF-c-induced MHC II antigen protein expression, although not to the level in the control group. The reason may be that Sertoli cells secrete TGF-b1, which inhibits MHC II protein expression. This inhibition is able to reduce the antigenicity of ECs substantially. Together with the suppression on INF-c secretion by lymphocyte, Sertoli cells are able to maximally inhibit MHC II antigen expression on the surface of ECs. Cytokine is also one of the important factors contributing to transplantation rejection [26]. Another important display of EC immunogenicity is the increased expression of inflammatory cytokines secreted by macrophages after induction by TNF-a including IL-6, IL-8 and sICAM, which can recruit leukocytes and cause damage to the graft [9,10]. There was no report that Sertoli cells can inhibit macrophage secretion of TNF-a, thus it is particularly important to inhibit TNF-a-induced up-regulation of inflammatory cytokine expression in ECs. Sertoli cells exert strong inhibition on the expressions of inflammatory factors such as IL-6, IL-8 and sICAM in co-cultured ECs. Since TNF-a induces increased expression of inflammatory factors in ECs by NF-jB pathway [9,10], we speculate that the effect of Sertoli cells on EC secretion of inflammatory factors may also be achieved by inhibiting NF-jB. This is one of the directions for our future research. MLC is the best detection method for immunogenicity of ECs co-cultured with Sertoli cells. This study showed that cultured ECs alone can stimulate spleen lymphocytes leading to their rapid proliferation. The reason is that ECs are strongly immunogenic. This result may indicate that Sertoli cells reduced the immunogenicity of co-cultured ECs. At the same time, TGF-b1 secreted by Sertoli cells has a broad inhibitory effect on lymphocytes [27]. This may also be the reason that the lymphocyte stimulation index showed no obvious increase in the co-cultured group. In summary, we found that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate posttransplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes. However, the effect of Sertoli cells on ECs still needs to be validated in vivo through co-transplantation. Furthermore, it is not clear what effect Sertoli cells have on ECs when the cells are in direct contact, and this needs to be investigated in future experiment. Nevertheless, our research provides a new approach for the application of ECs in cell transplantation. References [1] A. Pileggi, C. Ricordi, M. Alessiani, L. Inverardi, Factors influencing islet of Langerhans graft function and monitoring, Clin. Chim. Acta. 310 (2001) 3–16. [2] C. Beger, V. Cirulli, P. Vajkoczy, P.A. Halban, M.D. Menger, Vascularization of purified pancreatic islet-like cell aggregates (pseudoislets) after syngeneic transplantation, Diabetes 47 (1998) 559–565. [3] F.A. Merchant, K.R. Diller, S.J. Aggarwal, A.C. Bovik, Angiogenesis in cultured and cryopreserved pancreatic islet grafts, Transplantation 63 (1997) 1652–1660.
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