Hepatic Stellate Cells Induce Immunotolerance of Islet Allografts Z.-Y. Zhanga, Z.-Q. Zhoua, K.-B. Songb, S.-C. Kimb, and G.-W. Zhoua,* From the aDepartment of Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China; and the bDivision of Hepatobiliary and Pancreatic Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
ABSTRACT Activated hepatic stellate cells (HSCs) possess strong immune inhibitory activity. The present study highlighted the protective role of HSCs in islet transplantation. Recipients were randomly divided into 4 groups: a diabetic group, an HSC-alone group, an islet-alone transplant group, and a cotransplant group. Graft survival was compared among the 4 groups. Serum transforming growth factor b (TGFb), tumor necrosis factor a, interleukin1b, and interferon gamma expression levels were measured. The infiltration of lymphocytes was observed via hematoxylin and eosin staining, and immunohistochemical examinations were performed. Results showed that allogeneic HSCs protect islet allografts better than syngeneic HSCs. There was significant prolonged graft survival and a higher level of TGFb in the cotransplant group (P < .01). The infiltration of lymphocytes in the cotransplant group was notably less than in the islet-alone group (P < .01). The formation of desminpositive HSC packages was detected in the cotransplant group. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFb expression and forming a biological capsule around the graft.
T
YPE 1 diabetes is one of the most serious organ-specific autoimmune diseases that damages the islet b cells by abnormally secreting autoantibodies and inducing a variety of complications, seriously affecting health. Long-term follow-up suggests that complications of type 1 diabetes are not efficiently prevented by drug intake alone. Allogeneic islet transplantation is considered one of the most potentially therapeutic treatments for type 1 diabetes [1,2], but the adverse effects caused by long-term immunosuppression severely hamper the promotion of this option. Hence, attention has been drawn to the induction of immune tolerance. Hepatic stellate cells (HSCs) are a population of liver nonparenchymal cells in the space of Disse between the hepatocyte and hepatic sinusoidal endothelial cells. When activated, HSCs have anti-inflammatory activity primarily mediated by transforming growth factor b (TGFb) [3]. HSCs express very few key immune surface molecules in the quiescent stage. However, upon activation, HSCs upregulate TGFb messenger ribonucleic acid, causing TGFb oversecretion, which suppresses the activity of Th1 cells. Activated HSCs produce interleukin (IL)-10 to restrain macrophagocytes, and IL-6, which has dual immunomodulatory activity [4]. Kobayashi et al [5] and Yu et al [6]
reported that HSCs express intercellular adhesion molecule 1, major histocompatibility complex, B7, and B7-H1, and activated HSCs show strong inhibition of T-cell response via enhanced T-cell apoptosis. The purpose of the present study was to determine if HSCs serve a function in the graft protection of islet transplants. MATERIALS AND METHODS Animal Male BALB/c mice (6e8 weeks old, 24e26 g) were used as islet donors, and male C57BL/6 mice (6e8 weeks old, 23e27 g) were used as recipients. The mice were purchased from the Chinese Academy of Science (Shanghai, China) and were housed in laminar air flow cabinets under specific pathogen-free conditions. Mice were cared
This work was supported by grants from the Research and Innovation Project of Shanghai Municipal Education Commission (grant 11ZZ100) and the National Natural Science Foundation of China (grant 81170721). *Address correspondence to Guang-Wen Zhou, Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, 200233, Shanghai, China. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.03.009
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 46, 1594e1600 (2014)
HEPATIC STELLATE CELLS AND IMMUNOTOLERANCE for and handled according to the recommendations of the National Institute of Health Guidelines for Care and Use of Laboratory Animals. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Committee. Animals were fasted for 24 hours before experiments, but water was given.
1595 Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, United States) with 10% fetal calf serum, L-glutamine 2 mmol/L, and HEPES 25 mmol/L (Mediatech, Inc, Herndon, Va, United States).
Isolation and Culture of Mouse HSCs Mouse Islet Isolation Islets were isolated from BALB/c mice via pancreas perfusion, collagenase XI (Sigma-Aldrich Co LLC, St Louis, Mo, United States) digestion, and purification with Ficoll (GE Healthcare, Little Chalfont, United Kingdom) discontinuous gradient centrifugation [7]. Islets were identified by following the instructions of a previous study. Islet purity was assessed by dithizone (SigmaAldrich Co LLC, St Louis, Mo, United States) staining, and cell mass was counted by using a microscope. Islet function was assessed with the use of an insulin-releasing test. Islet viability was assessed by acridine orangeepropidium iodide (Sigma-Aldrich Co LLC, St Louis, Mo, United States) fluorescent staining; living cells exhibited green staining (acridine orange), whereas dead cells showed brownered staining (propidium iodide). Islets were cultured in
HSCs were isolated from BLAB/c mice and C57BL/6 mice via liver perfusion, collagenase IV (Sigma-Aldrich Co LLC) digestion, colation, lavation, and purification by 60% Percoll (GE Healthcare) gradient centrifugation. The stratosphere of the centrifugate was composed of a liver cell layer beneath the HSC layer. The HSC layer was collected and cultured. After 48 hours of culturing, immunocytochemical staining was performed, and cells were observed by using an inverted microscope. Cell yield was estimated as cell population ¼ (cell amount of 4 parts in globulimeter/4) 104 cell suspension volume (in milliliters). Each sample was estimated twice to obtain the average. HSC viability was assessed by trypan blue staining. HSCs viability (%) ¼ alive cell amount/total cell amount 100%. HSC purity was assessed by desmin immunocytochemical staining, and cells were counted according to the
Fig 1. Isolation and activation of hepatic stellate cells (HSCs) (representative figures from BALB/c mice). (A) HSCs cultured for 4 hours. (B) HSCs (synapse with bright lipid droplet) cultured for 24 hours. (C) Fully activated growth of HSCs. (D) HSCs cultured for 11 days showed a fibroblast-like morphology. (E) HSCs cultured for 5 days expressed desmin (red). The nucleus was labeled by 40 ,6-diamidino-2-phenylindole staining (blue). (F) HSCs cultured for 7 days expressed a-smooth muscle actin (green). The nucleus was labeled by 40 ,6diamidino-2-phenylindole staining (blue).
1596 population of desmin-positive cells. HSCs purity (%) ¼ desminpositive cell/total cell amount 100%.
Optimization of the HSC Dose A total of 1.5 105, 3 105, and 4.5 105 HSCs from either BALB/c mice or C57BL/6 mice were cotransplanted with 300 islet equivalents (IEQ; 1 IEQ ¼ 1150 mm islet) from BALB/c mice into the subcapsular space of the left kidney of C57BL/6 mice [7]. The optimized HSC dose was identified.
Islet Transplantation C57BL/6 mice were fasted for 18 hours, before being rendered diabetic by a single intraperitoneal injection of streptozotocin (Sigma-Aldrich Co LLC, St Louis, Mo, United States) at a dose of 200 mg/kg. At 72 hours after administration, mice with 3 consecutive random daily blood glucose readings >20 mmol/L were used as islet recipients. Recipients were randomly divided into 3 groups: the nontransplant diabetic group (n ¼ 8), the HSC transplant group (n ¼ 8), the islet-alone transplant group (n ¼ 9), and the HSC and islet cotransplant group (n ¼ 10). Blood glucose was monitored daily after transplantation. Transplantation and graft survival were considered successful if the nonfasting blood glucose level returned to normal and remained normal (20 mmol/L after a period of normoglycemia. The first of 2 consecutive days in which blood glucose readings were >20 mmol/L was defined as the date of islet graft failure. Seven days after transplantation, 3 recipients in each group underwent cervical dislocation, and their grafts were preserved for further testing.
Glucose Tolerance Test An intraperitoneal glucose tolerance test (IPGTT) was performed 3 days after normoglycemia was achieved following transplantation. Mice were fasted for 12 hours before administering an intraperitoneal injection of 50% dextrose solution at a dose of 6 g/kg. Blood glucose was measured before and after glucose injection at specific time points (0, 15, 30, 60, 90, 120, and 150 minutes) by a glucose meter through tail vein hemospasia.
Fig 2. Optimization of the hepatic stellate cell (HSC) dose to protect islet allografts; 3 105 HSCs and 4.5 105 HSCs collected from both BALB/c and C57BL/6 mice protected BALB/c islets after cotransplantation (HSCs þ 300 islet equivalent islets) (P < .01). The effect of 3 105 HSCs did not differ from the effect of 4.5 105 HSCs in protecting the islet graft. HSCs from BALB/c mice exhibited better protection of islets compared with HSCs from C57BL/6 mice (P < .01) (n ¼ 8 in each group).
ZHANG, ZHOU, SONG ET AL
Cytokine Measurement Serum interferon gamma, tumor necrosis factor a, IL-1b, and TGFb levels in the mice were measured 7 days after transplantation by using an enzyme-linked immunosorbent assay in accordance with the test kit instructions (Jingmei Bioscience Ltd, Shanghai, China).
Hematoxylin and Eosin Staining Islet graft specimens were fixed in 10% formalin solution for 24 hours and embedded in paraffin. Sections (3 mm) were dewaxed in dimethyl benzene and washed with gradient ethanol followed by distilled water. Specimens were stained with hematoxylin for 5 minutes, placed in hydrochloric acid for 30 seconds (alcoholation), soaked in distilled water for 15 minutes, stained with eosin, and then finalized after gradient ethanol dehydration.
Immunohistochemistry and Immunofluorescent Staining After dewaxing, sections were subjected to microwave antigen retrieval in citrate phosphate buffer (0.01 M). To block nonspecific reactivity and staining from endogenous peroxidase, sections were incubated in hydrogen peroxide (1%) for 20 minutes and in serumfree protein block (Dako Co, Tokyo, Japan) for 5 minutes. After rinsing, slides were incubated overnight at 4 C with insulin primary antibody (1:100) (Sigma-Aldrich Co LLC, St Louis, Mo, United States) desmin primary antibody (1:100) (Sigma-Aldrich Co LLC), and a-smooth muscle actin primary antibody (1:200) (SigmaAldrich Co LLC). Secondary antibodies and 3,30 -diaminobenzidine tetrahydrochloride chromogen solution was added to the slides (Dako Real Envision Detection System, Dako Denmark A/S, Glostrup, Denmark). Slides were counterstained with hematoxylin. Images were acquired by using a computerized image system (ScanScope XT, Vista, Calif, United States). Immunofluorescent staining used the same procedure as hematoxylin and eosin staining until the second antibody incubation. Sections were incubated in lucifugal places at room temperature for 60 minutes; 40 ,6-diamidino-2-phenylindole was added, and sections were finalized after PBST washing. Slides were observed under immunofluorescent microscope.
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Statistical Analysis Data are expressed as mean values SDs. The Student t test and one-way analysis of variance were used to compare mean values. Kaplan-Meier curves were used for survival analysis, and P < .05 was considered statistically significant.
RESULTS Purity and Activity of Freshly Isolated Islets and HSCs
A mean of 190.00 10.98 islets could be obtained from 1 donor. The purity of isolated islets was >90%, and the activity of islets was >95%. The insulin-releasing amount of islets under stimulation of low-concentration and highconcentration glucose was 3.65 0.49 and 10.40 0.80 mmol/L, respectively. The insulin amount in highconcentration glucose stimulation was 3 times more than that in low-concentration glucose stimulation, which was a statistically significant difference (P < .01). A mean of (4.90 0.40) 105 HSCs could be obtained from 1 mouse (BALB/c or C57BL/6). The purity of HSCs was >90%. The activity of HSCs was >95% (Fig 1).
Fig 4. Blood glucose levels were tested daily after transplantation in recipient mice. Mice in the cotransplant group (n ¼ 10) had a significantly longer period of normoglycemia than those in the islet-alone group (n ¼ 9) (P ¼.008).
Effects of Cotransplantation With Allogeneic HSCs
In this study, 3 105 HSCs and 4.5 105 HSCs collected from both BALB/C and C57BL/6 mice protected BALB/C islets after cotransplantation (HSCs þ 300 IEQ islets) (P < .01). The effect of 3 105 HSCs did not differ from the effect of 4.5 105 HSCs in protecting the islet graft. HSCs from BALB/c mice exhibited better protection of islets compared with HSCs from C57BL/6 mice (P < .01) (n ¼ 8 in each group) (Fig 2). Therefore, 3 105 HSCs from BLAB/c mice were selected to be cotransplanted with islets (3 105 HSCs þ 300 IEQ islets) for the succeeding experiments.
Twenty days after transplantation, blood glucose levels were better controlled in the islet-alone and cotransplant groups compared with the diabetic and HSC-alone groups (P < .01). The cotransplant group had a significantly lower blood glucose trend than the islet-alone group (P < .05). The diabetic group and the HSC-alone group did not have a period of normoglycemia, and the blood glucose level did not differ between groups (Fig 3). The blood glucose level was >20 mmol/L on day 20 in the islet-alone group, which was regarded as graft rejection; in the cotransplant group, the blood glucose level remained normal. The difference was statistically significant (P < .05). The survival of islets was monitored and represented by normoglycemic periods in recipients. The normoglycemia time in the cotransplant
Fig 3. The blood glucose level was tested daily after transplantation. From day 20, the blood glucose level in the islet-alone group was >20 mmol/L. The cotransplant group had a significantly lower blood glucose level than the islet-alone group (*P < .05), the diabetic group, and the hepatic stellate cell (HSC)-alone group (#P < .01). The blood glucose level did not differ between the diabetic group and the HSC-alone group.
Fig 5. The intraperitoneal glucose tolerance test of the islet graft. Compared with the diabetic group and the hepatic stellate cell (HSC)-alone group, the blood glucose levels in the islet-alone group and the cotransplant group were significantly lower (*P < .01). The mean blood glucose level in the islet-alone group was insignificant compared with the cotransplant group.
Optimization of HSC Dose to Protect Islet Allograft
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ZHANG, ZHOU, SONG ET AL
compared with the islet-alone group. The blood glucose levels in both the diabetic group and the HSC-alone group did not differ significantly (Fig 5). Expression of Cytokines In Vivo
Fig 6. The blood concentration of cytokines in the recipients. Transforming growth factor b (TGFb) levels in the cotransplant group were much higher than those in the islet-alone group, the hepatic stellate cell (HSC)-alone group, and the diabetic group (*P < .01). TGFb levels in the groups other than the cotransplant group did not differ significantly. The expression levels of interferon gamma, interleukin-1b (IL-1b), and tumor necrosis factor a (TNF-a) were not significantly different among the 4 groups.
group was significant longer than that of the islet-alone group (23.75 8.96 vs 11.90 6.92 days) (Fig 4). Evaluation of Islet Function In Vivo
IPGTT was performed at specific time points (0, 15, 30, 60, 90, 120, and 150 minutes) after intraperitoneal injection of dextrose injection. The cotransplant group and the isletalone group exhibited better glucose tolerance than the other groups (P < .01), but the average blood glucose level in the cotransplant group was statistically insignificant
Fig 7. Hematoxylin and eosin (HE) staining: (A and B) Islet-alone graft was detectable as small foci under the kidney capsule, with cell degeneration and apoptosis. Lymphocyte infiltrated around the graft. (C and D) Grafts in the cotransplant group exhibited nodules under the kidney capsule and were surrounded by a limited lymphocyte infiltration.
Cytokines were examined 1 week after transplantation. TGFb in the cotransplant group was much higher than that in the islet-alone group, the HSC-alone group, and the diabetic group (2292.31 50.87 pg/mL vs 1246.55 38.91, 1323.44 74.21, and 1280.65 55.44 pg/mL) (P < .01). TGFb in all groups except the cotransplant group did not differ significantly. The expression levels of interferon gamma, IL-1b, and tumor necrosis factor a were not significantly different among the 4 groups (Fig 6). Pathological Findings
Microscope images showed obvious lymphocyte infiltration around the islet grafts in the islet-alone group, whereas in the cotransplant group, lymphocyte infiltration was apparently much less. The grafts were in better morphological normality in the cotransplant group than in the islet-alone group (Fig 7). The cotransplant group expressed more insulin than the islet-alone group after transplantation (Figs 8A and 8B). The insulin immunofluorescence of the cotransplant group (Fig 8C) showed islet grafts surrounded by HSCs (Fig 8D), suggesting a tendency for islet grafts to be encapsulated by HSCs, which were desmin positive (Fig 8E). The nucleus of cells was labeled by using 40 ,6-diamidino-2-phenylindole staining (Fig 8F).
HEPATIC STELLATE CELLS AND IMMUNOTOLERANCE
DISCUSSION
Islets transplantation is regarded as one of the most potentially therapeutic treatments for type 1 diabetes [1,2], but the adverse effects caused by immunosuppressant drugs after transplantation have severely hindered the development of islet transplantation. Therefore, the induction of immune tolerance by using biological materials is critical. The present study demonstrated that HSC cotransplants with islets may prolong the allograft survival of islets. Allogeneic HSCs exhibited this trend more apparent than syngeneic HSCs. However, the mechanisms underlying the inconsistent protective function of allogeneic and syngeneic HSCs are still unknown. Recipients who underwent islet and allogeneic HSC cotransplants maintained normoglycemia for a much longer time than the recipients of islets alone. Blood glucose was better controlled in the cotransplant recipients than in the islet-alone recipients. These findings suggest that allogeneic HSCs protect the allograft from being rejected and play a vital function in immune
1599
regulation. The functional efficiency of islet was determined by using IPGTT tests in vivo. Results showed that there was no difference in the regulation of instant hyperglycemia in both groups, indicating that although the activated allogeneic HSCs protected the islet grafts from rejection, they could not improve the functional efficiency of the graft. However, interestingly, we found that cotransplant islets had better insulin-releasing and blood glucose regulation ability than the transplant of islets alone (based on bloodglucose monitoring and immunochemical staining). Previous reports have indicated that the costimulatory molecule B7-H1 was overexpressed when the HSCs were activated, thereby producing anti-inflammatory effects via the induction of various cytokines, in which HSCs overexpressed TGFb to inhibit macrophagocyte, CD4þ T cells, and memory Th1 cells [3]. Similar to the finding mentioned earlier, the results of our study revealed that the TGFb levels in the HSC-alone group were not significantly higher than those in the islet-alone group or the diabetic group. However, when HSCs were cotransplanted
Fig 8. (A and B) Insulin expression in the grafts. The (B) cotransplanted group expressed more insulin than the (A) isletalone group. (C) Insulin expression in the immunofluorescence (green) of the cotransplant group. (D) Islet grafts (green) were surrounded by hepatic stellate cells (red, arrow) in the cotransplant group. (E) Hepatic stellate cells surrounding the islet grafts were desmin positive (red). (F) The nucleus was labeled by using 40 ,6diamidino-2-phenylindole staining (blue).
1600
with islets, they were activated, thus secreting a large amount of TGFb. As a signal factor, TGFb shuts down the immune response and inflammatory reaction, and induces the formation of immune tolerance in transplantation [8,9]. Kobayashi et al [5] found that HSCs turned into myofibroblast-like cells when activated, and that they could induce apoptosis of activated T lymphocytes to suppress rejection of transplants. Yu et al [6] proved that when cultivated HSCs were activated, they could induce T-cell apoptosis. In line with these findings, we found that the TGFb level of the cotransplant recipients was significantly higher, proving the suppressive effect of TGFb on inflammatory response. Apart from the protective effect of TGFb on the islet graft, immunohistochemistry staining results of the cotransplant group exhibited much less lymphocyte infiltration surrounding the graft compared with the lymphocyte infiltration of the islet-alone group. The insulin expression level was also much higher in the cotransplant group. Furthermore, we found that, in immunofluorescent staining, the capsule cells surrounding the islets in the cotransplant group were desmin positive, suggesting that the capsule cells surrounding the islet grafts originated from the HSCs. These findings suggest that HSCs could effectively suppress lymphocyte infiltration in the islet graft, thus letting the grafts avoid immune rejection. This vital finding is in agreement with that of Chen et al [10]. In addition, we believe that the suppressive effect of TGFb on lymphocytes also blocks the lymphocyte infiltration. Although the precise mechanisms of protection and immune toleranceeinducing function of HSCs in islet transplantation were not clear, the capsule formed by HSCs that surrounded the graft provide a new insight into the
ZHANG, ZHOU, SONG ET AL
protective function of HSCs. This is probably a new way to promote immune tolerance in islet transplantation. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFb expression and forming a biological capsule around the graft.
REFERENCES [1] Bach JF. Immunotherapy of insulin-dependent diabetes mellitus. Curr Opin Immunol 2001;13:601e5. [2] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230e8. [3] Sato M, Suzuki S, Senoo H. Hepatic stellate cells: unique characteristics in cell biology and phenotype. Cell Struct Funct 2003;28:105e12. [4] Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001;21:417e26. [5] Kobayashi S, Seki S, Kawada N, et al. Apotosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells. Cell Tissue Res 2003;311: 353e64. [6] Yu MC, Chen CH, Liang X, et al. Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology 2004;40:1312e21. [7] Chen X, Zhang Z, Su C, et al. Protective effect of heme oxygenase- 1 to pancreas islet xenograft. J Surg Res 2010;164: 336e43. [8] Wahl SM, Chen W. TGF-beta: how tolerant can it be? Immunol Res 2003;28:167e79. [9] Dufour JM, Rajotte RV, Kin T, et al. Immunoprotection of rat islet xenografts by cotransplantation with sertoli cells and a single injection of antilymphocyte serum. Transplantation 2003;75: 1594e6. [10] Chen CH, Kuo LM, Chang Y, et al. In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology 2006;44:1171e81.
ABSTRACT Activated hepatic stellate cells (HSCs) possess strong immune inhibitory activity. The present study highlighted the protective role of HSCs in islet transplantation. Recipients were randomly divided into 4 groups: a diabetic group, an HSC-alone group, an islet-alone transplant group, and a cotransplant group. Graft survival was compared among the 4 groups. Serum transforming growth factor b (TGFb), tumor necrosis factor a, interleukin1b, and interferon gamma expression levels were measured. The infiltration of lymphocytes was observed via hematoxylin and eosin staining, and immunohistochemical examinations were performed. Results showed that allogeneic HSCs protect islet allografts better than syngeneic HSCs. There was significant prolonged graft survival and a higher level of TGFb in the cotransplant group (P < .01). The infiltration of lymphocytes in the cotransplant group was notably less than in the islet-alone group (P < .01). The formation of desminpositive HSC packages was detected in the cotransplant group. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFb expression and forming a biological capsule around the graft.
T
YPE 1 diabetes is one of the most serious organ-specific autoimmune diseases that damages the islet b cells by abnormally secreting autoantibodies and inducing a variety of complications, seriously affecting health. Long-term follow-up suggests that complications of type 1 diabetes are not efficiently prevented by drug intake alone. Allogeneic islet transplantation is considered one of the most potentially therapeutic treatments for type 1 diabetes [1,2], but the adverse effects caused by long-term immunosuppression severely hamper the promotion of this option. Hence, attention has been drawn to the induction of immune tolerance. Hepatic stellate cells (HSCs) are a population of liver nonparenchymal cells in the space of Disse between the hepatocyte and hepatic sinusoidal endothelial cells. When activated, HSCs have anti-inflammatory activity primarily mediated by transforming growth factor b (TGFb) [3]. HSCs express very few key immune surface molecules in the quiescent stage. However, upon activation, HSCs upregulate TGFb messenger ribonucleic acid, causing TGFb oversecretion, which suppresses the activity of Th1 cells. Activated HSCs produce interleukin (IL)-10 to restrain macrophagocytes, and IL-6, which has dual immunomodulatory activity [4]. Kobayashi et al [5] and Yu et al [6]
reported that HSCs express intercellular adhesion molecule 1, major histocompatibility complex, B7, and B7-H1, and activated HSCs show strong inhibition of T-cell response via enhanced T-cell apoptosis. The purpose of the present study was to determine if HSCs serve a function in the graft protection of islet transplants. MATERIALS AND METHODS Animal Male BALB/c mice (6e8 weeks old, 24e26 g) were used as islet donors, and male C57BL/6 mice (6e8 weeks old, 23e27 g) were used as recipients. The mice were purchased from the Chinese Academy of Science (Shanghai, China) and were housed in laminar air flow cabinets under specific pathogen-free conditions. Mice were cared
This work was supported by grants from the Research and Innovation Project of Shanghai Municipal Education Commission (grant 11ZZ100) and the National Natural Science Foundation of China (grant 81170721). *Address correspondence to Guang-Wen Zhou, Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, 200233, Shanghai, China. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.03.009
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1594
Transplantation Proceedings, 46, 1594e1600 (2014)
HEPATIC STELLATE CELLS AND IMMUNOTOLERANCE for and handled according to the recommendations of the National Institute of Health Guidelines for Care and Use of Laboratory Animals. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Committee. Animals were fasted for 24 hours before experiments, but water was given.
1595 Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, United States) with 10% fetal calf serum, L-glutamine 2 mmol/L, and HEPES 25 mmol/L (Mediatech, Inc, Herndon, Va, United States).
Isolation and Culture of Mouse HSCs Mouse Islet Isolation Islets were isolated from BALB/c mice via pancreas perfusion, collagenase XI (Sigma-Aldrich Co LLC, St Louis, Mo, United States) digestion, and purification with Ficoll (GE Healthcare, Little Chalfont, United Kingdom) discontinuous gradient centrifugation [7]. Islets were identified by following the instructions of a previous study. Islet purity was assessed by dithizone (SigmaAldrich Co LLC, St Louis, Mo, United States) staining, and cell mass was counted by using a microscope. Islet function was assessed with the use of an insulin-releasing test. Islet viability was assessed by acridine orangeepropidium iodide (Sigma-Aldrich Co LLC, St Louis, Mo, United States) fluorescent staining; living cells exhibited green staining (acridine orange), whereas dead cells showed brownered staining (propidium iodide). Islets were cultured in
HSCs were isolated from BLAB/c mice and C57BL/6 mice via liver perfusion, collagenase IV (Sigma-Aldrich Co LLC) digestion, colation, lavation, and purification by 60% Percoll (GE Healthcare) gradient centrifugation. The stratosphere of the centrifugate was composed of a liver cell layer beneath the HSC layer. The HSC layer was collected and cultured. After 48 hours of culturing, immunocytochemical staining was performed, and cells were observed by using an inverted microscope. Cell yield was estimated as cell population ¼ (cell amount of 4 parts in globulimeter/4) 104 cell suspension volume (in milliliters). Each sample was estimated twice to obtain the average. HSC viability was assessed by trypan blue staining. HSCs viability (%) ¼ alive cell amount/total cell amount 100%. HSC purity was assessed by desmin immunocytochemical staining, and cells were counted according to the
Fig 1. Isolation and activation of hepatic stellate cells (HSCs) (representative figures from BALB/c mice). (A) HSCs cultured for 4 hours. (B) HSCs (synapse with bright lipid droplet) cultured for 24 hours. (C) Fully activated growth of HSCs. (D) HSCs cultured for 11 days showed a fibroblast-like morphology. (E) HSCs cultured for 5 days expressed desmin (red). The nucleus was labeled by 40 ,6-diamidino-2-phenylindole staining (blue). (F) HSCs cultured for 7 days expressed a-smooth muscle actin (green). The nucleus was labeled by 40 ,6diamidino-2-phenylindole staining (blue).
1596 population of desmin-positive cells. HSCs purity (%) ¼ desminpositive cell/total cell amount 100%.
Optimization of the HSC Dose A total of 1.5 105, 3 105, and 4.5 105 HSCs from either BALB/c mice or C57BL/6 mice were cotransplanted with 300 islet equivalents (IEQ; 1 IEQ ¼ 1150 mm islet) from BALB/c mice into the subcapsular space of the left kidney of C57BL/6 mice [7]. The optimized HSC dose was identified.
Islet Transplantation C57BL/6 mice were fasted for 18 hours, before being rendered diabetic by a single intraperitoneal injection of streptozotocin (Sigma-Aldrich Co LLC, St Louis, Mo, United States) at a dose of 200 mg/kg. At 72 hours after administration, mice with 3 consecutive random daily blood glucose readings >20 mmol/L were used as islet recipients. Recipients were randomly divided into 3 groups: the nontransplant diabetic group (n ¼ 8), the HSC transplant group (n ¼ 8), the islet-alone transplant group (n ¼ 9), and the HSC and islet cotransplant group (n ¼ 10). Blood glucose was monitored daily after transplantation. Transplantation and graft survival were considered successful if the nonfasting blood glucose level returned to normal and remained normal (20 mmol/L after a period of normoglycemia. The first of 2 consecutive days in which blood glucose readings were >20 mmol/L was defined as the date of islet graft failure. Seven days after transplantation, 3 recipients in each group underwent cervical dislocation, and their grafts were preserved for further testing.
Glucose Tolerance Test An intraperitoneal glucose tolerance test (IPGTT) was performed 3 days after normoglycemia was achieved following transplantation. Mice were fasted for 12 hours before administering an intraperitoneal injection of 50% dextrose solution at a dose of 6 g/kg. Blood glucose was measured before and after glucose injection at specific time points (0, 15, 30, 60, 90, 120, and 150 minutes) by a glucose meter through tail vein hemospasia.
Fig 2. Optimization of the hepatic stellate cell (HSC) dose to protect islet allografts; 3 105 HSCs and 4.5 105 HSCs collected from both BALB/c and C57BL/6 mice protected BALB/c islets after cotransplantation (HSCs þ 300 islet equivalent islets) (P < .01). The effect of 3 105 HSCs did not differ from the effect of 4.5 105 HSCs in protecting the islet graft. HSCs from BALB/c mice exhibited better protection of islets compared with HSCs from C57BL/6 mice (P < .01) (n ¼ 8 in each group).
ZHANG, ZHOU, SONG ET AL
Cytokine Measurement Serum interferon gamma, tumor necrosis factor a, IL-1b, and TGFb levels in the mice were measured 7 days after transplantation by using an enzyme-linked immunosorbent assay in accordance with the test kit instructions (Jingmei Bioscience Ltd, Shanghai, China).
Hematoxylin and Eosin Staining Islet graft specimens were fixed in 10% formalin solution for 24 hours and embedded in paraffin. Sections (3 mm) were dewaxed in dimethyl benzene and washed with gradient ethanol followed by distilled water. Specimens were stained with hematoxylin for 5 minutes, placed in hydrochloric acid for 30 seconds (alcoholation), soaked in distilled water for 15 minutes, stained with eosin, and then finalized after gradient ethanol dehydration.
Immunohistochemistry and Immunofluorescent Staining After dewaxing, sections were subjected to microwave antigen retrieval in citrate phosphate buffer (0.01 M). To block nonspecific reactivity and staining from endogenous peroxidase, sections were incubated in hydrogen peroxide (1%) for 20 minutes and in serumfree protein block (Dako Co, Tokyo, Japan) for 5 minutes. After rinsing, slides were incubated overnight at 4 C with insulin primary antibody (1:100) (Sigma-Aldrich Co LLC, St Louis, Mo, United States) desmin primary antibody (1:100) (Sigma-Aldrich Co LLC), and a-smooth muscle actin primary antibody (1:200) (SigmaAldrich Co LLC). Secondary antibodies and 3,30 -diaminobenzidine tetrahydrochloride chromogen solution was added to the slides (Dako Real Envision Detection System, Dako Denmark A/S, Glostrup, Denmark). Slides were counterstained with hematoxylin. Images were acquired by using a computerized image system (ScanScope XT, Vista, Calif, United States). Immunofluorescent staining used the same procedure as hematoxylin and eosin staining until the second antibody incubation. Sections were incubated in lucifugal places at room temperature for 60 minutes; 40 ,6-diamidino-2-phenylindole was added, and sections were finalized after PBST washing. Slides were observed under immunofluorescent microscope.
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Statistical Analysis Data are expressed as mean values SDs. The Student t test and one-way analysis of variance were used to compare mean values. Kaplan-Meier curves were used for survival analysis, and P < .05 was considered statistically significant.
RESULTS Purity and Activity of Freshly Isolated Islets and HSCs
A mean of 190.00 10.98 islets could be obtained from 1 donor. The purity of isolated islets was >90%, and the activity of islets was >95%. The insulin-releasing amount of islets under stimulation of low-concentration and highconcentration glucose was 3.65 0.49 and 10.40 0.80 mmol/L, respectively. The insulin amount in highconcentration glucose stimulation was 3 times more than that in low-concentration glucose stimulation, which was a statistically significant difference (P < .01). A mean of (4.90 0.40) 105 HSCs could be obtained from 1 mouse (BALB/c or C57BL/6). The purity of HSCs was >90%. The activity of HSCs was >95% (Fig 1).
Fig 4. Blood glucose levels were tested daily after transplantation in recipient mice. Mice in the cotransplant group (n ¼ 10) had a significantly longer period of normoglycemia than those in the islet-alone group (n ¼ 9) (P ¼.008).
Effects of Cotransplantation With Allogeneic HSCs
In this study, 3 105 HSCs and 4.5 105 HSCs collected from both BALB/C and C57BL/6 mice protected BALB/C islets after cotransplantation (HSCs þ 300 IEQ islets) (P < .01). The effect of 3 105 HSCs did not differ from the effect of 4.5 105 HSCs in protecting the islet graft. HSCs from BALB/c mice exhibited better protection of islets compared with HSCs from C57BL/6 mice (P < .01) (n ¼ 8 in each group) (Fig 2). Therefore, 3 105 HSCs from BLAB/c mice were selected to be cotransplanted with islets (3 105 HSCs þ 300 IEQ islets) for the succeeding experiments.
Twenty days after transplantation, blood glucose levels were better controlled in the islet-alone and cotransplant groups compared with the diabetic and HSC-alone groups (P < .01). The cotransplant group had a significantly lower blood glucose trend than the islet-alone group (P < .05). The diabetic group and the HSC-alone group did not have a period of normoglycemia, and the blood glucose level did not differ between groups (Fig 3). The blood glucose level was >20 mmol/L on day 20 in the islet-alone group, which was regarded as graft rejection; in the cotransplant group, the blood glucose level remained normal. The difference was statistically significant (P < .05). The survival of islets was monitored and represented by normoglycemic periods in recipients. The normoglycemia time in the cotransplant
Fig 3. The blood glucose level was tested daily after transplantation. From day 20, the blood glucose level in the islet-alone group was >20 mmol/L. The cotransplant group had a significantly lower blood glucose level than the islet-alone group (*P < .05), the diabetic group, and the hepatic stellate cell (HSC)-alone group (#P < .01). The blood glucose level did not differ between the diabetic group and the HSC-alone group.
Fig 5. The intraperitoneal glucose tolerance test of the islet graft. Compared with the diabetic group and the hepatic stellate cell (HSC)-alone group, the blood glucose levels in the islet-alone group and the cotransplant group were significantly lower (*P < .01). The mean blood glucose level in the islet-alone group was insignificant compared with the cotransplant group.
Optimization of HSC Dose to Protect Islet Allograft
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compared with the islet-alone group. The blood glucose levels in both the diabetic group and the HSC-alone group did not differ significantly (Fig 5). Expression of Cytokines In Vivo
Fig 6. The blood concentration of cytokines in the recipients. Transforming growth factor b (TGFb) levels in the cotransplant group were much higher than those in the islet-alone group, the hepatic stellate cell (HSC)-alone group, and the diabetic group (*P < .01). TGFb levels in the groups other than the cotransplant group did not differ significantly. The expression levels of interferon gamma, interleukin-1b (IL-1b), and tumor necrosis factor a (TNF-a) were not significantly different among the 4 groups.
group was significant longer than that of the islet-alone group (23.75 8.96 vs 11.90 6.92 days) (Fig 4). Evaluation of Islet Function In Vivo
IPGTT was performed at specific time points (0, 15, 30, 60, 90, 120, and 150 minutes) after intraperitoneal injection of dextrose injection. The cotransplant group and the isletalone group exhibited better glucose tolerance than the other groups (P < .01), but the average blood glucose level in the cotransplant group was statistically insignificant
Fig 7. Hematoxylin and eosin (HE) staining: (A and B) Islet-alone graft was detectable as small foci under the kidney capsule, with cell degeneration and apoptosis. Lymphocyte infiltrated around the graft. (C and D) Grafts in the cotransplant group exhibited nodules under the kidney capsule and were surrounded by a limited lymphocyte infiltration.
Cytokines were examined 1 week after transplantation. TGFb in the cotransplant group was much higher than that in the islet-alone group, the HSC-alone group, and the diabetic group (2292.31 50.87 pg/mL vs 1246.55 38.91, 1323.44 74.21, and 1280.65 55.44 pg/mL) (P < .01). TGFb in all groups except the cotransplant group did not differ significantly. The expression levels of interferon gamma, IL-1b, and tumor necrosis factor a were not significantly different among the 4 groups (Fig 6). Pathological Findings
Microscope images showed obvious lymphocyte infiltration around the islet grafts in the islet-alone group, whereas in the cotransplant group, lymphocyte infiltration was apparently much less. The grafts were in better morphological normality in the cotransplant group than in the islet-alone group (Fig 7). The cotransplant group expressed more insulin than the islet-alone group after transplantation (Figs 8A and 8B). The insulin immunofluorescence of the cotransplant group (Fig 8C) showed islet grafts surrounded by HSCs (Fig 8D), suggesting a tendency for islet grafts to be encapsulated by HSCs, which were desmin positive (Fig 8E). The nucleus of cells was labeled by using 40 ,6-diamidino-2-phenylindole staining (Fig 8F).
HEPATIC STELLATE CELLS AND IMMUNOTOLERANCE
DISCUSSION
Islets transplantation is regarded as one of the most potentially therapeutic treatments for type 1 diabetes [1,2], but the adverse effects caused by immunosuppressant drugs after transplantation have severely hindered the development of islet transplantation. Therefore, the induction of immune tolerance by using biological materials is critical. The present study demonstrated that HSC cotransplants with islets may prolong the allograft survival of islets. Allogeneic HSCs exhibited this trend more apparent than syngeneic HSCs. However, the mechanisms underlying the inconsistent protective function of allogeneic and syngeneic HSCs are still unknown. Recipients who underwent islet and allogeneic HSC cotransplants maintained normoglycemia for a much longer time than the recipients of islets alone. Blood glucose was better controlled in the cotransplant recipients than in the islet-alone recipients. These findings suggest that allogeneic HSCs protect the allograft from being rejected and play a vital function in immune
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regulation. The functional efficiency of islet was determined by using IPGTT tests in vivo. Results showed that there was no difference in the regulation of instant hyperglycemia in both groups, indicating that although the activated allogeneic HSCs protected the islet grafts from rejection, they could not improve the functional efficiency of the graft. However, interestingly, we found that cotransplant islets had better insulin-releasing and blood glucose regulation ability than the transplant of islets alone (based on bloodglucose monitoring and immunochemical staining). Previous reports have indicated that the costimulatory molecule B7-H1 was overexpressed when the HSCs were activated, thereby producing anti-inflammatory effects via the induction of various cytokines, in which HSCs overexpressed TGFb to inhibit macrophagocyte, CD4þ T cells, and memory Th1 cells [3]. Similar to the finding mentioned earlier, the results of our study revealed that the TGFb levels in the HSC-alone group were not significantly higher than those in the islet-alone group or the diabetic group. However, when HSCs were cotransplanted
Fig 8. (A and B) Insulin expression in the grafts. The (B) cotransplanted group expressed more insulin than the (A) isletalone group. (C) Insulin expression in the immunofluorescence (green) of the cotransplant group. (D) Islet grafts (green) were surrounded by hepatic stellate cells (red, arrow) in the cotransplant group. (E) Hepatic stellate cells surrounding the islet grafts were desmin positive (red). (F) The nucleus was labeled by using 40 ,6diamidino-2-phenylindole staining (blue).
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with islets, they were activated, thus secreting a large amount of TGFb. As a signal factor, TGFb shuts down the immune response and inflammatory reaction, and induces the formation of immune tolerance in transplantation [8,9]. Kobayashi et al [5] found that HSCs turned into myofibroblast-like cells when activated, and that they could induce apoptosis of activated T lymphocytes to suppress rejection of transplants. Yu et al [6] proved that when cultivated HSCs were activated, they could induce T-cell apoptosis. In line with these findings, we found that the TGFb level of the cotransplant recipients was significantly higher, proving the suppressive effect of TGFb on inflammatory response. Apart from the protective effect of TGFb on the islet graft, immunohistochemistry staining results of the cotransplant group exhibited much less lymphocyte infiltration surrounding the graft compared with the lymphocyte infiltration of the islet-alone group. The insulin expression level was also much higher in the cotransplant group. Furthermore, we found that, in immunofluorescent staining, the capsule cells surrounding the islets in the cotransplant group were desmin positive, suggesting that the capsule cells surrounding the islet grafts originated from the HSCs. These findings suggest that HSCs could effectively suppress lymphocyte infiltration in the islet graft, thus letting the grafts avoid immune rejection. This vital finding is in agreement with that of Chen et al [10]. In addition, we believe that the suppressive effect of TGFb on lymphocytes also blocks the lymphocyte infiltration. Although the precise mechanisms of protection and immune toleranceeinducing function of HSCs in islet transplantation were not clear, the capsule formed by HSCs that surrounded the graft provide a new insight into the
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protective function of HSCs. This is probably a new way to promote immune tolerance in islet transplantation. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFb expression and forming a biological capsule around the graft.
REFERENCES [1] Bach JF. Immunotherapy of insulin-dependent diabetes mellitus. Curr Opin Immunol 2001;13:601e5. [2] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230e8. [3] Sato M, Suzuki S, Senoo H. Hepatic stellate cells: unique characteristics in cell biology and phenotype. Cell Struct Funct 2003;28:105e12. [4] Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001;21:417e26. [5] Kobayashi S, Seki S, Kawada N, et al. Apotosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells. Cell Tissue Res 2003;311: 353e64. [6] Yu MC, Chen CH, Liang X, et al. Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology 2004;40:1312e21. [7] Chen X, Zhang Z, Su C, et al. Protective effect of heme oxygenase- 1 to pancreas islet xenograft. J Surg Res 2010;164: 336e43. [8] Wahl SM, Chen W. TGF-beta: how tolerant can it be? Immunol Res 2003;28:167e79. [9] Dufour JM, Rajotte RV, Kin T, et al. Immunoprotection of rat islet xenografts by cotransplantation with sertoli cells and a single injection of antilymphocyte serum. Transplantation 2003;75: 1594e6. [10] Chen CH, Kuo LM, Chang Y, et al. In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology 2006;44:1171e81.
