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Immunosuppressive Function of Bone Marrow Mesenchymal Stem Cells on Acute Rejection of Liver Allografts in Rats
Immunosuppressive Function of Bone Marrow Mesenchymal Stem Cells on Acute Rejection of Liver Allografts in Rats Z.-F. Hong, X.-J. Huang, Z.-Y. Yin, W.-X. Zhao, and X.-M. Wang ABSTRACT Bone marrow mesenchymal stem cells (MSCs) demonstrate functions of immunologic regulation. However, little is known about the role of interferon-␥ (IFN-␥) on MSCs and whether MSCs alone can prevent allograft rejection. We purified MSCs, which were or were not treated with IFN-␥, to act as regulatory cells in mixed lymphocyte reactions. We measured their expression of PDL-1, MHC-I, MHC-II, CD40, CD54, and CD86. The MSCs stained with carboxyfluorescein diacetate-succinimidyl ester were used to detect homing in vivo. The MSCs were injected into an orthotopic liver transplantation model. The result suggested that IFN-␥ enhances expression of PDL-1, MHC-I, MHC-II, and CD54 and boosts immunosuppressive ability in vivo. The MSCs demonstrated homing to the liver, alleviating acute immunologic rejection of an hepatic graft in rats. We conclude that IFN-␥ may enhance the immunosuppressive function of MSCs to protect liver allografts in rats from acute immunologic rejection.
I
MMUNOLOGIC REJECTION continues to hinder the development of liver transplantation. Mesenchymal stem cells (MSCs) demonstrate immunologic regulatory functions. Interferon-␥ (IFN-␥) is an important cytokine in vivo; however, little is known about its effects on MSCs and
whether MSCs alone can alleviate acute allograft rejection. We examined the in vitro immunologic characteristics of MSCs treated or not treated with IFN-␥. An orthotopic liver transplantation (OLT) model was treated with MSCs to examine their role in vivo. METHODS Isolation and Culture of MSCs Bone marrow from male Brown Norway (BN) rats (Experimental Research Institute, Chinese Medical Academy of Science, Beijing, China) was used to create cultures in Dulbecco modified Eagle medium containing 10% fetal bovine serum (HyClone, Logan, Utah). The MSCs were purified using an adhesive screening method.1 In the experiment, we used the third-passage MSCs.
Detection of Immunologic Phenotype Flow cytometry was used to examine CD45, CD11b/c, CD90 (clones OX-1, OX-42, and OX7, respectively; Invitrogen Corp, Carlsbad, California), CD29 (clone HM1-1; BioLegend, Inc, San Diego, California),
Fig 1. Mesenchymal stem cells exhibited a homogeneous fusiform shape and were arranged in a swirl pattern at the third passage. © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 403– 409 (2009)
From the Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen, China. This study was supported by grant 30872484 from the National Natural Science Foundation of China and grant WKJ 2005-2-020 from the National Ministry of Health of China. Address reprint requests to Xiao-Min Wang, MD, Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.10.020 403
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Fig 2. Immunophenotyping. Mesenchymal stem cells tested positive for CD29 and CD90, negative for CD45 and CD11b/c, and weak for CD45 plus CD11b/c. Expression rates were as follows: CD29, 98.8% ⫾ 0.5% (A); CD45, 5.2% ⫾ 0.2% (B); CD90, 98.7% ⫾ 0.5% (C); CD11b/c, 0.7% (D); CD90 plus CD29, 96.7% ⫾ 1.0% (E); and CD45 plus CD11b/c, 0.1% (F), demonstrating that cell purity was high. PE indicates phycoerythrin. CD54 (clone A-19; Antigenix America Inc, Huntington Station, New York), PDL-1 (H-130:sc-50298; Santa Cruz Biotechnology, Inc, Santa Cruz, California), CD40 (clone HM40-3; BD Biosciences, San Jose, California), CD80 and CD86 (clones 3H5 and 24F, respectively; eBio-
science, Inc, San Diego, California), and MHC-I and MHC-II (clones OX-18 and OX-76, respectively; AbD Serotec, Kidlington, England). The isotype control included mouse IgG1 (fluoroescein isothiocyanate; Invitrogen Corp) and IgG2 (phycoerythrin; Invitrogen Corp).
Fig 3. At 3 days, expression rates of PDL-1, CD54, MHC-I, and MHC-II were 12.3% ⫾ 3.5%, 53.9% ⫾ 11.4%, 4.1% ⫾ 0.8%, and 0.5% ⫾ 0.3%, respectively, in mesenchymal stem cells (MSCs) treated with 0 U/mL of interferon-␥ (IFN-␥) and 42.8% ⫾ 5.4%, 91.7% ⫾ 5.4%, 91.7% ⫾ 4.2%, 9.6% ⫾ 0.7%, and 5.0% ⫾ 0.8%, respectively in MSCs treated with 200 U/mL of IFN-␥. There was statistical significance between treatment groups (P ⬍ .01, rank sum test), in the same immunologic relative molecules. Interferon-␥ can upregulate expression of CD54, PDL-1, MHC-I, and MHC-II. CD40, CD80, and CD86 were negative with or without treatment.
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Mixed Lymphocyte Reaction
Hepatic Tissue Pathologic Features
A one-way mixed lymphocyte reaction was performed in 12-well clusters containing 2 mL of Dulbecco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin or streptomycin (HyClone). The MSCs acted as regulatory cells, and BN rat T cells as responders (2 ⫻ 105/well), as in a previous report,2 and concanavalin A, 5 g/mL (Sigma, St Louis, Mo) was the stimulator. The T-cell suspension was obtained by mechanical shredding of the spleen, and was purified using a nylon fiber column. The cell purity was greater than 90% as detected using CD3. The experimental group included a reactive cell plus stimulator plus regulatory cell; the control group, a reactive cell plus stimulator; and the background group, a reactive cell plus a regulatory cell. Seven homeoplastic specimen wells were set in each group. After six days, lymphocytes were collected and were counted using a hemodynameter. The inhibition rate was defined as follows: (Experiment group cell number ⫺ Background group cell number)/(Control group cell number ⫺ Background group cell number).
Paraffin sections prepared from liver were stained with hematoxylineosin for examination under light microscopy. Immunologic rejection was graded according to the Banff system.
Statistical Analysis Data were analyzed using commercially available software (SPSS version 13.0; SPSS, Inc, Chicago, Illinois). The mean value was used when multiple data were available. The t test was used for bivariable analysis, and the rank sum test for multivariable data.
RESULTS Culture and Immunologic Phenotype of MSCs
The MSCs exhibited a homogeneous fusiform shape with a swirl pattern at the third passage (Fig 1). The MSCs highly expressed CD29 and CD90 but only rarely expressed CD45 and did not express CD11b/c (Fig 2).
Establishment of OLT Models Role of IFN-␥ in MSCs
Orthotopic liver transplantation was performed using the two-cuff technique of Kamada and Calne.3 Male Lewis rats (Experimental Animal Research Institute, Chinese Medical Academy of Science) served as donor and female BN rat organ recipients. The models were considered successful if recipients survived for longer than 72 hours after surgery.
Because IFN-␥ is an important factor in inflammatory reactions, MSCs were treated with and without a stimulus of IFN-␥ (PeproTech Inc, Rocky Hill, New Jersey), 200 U/mL, at 3 days, as previously reported.2,4,5
Cell Homing In Vivo
Immunologically Relevant Cell-Surface Molecules
Fifteen healthy BN hosts were injected with 5 ⫻ 106 MSCs stained with carboxyfluoroscein diacetate–succinimidyl ester, a nonfluorescent dye that passively diffuses into the cytoplasm of cells. Once inside the cells, it is cleaved by intracellular esterase to become flueorescent and can be detected using a fluorescence microscope.
MHC-I, MHC-II, CD40, CD80, and CD86 costimulatory molecules are crucial in antigen presentation; CD54 is essential for direct contact between cells; and PD-L1 is important in immunologic tolerance. The expression of
Specimen Collection In general, acute rejection is apparent at day 10 to 14 after transplantation; therefore, rats were killed at 14 days after the procedure. Blood, liver, and spleen were preserved in liquid nitrogen.
Detection of His19 Epitope His19 epitope expression by splenocytes was detected using His19 monoclonal antibody (eBioscience) and flow cytometry. His19 reacts with an epitope of all rat MHC II molecules except the RT1n, which is present in BN strains.
Measurement of the Sex-Determining Region Y gene Peripheral blood mononuclear cells (PBMCs) were prepared using density gradient centrifugation with a polysaccharide (Ficoll; Sigma). After DNA was extracted from the PBMCs, a polymerase chain reaction was used to detect the SRY gene. The forward primer was 5=-CTCTGCTCCTACCTATGCCAACA-3, and the reverse primer was 5=-GGAATGCCTGCAAGATCCTACTG-3=. The PBMCs from male Lewis rats was the positive control, and PBMCs from female BN rats was the negative control.
Hepatic Function Rat serum was used to detect blood alanine aminotransferase and blood total bilirubin using an automated biochemical analyzer.
Fig 4. The mesenchymal stem cell (MSC)–spleen ratio ⫽ (Number of MSCs)/(Number of lymphocytes). The MSCs were treated with 0 U/mL of interferon-␥ (⫺IFN-␥) or 200 U/mL of IFN-␥ (⫹IFN-␥) at 3 days and then acted as regulatory cells. Inhibition rates of ⫺IFN-␥ were 3.94% ⫾ 1.9%, 4.8% ⫾ 2.2%, 22.6% ⫾ 8.3%, 32.3% ⫾ 7.4%, and 59.5% ⫾ 10.5%, respectively, and for ⫹IFN-␥ were 3.87% ⫾ 0.6%, 4.2% ⫾ 0.5%, 30.8% ⫾ 3.3%, 49.9% ⫾ 5.6%, and 77.9% ⫾ 4.8%, respectively, in the 1/100, 1/80, 1/40, 1/20, and 1/10 groups. There was statistical significance between any two levels in the ⫺IFN-␥ or the ⫹IFN-␥ groups (P ⬍ .01) except between the 1/100 and 1/80 levels (P ⬎ .01) The inhibition effect of MSCs was dose-related, and a certain amount of MSCs was essential to inhibit proliferation of lymphocytes. Comparison of the same MSC-splenocyte ratio in ⫺IFN-␥ and ⫹IFN-␥ demonstrated that IFN-␥ can enhance the inhibition rate of MSCs to the proliferation of lymphocytes (*P ⬍ .01) except at the 1/80 and 1/100 levels (†P ⬎ .01).
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Fig 5. Cell homing in vivo. A, Frozen sections were made from liver from rats injected with mesenchymal stem cells stained with CFDA-SE (carboxyfluorescein diacetate–succinimidyl ester). The sections were then stained with DAPI (4=,6-diamidine-2-phenylindole dihydrochloride; Roche Applied Science, Indianapolis, Indiana) and observed with a fluorescence microscope with blue excitation light (4) and green excitation light (5). (Original magnification ⫻40.) B, Frozen sections from healthy liver were made negative (controls) with blue excitation light (1) and green excitation light (2). 3, Overlay of 1 and 2, and 6, overlay of 4 and 5. Green fluorescence is seen in the liver tissue, especially around the portal areas. (Original magnification ⫻100.)
MHC-I, CD54, and PDL-1 by MSCs was upregulated when the cells were treated with IFN-␥. MHC-II was negative but became weakly positive after IFN-␥ stimulus. CD40, CD80, and CD86 were negative whether they were or were not treated with IFN-␥ (Fig 3). One-Way MLR
To evaluate the dose-related effects on inhibitory ability, five MSC groups were constructed in which the MSC-spleen ratios were 1/100, 1/80, 1/40, 1/20, 1/10, for MSC groups 1 through 5, respectively (Fig 4). The MSCs treated or not treated with IFN-␥ at 3 days served as regulatory cells. We observed that an MSC-spleen ratio larger than 1/80 was essential to inhibit lymphocyte proliferation. The inhibitory ability of MSCs can be enhanced by IFN-␥, an observation cosistent with that of Ryan et al.5
Cell Homing In Vivo
Mesenchymal stem cells stained with carboxyfluorescein diacetate-succinimidyl ester were found in liver tissue, especially around portal areas (Fig 5). Role of MSCs in Alleviating Immunologic Rejection of Allografts
To examine the role of MSCs in alleviating acute allograft rejection, 15 animals were injected with 5 ⫻ 106 MSCs, as in previous reports,6,7 and 15 control animals were injected with saline solution. The general state of health, chimera formation, hepatic function, and liver pathologic features were observed after the procedure. General Health
The experimental and control animals all survived longer than 12 days. Compared with experimental models, control
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the female recipient was heterozygous and not homozygous. That is, chimerism occurred. The His19 epitope is present in Lewis rats but not BN rats. A His19 epitope in a BN rat recipient that received a male Lewis rat liver transplant would indicate that the female BN recipient was heterozygous and not homozygous. In our study, the experimental models but not the control models exhibited the SRY gene (Fig 6). Splenocytes were His19-positive in the experimental group (14.9% ⫾ 1.0%) but negative in the control group (Fig 7), demonstrating generation of chimerism. Hepatic Function and Liver Pathologic Features
Compared with the control group, hepatic function in the experimental group was much improved (Fig 8). Severe acute rejection (class III) was observed in the control group, and mild to moderate acute rejection (class I or II) in the cell transplantation group (Fig 9). Fig 6. SRY gene detection. ⫺MSC represents peripheral blood monocytes (PBMCs) from the untreated control group; ⫹MSC, PBMCs from the experimental group treated with mesenchymal stem cells; positive control, PBMCs from male Lewis rats; and negative control, PBMCs from female Brown Norway rats.
models demonstrated decreased appetite and activity, and serious jaundice. Chimera Formation
In general, the SRY gene is present in male but not female rats. The SRY gene present in a female BN recipient that received a male Lewis rat liver transplant would indicate that
DISCUSSION
Mesenchymal stem cells are defined by a composite of morphologic features, phenotype, and functional characteristics. Our experiment showed that the expression rates of both CD29 and CD90 were greater than 90%, and simultaneous expression rates of CD29 and CD90 were greater than 96%. After treatment with IFN-␥, CD11b/c, CD40, CD80, and CD86 were negative and CD45, CD54, MHC-I, and PDL-1 were positive. MHC-II was negative but became weakly positive after treatment with IFN-␥. The cells were fusiform and arranged in a swirl pattern, consistent with findings of previous reports.8 –11 Not only can MSCs differ-
Fig 7. Expression of His19. Spleen single-cell suspensions from the experimental model treated with mesenchymal stem cells (A) and the untreated control model (B) were used to detect His19 epitope using flow cytometry. Mouse IgG1 was used as isotype control. The rate of His19-positive splenocytes was 14.9% ⫾ 1.0% in the experimental group but was negative in the control group.
Fig 8. Hepatic function. A, Alanine aminotransferase levels (ALT) in the untreated control group and in the experimental group treated with mesenchymal stem cells were 783.34 ⫾ 87.64 and 145 ⫾ 8.3 U/L, respectively. There was statistical significance between groups (P ⬍ .01). B, Total bilirubin (TBIL) levels in the untreated control group and in the experimental group treated with mesenchymal stem cells were 145 ⫾ 8.3 and 89.54 ⫾ 7.8 mol/L, respectively. There was statistical significance between groups (P ⬍ .01).
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Fig 9. A, Severe acute rejection (class III) was demonstrated in liver tissue from the untreated control group. The liver cells were disorderly arranged and balloonlike. The portal area was occupied by inflammatory cells, which extended to the liver parenchyma. There was endometrial vascular damage. Many flakes of liver cell necrosis were noted. B and C, Mild to moderate acute rejection (class II and I, respectively) was demonstrated in liver tissue from the experimental group treated with mesenchymal stem cells. A medium number of monocytes infiltrated the portal area; however, the inflammatory cells were confined to it. The liver cells were orderly arranged and slightly swollen.
entiate into mesoblastic cells, they can also differentiate into other blastodermal cells such as endothelial cells and hepatocytes.12,13 Both CD45 and CD11b/c antigens are characteristic of hematopoietic cells. Thus, MSCs are stem cells but not hematopoietic stem cells.
Our experimental results suggest that rats that underwent OLT and were treated with MSCs exhibited milder immunologic rejection of liver grafts. Mesenchymal stem cells have both vicarious and immunologic roles. Vicariously, MSCs survive and differentiate to serve in repair and
MESENCHYMAL STEM CELLS AND LIVER ALLOGRAFT REJECTION
regeneration.10,13,14 The MSCs stained with carboxyfluorescein diacetate–succinimidyl ester settle in the liver, especially around the portal areas. Immunologically, MSCs have immunosuppressive functions. First, MSCs do not express stimulating elements such as CD40, CD80, CD86, and MHC-II. The MSCs not only fail to induce T-cell activation but also evade dissolution by CD8-positive cytotoxic T lymphocytes,15,16 allowing them to survive. Second, MSCs inhibit the cellular immune response, acting as regulatory cells inhibiting lymphocyte proliferation in vivo. The inhibitory effects of MSCs is dose-related. Third, MSCs can induce formation of a chimera. In our study, chimera formation was apparent in models treated with MSCs but not models not treated with MSCs. This suggests that donor and recipient cells can coexist peacefully in vivo, consistent with the findings of Itakura et al.17 There are many explanations for the immunosuppressive mechanisms of MSCs; however, little is known about the contribution of direct contact between MSCs and immunologic cells. The MSCs inhibit T-cell proliferation primarily by direct contact between cells but not by inducing apoptosis in an MHC-dependent manner.18,19 CD4- and CD8positive T cells both bind to MSCs as found at flow cytometry analysis. We observed that MSCs highly express PDL-1, CD54, and MHC-I, especially after treatment with IFN-␥. The MSCs may connect with T cells via CD54. Augello et al18 noted that MSCs inhibit lymphocyte proliferation by activating the programmed death-1 pathway. The MSCs may highly express CD54 and PDL-1 with stimulation by IFN-␥ in vivo during OLT. Interferon-␥ enhances the immunologic suppression of MSCs in other ways. Ren et al20 reported that the immunosuppressive function of MSCs was elicited by proinflammatory cytokines such as IFN-␥, tumor necrosis factor–␣, and interleukin-1. Interferon-␥ induced indoleamine 2,3dioxygenase, nitric oxide, and expression of other immunosuppressive molecules, strengthening immunologic regulation. REFERENCES 1. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow: analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6:230, 1968 2. Klyushnenkova E, Mosca JD, Zernetkina V, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci 12:47, 2005 3. Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery 93:64, 1983
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4. Krampera M, Cosmi L, Angeli R, et al. Role for interferon-␥ in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 24:386, 2006 5. Ryan JM, Barry F, Murphy JM, et al. Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol 149:353, 2007 6. Le Blanc K, Samuelsson H, Gustafsson B, et al. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21:1733, 2007 7. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30:42, 2002 8. Stagg J. Immunoregulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 69:1, 2007 9. Sylwia B, Danuta J, Marcin M. Mesenchymal stem cells: characteristics and clinical applications. Folisa Histochem Cytobiolog 44:215, 2006 10. Carvalho KA, Oliveira L, Malvezzi M, et al. Immunophenotypic expression by flow cytometric analysis of cocultured skeletal muscle and bone marrow mesenchymal stem cells for therapy into myocardium. Transplant Proc 40:842, 2008 11. Guarita-Souza LC, Carvalho KA, Simeone BR, et al. Functional outcome of bone marrow stem cells: mononuclear versus mesenchymal stem cells after cellular therapy in myocardial scar in Wistar rats. Transplant Proc 38:1953, 2006 12. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337, 2002 13. Kao CL, Lin HT, Chen YW, et al. Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells. Transplant Proc 39:3444, 2007 14. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6:1282, 2000 15. Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 76:1208, 2003 16. Angoulvant D, Clerc A, Benchalal S, et al. Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens. Biorheology 41:469, 2004 17. Itakura S, Asari S, Rawson J, et al. Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat. Am J Transplant 7:336, 2007 18. Augello A, Tasso R, Negrini SM, et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death-1 pathway. Eur J Immunol 35:1482, 2005 19. Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75:389, 2003 20. Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell– mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2:141, 2008
I
MMUNOLOGIC REJECTION continues to hinder the development of liver transplantation. Mesenchymal stem cells (MSCs) demonstrate immunologic regulatory functions. Interferon-␥ (IFN-␥) is an important cytokine in vivo; however, little is known about its effects on MSCs and
whether MSCs alone can alleviate acute allograft rejection. We examined the in vitro immunologic characteristics of MSCs treated or not treated with IFN-␥. An orthotopic liver transplantation (OLT) model was treated with MSCs to examine their role in vivo. METHODS Isolation and Culture of MSCs Bone marrow from male Brown Norway (BN) rats (Experimental Research Institute, Chinese Medical Academy of Science, Beijing, China) was used to create cultures in Dulbecco modified Eagle medium containing 10% fetal bovine serum (HyClone, Logan, Utah). The MSCs were purified using an adhesive screening method.1 In the experiment, we used the third-passage MSCs.
Detection of Immunologic Phenotype Flow cytometry was used to examine CD45, CD11b/c, CD90 (clones OX-1, OX-42, and OX7, respectively; Invitrogen Corp, Carlsbad, California), CD29 (clone HM1-1; BioLegend, Inc, San Diego, California),
Fig 1. Mesenchymal stem cells exhibited a homogeneous fusiform shape and were arranged in a swirl pattern at the third passage. © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 403– 409 (2009)
From the Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen, China. This study was supported by grant 30872484 from the National Natural Science Foundation of China and grant WKJ 2005-2-020 from the National Ministry of Health of China. Address reprint requests to Xiao-Min Wang, MD, Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.10.020 403
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Fig 2. Immunophenotyping. Mesenchymal stem cells tested positive for CD29 and CD90, negative for CD45 and CD11b/c, and weak for CD45 plus CD11b/c. Expression rates were as follows: CD29, 98.8% ⫾ 0.5% (A); CD45, 5.2% ⫾ 0.2% (B); CD90, 98.7% ⫾ 0.5% (C); CD11b/c, 0.7% (D); CD90 plus CD29, 96.7% ⫾ 1.0% (E); and CD45 plus CD11b/c, 0.1% (F), demonstrating that cell purity was high. PE indicates phycoerythrin. CD54 (clone A-19; Antigenix America Inc, Huntington Station, New York), PDL-1 (H-130:sc-50298; Santa Cruz Biotechnology, Inc, Santa Cruz, California), CD40 (clone HM40-3; BD Biosciences, San Jose, California), CD80 and CD86 (clones 3H5 and 24F, respectively; eBio-
science, Inc, San Diego, California), and MHC-I and MHC-II (clones OX-18 and OX-76, respectively; AbD Serotec, Kidlington, England). The isotype control included mouse IgG1 (fluoroescein isothiocyanate; Invitrogen Corp) and IgG2 (phycoerythrin; Invitrogen Corp).
Fig 3. At 3 days, expression rates of PDL-1, CD54, MHC-I, and MHC-II were 12.3% ⫾ 3.5%, 53.9% ⫾ 11.4%, 4.1% ⫾ 0.8%, and 0.5% ⫾ 0.3%, respectively, in mesenchymal stem cells (MSCs) treated with 0 U/mL of interferon-␥ (IFN-␥) and 42.8% ⫾ 5.4%, 91.7% ⫾ 5.4%, 91.7% ⫾ 4.2%, 9.6% ⫾ 0.7%, and 5.0% ⫾ 0.8%, respectively in MSCs treated with 200 U/mL of IFN-␥. There was statistical significance between treatment groups (P ⬍ .01, rank sum test), in the same immunologic relative molecules. Interferon-␥ can upregulate expression of CD54, PDL-1, MHC-I, and MHC-II. CD40, CD80, and CD86 were negative with or without treatment.
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Mixed Lymphocyte Reaction
Hepatic Tissue Pathologic Features
A one-way mixed lymphocyte reaction was performed in 12-well clusters containing 2 mL of Dulbecco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin or streptomycin (HyClone). The MSCs acted as regulatory cells, and BN rat T cells as responders (2 ⫻ 105/well), as in a previous report,2 and concanavalin A, 5 g/mL (Sigma, St Louis, Mo) was the stimulator. The T-cell suspension was obtained by mechanical shredding of the spleen, and was purified using a nylon fiber column. The cell purity was greater than 90% as detected using CD3. The experimental group included a reactive cell plus stimulator plus regulatory cell; the control group, a reactive cell plus stimulator; and the background group, a reactive cell plus a regulatory cell. Seven homeoplastic specimen wells were set in each group. After six days, lymphocytes were collected and were counted using a hemodynameter. The inhibition rate was defined as follows: (Experiment group cell number ⫺ Background group cell number)/(Control group cell number ⫺ Background group cell number).
Paraffin sections prepared from liver were stained with hematoxylineosin for examination under light microscopy. Immunologic rejection was graded according to the Banff system.
Statistical Analysis Data were analyzed using commercially available software (SPSS version 13.0; SPSS, Inc, Chicago, Illinois). The mean value was used when multiple data were available. The t test was used for bivariable analysis, and the rank sum test for multivariable data.
RESULTS Culture and Immunologic Phenotype of MSCs
The MSCs exhibited a homogeneous fusiform shape with a swirl pattern at the third passage (Fig 1). The MSCs highly expressed CD29 and CD90 but only rarely expressed CD45 and did not express CD11b/c (Fig 2).
Establishment of OLT Models Role of IFN-␥ in MSCs
Orthotopic liver transplantation was performed using the two-cuff technique of Kamada and Calne.3 Male Lewis rats (Experimental Animal Research Institute, Chinese Medical Academy of Science) served as donor and female BN rat organ recipients. The models were considered successful if recipients survived for longer than 72 hours after surgery.
Because IFN-␥ is an important factor in inflammatory reactions, MSCs were treated with and without a stimulus of IFN-␥ (PeproTech Inc, Rocky Hill, New Jersey), 200 U/mL, at 3 days, as previously reported.2,4,5
Cell Homing In Vivo
Immunologically Relevant Cell-Surface Molecules
Fifteen healthy BN hosts were injected with 5 ⫻ 106 MSCs stained with carboxyfluoroscein diacetate–succinimidyl ester, a nonfluorescent dye that passively diffuses into the cytoplasm of cells. Once inside the cells, it is cleaved by intracellular esterase to become flueorescent and can be detected using a fluorescence microscope.
MHC-I, MHC-II, CD40, CD80, and CD86 costimulatory molecules are crucial in antigen presentation; CD54 is essential for direct contact between cells; and PD-L1 is important in immunologic tolerance. The expression of
Specimen Collection In general, acute rejection is apparent at day 10 to 14 after transplantation; therefore, rats were killed at 14 days after the procedure. Blood, liver, and spleen were preserved in liquid nitrogen.
Detection of His19 Epitope His19 epitope expression by splenocytes was detected using His19 monoclonal antibody (eBioscience) and flow cytometry. His19 reacts with an epitope of all rat MHC II molecules except the RT1n, which is present in BN strains.
Measurement of the Sex-Determining Region Y gene Peripheral blood mononuclear cells (PBMCs) were prepared using density gradient centrifugation with a polysaccharide (Ficoll; Sigma). After DNA was extracted from the PBMCs, a polymerase chain reaction was used to detect the SRY gene. The forward primer was 5=-CTCTGCTCCTACCTATGCCAACA-3, and the reverse primer was 5=-GGAATGCCTGCAAGATCCTACTG-3=. The PBMCs from male Lewis rats was the positive control, and PBMCs from female BN rats was the negative control.
Hepatic Function Rat serum was used to detect blood alanine aminotransferase and blood total bilirubin using an automated biochemical analyzer.
Fig 4. The mesenchymal stem cell (MSC)–spleen ratio ⫽ (Number of MSCs)/(Number of lymphocytes). The MSCs were treated with 0 U/mL of interferon-␥ (⫺IFN-␥) or 200 U/mL of IFN-␥ (⫹IFN-␥) at 3 days and then acted as regulatory cells. Inhibition rates of ⫺IFN-␥ were 3.94% ⫾ 1.9%, 4.8% ⫾ 2.2%, 22.6% ⫾ 8.3%, 32.3% ⫾ 7.4%, and 59.5% ⫾ 10.5%, respectively, and for ⫹IFN-␥ were 3.87% ⫾ 0.6%, 4.2% ⫾ 0.5%, 30.8% ⫾ 3.3%, 49.9% ⫾ 5.6%, and 77.9% ⫾ 4.8%, respectively, in the 1/100, 1/80, 1/40, 1/20, and 1/10 groups. There was statistical significance between any two levels in the ⫺IFN-␥ or the ⫹IFN-␥ groups (P ⬍ .01) except between the 1/100 and 1/80 levels (P ⬎ .01) The inhibition effect of MSCs was dose-related, and a certain amount of MSCs was essential to inhibit proliferation of lymphocytes. Comparison of the same MSC-splenocyte ratio in ⫺IFN-␥ and ⫹IFN-␥ demonstrated that IFN-␥ can enhance the inhibition rate of MSCs to the proliferation of lymphocytes (*P ⬍ .01) except at the 1/80 and 1/100 levels (†P ⬎ .01).
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Fig 5. Cell homing in vivo. A, Frozen sections were made from liver from rats injected with mesenchymal stem cells stained with CFDA-SE (carboxyfluorescein diacetate–succinimidyl ester). The sections were then stained with DAPI (4=,6-diamidine-2-phenylindole dihydrochloride; Roche Applied Science, Indianapolis, Indiana) and observed with a fluorescence microscope with blue excitation light (4) and green excitation light (5). (Original magnification ⫻40.) B, Frozen sections from healthy liver were made negative (controls) with blue excitation light (1) and green excitation light (2). 3, Overlay of 1 and 2, and 6, overlay of 4 and 5. Green fluorescence is seen in the liver tissue, especially around the portal areas. (Original magnification ⫻100.)
MHC-I, CD54, and PDL-1 by MSCs was upregulated when the cells were treated with IFN-␥. MHC-II was negative but became weakly positive after IFN-␥ stimulus. CD40, CD80, and CD86 were negative whether they were or were not treated with IFN-␥ (Fig 3). One-Way MLR
To evaluate the dose-related effects on inhibitory ability, five MSC groups were constructed in which the MSC-spleen ratios were 1/100, 1/80, 1/40, 1/20, 1/10, for MSC groups 1 through 5, respectively (Fig 4). The MSCs treated or not treated with IFN-␥ at 3 days served as regulatory cells. We observed that an MSC-spleen ratio larger than 1/80 was essential to inhibit lymphocyte proliferation. The inhibitory ability of MSCs can be enhanced by IFN-␥, an observation cosistent with that of Ryan et al.5
Cell Homing In Vivo
Mesenchymal stem cells stained with carboxyfluorescein diacetate-succinimidyl ester were found in liver tissue, especially around portal areas (Fig 5). Role of MSCs in Alleviating Immunologic Rejection of Allografts
To examine the role of MSCs in alleviating acute allograft rejection, 15 animals were injected with 5 ⫻ 106 MSCs, as in previous reports,6,7 and 15 control animals were injected with saline solution. The general state of health, chimera formation, hepatic function, and liver pathologic features were observed after the procedure. General Health
The experimental and control animals all survived longer than 12 days. Compared with experimental models, control
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the female recipient was heterozygous and not homozygous. That is, chimerism occurred. The His19 epitope is present in Lewis rats but not BN rats. A His19 epitope in a BN rat recipient that received a male Lewis rat liver transplant would indicate that the female BN recipient was heterozygous and not homozygous. In our study, the experimental models but not the control models exhibited the SRY gene (Fig 6). Splenocytes were His19-positive in the experimental group (14.9% ⫾ 1.0%) but negative in the control group (Fig 7), demonstrating generation of chimerism. Hepatic Function and Liver Pathologic Features
Compared with the control group, hepatic function in the experimental group was much improved (Fig 8). Severe acute rejection (class III) was observed in the control group, and mild to moderate acute rejection (class I or II) in the cell transplantation group (Fig 9). Fig 6. SRY gene detection. ⫺MSC represents peripheral blood monocytes (PBMCs) from the untreated control group; ⫹MSC, PBMCs from the experimental group treated with mesenchymal stem cells; positive control, PBMCs from male Lewis rats; and negative control, PBMCs from female Brown Norway rats.
models demonstrated decreased appetite and activity, and serious jaundice. Chimera Formation
In general, the SRY gene is present in male but not female rats. The SRY gene present in a female BN recipient that received a male Lewis rat liver transplant would indicate that
DISCUSSION
Mesenchymal stem cells are defined by a composite of morphologic features, phenotype, and functional characteristics. Our experiment showed that the expression rates of both CD29 and CD90 were greater than 90%, and simultaneous expression rates of CD29 and CD90 were greater than 96%. After treatment with IFN-␥, CD11b/c, CD40, CD80, and CD86 were negative and CD45, CD54, MHC-I, and PDL-1 were positive. MHC-II was negative but became weakly positive after treatment with IFN-␥. The cells were fusiform and arranged in a swirl pattern, consistent with findings of previous reports.8 –11 Not only can MSCs differ-
Fig 7. Expression of His19. Spleen single-cell suspensions from the experimental model treated with mesenchymal stem cells (A) and the untreated control model (B) were used to detect His19 epitope using flow cytometry. Mouse IgG1 was used as isotype control. The rate of His19-positive splenocytes was 14.9% ⫾ 1.0% in the experimental group but was negative in the control group.
Fig 8. Hepatic function. A, Alanine aminotransferase levels (ALT) in the untreated control group and in the experimental group treated with mesenchymal stem cells were 783.34 ⫾ 87.64 and 145 ⫾ 8.3 U/L, respectively. There was statistical significance between groups (P ⬍ .01). B, Total bilirubin (TBIL) levels in the untreated control group and in the experimental group treated with mesenchymal stem cells were 145 ⫾ 8.3 and 89.54 ⫾ 7.8 mol/L, respectively. There was statistical significance between groups (P ⬍ .01).
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Fig 9. A, Severe acute rejection (class III) was demonstrated in liver tissue from the untreated control group. The liver cells were disorderly arranged and balloonlike. The portal area was occupied by inflammatory cells, which extended to the liver parenchyma. There was endometrial vascular damage. Many flakes of liver cell necrosis were noted. B and C, Mild to moderate acute rejection (class II and I, respectively) was demonstrated in liver tissue from the experimental group treated with mesenchymal stem cells. A medium number of monocytes infiltrated the portal area; however, the inflammatory cells were confined to it. The liver cells were orderly arranged and slightly swollen.
entiate into mesoblastic cells, they can also differentiate into other blastodermal cells such as endothelial cells and hepatocytes.12,13 Both CD45 and CD11b/c antigens are characteristic of hematopoietic cells. Thus, MSCs are stem cells but not hematopoietic stem cells.
Our experimental results suggest that rats that underwent OLT and were treated with MSCs exhibited milder immunologic rejection of liver grafts. Mesenchymal stem cells have both vicarious and immunologic roles. Vicariously, MSCs survive and differentiate to serve in repair and
MESENCHYMAL STEM CELLS AND LIVER ALLOGRAFT REJECTION
regeneration.10,13,14 The MSCs stained with carboxyfluorescein diacetate–succinimidyl ester settle in the liver, especially around the portal areas. Immunologically, MSCs have immunosuppressive functions. First, MSCs do not express stimulating elements such as CD40, CD80, CD86, and MHC-II. The MSCs not only fail to induce T-cell activation but also evade dissolution by CD8-positive cytotoxic T lymphocytes,15,16 allowing them to survive. Second, MSCs inhibit the cellular immune response, acting as regulatory cells inhibiting lymphocyte proliferation in vivo. The inhibitory effects of MSCs is dose-related. Third, MSCs can induce formation of a chimera. In our study, chimera formation was apparent in models treated with MSCs but not models not treated with MSCs. This suggests that donor and recipient cells can coexist peacefully in vivo, consistent with the findings of Itakura et al.17 There are many explanations for the immunosuppressive mechanisms of MSCs; however, little is known about the contribution of direct contact between MSCs and immunologic cells. The MSCs inhibit T-cell proliferation primarily by direct contact between cells but not by inducing apoptosis in an MHC-dependent manner.18,19 CD4- and CD8positive T cells both bind to MSCs as found at flow cytometry analysis. We observed that MSCs highly express PDL-1, CD54, and MHC-I, especially after treatment with IFN-␥. The MSCs may connect with T cells via CD54. Augello et al18 noted that MSCs inhibit lymphocyte proliferation by activating the programmed death-1 pathway. The MSCs may highly express CD54 and PDL-1 with stimulation by IFN-␥ in vivo during OLT. Interferon-␥ enhances the immunologic suppression of MSCs in other ways. Ren et al20 reported that the immunosuppressive function of MSCs was elicited by proinflammatory cytokines such as IFN-␥, tumor necrosis factor–␣, and interleukin-1. Interferon-␥ induced indoleamine 2,3dioxygenase, nitric oxide, and expression of other immunosuppressive molecules, strengthening immunologic regulation. REFERENCES 1. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow: analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6:230, 1968 2. Klyushnenkova E, Mosca JD, Zernetkina V, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci 12:47, 2005 3. Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery 93:64, 1983
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4. Krampera M, Cosmi L, Angeli R, et al. Role for interferon-␥ in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 24:386, 2006 5. Ryan JM, Barry F, Murphy JM, et al. Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol 149:353, 2007 6. Le Blanc K, Samuelsson H, Gustafsson B, et al. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21:1733, 2007 7. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30:42, 2002 8. Stagg J. Immunoregulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 69:1, 2007 9. Sylwia B, Danuta J, Marcin M. Mesenchymal stem cells: characteristics and clinical applications. Folisa Histochem Cytobiolog 44:215, 2006 10. Carvalho KA, Oliveira L, Malvezzi M, et al. Immunophenotypic expression by flow cytometric analysis of cocultured skeletal muscle and bone marrow mesenchymal stem cells for therapy into myocardium. Transplant Proc 40:842, 2008 11. Guarita-Souza LC, Carvalho KA, Simeone BR, et al. Functional outcome of bone marrow stem cells: mononuclear versus mesenchymal stem cells after cellular therapy in myocardial scar in Wistar rats. Transplant Proc 38:1953, 2006 12. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337, 2002 13. Kao CL, Lin HT, Chen YW, et al. Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells. Transplant Proc 39:3444, 2007 14. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6:1282, 2000 15. Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 76:1208, 2003 16. Angoulvant D, Clerc A, Benchalal S, et al. Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens. Biorheology 41:469, 2004 17. Itakura S, Asari S, Rawson J, et al. Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat. Am J Transplant 7:336, 2007 18. Augello A, Tasso R, Negrini SM, et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death-1 pathway. Eur J Immunol 35:1482, 2005 19. Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75:389, 2003 20. Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell– mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2:141, 2008