T Cell Apoptosis in Human Heart Allografts
1998; Elsevier BV; Volume: 153; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65696-9
ISSN1525-2191
AutoresEls van Hoffen, D.F. van Wichen, Jaklien C. Leemans, Richard A.J.F. Broekhuizen, Annette H. Bruggink, Mark de Boer, Nicolaas de Jonge, Hans Kirkels, Piet J. Slootweg, F.H.J. Gmelig-Meyling, Roel A. de Weger,
Tópico(s)Transplantation: Methods and Outcomes
ResumoIt is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation. It is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation. Despite improved immunosuppressive regimens, human heart transplantation is still complicated by acute rejection episodes. Acute rejection, a T-cell-mediated process, occurs most frequently during the first months after transplantation. Most studies have concentrated on the induction of cytotoxicity against the graft and the production of cytokines within the graft. However, little is known about the immune-regulatory mechanisms occurring within the transplanted organ. During rejection, T cells enter the graft. Activation of these T cells requires two signals. In addition to the interaction between the T cell receptor and the major histocompatibility complex on the antigen-presenting cell (APC), a second signal is required, which is provided through co-stimulatory molecules, present on both the T cell and on the APC.1Hathcock KS Laszlo G Pucillo C Linsley P Hodes RJ Comparative analysis of B7–1 and B7–2 costimulatory ligands: expression and function.J Exp Med. 1994; 180: 631-640Crossref PubMed Scopus (644) Google Scholar, 2Ding L Shevach EM Activation of CD4+ T cells by delivery of the B7 costimulatory signal on bystander antigen-presenting cells (trans-costimulation).Eur J Immunol. 1994; 24: 859-866Crossref PubMed Scopus (84) Google Scholar The two most common pathways of co-stimulation are mediated by B7-1/B7-2 on the APC and CD28 or CTLA4 on the T cell and by CD40 on the APC and CD40 ligand (CD40L) on the T cell. Absence of a co-stimulatory signal during primary activation will lead to a state of anergy, in which the T cells are unable to respond to a renewed antigen challenge. This anergic state can result in apoptosis of the T cell.3Boussiotis VA Freeman GJ Gray G Gribben J Nadler LM B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance.J Exp Med. 1993; 178: 1753-1763Crossref PubMed Scopus (215) Google Scholar, 4June CH Bluestone JA Nadler LM Thompson CB The B7 and CD28 receptor families.Immunol Today. 1994; 15: 321-331Abstract Full Text PDF PubMed Scopus (187) Google Scholar The role of this pathway in anergy induction toward allografts has been shown in rodents and primates; blocking the co-stimulatory pathway by treatment with CTLA4 Ig or anti-CD40L leads to prolonged or permanent acceptance of the allograft.5Kirk AD Harlan DM Armstrong NN Davis TA Dong Y Gray GS Hong X Thomas D Fechner Jr, JH Knechtle SJ CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates.Proc Natl Acad Sci USA. 1997; 94: 8789-8794Crossref PubMed Scopus (827) Google Scholar, 6Lakkis FD Konieczny BT Saleem S Baddoura FK Linsley PS Alexander DZ Lowry RB Pearson TC Larsen CP Blocking the CD28–B7 T cell costimulation pathway induces long term cardiac allograft acceptance in the absence of IL-4.J Immunol. 1997; 158: 2443-2448PubMed Google Scholar Human lymphocytes can be anergized in vitro, using the same treatment.7Van Gool SW de Boer M Ceuppens JL The combination of anti-B7 monoclonal antibody and cyclosporin A induces alloantigen-specific anergy during a primary mixed lymphocyte reaction.J Exp Med. 1994; 179: 715-720Crossref PubMed Scopus (51) Google Scholar Anergy inductionin vivo may eventually lead to donor-specific nonresponsiveness, resulting in a reduction of the number of rejection episodes later after transplantation. In heart transplant recipients, this nonresponsiveness has been shown to be accompanied by a reduction in the frequency of donor-specific precursor cytotoxic T lymphocytes.8Hu H Robertus M De Jonge N Gmelig-Meyling FHJ Van der Meulen A Schuurman HJ Doornewaard H Van Prooijen HC De Weger RA Reduction of donor-specific cytotoxic T lymphocyte precursors in peripheral blood of allografted heart recipients.Transplantation. 1994; 58: 1263-1268PubMed Google Scholar The cytotoxic T cells are effector cells in the rejection process, causing tissue damage inside the graft. The cytotoxicity can be mediated via the secretion of granzyme and perforin but also via the interaction between Fas on the target cell and Fas-ligand (FasL) on the T cell. Both mechanisms induce apoptosis in the target cell.9Nagata S Golstein P The Fas death factor.Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3957) Google Scholar, 10Kummer JA Wever PC Kamp AM ten Berge IJM Hack CE Weening JJ Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection.Kidney Int. 1995; 47: 70-77Crossref PubMed Scopus (80) Google Scholar Fas is expressed constitutively on several cell types, including mouse heart tissue, but also on T cells.10Kummer JA Wever PC Kamp AM ten Berge IJM Hack CE Weening JJ Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection.Kidney Int. 1995; 47: 70-77Crossref PubMed Scopus (80) Google Scholar After activation of the T cell, Fas expression is up-regulated. At the same time, FasL expression is induced. FasL can induce apoptosis in a Fas-expressing target cell, including the T cell itself. Therefore, the Fas/FasL pathway is not only involved in cytotoxicity, but has also been described as a pathway to down-regulate an ongoing immune response, so-called activation-induced cell death (AICD).9Nagata S Golstein P The Fas death factor.Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3957) Google Scholar, 11Alderson MR Tough TW Davis-Smith T Braddy S Falk B Schooley KA Goodwin RG Smith CA Ramsdell F Lynch DH Fas Ligand mediates activation-induced cell death in human T lymphocytes.J Exp Med. 1995; 181: 71-77Crossref PubMed Scopus (864) Google Scholar The process of apoptosis is strictly regulated. Two important regulating proteins are Bcl2 and Bax, both members of the Bcl2 gene family. Bcl2 protects, whereas Bax induces, apoptosis. Both molecules are localized in the inner mitochondrial membranes, the endoplasmic reticulum, and the perinuclear membrane.12Jacobson MD Burne JF King MP Miyashita T Reed JC Raff MC Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA.Nature. 1993; 361: 365-369Crossref PubMed Scopus (703) Google Scholar, 13Oltvai ZN Milliman CL Korsmeyer SJ Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5823) Google Scholar The different family members can homo- and heterodimerize with one another. As long as heterodimers are present in excess, Bcl2 prevents the induction of apoptosis. However, when the expression of Bax increases, resulting in the formation of Bax homodimers, this can lead to the induction of the apoptotic pathway.14Nag B Kendrick T Arimilli S Yu SCT Sriram S Soluble MHC II-peptide complexes induce antigen-specific apoptosis in T cells.Cell Immunol. 1996; 170: 25-33Crossref PubMed Scopus (33) Google Scholar, 15Osborne BA Apoptosis and the maintenance of homeostasis in the immune system.Curr Opin Immunol. 1996; 8: 245-254Crossref PubMed Scopus (163) Google Scholar Therefore, the stochastic ratio of these inhibitors and activators inside a cell determines the sensitivity of a cell to undergo programmed cell death. Many of the above mentioned processes are well studied in in vitro experiments. However, little is known about the occurrence of these immune-regulatory mechanisms within the graft in vivo after transplantation. Therefore, we used endomyocardial biopsies (EMBs) from heart transplantation patients to study the role of T cells and macrophages in the processes of co-stimulation and apoptosis inside the graft. We developed a double-immunofluorescence technique with a high sensitivity, which can be used for confocal laser scan microscopy (CLSM). This enabled us to link the expression of certain molecules on certain cells with the phenotype of those cells. To study the role of co-stimulatory molecules on macrophages and lymphocytes in the activation of T cells, the expression of CD28, CTLA4, and CD40L on T cells and B7-1, B7-2, and CD40 on APCs was analyzed in relation to different grades of rejection. To analyze whether AICD plays a role in T cell regulation, apoptosis was evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL) method and DNA fragmentation. For the same reason, the expression of Fas and FasL and Bcl2 and Bax and the production of granzyme B and perforin was studied to evaluate which mechanism may be responsible for the apoptosis and tissue destruction during different grades of rejection. This study indicates that lymphocytes, infiltrating the cardiac graft during acute rejection, are actively down-regulated via AICD) by macrophages and by other lymphocytes in the infiltrate. This may affect the balance between T cell activation and inactivation, thereby influencing the immunological status of the patient after heart transplantation. EMBs were obtained from patients during the first months after allogeneic heart transplantation. Paraffin-embedded biopsies were histopathologically examined for rejection, according to the criteria of the International Society for Heart and Lung Transplantation.16Billingham ME Cary NRB Hammond ME Kemnitz J Marboe C McCallister HA Snovar DC Winters GL Zerbe A A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group.J Heart Transplant. 1990; 9: 587-593PubMed Google Scholar Extra biopsies were snap-frozen in liquid nitrogen for research purposes. For this study, frozen and paraffin-embedded biopsies were used with grades of rejection varying from grade 0 (no rejection) to grade 3B (severe rejection). As grading of the rejection is not feasible on frozen tissue, the number of CD3+ T cells per mm2 was counted, as reported previously.17Giordano C Stassi G De Maria R Todaro M Richiusa P Papoff G Ruberti G Bagnasco M Testi R Galluzzo A Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis.Science. 1997; 275: 960-963Crossref PubMed Scopus (531) Google Scholar Biopsies were grouped according to these T cell numbers, as follows: −, no cells; ±, 1 to 100 cells/mm2; +, 101 to 200 cells/mm2; ++, >200 cells/mm2. These groups generally correlated with grading on the parallel paraffin-embedded EMBs. Absolute numbers of macrophages are difficult to determine, because they intermingle with surrounding cells due to their irregular shape. Therefore, the numbers of macrophages were semiquantitatively correlated to the T cell numbers and are indicated as follows: ±, few macrophages; +, intermediate number of macrophages; ++, high number of macrophages. Their number increased corresponding with the number of T cells. Because EMBs are too small for elaborate studies, heart tissue with rejection grade 3A or 3B, obtained from four patients after autopsy, was used to develop the methodology and for the double-staining experiments. Antibodies were titrated on tonsil and heart tissue to obtain optimal dilutions (Table 1). For a basic evaluation, antibodies were applied using conventional immunoperoxidase staining. In brief, paraffin sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 1.5% H2O2 in methanol for 30 minutes. For antigen retrieval, sections were boiled for 15 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) or, only in the case of FasL, predigested with 2.5 × 106 U/L pepsin in 0.1 mmol/L glycine buffer (pH 2.0). Frozen sections were fixed in acetone for 10 minutes. After washing in PBS/Tween-20, sections were preabsorbed using 10% normal horse serum or normal goat serum for 15 minutes and incubated with the primary antibody, an diluted in PBS/1% bovine serum albumin (BSA), for 1 hour. The sections were washed and incubated with biotinylated horse anti-mouse antibody (1:500; lot F0425, Vector Laboratories, Burlingame, CA) or biotinylated goat anti-rabbit antibody (1:500; lot F505, Vector) in PBS/1% BSA for 30 minutes, washed again in PBS/Tween-20, and then incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:400; lot 14022721, Boehringer Mannheim, Mannheim, Germany) in PBS/1% BSA for 30 minutes. After development of the peroxidase with diaminobenzidine (DAB)/0.03% H2O2, the sections were counterstained with hematoxylin and embedded in dibutylphthalate polystyrene xylene (DPX). Omission of the primary antibody and an isotype-matched control antibody replacing the primary antibody served as negative controls. Only results that stand up to these controls are presented.Table 1Antibodies Used for ImmunohistochemistryCell markers used for double stainingAntibodyDilutionLot/cloneCD3a,bCD4aCD8aCD68cUlexdTUNELCo-stimulatory moleculesCD28e1:2505xCD28f1:5M014631CTLA4f1:10M014748CTLA4g1:100011D4CD40Lh1:25M91CD80i1:200B7-24xxCD86f1:502331/Fun1xxCD40j1:20005D12xxApoptosis and cytotoxicityBaxe1:101xxxxxBax*Polyclonal rabbit antibody.†Used on paraffin-embedded tissue.k1:51089501-4Bcl2†Used on paraffin-embedded tissue.c1:100105xFasl1:100ws103FasLm1:40C047xxxxxActin SMAd1:400029f4909xDesmin*Polyclonal rabbit antibody.n1:20036-03xTUNELxxxxPerforin†Used on paraffin-embedded tissue.o1:10303325Granzyme B†Used on paraffin-embedded tissue.p1:500GrB7xCD57†Used on paraffin-embedded tissue.e1:2503.1xIndicated is which antibodies are used in different combinations in fluorescent double labeling (x). Dilutions are mentioned for single staining in conventional immunohistochemistry. For double immunofluorescence, dilutions were adapted to obtain proper signals. CD3, CD4, CD8, and CD68 were FITC conjugated, andUlex (lot 534033) was TRITC conjugated. Cell markers and antibodies were obtained from the following sources:aCD3, lot 50527; CD4, lot 60442; CD8, lot 60992, Becton Dickinson, San Jose, CA; bCD3 in double-staining on paraffin sections: polyclonal rabbit antibody, 1:200, lot 102, Dako, Glostrup, Denmark; cCD68, lot 125(101), Dako;dSigma, St. Louis, MO;eImmunotech/Coulter, Marseille, France;fPharmingen, San Diego, CA;gBristol-Meyers, Seattle WA;hImmunex, Seattle WA;iInnogenetics, Gent, Belgium;jPanGenetics, Amsterdam, The Netherlands;kOncogene Research Products/Calbiochem, Cambridge, MA; l6th International Workshop HLDA;mSanta Cruz Biotechnology, Santa Cruz, CA;nEuro-Diagnostics, Apeldoorn, The Netherlands;oEndogen, Cambridge, MA;pgift from Dr. Kummer, Amsterdam, The Netherlands.* Polyclonal rabbit antibody.† Used on paraffin-embedded tissue. Open table in a new tab Indicated is which antibodies are used in different combinations in fluorescent double labeling (x). Dilutions are mentioned for single staining in conventional immunohistochemistry. For double immunofluorescence, dilutions were adapted to obtain proper signals. CD3, CD4, CD8, and CD68 were FITC conjugated, andUlex (lot 534033) was TRITC conjugated. Cell markers and antibodies were obtained from the following sources:aCD3, lot 50527; CD4, lot 60442; CD8, lot 60992, Becton Dickinson, San Jose, CA; bCD3 in double-staining on paraffin sections: polyclonal rabbit antibody, 1:200, lot 102, Dako, Glostrup, Denmark; cCD68, lot 125(101), Dako;dSigma, St. Louis, MO;eImmunotech/Coulter, Marseille, France;fPharmingen, San Diego, CA;gBristol-Meyers, Seattle WA;hImmunex, Seattle WA;iInnogenetics, Gent, Belgium;jPanGenetics, Amsterdam, The Netherlands;kOncogene Research Products/Calbiochem, Cambridge, MA; l6th International Workshop HLDA;mSanta Cruz Biotechnology, Santa Cruz, CA;nEuro-Diagnostics, Apeldoorn, The Netherlands;oEndogen, Cambridge, MA;pgift from Dr. Kummer, Amsterdam, The Netherlands. As positive control tissues, tonsil was used as a normal lymphoid tissue, a muscle biopsy from a polymyositis patient was used as a control for inflammation in muscle tissue in comparison with heart muscle, and resected thyroid from a patient with Hashimoto's thyroiditis was used, as in this disease thyrocytes have been described to constitutively express both Fas and FasL.17Giordano C Stassi G De Maria R Todaro M Richiusa P Papoff G Ruberti G Bagnasco M Testi R Galluzzo A Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis.Science. 1997; 275: 960-963Crossref PubMed Scopus (531) Google Scholar As negative control, healthy nontransplanted heart tissue obtained after autopsy was used. Using the tyramide signal amplification (TSA) Direct- and Indirect kit (NEL 701/700, DuPont/NEN Life Science Products, Boston, MA), we developed a double-fluorescence technique that enabled us to combine any two monoclonal antibodies, provided that one of them is fluorescein isothiocyanate labeled. The kits can also be used for single-fluorescence techniques. The combinations of antibodies used in double-staining experiments are indicated in Table 1. For double staining, frozen sections were fixed in acetone for 10 minutes and washed in PBS/Tween-20. The sections were incubated with the primary antibody, eg, a mouse antibody, diluted in PBS/1% human serum albumin (HSA) for 1 hour. The sections were washed three times for 5 minutes each in Tris-buffered saline (TBS)/Tween-20 buffer (0.1 mmol/L TBS, pH 7.5/0.01% Tween-20), and incubated with, in the case of a mouse antibody, HRP-conjugated rabbit anti-mouse antibody (RAMPO; 1:200, lot 020, Dako, Glostrup, Denmark) in TBS/10% human AB serum. After washing in TBS/Tween-20, the amplification of the signal was performed using the TSA Indirect kit, with biotinyl-conjugated tyramide diluted 1:50, according to the manufacturer's instructions, for 8 minutes. The sections were washed in TBS, incubated with Texas-Red-conjugated streptavidin (1:250 in TBS; lot NEL 721, DuPont/NEN Life Science Products) for 30 minutes, and washed again in TBS. The residual RAMPO activity was blocked with PBS/1% H2O2 for 20 minutes. Sections were washed and preincubated with 10% normal mouse serum in TBS. A 1-hour incubation followed with a second FITC-labeled antibody of interest in PBS/1% HSA. The sections were washed in TBS/Tween-20 and incubated with HRP-conjugated rabbit-anti-FITC (1:200; lot 015(201), Dako) for 30 minutes. After washing, the FITC signal was amplified using the TSA Direct kit, with FITC-conjugated tyramide diluted 1:50, according to the manufacturer's instructions, for 8 minutes. After a final washing step in TBS/Tween-20, sections were embedded in Vectashield. Double staining of granzyme B or CD57 with CD3 was performed on paraffin sections. Sections were deparaffinized, rehydrated, and boiled for 15 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were preabsorbed with normal horse serum and incubated with primary antibodies diluted in PBS/1% BSA. After washing, an incubation with biotinylated horse anti-mouse was performed, followed by an incubation with tetramethylrhodamine isothiocyanate (TRITC)-conjugated streptavidin. Then the sections were washed and incubated with a polyclonal anti-CD3 antibody (1:40; Dako), followed by incubation with HRP-conjugated swine anti-rabbit antibody (1:50; Dako). This signal was visualized and amplified using the TSA Direct kit, resulting in a FITC labeling. The TUNEL method was used, as described elsewhere,18Gavrieli Y Sherman Y Ben-Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9100) Google Scholar to analyze apoptosis in EMB sections. For the longitudinal study of three patients, paraffin-embedded tissue sections were deparaffinized and rehydrated. Sections were then treated with proteinase K (20 μg/ml; Boehringer Mannheim) for 30 minutes. After washing in double-distilled water, endogenous peroxidase was blocked using PBS/2% H2O2 for 10 minutes. Sections were then presoaked in TdT buffer (0.5 mmol/L cacodylate, 1 mmol/L CoCl, 0.5 mmol/L dithiothreitol, 0.05% BSA, 0.15 mol/L NaCl) for 10 minutes. Frozen sections were fixed in acetone for 10 minutes at room temperature (RT) and then presoaked in TdT buffer. Sections were then incubated for 2 hours at 37°C in 25 μl of TdT solution, containing 1X terminal transferase buffer (Promega, Madison, WI), 0.5 nmol of biotin-dUTP (Boehringer Mannheim), and 5 to 10 U of TdT (Promega). After the TdT reaction, slides were soaked in TdT blocking buffer (300 mmol/L NaCl, 30 mmol/L tri-sodium citrate-2-hydrate), incubated with HRP-conjugated streptavidin for 30 minutes at RT, and developed for 10 minutes in phosphate-buffered citrate (pH 5.8) containing 0.6 mg/ml DAB. Nuclei were counterstained with hematoxylin. For CLSM, sections were incubated with FITC-conjugated streptavidin (1:20; lot 14594222-09, Boehringer Mannheim) for 30 minutes at RT. Nuclei were counterstained with 4 μg/ml propidium iodide, washed in PBS, and mounted in Vectashield. Positive controls were obtained by a DNAse I treatment for 60 minutes at 37°C (40 U/ml; Boehringer Mannheim). For negative controls, TdT was replaced by distilled water in the TdT solution. Frozen sections were fixed in acetone for 10 minutes. After washing in PBS/Tween-20, sections were incubated for 1 hour with an unlabeled or FITC-conjugated primary antibody for phenotype analysis, diluted in PBS/1% HSA. After fixation in 4% formaldehyde for 10 minutes, the TUNEL reaction was performed as described above, using biotin-16-dUTP. The TUNEL reaction was developed using TRITC-conjugated streptavidin (1:10; lot A286-N596B, Southern Biotechnology Associates, Birmingham, AL). After washing, sections were incubated with secondary antibodies for 30 minutes. In the case of FITC-conjugated primary antibodies, the HRP-conjugated rabbit anti-FITC antibody was used. If primary antibodies were unlabeled, a RAMPO (1:100) or a HRP-conjugated swine anti-rabbit antibody (SWARPO, 1:100; lot 035(101), Dako) was used. For smooth muscle actin and desmin, a FITC-conjugated rabbit anti-mouse antibody (1:40; lot 082, Dako) and a FITC-conjugated horse anti-rabbit antibody (1:20; lot PK17-02-F10, CLB, Amsterdam, The Netherlands) was used, respectively. Except for these last two, all signals were amplified using the TSA Direct kit for 12 minutes, resulting in amplification of the FITC signal. All sections were washed and mounted in Vectashield. From postmortem heart tissue without and with rejection (grade 3A/3B), 20 frozen sections of 10 μm were resuspended in 20 μl of solution A (10 mmol/L EDTA, 0.5 mmol/L Tris/HCl, 0.5% Sarcosyl, K 0.5 μg/ml proteinase) and incubated at 50°C for 60 minutes. Then 10 μl of DNAse-free RNAse was added (containing 5 μg of RNAse A, Boehringer Mannheim), followed by an incubation for 60 minutes at 50°C. Then 20 μl of solution B (40% sucrose containing Orange G, 10 mmol/L EDTA, and 1% low-melting-point agarose) was added at 56°C, and samples were loaded on a 1.5% agarose gel containing ethidium bromide. After polymerization of the samples, the gel was run and visualized under ultraviolet light. In situ hybridization was performed for FasL using the same procedure as described previously.19Van Hoffen E Van Wichen D Stuij I De Jonge N Klöpping C Lahpor J Van Den Tweel J Gmelig-Meyling F De Weger R In situ expression of cytokines in human heart allografts.Am J Pathol. 1996; 149: 1991-2003PubMed Google Scholar The following primers for FasL were used in a polymerase chain reaction for the synthesis of the digoxigenin (Dig)-dUTP-labeled probe: 5′ primer, 5′-CAAGTCCAACTCAAGGTCCATGCC-3′; 3′ primer, 5′-CAGAGAGAGCTCAGATACGTTTGAC-3′. The probe was sequenced to confirm that the specific FasL product was amplified. After overnight hybridization of the probe on tissue sections, the signal was visualized using a mouse anti-Dig monoclonal antibody (1:50; lot 13026924-01, Boehringer Mannheim). The signal was amplified with RAMPO and SWARPO and developed with DAB. As negative controls, sections were pretreated overnight with RNAse T1 (100 U/ml; Boehringer Mannheim). The Mann-Whitney U test was used to compare the expression of molecules or apoptosis in different groups of cells or biopsies.20Armitage P Berry G Statistical Methods in Medical Research. ed 2. Blackwell Scientific Publications, Oxford1987: 411-417Google Scholar P values <0.05 were considered to be statistically significant. To study the in situ expression of co-stimulatory molecules during acute rejection of human heart allografts, we used 32 frozen EMBs from 17 patients, with and without signs of rejection. As described previously, the mononuclear cell infiltrate during acute rejection consists mainly of T cells and macrophages. The ratio CD4+ to CD8+ T cells is ∼2:1.19Van Hoffen E Van Wichen D Stuij I De Jonge N Klöpping C Lahpor J Van Den Tweel J Gmelig-Meyling F De Weger R In situ expression of cytokines in human heart allografts.Am J Pathol. 1996; 149: 1991-2003PubMed Google Scholar Expression of co-stimulatory molecules was studied by single- and double-immunofluorescence techniques, using tyramide amplification, and detection via CLSM. On T cells, no or hardly any expression of CD28 (Figure 1A), and no expression of CTLA4 and CD40L was observed (not shown). These molecules were detected on T cells in control tonsil tissue (not shown). Only when a severe rejection infiltrate was present did a small proportion of T cells express CD28. In some Quilty lesions (mononuclear cell infiltrates in the endomyocardium not related to rejection),21Joshi A Masek MA Brown BW Weiss LM Billingham ME "Quilty" revisited: a 10-year perspective.Hum Pathol. 1995; 26: 547-557Abstract Full Text PDF PubMed Scopus (55) Google Scholar expression of CD28 was observed (not shown). In positive control tissues containing activated T cells, taken from normal tonsil, polymyositis (Figure 1B), and Hashimoto's thyroiditis patients, an abundant expression of CD28 on T cells was observed. B7-1 (CD80) and B7-2 (CD86) were present on the majority of the macrophages, as confirmed by double-fluorescent staining with CD68 (Figure 1C). CD40 was detectable both on macrophages and on endothelium (not shown). Despite the absence of co-stimulatory molecules on T cells, the T cells do make close contact with APCs, as shown by CLSM (Figure 1D). These data were (semiquantified in relation to th
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