Enhanced apoptosis of peripheral blood mononuclear cells in cardiac transplanted patients undergoing chronic immunosuppressive treatment

Transplant Immunology 10 (2002) 269–275

Enhanced apoptosis of peripheral blood mononuclear cells in cardiac transplanted patients undergoing chronic immunosuppressive treatment M. Di Renzoa,*, P.L. Capecchia,b, A. Camurrid, F. Di Ciollac, M. Maccherinic, G. Lisic, G. Pompellaa, A.L. Pasquia, A. Auteria, M.P. Abbracchiod, F. Laghi Pasinia,b a

Istituto di Semeiotica Medica, Policlinico Le Scotte, Viale Bracci, University of Siena, 53100 Siena, Italy U.O. di Immunologia Clinica, Policlinico Le Scotte, Viale Bracci, University of Siena, 53100 Siena, Italy c Istituto di Cardiochirurgia, Policlinico Le Scotte, Viale Bracci, University of Siena, 53100 Siena, Italy d Dipartimento di Farmacologia, Via Balzaretti 9, University of Milan, 20133 Milan, Italy

b

Received 14 January 2002; accepted 22 March 2002

Abstract Apoptosis plays a major role in tissue transplantation because intact T-cell-apoptosis pathways are required for the induction of tolerance to allografts. Moreover, immunosuppressive agents commonly used in clinical transplantation medicine promote lymphocyte apoptosis inhibiting the expression and production of cytokines involved in lymphocyte survival. The aim of our study was to evaluate peripheral blood mononuclear cells (PBMC) spontaneous apoptosis in patients undergoing chronic immunosuppressive treatment after cardiac transplantation. PBMC obtained from patients (ns31) and controls matched for age and sex (ns25) were cultured for 72 h and apoptosis was evaluated by quantification of fragmented DNA, staining with Hoechst 33258 dye and annexin V binding. We also investigated Fas expression and FasL mRNA expression as well as the ability of an IgM anti-Fas antibody to induce apoptosis. Finally, we evaluated IL2 production induced by PHA and the ability of IL2 to prevent apoptosis. In patients, PBMC underwent enhanced spontaneous apoptosis in comparison with controls. However, we could not find any difference between patients and normals as regards the expression of Fas and of FasL mRNA, even if the cross-linking of the Fas molecule induced apoptosis in PBMC from patients, whereas it failed to induce cell death in normals. We also found that IL2 production was significantly decreased in patients and that the addition of IL2 to the culture medium reduced PBMC spontaneous apoptosis. Our findings suggest that in cardiac transplanted patients PBMC undergo enhanced spontaneous apoptosis, which may contribute to prevent allograft rejection. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Transplantation; Apoptosis; FasyFasL; IL2

1. Introduction Apoptosis or programmed cell death (PCD) is a mechanism of cell death with distinct morphological features which results in the phagocytosis of the dying cell without the release of its content w1x. Recently T and B cell PCD has been extensively studied because it plays an important role in the development and maintenance of the immune system w2x where it is tightly regulated by a number of gene products which either promote cell death or extend cell survival w3x. Apoptosis also plays a major role in tissue transplantation both in *Corresponding author. Tel.: q39-0577585741; fax: q390577286202. E-mail address: [email protected] (M. Di Renzo).

graft rejection and in transplantation tolerance w4,5x: intact T-cell-apoptosis pathways are required for the induction of tolerance through MHC barriers w6x and treatments that enhance the induction of apoptosis in the responding T cells such as costimulator block plus rapamycin promote tolerance w7x. Therefore the induction of T cell apoptosis seems to be essential for longlasting stable immune tolerance to allografts. Moreover, it is well-known that immunosuppressive agents such as glicocorticoids (GC) and cyclosporin A (CsA) inhibit the expression and production of several cytokines involved in the regulation of lymphocyte proliferation or differentiation as well as in lymphocyte survival w8x. It is also known that immunosuppressive agents induce apoptosis in T-cell hybridomas, thymo-

0966-3274/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 2 . 0 0 0 7 5 - 8

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cytes and peripheral blood mononuclear cells (PBMC) w9–11x: in particular GC are able to induce in vitro PCD of mitogen-activated PBMC through a downregulation of IL2 production as well as of CD3 molecules w12,13x; controversial results have been reported as regards CyA because CyA blocks activation-induced apoptosis in T-cell lines w14x, whereas it induces apoptosis of both resting and mitogen-activated human lymphocytes w11,15x. 2. Objective On the basis of these premises, we investigated whether in cardiac transplanted patients undergoing chronic immunosuppressive treatment PBMC undergo enhanced spontaneous apoptosis in comparison with normal subjects. We also tried to clarify the mechanisms involved in the accelerated PCD, examining both the FasyFasL pathway which mediates apoptosis of activated lymphocytes w16x and production of IL2 which promotes T cell survival w17x. 3. Materials and methods 3.1. Patients Thirty one cardiac transplanted patients, age range 30–70, who had undergone cardiac transplantation in a period of time ranging from 3 months to 4 years before being evaluated, were enrolled in the study after informed consent was obtained. Twelve patients were receiving GC, CsA and azathioprine, 8 patients were receiving GC and CsA, 2 patients were receiving CsA and azathioprine, 9 patients were receiving only CsA. All patients were free from acute or exacerbated chronic bacterial or viral infections when studied as well as from acute rejection evaluated through endomyocardial biopsy or chronic rejection evaluated through coronarography. Twenty six healthy blood donors matched for age and sex were studied in parallel with the patients. 3.2. Cell isolation and culture PBMC were isolated from the patients and the controls heparinized whole blood by density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). PBMC were cultured in RPMI 1640 medium supplemented with fetal calf serum (10%), L-glutamine (2 mM) and penicillin–streptomycin (100 Uyml, 100 mgyml, Gibco, Paisley, UK). In some experiments, cardiac transplanted patients PBMC were cultured with human recombinant IL2 (100 IUyml, Pharmingen, San Diego, CA). 3.3. Apoptosis assays After 72 h of culture, PBMC viability was assessed by Trypan blue exclusion while PBMC spontaneous

apoptosis was evaluated by three different methods. For measuring intracellular histone-associated DNA fragments of PBMCs an ELISA was used according to manufacturer’s instructions (Cell Death Detection ELISAPLUS, Roche Molecular Biochemicals, Mannheim, Germany). PBMCs (20 000) were obtained from the culture and pelleted. The cells were lysed and the supernatant containing the nuclear-derived histone-associated DNA fragments was used for ELISA. The optical density (OD) 405 nm gave the degree of DNA fragmentation. For identification of apoptosis and necrosis of PBMCs, the binding of Annexin V and the uptake of propidium iodide were measured by flow cytometry (PAS, Partec, Germany) using the apoptosis detection kit annexin V-FITC (Dako, Glostrup, Denmark). Forward light scatter characteristics of living cells were used to delete debris from the analyses. Logarithmic fluorescence intensity of annexin V-FITC was plotted vs. the fluorescence intensity of propidium iodide in a dot plot. Data from 10 000 PBMCs were analysed for each plot. Finally, the morphologic features of PBMC were observed by fluorescence microscopy: cells were collected by centrifugation, fixed with 4% paraformaldehyde and allowed to mechanically re-attach to glass coverslips coated with poly-D-lysine. Nuclear chromatin was stained with the Hoechst 33 258 dye (Sigma Chemical CO, St Louis, MO) and analysed with a fluorescence light microscope equipped with a UV filter. Nuclear fragmentation andyor marked condensation of the chromatin with reduction of nuclear size were considered features of apoptotic cells. 3.4. Fas expression PBMC were washed twice with PBS and stained with FITC-labelled anti-CD3 Ab (Becton Dickinson, Mountain View, CA) and PE-labelled anti-Fas (Becton Dickinson) for 30 min at 4 8C. FITC-labelled and PE-labelled mouse IgG were used as isotype-matched background control. Following incubation, the cells were washed twice with PBS and 5000 cells were acquired using flow cytometry (FACScan, Becton Dickinson). Lymphocytes were gated and percentage of double-positive cells (CD3qFasq) was determined. 3.5. Reverse transcriptase-PCR analysis of FasL Total RNA was extracted from freshly isolated PBMC using Trizol (Gibco). One microgram of RNA was reverse transcribed at 42 8C for 45 min in 20 ml buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 1 mM each of dATP, dCTP, dGTP, and dTTP, 0.1 mg oligo(dT)15 (Gibco) and 200 U MMLV Reverse Transcriptase (Gibco). Reactions were stopped by heat inactivation at 99 8C for 5 min and chilled on ice. Subsequently 1 ml of the cDNA products

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same conditions. The sequence of the primers (Pharmacia) were (in a 59–39 orientation) as follows:bactin: 59-TGACGGGGTCACCCACACTGTGCCCATCTA and 39-CTAGAAGCATTGCGGTGGACGATGGAGGG; FasL: 59-ATTCTTTGTTACAGGCACCG e 39GAGTTGATTGTCAGGAAGCA. Amplified products were analyzed by 1.5% agarose gel electrophoresis and visualized under UV rays after ethidium bromide staining. The FasL product was 696 bp, the b-actin product was 661 bp. The bands were quantified using the Imagequant program and the ratio b-actinyFasL was calculated. 3.6. Fas-induced apoptosis

Fig. 1. PBMC from cardiac transplanted patients undergo enhanced PCD in comparison with normal subjects after 72 h of culture. PCD was evaluated by ELISA detection of intracellular histone-associated DNA fragments and expressed as optical density (OD). Horizontal lines represent the median values (**P-0.01).

PBMC freshly isolated were stimulated for 16 h with 1 mgyml of CH-11 (Upstate, Lake Placid, NY), an antiFas IgM antibody or with a control IgM (Upstate). Then PBMC apoptosis was evaluated by ELISA (Cell Death Detection ELISAPLUS, Roche Molecular Biochemicals) as previously described. 3.7. IL2 production

was amplified by PCR in 25 ml of 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, in the presence of 6.5 pmol each of the sequence specific primers and 1.25 U of Taq polymerase (FastTaq, Roche Molecular Biochemicals). Amplification was performed for 32 cycles after 10 min initial denaturation at 95 8C. Each cycle of PCR included 1 min of denaturation at 95 8C, 1 min of annealing at 58 8C (FasL) and 1 min of extensiony synthesis at 72 8C in a DNA thermal cycler (Omn-E, Hybaid, Ashford, UK). All PCR cycles were terminated with a final extension step at 72 8C for 10 min. As a control b-actin amplification was performed under the

Cell culture media harvested after 16 h of stimulation with PHA (5 mgyml, Sigma) were tested for IL2 production by ELISA using ELISA microtiter plates (Corning Easy Wash, Celbio, Milano, Italy) coated overnight with 2 mgyml anti-IL-2 capture mAb (Pharmingen) in 0.1 M Na2HPO4 pH 9 buffer and blocked with PBSyTween. A biotin-labelled anti-IL-2 detecting antibody (Pharmingen) at 1 mgyml in PBSy10% fetal calf serum was used. The plates were developed using avidin-HRP (Vector, Burlingame, CA) and 2,2 azinobis substrate (Sigma). The lower limit of detection was 15.6 pgyml. 3.8. Statistical analysis The Mann–Whitney U-test was used to compare data obtained in cardiac transplanted patients to those obtained in normal controls. 4. Results 4.1. Cardiac transplanted patients’ PBMCs undergo enhanced spontaneous apoptosis in vitro

Fig. 2. PBMC from cardiac transplanted patients (j) undergo enhanced PCD in comparison with normal subjects (3) after 72 h of culture. Median percentage of apoptotic cells evaluated by annexin V binding in flow cytometry and by Hoechst 33 258 staining in fluorescence microscopy (*P-0.05).

Non stimulated PBMCs of 31 cardiac transplanted patients and 26 normal subjects were cultured in complete medium and apoptosis was evaluated after 72 h of culture. In comparison with normal subjects, cardiac transplanted patients showed an enhanced spontaneous PCD, whereas PBMC viability assessed by Trypan blue exclusion was not reduced (data not shown): Fig. 1 shows that the amount of intracellular histone-associated

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cardiac transplanted patients than in normals (P-0.05; Fig. 2). 4.2. FasyFasL expression in cardiac transplanted patients

Fig. 3. Percentage of Fas expression within T cells from 21 cardiac transplanted patients and 15 normal subjects. n.s., not significant. Horizontal lines represent the median values.

DNA fragments evaluated in ELISA was higher in PBMC from cardiac transplanted patients than in controls (P-0.01). Further confirmation of apoptosis was made by examining Annexin V binding in flow cytometry and nuclear morphology after staining with Hoechst 33258. Both the percentage of Annexin Vbinding cells and the percentage of cells with apoptotic features in fluorescence microscopy were higher in

To clarify whether an increased Fas expression might contribute to the accelerated PBMC apoptosis, we analyzed and compared freshly isolated PBMC from cardiac transplanted patients and normal subjects as regards Fas expression on T cells by flow cytometry. As shown in Fig. 3, both patients’ and normals’ T cells had a variable expression of Fas on T cells. Due to this variability, we did not find any significant difference between cardiac transplanted subjects and normals. We also investigated FasL mRNA expression in PBMC by RT-PCR. As shown in Fig. 4, the expression of FasL in cardiac transplanted patients was not significantly different from the one expressed in normals (the median beta-actiny FasL ratio was 1.6 range 0.7–1.8 in 16 cardiac transplanted patients and 1.5 range 0.8–1.9 in 13 controls). 4.3. Fas-induced cell death in cardiac transplanted patients To determine whether cardiac transplanted patients showed an enhanced susceptibility of PBMC to undergo Fas-induced apoptosis in spite of the normal Fas expression and the normal FasL mRNA expression, we treated the PBMC of 10 patients with anti-Fas (1 mgyml) mAb

Fig. 4. FasL mRNA expression in lymphocytes from cardiac transplanted patients and normal subjects C, negative control; MW, molecular weight marker.

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Fig. 5. Fas cross-linking with CH-11, an anti-Fas IgM, induces cell death in PBMC from 11 cardiac transplanted patients (Z), but not in 6 normal controls (j) (P-0.05). Control IgM has no effect in both groups. Cell death was evaluated by ELISA detection of intracellular histone-associated DNA fragments and expressed as optical density (OD). Data represent the median values.

or its isotype-matched control for 16 h. In the same experiment we used cultured Jurkat T cells as a positive control. The treatment with the isotype-matched control failed to induce PCD, whereas the treatment with antiFas induced apoptosis in PBMC from cardiac transplanted patients (P-0.05; Fig. 5). Both treatments were not able to induce cell death in normal PBMC (Fig. 5). 4.4. Cardiac transplanted patients show a reduced IL2 production and IL2 enhances cell survival Since immunosuppressive agents inhibit the expression and production of several cytokines such as IL2 and a reduced IL2 production might contribute to the accelerated PBMC apoptosis in our cardiac transplanted patients who were receiving an immunosuppressive treatment to prevent graft rejection, PBMC IL2 production was investigated. We found that PBMC from cardiac transplanted patients produced lower levels of IL-2 than normal subjects after PHA stimulation (P-0.05, Fig. 6). Moreover, addition of IL-2 to cell medium at the beginning of the cell culture significantly reduced PBMC spontaneous apoptosis after 72 h (Fig. 7).

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Fig. 6. In 18 cardiac transplanted patients PBMC showed a significantly reduced production of IL2 after stimulation with PHA in comparison with 17 normal subjects (P-0.05). Horizontal lines represent the median values.

example, systemic administration of bacterial superantigens into some mouse strains results in expansion of antigen-reactive T cells with subsequent massive apoptosis of those T cells and acquisition of specific tolerance to the superantigen w18x. Therefore, apoptosis of allograft-specific T cells might induce a long-lasting stable tolerance to the allograft. This requirement of T cell PCD in the induction of tolerance to allografts has been shown in several animal models w19x: for example IL2KO mice which display a gross defect in PCD are resistant to allograft tolerance induced by costimulation blockage or rapamycin treatment w20x. Similarly, Bcl-xl transgenic mice whose T cells do not need costimulation

5. Discussion In this study, we found that cardiac transplanted patients undergoing chronic immunosuppressive treatment showed an enhanced PBMC spontaneous apoptosis in vitro. Although the precise nature of T cell apoptosis in the acquisition of peripheral tolerance remains to be firmly defined, there are certain situations where T cell apoptosis is absolutely critical in immune tolerance. For

Fig. 7. IL2 addition to the cell medium reduces PBMC spontaneous apoptosis in 13 cardiac transplanted patients (P-0.05). Cell death was evaluated by ELISA detection of intracellular histone-associated DNA fragments and expressed as optical density (OD). Data represent the median values.

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to extend cell survival, quickly reject cardiac allografts in spite of costimulation blockage or rapamycin treatment which induce tolerance in wild-type animals w6x. Apoptosis of graft-infiltrating T cells has also been demonstrated in human renal allografts, where it might be a down-regulatory process of activated T cells eliminating potentially self-damaging T cells because the kidneys showing evidence of lymphocyte apoptosis have a beneficial recovery from acute rejection w21x. There are two distinct mechanisms of T cell apoptosis: the so-called activation-induced cell death, which is largely mediated through members of the TNF-receptor family and the so-called passive cell death which occurs when activated T cells are deprived of growth factors w22x. Cardiac allograft implies a persistent exposure of the immune system to the alloantigens of the graft. It is likely that in our cardiac transplanted patients PBMC are chronically activated in vivo due to this persistent exposure and therefore PBMC are predisposed to the activation-induced cell death. This is the reason why we first investigated the FasyFasL pathway which plays a major role in activation-induced cell death w23x. Our data demonstrated that Fas protein and FasL mRNA expression were not enhanced in PBMC from patients; however, the same cells underwent apoptosis after Fas cross-linking, showing the phenotype of in vivo activated cells, because resting PBMC are resistant to Fasinduced cell death whereas chronically activated PBMC become sensitive w24x. Moreover, it is important to underline the fact that the susceptibility to Fas-induced cell death is usually related to the expression of FasL protein and not to FasL mRNA that we have not found increased in our patients, whereas the protein level of the same molecule might actually be enhanced w25x. In the cardiac transplanted patients that we studied the increased PBMC apoptosis may be also due to the immunosuppressive treatment that they were receiving to prevent rejection, even if we were not able to correlate the degree of apoptosis with the type of immunosuppressive drugs taken or with the time from the transplantation procedure which implies a stronger immunosuppression the closer the date of transplantation. Actually, the two hypotheses, that is the activationinduced cell death due to chronic exposure to alloantigens and the enhanced cell death due to the immunosuppressive treatment are not mutually exclusive because several studies have shown that both GC and CsA are able to promote PBMC apoptosis in vitro after mitogen activation w15x. Moreover, GC-induced cell death seems to be partially due to an involvement of the FasyFasL pathway, because GC-induced apoptosis of PBMC blasts may be reduced neutralizing the Fas receptor w26x. However, another hypothesis is that the enhanced PBMC apoptosis of our cardiac transplanted patients is

a passive cell death due to cytokine withdrawal and in particular to IL2 withdrawal. It is well-known that both GC and CsA exert part of their immunosuppressive action inhibiting the expression and production of IL2 w8,27x which plays a complex role in T cell apoptosis. IL2 plays a major role in activation-induced cell death where it promotes the lymphocyte progression through the cell cycle and therefore the susceptibility to activation-induced cell death w28x. However, IL2 is also able to promote survival of resting human T cells, especially in patients with autoimmune diseases and with primary or secondary immunodeficiencies w29–31x; moreover, IL2 rescues T cells from GC-induced apoptosis w27x, upregulating the expression of antiapoptotic genes w32x. In our patients, PBMC IL2 production was significantly decreased in comparison with normals, probably thanks to the immunosuppressive treatment. Moreover, the addition of IL2 to the culture medium promoted cell survival, confirming the role of IL2 withdrawal in the enhanced PCD. In conclusion, both mechanisms, the activationinduced cell death and the passive cell death, seem to contribute to the enhanced PBMC apoptosis in cardiac transplanted patients undergoing immunosuppressive treatment. Moreover, it is important to underline that all patients studied were free from acute or chronic rejection. Therefore, the enhanced PCD seems to contribute to tolerance, even if further studies comparing PBMC apoptosis in a group of patients with no rejection to PBMC apoptosis in a group of patients with on-going rejection will be needed to state it definitely. References w1x Wyllie AH. Apoptosis. Br J Cancer 1993;67(2):205 –208. w2x Abbas A. Die and let live: eliminating dangerous lymphocytes. Cell 1996;84(5):655 –657. w3x Mountz JD, Zhou T, Wu J, Wang W, Su X, Cheng J. Regulation of apoptosis in immune cells. J Clin Immunol 1995;15(1):1 – 16. w4x Zavazava N, Kabelitz D. Alloreactivity and apoptosis in graft rejection and transplantation tolerance. J Leuk Biol 2000;68(2):167 –174. w5x Ferguson TA, Green DR. T cells are just dying to accept grafts. Nature Med 1999;5(11):1231 –1232. w6x Wells DA, Li XC, Li Y, et al. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nature Med 1999;5(11):1303 –1307. w7x Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nature Med 1999;5(11):1298 –1302. w8x Allison AC. Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 2000;47(2-3):63 –83. w9x Tuosto L, Cundari E, Montani MS, Piccolella E. Analysis of susceptibility of mature human T-lymphocytes to dexamethasone-induced apoptosis. Eur J Immunol 1994;24(5):1061 – 1065. w10x Cohen JJ. Glucocorticoid-induced apoptosis in the thymus. Semin Immunol 1992;4(6):363 –369.

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