| | PUVA apoptotic response in activated and resting human lymphocytesAbstract In extracorporeal photopheresis (ECP) collected cells are treated by 8 methoxypsoralen and UVA (PUVA) which induced apoptosis. The mechanism of action of reinfused cell is unclear. A vaccination model postulates an efficient presentation of apoptotic alloreactive cells to the patient immune system. The efficiency may depend upon a predominence of apoptotic alloreactive cells after PUVA. Such selectivity could result from their activation. We studied apoptosis in resting and PHA-activated lymphocytes. Both were equally susceptible. Changes in early apoptosis were possibily missed. We evaluated the effect of preincubation before PUVA. During preincubation monocyte could affect lymphocytes susceptibility to apoptosis as an increase of number of apoptotic cells was observed after 72 hours in stimulated and resting cells. Our findings do not preclude a selectivity of other PUVA effects since expression of membrane marker also targets to PUVA is modified by activation.
1. Introduction  In extracorporeal photopheresis (ECP), leucopheresed mononuclear cell are first exposed to PUVA (incubation with 8 methoxypsoralen (8MOP) followed by UVA irradiation) and returned to the patient. Treated cells infusion produces an immunomodulatory effect used to treat cutaneous T-cell lymphoma [1] and other T mediated diseases [2] to prevent or reverse acute and chronic allograft rejection [3] or to treat graft versus host disease (GvHD) [4]. Various mechanisms have been proposed to explain the action of ECP, one of which being the so called T cell vaccination [5]. It can result from the combined effects of lymphocyte apoptosis and monocyte activation both inducible by 8MOP and UVA. The number of injected cells is low comparatively to the total lymphocyte number of the patient yet the effect of this small number apparently last for months in some patients at least. Their direct immunosuppressive action would not suffice. A vaccination model was explored by Berger in cancer therapy while this study was being conducted [6]. Apoptosis is a form of cell death distinct from necrosis and apoptotic cells evoke no inflammatory response [7]. Different cell types are susceptible in vitro to PUVA-induced apoptosis including blood mononuclear cells from Sezary syndrome patients or healthy donors [8]. The ability of ECP to induce apoptosis in lymphoid cells was first reported by Marks and Fox [9] after distinctive changes in morphology and DNA fragmentation in cultured peripheral blood mononuclear cells (PBMC) exposed in vitro to ECP-like conditions. Later papers have confirmed their findings [8], [10], [11], [12], [13], [14]. Culturing the cells from patients treated by ECP produced the same results [14], [15], [16]. Apoptosis has also been observed in histological examination of skin samples from ECP-treated patients [17], [18]. Sezary clones and various leukemic human-derived cell lines [10] are susceptible to PUVA apoptosis. PHA activated lymphocytes are not resistant to PUVA [13]. However the magnitudes of apoptosis in stimulated compared to unstimulated cultures has not been established. Monocytes are resistant to ECP-induced apoptosis and retain recognition and phagocytosis of ECP-induced apoptotic PBL [8]. Furthermore monocytes treated by ECP produce larger amounts of TNFalpha which is involved in cytotoxic T cell response [6], [19]. In addition, ECP induces both the differentiation of monocytes to dendritic cells which are the most efficient antigen-presenting cells for the induction of antitumor immunity [6]. Apoptosic fragments are phagocytized by antigen presenting cells and able to induce a proliferation of normal alloreactive lymphocytes [8]. In this way, antigens from apoptotic cells may be presented by antigen presenting cell, inducing a cytotoxic T cell response. Membrane markers including alloreactive T cell receptors (TCR) may be prepared and presented by antigen presenting cells. A patient immune response against TCR idiotypes would ensue providing that alloreactive apoptotic T cell antigen predominate among antigen from all apoptotic cells. A selective effect of PUVA has been postulated. The response would eliminate alloreactive T cells, the so-called T vaccination. It could be related to their activated state. Such selectivity is consistant with the accepted mechanism of UVA-8MOP potentiation. 8MOP-induced DNA crosslinks and thus would sensitize preferentialy activated cells [2], [20]. However the selectivity has not been studied so far. In the present work, procedure for treating cell cultures were derived from ECP. PUVA apoptotic response kinetics had to be established for their proper measurement and for better understanding of the fate after reinjection of ECP cells. The dimorphism of treated population (adhering monocyte and nonadhering) had to be considered. We also assessed to what extended treated cells can still proliferate in vitro hence presumably in patients. 2. Materials and methods 2.1. Cell preparation and culture Blood samples were collected from healthy volunteers. PBMC were isolated by sedimentation on Ficoll–Hypaque gradient according to the manufacturer recommendations (Milieu de séparation lymphocytaire, MSL, d=1.077 g/l, Eurobio, Les Ulis, France). Freshly isolated PBMC were incubated RPMI 1640 (without phenol red) supplemented with 1% L glutamine (both from Sigma, St Louis, MO, USA) and 10% fetal calf serum (SVF, Valbiotech, Paris, France). Cultures of 106 cells/ml, 10 ml per 75 cm3 culture vials (Falcon Becton Dickinson, USA) were incubated at 37 °C in humidified atmosphere with 5% CO2. Lymphocytes, monocytes and contaminating red blood cells (RBC) were counted in the automated ST Coulter (Coulter Corporation, Miami, USA). For proliferation assay, cells were incubated with phytohaemaglutimine (PHA, Sigma Chemical C, 5 μg/ml). Proliferative response to PHA was measured at 24 and 72 h by incorporation of tritiated thymidine (3[H]-TdR (methy-3H) thymidine; Amsterdam Int, Little Chalfont) at 0.5 uCi per well for 8 h. Labelled cells were harvested on a multiple samples automated cell harverster. Their beta emission was measured in a beta counter (LS 1801, Beckman, Irvin, CA). PHA was present during all the preincubation time. 2.2. PUVA treatment of cultured cells After 24 or 72 h of culture, unstimulated PBMC and PHA-activated PBMC were submitted to PUVA therapy. 8MOP (8MOP, C12 H8 O4, Sigma Chemical Co) was added to the cultures at 200 ng/ml final concentration 20 min before irradiation. 8MOP was diluted extemporaneously in phosphate buffer saline (PBS) (PBS, Dubecco, Gibco, Paysley, Scotland) from a stock solution at 1 mg/ml. UVA irradiation was performed by the Biogenic® apparatus (Vilbert Lourmat, Marne la Vallée, France). In this device, two UV light sources (360 nm peak output, distributed as 1 upper and 1 lower source) each with 2 sets of 4 tubes mounted perpendicularly provide an homogeneous irradiation to the target surface. Two UV sensors control the UV intensity received from each source. These sensors convey the information to a microprocessor which sums up in real time the energy received by the target. When upper and lower energy sums meet the energy requirement, the microprocessor switches off the corresponding source. The dose programmed for the present PUVA treatment was 2 J/cm2. Temperature had to be maintained at 25 °C and the samples protected from light before and after irradiation. Contaminant RBC should be fewer than 0.5% [2], [20]. Controls were incubated with 8MOP without UVA or exposed to UVA without 8MOP or incubated under the same conditions but without both. 2.3. Apoptosis evaluation Dose response and kinetic studies of PUVA-induced apoptosis were performed at 4, 24 and 48 h after treatment. Two complementary methods were used to measure apoptosis. Cell suspensions were analysed by fluorescence microscopy after staining by Hoechst 33342 (Sigma Chemical Co) at 10 μg/ml as described by Darzynkiewicz [21], under an epifluorecence microscope using a X 100 oil immersion to assess nuclear changes. Nuclear fragmentation and/or marked condensation of chromatin with reduction in nuclear size were counted as typical features of apoptotic cells. At least 200 cells were examined for each experiment. Hoechst 33342 staining detects morphologic changes of chromatin wich can be considered as a later stage in the apoptosis process. Apoptotic cell number was also mesured by cytofluorimetry after staining by Annexin V-FITC (Boerheingenr Ingelheim, Vienna, Austria) as recommended by the manufacturer. Annexin V binds the phosphatidyl serine witch is translated from the inner layer of the plasma membrane to the outer layer at an early stage of apoptosis. Propidium iodide (PI) was used for excluding necrotic cells. The procedure followed Koopman [22]. Briefly, cells were washed twice in PBS then resuspended 1×106/ml in 100 μl Annexin-binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Annexin V-FITC and PI were added to a final concentration of 5 μl/ml each. Cells suspensions were incubated for 15 min at room temperature in the dark and diluted in 300 μl of binding buffer for flow cytometry in a FACS EPICS XL (Coulter Corporation, Miami, USA) with 488 nm argon laser and emission filters were BP525 for FTIC and BP620 for PI. A minimum of 10,000 cells per sample were analysed. Cell debris were excluded by forward-light-scatter and side scatter thresholds setting. Bivariate analyses of cells population were performed throughout. Counts of viable cells by trypan blue exclusion and measurement of 3[H]-TdR incorporation over the last 8 h were performed on the same suspension at indicated times. 2.4. Statistical analysis At least three measurements were made for each experiment. Apoptosis was measured as the proportion of apoptotic cells in culture. To take into account any apoptotic cell background in controls the proportion was correct by the formula Data are given as the mean ± standard deviation. Comparisons between control and treated cultures were made by Mann and Whitney test ( α=0,05). 3. Results 3.1. Cell populations before culture PBMC obtained after Ficoll sedimentation contained 84%±4% lymphocytes and 11%±1% monocytes. The RBC of the suspension was 0.07%±0.1%. 3.2. Apoptosis of resting PBMC treated after 24 or 72 h of culture After 24 h incubation, cells had been treated with PUVA, UVA alone, or 8MOP alone. Control treated cells did not received UVA nor 8MOP. The number of apoptotic cells observed by nuclear changes at fluorescence microscopy 4 h after PUVA, UVA or 8MOP were not different from control. It increased significantly at 24 h after PUVA (p=0.02,n=4) but no change was observed for UVA and 8MOP alone. At 48 h the number of apoptotic cells was significantly higher than in control (p=0.02,n=4). At that time, UVA alone began to produce a significant (p=0.05, n=3) but much smaller apoptotic cell population (17%±5% versus 88%±11% p=0.05,n=3). No significant apoptosis occurred after 8MOP alone. (Fig. 1A). After 72 h incubation before treatment the same pattern and a significant increase in the number of apoptotic cells was observed at 24 and 48 h while 8MOP alone remained ineffective. PUVA-induced significatively more apoptosis than UVA (81%±18% versus 56%±14%, p=0.05, n=4 at 24 hours; 97%±1% versus 69%±12%, p=0.05, n=3 at 48 h) (Fig. 1B). Flow cytometric analysis confirmed those results. The number of Annexin V positive/PI negative cells at 24 h after PUVA and UVA were significantly greater than in control (PUVA: 79%±1%, UVA: 61%±6%, control 11%±8%, p=0.05, n=3, and p=0.05, n=3 respectively) and reached to 98% at 48 h. The number of Annexin V positive cells at 24 and 48 h after 8MOP was not different from controls. (Fig. 2A). 3.3. Apoptosis in activated PBMC treated after 24 or 72 h of culture Cells stimulated by PHA for 24 h were treated with PUVA, UVA alone, or 8MOP alone. Control cells received neither UVA nor 8MOP. The number of apoptotic cells by Hoechst staining at 4 h after treatment was not different in treated and in control cells. This number increased significantly to 73%±19% at 24 h and 96%±5% at 48 h post PUVA whereas no significant apoptosis was observed with UVA or 8MOP alone. (Fig. 3(A)). Cells stimulated by PHA for 72 h were also treated with PUVA, UVA alone, or 8MOP alone. 72 h control cells were also included. The number of apoptotic cells by Hoechst staining 4 h after of PUVA was significantly greater than that of control whereas no significant apoptosis occurred at 4 h after UVA alone or 8MOP alone. A significant increase in apoptosis was observed at 24 and 48 h after PUVA, UVA alone but not after 8MOP alone. PUVA-induced consistently more apoptosis than UVA alone at 24 h post irradiation (respectively 95%±3% versus 64%±14%, p=0.02, n=4) and at 48 h post irradiation (99%±1% versus 75%±9%, p=0.02, n=4) (Fig. 3(B)). The number of Annexin V positive cells observed at 24 h after PUVA or UVA was greater than in control (90%±7% and 70%±9% respectively versus 48%±3%, p=0.05, n=3 and n=2). The number increased to 97% at 48 h after PUVA. At 24 h, 8MOP did not make any difference from control (47% versus 48%). (Fig. 2(B)). 3.4. Comparison of PUVA-induced specific apoptosis between resting and PHA-activated PBMC A pairwise comparison of stimulated and unstimulated cultures from each treatment group showed that PHA stimulation did not increased apoptotic responses to PUVA or UVA. In 24 h cultures, the number of apoptotic cells by Hoechst staining at 4 h after PUVA was 1.95%±1% in unstimulated cultures versus 8%±15% in PHA-stimulated culture (p=0.77, n=4). Measurements 24 h after PUVA were 50±14% versus 69%±22% (p=0.127, n=5). Measurements at 48 h after PUVA were 88%±11% versus 95%±6% (p=0.25, n=4). (Fig. 4(A)). After 72 h incubation before treatment stimulated and unstimulated cultures showed almost equal number of apoptosic cells by Hoechst staining at 4, 24 and 48 h: respectively 16%±15% versus 26%±20% (p=0.25, n=5), 78%±20% versus 93%±4% (p=0.17, n=5), 96%±1% versus 99%±1% (p=0.13, n=3) respectively Fig. 4(B). By fluorocytometry, no difference in the number of apoptotic cells was found between unstimulated and PHA-stimulated cultures 24 h after of PUVA: 77%±3% versus 81%±12% (p=0.25, n=3) (Fig. 5). 3.5. PHA response of treated PBMC PUVA treatment of PHA incubated cells decreased the proliferative response of the PBMC by 95% and 83% (24 or 72 h respectively incubation). UVA alone decreased the proliferative response by 34% and 59% respectively. In contrast, treatment with 8MOP produced no decrease but rather a slight enhancement of thymidine uptake (Table 1). 4. Discussion Our results establish that stimulated and resting, nonadherent lymphocytes are equally susceptible to apoptosis induction by PUVA. No differences between then reached significance. However, the results show consistently a larger number of apoptotic cells in stimulated compared to unstimulated cultures. Susceptibility to apoptosis induction was increased by preincubation before treatment both in stimulated and unstimulated cultures. This effect is more apparent with UVA alone and absent with 8MOP and in controls. It does not appear to be due to total length of culture time. This finding could result from the action of adherent cells present in the culture. A possible selective effect on stimulated cells between 4 and 24 h could have been missed by Hoechst staining for lack of intermediate measurements. Furthermore the earlier time for Annexin V results was 24 h. Bladon et al. have established that ECP induces both immediate and delayed apoptosis. They used Annexin method for detecting irreversible preprogrammed cell death [16]. Two tests which determine different cellular features of apoptosis had been chosen for this work for their complementarity before Blandon paper. Yet our time sampling schedule did not allow the detection of early apoptosis. The number of apoptotic cells (Annexin V+/PI−) observed in fluorocytometry was greater than that observed by fluorescence microscopy. The former may detected cells in earlier stage of apoptosis. The methods employed in the present work appear to be sound. Our results are supported by others. Fluorescence microscopy results are in keeping with those of Yoo [8] and Vowels [10] by fluorescence microscopy with acridine orange and those of Enomoto [11] who used in situ nick translatation TUNEL. Fluorocytometry results with Annexin V/PI are in keeping with those of Blandon [16]. Delay in PUVA-induced apoptosis was reported by Marks [9], Vowels [10], Enomoto [11], Godar [12], Legitimo [13]. They all observed a delayed apoptosis occuring within 24–48 h after irradiation. The almost complete inhibition of thymidine uptake after PUVA rules out any significant expension after reinfusion of treated cells in the patient. 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J. Pharm. Pharmacol. 1991;43(1):63–64. MEDLINE a Service de Néphrologie (Pr Deteix) Henri Dunant BP69 63003, CHU Clermont-Ferrand Cedex, France b Unité d’Immunologie Clinique et laboratoire d’Immunologie B (Pr B Sauvezie), 63003 CHU Clermont-Ferrand Cedex, France c INSERM U503 (Pr JP Revillard)CHU, 69000, HCL Lyon, France d Unité Bioclinique (Pr F Demeocq), Departement de pédiatrie, 63003 CHU Clermont-Ferrand Cedex, France  Corresponding author. Tel.: +33-4-73-75-14-25; fax: +33-4-73-75-11-83
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