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Piłat A i wsp. Increased apoptosis of mononuclear cells in atopic patients... 209 Increased apoptosis of mononuclear cells in atopic patients – the effect of pollen season and stimulation with specific antigen Zwiększona apoptoza komórek jednojądrzastych u atopowych pacjentów – wpływ sezonu pylenia i pobudzenia swoistym antygenem ANNA PIŁAT 1, JANINA GRZEGORCZYK 2, MAREK L. KOWALSKI 3 Central University Clinical Hospital, Lodz, Poland Department of Microbiology and Laboratory Medical Immunology, Medical University of Lodz, Poland, 3 Department of Clinical Immunology and Allergy, Medical University of Lodz, Poland 1 2 Study supported by Medical University of Lodz research grant No 502-11-389 (22). Streszczenie Summary Wprowadzenie. Zwiększona aktywność proliferacyjna komórek mononuklearnych (MN), stwierdzana we krwi obwodowej u chorych z alergią atopową, może być związana ze zmienionym czasem przeżycia komórek. Introduction. Elevated proliferative activity of mononuclear cells (MNCs) in peripheral blood of atopic patients may be associated with shorter cell survival. Cel pracy. Ocena apoptozy komórek jednojądrowych krwi obwodowej od chorych z alergią atopową, przed i w czasie sezonu pylenia, w porównaniu do osób nieatopowych. Materiały i metody. Komórki MN, wyizolowano z krwi 15 pacjentów z sezonowym nieżytem nosa i astmą oskrzelową, przed i w czasie sezonu pylenia oraz od 16 osób nieatopowych. Apoptozę oceniano w mikroskopie fluorescencyjnym, po wybarwieniu oranżem akrydyny i bromkiem etydyny oraz potwierdzono poprzez analizę fragmentacji DNA. Badano zarówno komórki adherentne, jak i nieadherentne. Wyniki. Zarówno przed jak i w czasie sezonu pylenia, komórki MN od pacjentów z alergią, wykazywały istotnie nasiloną spontaniczną apoptozę po 24, 48 i 72 godzinach hodowli. Komórki MN od chorych atopowych, wykazywały również zwiększoną apoptozę po inkubacji z konkanawaliną A. W czasie sezonu pylenia u osób uczulonych, apoptoza limfocytów znamiennie wzrastała (p<0,05). Inkubacja nieadherentnych komórek MN z alergenem tymotki, nasilała apoptozę komórek przed sezonem pylenia (średnio 33% w 48 godzinie) i istotnie hamowała apoptozę limfocytów w czasie sezonu pylenia (średnio 27% w 48 godzinie). Wnioski. Komórki MN od osób uczulonych na pyłki, wykazują nasiloną apoptozę, a proces ten jest modulowany zarówno przez naturalną ekspozycję, jak i dodawany in vitro alergen. Słowa kluczowe: apoptoza, alergia na pyłki, astma oskrzelowa, nieżyt nosa, komórki mononuklearne, zapalenie alergiczne © Alergia Astma Immunologia 2012, 17 (4): 209-215 www.alergia-astma-immunologia.eu Przyjęto do druku: 04.01.2013 Aim. Assessment of the apoptosis of peripheral blood mononuclear cells (MNCs) from atopic patients before and during symptomatic pollen season as compared to non-atopic controls. Materials and methods. MNCs were obtained from 15 pollen-sensitive patients with seasonal rhinitis/asthma (SR/A) before and during the pollen season, and from 16 non-atopic controls. Apoptosis was assessed by fluorescent microscopy after staining with acridine orange and ethidium bromide, and confirmed by DNA fragmentation analysis. Both adherent and non-adherent MNCs were analysed. Results. Both before and during the pollen season, MNCs from pollensensitive patients, as compared to controls, demonstrated significantly enhanced spontaneous apoptosis at 24h, 48h and 72h of culture. MNCs isolated from atopic patients demonstrated also significantly enhanced ConA induced apoptosis as compared to non-atopic patients. During the pollen season in allergic patients, spontaneous apoptosis of MNCs and lymphocytes was significantly higher as compared with preseasonal values (p<0.05). Incubation of non-adherent MNCs with timothy allergen increased the cell apoptosis before the grass pollen season (on average by 33% at 48h), but significantly inhibited apoptosis of lymphocytes studied during the pollen season (on average by 27% at 48 h). Conclusion. Peripheral blood mononuclear cells from asymptomatic pollen-sensitive patients exhibit increased apoptosis, and this process is modulated by in vitro stimulation with specific allergen and by the exposure during natural pollen season. Keywords: apoptosis, pollen allergy, asthma, rhinitis, mononuclear cells, allergic inflammation Adres do korespondencji / Address for correspondence Marek L. Kowalski M.D. Department of Immunology, Rheumatology and Allergy, Faculty of Medicine, Medical University of Lodz 251 Pomorska St., 92-213 Lodz, Poland Tel: +48 42 678 15 36, Fax: +48 42 678 22 92 210 Alergia Astma Immunologia 2012, 17 (4): 209-215 INTRODUCTION Mononuclear cells isolated from peripheral blood of atopic patients (specifically T lymphocytes) are activated and release cytokine profile, which is different from nonatopic, cells [1-5]. This profile of cytokines is corresponding to TH2 type response with enhanced generation of IL-4, IL-5 and little production of interferon gamma. More recently a central role for T regulatory cells (including Th17 and Th9 cells) for control and maintenance of allergic inflammation has been implicated [6,7]. During allergen specific immunotherapy the profile of cytokines can be shifted from Th2 to a Th1-dominated immune response in parallel with activation of regulatory T cell subsets and reduction of the antigen-induced activation of peripheral blood lymphocytes (PBL) [8]. Peripheral blood leukocyte activation and increased cytokine generation may parallel allergen induced upper and or lower airway symptoms, and is alleviated during clinical improvement suggesting, that the activity of PBLs may be related to the activity of allergic inflammation in the airway mucosa [9-11]. Antigen stimulated lymphocytes, depending on the state of preactivation and the microenvironment may either proliferate or undergo apoptosis (programmed cell death). Mechanisms involved in lymphocyte death and elimination have critical role in the immune response providing a homeostatic mechanism for controlling the magnitude of an immune system activation [12-14]. Elimination of pathogenic, allergen-specific T(H)2 cells is an essential step in induction of tolerance during natural exposure or following allergen specific immunotherapy [15,16]. Although defective apoptosis of lymphocytes has been implicated in the pathomechanisms of autoimmune diseases, little is known on the apoptotic activity of PBL in atopic diseases. Increased apoptosis of T lymphocytes was reported in patients with bronchial asthma and, decreased serum levels of soluble Fas (sFAS) was found in patients with allergic rhinitis suggesting, an impairment of apoptotic pathways in allergy [1719]. In our previous study an increased spontaneous and mitogen-induced apoptosis of MNC from peripheral blood of atopic patients was found and serum levels of sFas and ICE/caspase-1 were correlated with apoptosis, suggesting abnormal regulation of apoptotic process in peripheral blood mononuclear cells [20]. The goal of our study was to assess the apoptosis of peripheral blood mononuclear cells from atopic patients before and during symptomatic pollen season as compared to control non-atopic subjects. In addition the effect of stimulation with specific, grass pollen antigen of apoptosis of MNC was studied before and that during the pollen season. PATIENTS AND METHODS Patients The study included 15 patients with seasonal rhinitis or seasonal rhinitis and atopic bronchial asthma, diagnosed for at last two years [8 male and 7 female; mean age 38 years (range 20-56)]. All patients had positive prick tests to a battery of grass pollens including the timothy allergen. None of the patients had taken oral corticosteroids. Antihistamine drugs were stopped at least 72 hours before obtaining their blood samples (Table I). Patients were studied twice: in January-March, before grass pollen season and in May-July during the pollen season. The control group comprised 16 healthy subjects without any history concerning respiratory tract and with negative skin prick tests to a battery of inhalant allergen. Cells isolation and culture Mononuclear cells were isolated with Boyüm’s method [14,21]. Shortly: 20 ml of heparinized venous blood were mixed with PBS in 1:3 proportion, then cautiously stratified on Histopaque 1,077 g/cm3 (Sigma) and centrifuged at 400x g for 20 min. The ring which was formed on the borderline of the phases was carefully collected and rinsed in phosphate buffer (pH=7,4 without Ca++ and Mg++). Finally suspended in the medium supplemented with 0,3% albumin, Ca++ and Mg++ and 0,036% glucose so that the Table I. Characteristic of patients Patient No F/M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 F M F M M M F M M F M F F F M Atopic patients Age in years 24 35 43 26 56 25 21 20 22 46 27 38 26 33 28 Clinically diagnosis Rhinitis Asthma + Rhinitis Rhinitis Rhinitis Rhinitis Rhinitis Asthma + Rhinitis Asthma + Rhinitis Rhinitis Asthma + Rhinitis Rhinitis Asthma + Rhinitis Rhinitis Rhinitis Asthma + Rhinitis Healthy subjects Patient No F/M Age in years 1 F 32 2 F 28 3 F 24 4 F 33 5 F 37 6 F 42 7 F 28 8 F 32 9 F 34 10 F 29 11 M 43 12 F 49 13 M 30 14 M 23 15 M 24 16 F 26 Piłat A i wsp. Increased apoptosis of mononuclear cells in atopic patients... number of cells equals 2x106/ml. One aliquot of MNC suspension was cultured and the second fractionated into lymphocytes and monocytes according to following procedure: five millilitre aliquots of mononuclear cells were incubated in plastic dishes at 37o C in 5% C02 for 1h. Non adherent cells were removed by vigorous washing, and the remaining adherent cells by scraping with a rubber policeman. The adherent cells were typically >65% monocytes, as assessed by immunofluorescence method with monoclonal antibody CD14 [15,22]. Unfractionated peripheral blood mononuclear cells, lymphocytes and monocytes from 15 asthmatic subjects and 7 non-atopic persons were cultured according to Theuson’s method [16,23]. After the cells were preincubated for 4h with Con A (10 µg/ml), timothy antigen (1, 10, 100 ng/ml) or medium and then centrifuged at 400 x g for 20 min., washed twice in PBS without Ca++ and Mg++ , suspended in the primary medium volume and cultured for 24h, 48h, 72h under the same conditions. Assessment of apoptosis The MNC were removed from culture after 24h, 48h and 72h to study apoptosis by morphology after fluorescent staining, as described McGahon [17,24]. In brief: 25 µl aliquots of MNC were mixed with PBS solution containing 100 µg/ml acridine orange base (Sigma) and 100 µg/ml ethidium bromide (Sigma). Stained MNC were transferred to a glass slide and examined by means of an epifluorescence microscope using the filter with the length of wave 490–525 nm. A minimum of 200 cells were assessed and the number of apoptotic, viable and necrotic cells were recorded. The percentage of apoptotic cells (apoptotic index) was calculated as follows: total number of cells with apoptotic nuclei % Apoptotic cells = ---------------------------------------x 100 total number of cells counted (viable + apoptotic + necrotic cells) DNA Gel Electrophoresis Fragmentation of DNA was assessed by method according to Hermann [18,25]. MNCs were removed from culture after 72 hours, washed with PBS and pelleted by centrifugation. The cell pellets were then treated for 10 s with lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM TrisHCL, pH 7,5; 10 µl per 106 cells). After centrifugation the supernatants were transfered to 1% SDS and treated for 2 hours with RNAse A at 56o C. Followed by digestion with proteinase K for at lest 2 hourse at 37oC. After addition of 1⁄2 vol. 10 M ammonium acetate the DNA was precipitated with 2,5 vol. ethanol and separated by electrophoresis in 1% agarose gell containing ethidium bromide. The samples were visualised by UV light and analysed by gel analysing system (Vilber Lourmat). Statistical analysis Data are shown as mean±SD. Statistical differences were assessed using the Student’s d-Test and t-Test, preceded by evaluation of normality with F-Test. P-Values<0,05 were considered significant. 211 RESULTS Spontaneous apoptosis Unfractionated mononuclear cells (lymphocytes + monocytes) and isolated from MNC lymphocytes and monocytes, demonstrated apoptosis immediately after isolation (To) and the percentage of apoptotic cells increased with the time of incubation in both groups. MNCs isolated from atopic patients before and during the pollen season demonstrated significantly enhanced apoptosis, as compared to controls, at 24h, at 48h and at 72h in patients before and during the pollen seasonal and controls respectively of culture (Fig. 1a). Apoptotic index for non-adherent MNCs (lymphocytes) was significantly higher in atopic patients studied before the pollen season or during the pollen season as compared to non-allergic subjects after 24, 48 and 72hours of culture (p<0,05) (Fig. 1b). In atopic patients the percentages of apoptotic unfractionated MNCs analysed during the pollen season were significantly higher as compared with the cells isolated before the pollen season at 48h and 72h (Fig 1a, 1b). Effect of specific allergen on apoptosis of MNC Unfractionated MNC from allergic patients incubated with timothy allergen before the season demonstrated a significant increase in the apoptotic index. No influence of allergen on apoptosis of MNC was observed during the pollen season (Fig. 2a, b). Incubation of non-adherent MNCs (lymphocytes) with specific allergen increased the cell apoptosis before the grass pollen season, but significantly inhibited apoptosis of lymphocytes isolated during the pollen season, at 48h (Fig. 3a, b). We did not observe any effect of allergen on apoptosis of monocytes. Incubation of mononuclear cells (unfractionated and fractionated) from non-atopic subjects with timothy allergen did not affect apoptotic index. Stimulation in vitro by ConA Incubation of cells with Con A (10 µg/ml ) resulted in a significant increase in the proportion of apoptotic cells in all cell population. Non-adherent MNCs (lymphocytes) isolated from atopic patients as compared to non-atopic subjects, demonstrated significantly enhanced of ConA stimulated apoptosis at 24h and 72h (Fig. 4a, b). The percentages of apoptotic monocytes in allergic patients, both before and during the season, were significantly higher as compared to non-allergic subjects only at 72h. In allergic patients the apoptotic index for MNCs during the pollen season, was not different as compared with preseasonal values. DNA fragmentation The presence of apoptosis of mononuclear cells was confirmed by DNA ladder. The fragmentation of DNA was observed after 72hrs of culture of MNC, only in 4 atopic patients with percentage of apoptotic MNC above 44% as assessed by fluorescence microscope. We did not observe any DNA fragmentation in cells from atopic patients with apoptotic index ≤40% or in non-atopic subjects (Table II). 212 Alergia Astma Immunologia 2012, 17 (4): 209-215 1a 1b Fig. 1. Spontaneous apoptosis of peripheral blood mononuclear cells (a) and lymphocytes (b) from healthy subjects (H) (n=16) and grasspollen-sensitive patients examined before (BS) and during the pollen season (S) (n=15). Results are expressed as the percentage of apoptotic cells (mean+SD). The statistical differences was assessed using the Student’s t-Test and d-Test. P-value less 0,05 was considered significant Fig. 1a. Unfractionated MNC (Lymphocytes + Monocytes) Fig. 1b. Lymphocytes 2a 2b Fig. 2. Effect of specific allergen on apoptosis of unfractionated mononuclear cells (lymphocytes+monocytes) from grass-pollen-sesitive patients (n=6) examined before (a) and during the pollen season (b). Results are expressed as a mean apoptotic index±SEM. The statistical differences between timothy allergen-treated and medium-treated cells was determined by Student’s d-Test; * p<0,05 as compared to control Fig. 2a. Monocytes before the pollen season Fig. 2b. Monocytes during the pollen season 3a 3b Fig. 3. Effect of specific allergen on apoptosis of lymphocytes from grass-pollen-sesitive patients (n=6) examined before (a) and during the pollen season (b). Results are expressed as a mean apoptotic index±SEM. The statistical differences between timothy allergen-treated and medium-treated cells was determined by Student’s d-Test; * p<0,05 as compared to control Fig. 3a. Lymphocytes before the pollen season Fig. 2b. Lymphocytes during the pollen season Piłat A i wsp. 213 Increased apoptosis of mononuclear cells in atopic patients... 4a 4b Fig. 4. Comparison ConA stimulated apoptosis of lymphocytes from healthy subjects (H=7) and grass-pollen-sensitive patients (A=15) examined before (a) or during the pollen season (b). Results are expressed as the percentage of apoptotic cells (mean+SD). The statistical differences was determined by Student’s t-Test.; * p<0,05 Fig. 4a. Before the pollen season Fig. 4b. During the pollen season Table II. Assesment of apoptosis by morphology and electrophoresis in 1% agarose gel (fragmentation of DNA) Patient No Atopic patients (n=5) Healthy subjects (n=7) 4 5 10 11 13 4 5 8 9 11 12 14 Morphological analysis of apoptosis (apoptotic index - %) 46% 63% 46% 44% 40% 22% 32% 20% 18% 30% 27% 31% DISCUSSION Our study demonstrated, that isolated peripheral blood mononuclear cells from both atopic patients and non-atopic subjects undergo apoptosis directly after isolation, and the percentage of apoptotic cells increases with time of culture. MNCs from atopic patients before and during the pollen season demonstrated significantly enhanced spontaneous apoptosis as compared to non-allergic subjects. Moreover, significantly higher percentage of apoptotic mononuclear cells was present in allergic patients during the pollen season as compared with preseasonal values. Apoptosis was assessed with morphologic fluorescent method and validated by DNA fragmentation analysis. Although the fragmentation of DNA was observed only in those patients with the highest percentage of apoptotic cells ( ≥44%,) these data are consisted with previous observation [26]. Incubation of non-adherent MNCs from grass pollen sensitive patients with timothy allergen increased the cell apoptosis before the grass pollen season, but significantly inhibited apoptosis of cells during the pollen season. The apoptosis of MNCs from non-atopic patients was not chan- Fragmentation of DNA + + + + _ _ _ _ _ _ _ _ ged after allergen challenge. Non specific stimulation by mitogen (ConA) significantly increased of apoptotic process in atopic patients and non-atopic subjects as compared to spontaneous apoptosis both before and during the pollen season. This study suggests, that the lifespan of mononuclear cells in peripheral blood is different in atopic and non atopic subjects and that in patients with clinical allergy lymphocyte apoptosis varies depending on patient’s clinical status. In our earlier study an increased spontaneous and ConA induced apoptosis of peripheral blood lymphocytes from house-dust mite sensitive patients with perennial allergic rhinitis and/or bronchial asthma was demonstrated [20]. Increased spontaneous and Dex-induced apoptosis of lymphocytes in atopic patients with bronchial asthma was reported previously by Ho et al. [17] although the authors did not analysed an atopic status of their patients. Apoptosis or programmed cell death is one of the mechanisms for controlling proliferation and survival of cells during allergic inflammation [27,28]. The degree of apoptosis of immunologically active cells is determined by either 214 Alergia Astma Immunologia 2012, 17 (4): 209-215 intrinsic (genetic) or micro environmental factors inducing or suppressing cell proliferation and apoptosis. This process is not only able to regulate immune response to allergen but also cell number and activity at the inflammatory site affecting allergic disease activity [29]. Increased apoptosis of lymphocytes found in atopic patients could be associated with profile of released cytokines which in atopics corresponds to Th2 type and includes among others IL-4, IL-5 [30,31]. However, our asymptomatic pollen-sensitive patients, at the time of the study were in stable condition and were not taking any oral medication suggesting, that increased apoptosis of lymphocytes was related to patients’ atopic status rather than disease activity. On the other hand natural exposure to allergen during the pollen season significantly increased spontaneous apoptosis of lymphocytes, suggesting that local inflammation in the airways, probably by released to systemic circulation cytokines, may affect apoptotic activity of peripheral blood lymphocytes. Increased apoptosis of lymphocytes in peripheral blood of allergic patients reported in our study and higher apoptotic susceptibility of T lymphocytes of asthmatic patients to DEX treatment reported by Ho et al. [17] are in contrast to decreased apoptosis observed at the site of inflammation in the bronchial mucosa of asthmatic patients [13]. In patients with asthma, specifically during allergy season, a different activity of apoptotic molecules belonging to Fas-FasL system have been reported [17,32]. A negative correlation between serum level of Fas molecule and peripheral blood MNCs apoptotic index was observed in house-dust mite sensitive patients [20]. In earlier study a resistance to Fas-dependent apoptosis was demonstrated and related to altered antigen-driven, accessory cell-dependent signalling and ineffective activation of Fas signal transduction in asthma, confirming the role for soluble serum proteins in controlling of the lymphocytes apoptosis in asthma [18,33,34]. 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