@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2015;21(2):91-96

Background
… [1, 2] MSC "in vitro" leads to hydrogen peroxide production inhibition in activated neutrophils. Therefore, it could potentially limit respiratory burst intensity under inflammatory conditions [3]. Interleukin-10 is an anti-inflammatory cytokine implicated in many repression processes [4]. Interleukin -6 (IL-6) is another cytokine that could be generated by MSC and can affect MSC immunological effects. It can, also, protect neutrophils against apoptosis [3]. … [5]
Previous Studies
MSC may protect apoptosis induction in lymphocytes, and can protect thymocyte and centroblasts against spontaneous apoptosis and protect them against induced death through Fas pathway [6]. Estrogen's effect on cytokine production via peripheral blood mononuclear cells has been investigated with or without cell culture supernatant. Treatment with estrogen has shown significant increase in IL-10 by peripheral blood mononuclear cell (PBMC). Estrogen plays a stimulating role in the immune system, as well as a suppressive role [7]. Estrogen prevents apoptosis in breast cancer cell lines, and on the other hand it increases Bcl2 protein expression. This protein has anti-apoptotic effect [8]. … [9]
Aim(s)
The aim of this study was to evaluate the effect of supernatant from mesenchymal cells in male rats with estrogen on the function and the survival of neutrophils.
Research type
This is an experimental study.
Research society, place and time
6-8 weeks rats were studied.
Sampling method and number
Non-declared
Used devices&materials
At the stage of isolation and culture of mesenchymal stem cells, mesenchymal cells was extracted from the bone marrow of the femur and tibia of mice with flushing a syringe containing DMEM (Sigma, USA), and after cell counting, it was cultivated with FBS 15% (fetal bovine serum) at 37˚C and 5% CO2. The first culture change was done after 72 hours and then every 3 days. In the phase of separation of neutrophils, Rezapour & Majidi method [10] was used. … [11-13] In the adjacency of bone marrow mesenchymal stem cells supernatant treated with beta estradiol by neutrophil cells, the supernatant was transferred to a flat bottom 24-well plate with floors covered with the supernatant. Then 5 × 106ml neutrophil cells with FBS 10% were added to each well containing supernatant, and then, they underwent 4-hour incubation at 37˚C in 5% CO2 and 80% humidity. At the neutrophil survival measurement stage at the presence of mesenchymal cells, to measure neutrophil survival at the presence of supernatant of mesenchymal cells, annexin propidium iodide kit (Sigma Aldrich, Cat NO: 51-6710AK; USA) was used. At the phagocytosis neutrophil testing stage, rat serum was isolated and was opsonized by yeast. This yeast, as the antigen supernatant, was placed in the adjacency to neutrophils which itself had been in the adjacency to mesenchymal stem cells which had been treated with estrogen. At the yeast preparing stage, the candida albicans yeast was used. The yeast had been cultured 24 hours ago to be fresh. Then, some of this culture was dissolved in PBS. 1 × 106 per ml was incubated 30min at 37˚C in RPMI medium (Sigma Aldrich; USA) containing10% of fresh rat serum [14]. In the apoptosis process, neutrophil were placed in 4-hour adjacency to supernatant of Mesenchymal cells treated or not treated with estrogen. After centrifugation, 10-landa acridine orange was added, and it was incubated at 37˚C and 30 minutes in the darkness. Then, some medium were put on that and it was centrifuged twice. The supernatant was poured and 10-landa PI was added to the plate cell of the bottom of solution and it underwent 5-minute incubation in darkness. Then some culture was poured on it and was centrifuged. Live and dead cells (necrosis and apoptosis) were investigated by a fluorescent microscope [3]. In the phagocytosis test, the Candida Albicans yeast was used. The yeast had been cultured 24 hours ago to be fresh. Then some of this culture was dissolved in PBS and centrifuged. In order to opsonization, 1 × 106 per ml underwent 30-minute incubation with RPMI culture containing10% fresh male rat serum and at 37˚C [14]. 100-landa neutrophil suspension was placed in vicinity of supernatant of MSCs treated with estrogen as well as the controlled sample 2 hours. Then, suspension was placed on sterile glass slides and 100 µl of opsonized yeast was added to it in order to make neutrophils-yeast access. This slide was incubated one hour at 37˚ C. The slide was examined under a light microscope and the percentage of neutrophils which had phagocytized the yeast was calculated [14]. In the Nitro Blue Tetrazolium (NBT) test, 15µl of neutrophils which had carried out the phagocytosis with yeast with 4 × 106 per ml density was mixed with 15µl NBT solution which had been prepared simultaneously with this test through combination of Zaymosan (Sigma, USA) and NBT powder (Sigma, USA) and RPMI medium (Sigma, USA) in a micro tube. This solution was incubated one hour at 37˚C and after this step, 400µl dimethylformamide (Sigma, USA) was added to this suspension. Then, the micro tubes were centrifuged. 200ml of the supernatant of both controlled samples and the samples which was in the adjacency to estrogen were taken and were poured to the sinks of ELISA plate. The optical density of supernatant was read at 520nm wavelength with an ELISA reader [14]. Data were analyzed using SPSS 18 software. Independent T-test, ANOVA and Tukey test were used for the mesenchymal cell supernatant group. … [15]
Findings by Text
The rate of apoptosis in the supernatant treated with 10nM estrogen dose (52.9+ ± 2.1) increased significantly in comparison with the control group (41.16± 2.1). Group treated with estrogen by 20nM dose (62.4 ± 4.2) showed significant difference compared with the control group. However, there was no significant difference between 20nM and 10nM doses. The phagocytosis percentage of mesenchymal cells in the supernatant after treated with10nM and 20nM estrogen had significant increase compared to control group (15.7 ± 1.1). There was a significant difference between 20nM (3.9 ± 0.4) and 10nM (9.1 ± 1.3) doses. The rate of respiratory burst of neutrophils exposed to supernatant of mesenchymal cells in treated group with 10nM estrogen dose (0.504 ± 0.038) had a significant difference compared to control group (0.613 ± 0.021). The group treated by 20nM estrogen dose (0.472 ± 0.029) as compared to control group showed a significant decrease. However, this difference was not significant between 20nM and 10nM estrogen doses.
Main comparison to the similar studies
Mesenchymal stem cells resident in the tissue play important roles in calling neutrophils and increasing their inflammatory capabilities following bacterial challenge [16]. … [17-23] Estrogen is effective on immune function [24, 25]. Mediatory functions of immune system on mesenchymal stem cells are set by cell-cell contact or soluble factors [7]. Estradiol and progesterone increase the survival of neutrophils that this operation is done through apoptosis delay by a reduction in the activity of caspase 3 and 9 [26]. The presence of bone marrow mesenchymal stem cells from healthy individuals adjacent to neutrophil cells with 1/500 proportion has significantly reduced apoptosis in resting neutrophils, activated neutrophils with interleukin -8 and activated neutrophils with tripeptide, N-formyl, L-methionine - L-lucil, and L-phenylalanine [27]. A similar function has been reported related to mesenchymal stem cells [18]. IL-6 produced by MSCs leads to more survival and inhibits apoptosis in the neutrophil cells [14]. IL-6 reduces Bax pre-apoptosis protein levels and mcl-1 anti-apoptosis protein in the neutrophil cells [26, 27]. Estrogen affects rat mesenchymal cells and inhibits apoptosis activity through an increase in BCL-2 and BCL-XL genomic molecules expressions [26]. Apoptosis decrease was observed in neutrophil exposed to the supernatant cells as well as mesenchymal cells treated with estrogen. Treatment of mesenchymal cells with estrogen increased neutrophils survival and this effect is done through caspase-3 and 9 activity reduction. Also, mesenchymal cells can protect thymocyte and centroblast from spontaneous apoptosis and protect them against death induced by Fas pathway [26]. IL-6 production causes anti-apoptotic effects that affect maintaining and strengthening of neutrophil function. Mesenchymal cells treatment and its supernatant with estrogen increased phagocytosis in neutrophils. In the supernatant of mesenchymal cells, IL-6 inhibits oxygen free radical production and development of respiratory burst of neutrophils cells treated or not treated with f-MLP [14]. NBT reduction test was observed in relation to the supernatant. Estrogen treatment of bone marrow mesenchymal stem cells following direct cells following direct cell proximity led to a significant increase in phagocytosis capability and respiratory burst in neutrophils cells.
Suggestions
Neutrophil interaction with supernatant from mesenchymal cells should be considered in interfering and hormonal therapy approaches. Mesenchymal cells adjacent to estrogen have significant effects on neutrophil function, and should be used to treat diseases associated with this function and physiological and pathological neutrophil responses in interaction with mesenchymal cells.
Limitations
Non-declared
Conclusions
Supernatant of MSCs, in the proximity to estrogen, increases phagocytosis capability and respiratory burst of the neutrophils.
Acknowledgments
The personnel of Immunology and Cell Culture Center of the Faculty of Veterinary in of Urmia University are appreciated.
Conflict of interest
Non-declared
Ethical Permissions
This study was in accordance with the guidelines of Iran Society for the Prevention of Cruelty to Animals.
Funding Sources
This study which is based on a graduate thesis was carried out in collaboration with the Cell Culture Center at the Faculty of Veterinary, University of Urmia.