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Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells
Research in Veterinary Science 107 (2016) 80–87
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Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells Tomasz Maślanka a,⁎, Małgorzata Chrostowska b, Iwona Otrocka-Domagała c, Anna Snarska d, Mateusz Mikiewicz d, Monika Zuśka-Prot a, Agnieszka Jasiecka a, Hubert Ziółkowski a, Włodzimierz Markiewicz a, Jerzy J. Jaroszewski a a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 13, 10-719 Olsztyn, Poland Wipasz S.A., Wadąg 9, 10-373 Olsztyn, Poland c Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 13, 10-719 Olsztyn, Poland d Department of Internal Medicine, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 14, 10-719 Olsztyn, Poland b
a r t i c l e
i n f o
Article history: Received 16 February 2016 Received in revised form 5 May 2016 Accepted 22 May 2016 Keywords: NK cells PGE2 EP receptor Apoptosis Proliferation Cattle
a b s t r a c t The aim of this research was to determine whether prostaglandin E2 (PGE2) affects bovine NK cells in respect of their counts, apoptosis and proliferation, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) these effects. We here report that long-term, but not short-term, exposure of bovine peripheral blood mononuclear cells to PGE2 at 10−5 M, 10−6 M and 10−7 M, but not at 10−8 M, caused a significant increase in the percentage of early apoptotic cells among NK cell subset. Moreover, PGE2 at 10−5 M and 10−6 M, but not at 10−7 M and 10−8 M, induced a considerable decrease in the absolute count of NK cells. The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors did not prevent the PGE2-induced apoptosis and depletion of NK cells. The results suggest that the proapoptotic effect of PGE2 is secondary in character and the induction of this effect is not mediated through EP receptors. Furthermore, the studies demonstrated that PGE2 at 10−5 M and 10−6 M, but not at 10−7 M and 10−8 M, highly significantly reduced the percentage of proliferating NK cells. The EP1, EP1/2 and EP3 receptor antagonists were unable to block this effect significantly, whereas the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of NK cells proliferation. These results indicate that PGE2 at certain concentrations may impair the proliferation of NK cells and this effect is mediated via the EP4 receptor. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction NK cells are effector lymphocytes of the innate immune system that represent a first line of defence against intracellular pathogens and tumors. NK cells perform two major functions. The first one is to recognize and lyse tumor cells and virally infected cells in the absence of previous sensitization (French and Yokoyama, 2003; Wu and Lanier, 2003); in addition to cytotoxicity, another important action of NK cells is to control intracellular pathogens by the production of large amounts of IFNγ (Goff et al., 2006; Bastos et al., 2008). The second function of NK cells is to regulate the innate and adaptive immune responses by secreting various chemokines (Taub et al., 1995; Inngjerdingen et al., 2001) or cytokines (Biron et al., 1999; Cooper et al., 2001). Several studies have indicated a role of NK cells in important infectious diseases of cattle (Boysen and Storset, 2009).
⁎ Corresponding author. E-mail address: [email protected] (T. Maślanka).
http://dx.doi.org/10.1016/j.rvsc.2016.05.009 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2) is typically perceived as a pro-inflammatory mediator. However, this eicosanoid actually shows a “Janus face” in terms of its effect on an inflammatory reaction because it exerts both pro- and anti-inflammatory effects (Konya et al., 2013). The effects of PGE2 are mediated via four distinct G protein-coupled E-prostanoid (EP) receptors, referred to as EP1, EP2, EP3 and EP4 receptors (Kalinski, 2012). It has been demonstrated that all these receptor subtypes are expressed by murine NK cells (Holt et al., 2011). Recently, we have found that prolonged exposure of bovine peripheral blood mononuclear cells (PBMCs) to meloxicam, i.e. a drug from the group of non-steroidal anti-inflammatory drugs (NSAIDs), raised the absolute count of NK cells and promoted the proliferation of mitogen-stimulated NK cells (Maślanka, 2013a). The major mechanism of action of NSAIDs consists in reducing the synthesis of PGE2 via the inhibition of cyclooxygenase-2 (COX-2), i.e. an enzyme that converts arachidonic acid into PGE2. Taking above into consideration, we have hypothesized that PGE2 compromises the proliferation of bovine NK cells in cattle. Our preliminary studies demonstrated that PGE2 at 10−6 M reduced the proliferation of NK cells derived from peripheral blood of cattle (Maślanka,
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2013b). This finding was a starting point for the current research. Whereas the role of NK cells in the immune system is gaining importance, we have concluded that broader research on the effect of this eicosanoid on bovine NK cells is recommendable. The primary objective of our study has been to determine at what concentrations PGE2 is capable of producing an inhibitory effect on the proliferation of NK cells in cattle and to identify which one(s) of EP receptor subtype(s) mediate(s) this effect. During our investigation, we observed that exposure of NK cells to PGE2 led to their depletion. This result implicated that PGE2 could induce the apoptosis of NK cells, hence the subsequent purpose of our study was to verify this hypothesis. As the experiments completed thus far have shown that PGE2 exerts the proapoptotic effect on NK cells, a study has been undertaken to identify which of the EP receptor subtype(s) mediate(s) this effect. While the results have a cognitive value, they can also contribute to certain practical solutions, because medications that inhibit production of PGE2 (i.e. NSAIDs) are used for the treatment of symptoms associated with various infections. 2. Materials and methods 2.1. Animals Blood was drawn from the jugular vein of clinically healthy HolsteinFriesian heifers from a farm at the Research Station of the University of Warmia and Mazury, located in Bałdy (Poland). The animals were housed and treated in accordance with the rules of the Local Ethics Commission for Animal Experiments in Olsztyn (affiliated to the National Ethics Commission for Animal Experimentation, Polish Ministry of Science and Higher Education). The study was approved by the above mentioned Local Ethics Commission. 2.2. Isolation of PBMCs and culture conditions Blood was collected into 10 mL heparinized sterile vacutainer tubes [Becton Dickinson (BD) Biosciences, San Jose, CA, USA]. PBMCs were isolated as previously described (Maślanka, 2013a; Maślanka et al., 2014), adjusted to a final concentration of 4 × 106 (evaluation of apoptosis) or 2 × 106 cells/mL (the remaining parameters) in complete medium [RPMI 1640, 10% FBS, 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate and 10 U/mL penicillin/streptomycin (all from Sigma-Aldrich)] and seeded in 24-well plates in 1 mL aliquots. In order to determine whether PGE2 affects the apoptosis and absolute count of NK cells, PBMCs were cultured with medium alone or in the presence of PGE2 (10−5 M, 10−6 M, 10−7 M and 10−8 M; Sigma-Aldrich) for 24 h and for 7 days. To evaluate the effect of PGE2 on the proliferation of NK cells, PBMCs were incubated without (medium) or with PGE2 (10−5 M, 10−6 M, 10−7 M and 10−8 M) for 24 h with concomitant stimulation with concanavalin A (Con A; 5 μg/mL; Sigma-Aldrich) in the presence of 5-bromo-2′-deoxyuridine (BrdU; APC BrdU Flow Kit, BD Biosciences) at a final concentration of 60 μM in cell culture medium during the last 8 h. Two experiments were performed, using 5 different animals in each one. Since PGE2 was dissolved in ethanol, an adequate volume of the solvent was added to control wells; all wells contained the same amount of ethanol. For the identification of EP receptor subtype(s) responsible for the PGE2-induced apoptosis and reduction in the number of NK cells, PBMCs were exposed only to PGE2 (10−6 M) or were pretreated with EP1 (SC-19220; Sigma-Aldrich) or EP1/2 (AH-6809; Sigma-Aldrich) or EP3 (L-798,106; Tocris Bioscience, Bristol, UK) or EP4 (L-161982; Tocris Bioscience) receptor antagonists (all at a final concentration of 10−5 M) for 1 h and then were exposed to PGE2 (10−6 M) for 7 days. PBMCs incubated in medium alone served as the control. For the identification of EP receptor subtype(s) responsible for the PGE2-induced inhibition of the proliferation of NK cells, PBMCs were exposed only to PGE2 (10−6 M) or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor
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antagonists (all at a final concentration of 10− 5 M) for 1 h and then were treated with PGE2 (10−6 M) for 24 h with concomitant stimulation with Con A (5 μg/mL) in the presence of BrdU at a final concentration of 60 μM in cell culture medium during the last 8 h. PBMCs incubated in medium alone served as the control. Moreover, to exclude the possibility that EP receptor antagonists alone could affect the evaluated parameters, PBMCs were cultured only in the presence of these agents. Two experiments were performed, using 5 different animals in each one. PGE2 and all antagonists of the EP receptors were dissolved in ethanol and DMSO, respectively. Therefore, the same amount of DMSO/ethanol and DMSO was added to control wells and wells treated only with PGE2, respectively. 2.3. Flow cytometry 2.3.1. Extracellular staining for NK cells Cells were removed from the wells by pipetting and rinsing with FACS buffer [FB; 1 × Dulbecco's PBS (Sigma-Aldrich) devoid of Ca2 + and Mg2+ with 2% (v/v) heat-inactivated FBS] and transferred into individual tubes and centrifuged. After additional washing in 2 mL FB, the cells were re-suspended in FB and stained with AF488-conjugated mouse anti-bovine CD335 mAb (1:20; NK cell marker; AKS1, IgG1; Serotec, Oxford, UK). After 45 min incubation (on ice and in the dark), the cells were washed in 2 mL FB. 2.3.2. Staining for apoptosis evaluation Cells stained with AF488-conjugated mouse anti-bovine CD335 mAb (as described above) were washed once in 1 mL of 1× Annexin V binding buffer (BD Biosciences). The supernatants were removed by centrifugation and the cells were suspended in 1× Annexin V binding buffer. A 5 μL of PE-conjugated Annexin V and 5 μL of 7-AAD (both from from BD Biosciences) were added to the cells. The cells were mixed gently and incubated for 15 min at RT in the dark, and then diluted with 400 μL of 1× Annexin V binding buffer and analyzed by flow cytometry within 1 h. 2.3.3. Staining for determination of proliferating cells PBMCs were labeled for NK cells (as described above), and thereafter stained for incorporated BrdU according to the manufacturer's procedure (APC BrdU Flow Kit, BD Biosciences). 2.3.4. FACS acquisition and analysis Flow cytometry analysis was performed using a FACSCanto II cytometer (BD Biosciences). The data were acquired by FACSDiva version 6.1.3 software (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc., Stanford, USA). The cytometry setup and tracking beads (BD Biosciences) were used to initialize PMT settings. Unstained and single fluorochrome-stained samples were used to set fluorochrome compensation levels. The entire volume of each sample was always acquired. The absolute counts of NK cells (i.e. number of NK cells per sample) were calculated using the dual platform method, i.e. the absolute cell count was determined by calculating the data obtained from cell counting chamber (a cell count per well) by the percentage of NK (i.e. CD335+ ) cells (the data from flow cytometric immunophenotyping). 2.4. Statistical analyses Results were the mean (± SD) of two independent experiments with 5 animals per experiment (overall n = 10). Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Differences were deemed significant when the P values were b0.05. SigmaPlot Software Version 12.0 (Systat Software Inc., San Jose, USA) was used for statistical analysis and plotting graphs.
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3. Results Exposure to PGE2 exerts proapoptotic effect on NK cells and induces their depletion
The effect of PGE2 on the apoptosis and absolute count of NK cells was assessed 24 h and 7 days after PBMCs exposure to this eicosanoid. No effect of PGE2 (at all used concentrations) on the apoptosis and absolute count of NK was demonstrated at the first time-point
Fig. 1. The effect of PGE2 on the apoptosis and absolute count of NK cells. PBMCs were cultured without (medium) or with PGE2 (10−5–10−8 M) for 7 days. The results are expressed as a percentage of early apoptotic (A) and late apoptotic/necrotic cells (B) within the NK cell population. The absolute count represents the number of NK cells per sample (C). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.05, **P b 0.01, ***P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). The dot plot of Annexin V versus 7-AAD was used for the assessment of apoptosis. Annexin V− 7-AAD− cells were considered viable; Annexin V+ 7-AAD− cells were deemed early-apoptotic; and double positive cells (Annexin V+ 7-AAD+) were considered to be a mixture of late apoptotic and primary necrotic cells (D). Examples of cytograms (density plots) showing percentages of early apoptotic, late apoptotic/necrotic and viable NK cells (E).
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Fig. 2. The effect of the blockade of PGE2 receptors (EP) on the apoptosis and absolute count of NK cells. To identify the EP receptor subtype(s) responsible for PGE2-induced apoptosis of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were exposed to PGE2 for 7 days. PBMCs incubated in medium alone served as the control. The results are expressed as a percentage of early apoptotic (A) and late apoptotic/necrotic cells (B) within the NK cell population. The absolute count represents the number of NK cells per sample (C). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Examples of cytograms (density plots) showing percentages of early apoptotic (Annexin V+ 7-AAD−), late apoptotic/necrotic (Annexin V+ 7AAD+) and viable (Annexin V− 7-AAD−) NK cells (D).
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(data not shown). It was found that 7-day-long exposure of PBMCs to PGE2 at 10− 5 M, 10− 6 M and 10− 7 M, but not at 10− 8 M, caused a significant increase in the percentage of early apoptotic (Fig. 1A and E) and of a mixed population of late apoptotic/necrotic cells among NK cells (Fig. 1B and E). PGE2 at 10−5 M and 10−6 M induced a considerable decrease in the absolute count of NK cells, whereas at lower concentrations it did not affect this parameter (Fig. 1C). The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors does not prevent PGE2-induced cell death To identify the EP receptor subtype(s) responsible for PGE2-induced apoptosis and depletion of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists and then were exposed to PGE2 for 7 days. The EP1, EP1/2, EP3 and EP4 antagonists were unable to abolish the PGE2-induced apoptosis and depletion of NK cells (Fig. 2). This is proven by the fact that the percentage of early apoptotic (Fig. 2A and D) and late apoptotic/necrotic NK cells (Fig. 2B and D) and the absolute number of NK cells (Fig. 2C) from samples pretreated with EP1, EP1/2, EP3 and EP4 antagonists were not different compared to those in samples exposed only to PGE2, while they were significantly higher (the percentage of early apoptotic and late apoptotic/necrotic NK cells) or lower (the absolute number of NK cells) than the control values. Thus, the blockade of any of the EP-receptor subtypes did not prevent the PGE2-induced apoptosis of NK cells. The percentage of early apoptotic and late apoptotic/necrotic NK cells, as well as the absolute number of NK cells from samples treated only with EP receptor antagonists did not differ substantially from the control values (data not shown). PGE2 impairs proliferation of NK cells The effect of PGE2 on the proliferation of NK cells was determined in PBMCs incubated with or without this eicosanoid for 24 h with the accompanying Con A stimulation in the presence of BrdU. These studies demonstrated that PGE2 at 10−5 M and 10− 6 M, but not at 10− 7 M
and 10−8 M, highly significantly reduced the percentage of proliferating NK cells (Fig. 3). The selective blockade of EP4 receptors prevents PGE2-induced inhibition of proliferation of NK cells To identify the EP receptor subtype(s) responsible for PGE2-induced inhibition of proliferation of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were treated with PGE2 for 24 h with concomitant stimulation with Con A in the presence of BrdU. The EP1, EP1/2 and EP3 antagonists were unable to block significantly the PGE2-induced inhibition of NK cell proliferation. This is proven by the fact that the percentage of proliferating NK cells from samples pretreated with EP1, EP1/2 and EP3 antagonists did not differ significantly from the values of these parameters obtained in samples exposed only to PGE2, while being considerably lower than the control values (Fig. 4). On the contrary, the percentage of proliferating NK cells from samples pretreated with the EP4 antagonist were higher than the values of this parameter obtained from samples exposed only to PGE2, but they did not differ significantly from the control values (Fig. 4). Thus, the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of proliferation of NK cells. The percentage of BrdU+ cells among NK cells in samples treated only with EP receptor antagonists showed no difference when compared with the control values (data not shown). 4. Discussion There are various levels of PGE2 attained at inflammatory sites, depending on the location of inflammation, type of disease as well as its course and stage. Literature indicates that PGE2 can reach concentrations of the order of 10−6–10−9 M (Nygård et al., 1992; Anderson et al., 1996; Hinson et al., 1996; Hirata et al., 2001). Thus, the concentrations of 10−6–10−8 M applied in our research mimic a real inflammatory levels of PGE2.
Fig. 3. The effect of PGE2 on the proliferation of NK cells. To evaluate the effect of PGE2 on proliferation NK cells, PBMCs were incubated without (medium) or with PGE2 (10−5–10−8 M) for 24 h with concomitant stimulation with Con A in the presence of 5-bromo-2′-deoxyuridine (BrdU) in cell culture medium during the last 8 h. The results are expressed as a percentage of BrdU-incorporating cells among the NK cell population (A). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Dot plot cytograms demonstrate the distribution of BrdU-positive and -negative cells within NK cells (B).
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Fig. 4. The effect of the blockade of PGE2 receptors (EP) on the proliferation of NK cells. To identify the EP receptor subtype(s) responsible for PGE2-induced inhibition of proliferation of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were treated with PGE2 for 24 h with concomitant stimulation with Con A in the presence of 5-bromo-2′-deoxyuridine (BrdU) in cell culture medium during the last 8 h. The results are expressed as a percentage of BrdU-incorporating cells among the NK cell population (A). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Dot plot cytograms demonstrate the distribution of BrdU-positive and -negative cells within NK cells (B).
In the current experiments, average percentages of early apoptotic cells and a mixed population of late apoptotic/necrotic cells among NK cells in the control samples were 11.16% and 8.46%, respectively. These are normal levels because, under in vitro conditions, a certain pool of cells continually undergoes apoptosis, which is associated with the specific character of cell culture. Short-term treatment with PGE2 did not affect the apoptosis or count of NK cells, whereas 7-day exposure of PBMCs to this eiconasoid in the concentrations 10−5, 10−6 and 10−7 M, but not 10−8 M, induced a significant increase in the percentage of early apoptotic and mixed population of late apoptotic/necrotic cells within NK cells, but the intensity of this effect diminished as the concentration of PGE2 was lowered. This relates to the results of investigations into the effect of PGE2 on counts of NK cells, where it was revealed that exposure of PBMCs to PGE2 in the concentrations 10−5 and 10− 6 M led to a significant depletion of NK cells, with the effect being more distinctly expressed at a higher concentration of the eiconasoid. Such results implicate that PGE2 produces a proapoptotic effect on bovine NK cells, and (a) this effect does not appear promptly, (b) induction of this effect requires a relatively high concentration of PGE2, and (c) the intensity of this effect is dependent on the concentration of PGE2. Thus, the data obtained from this study suggest that in cattle with inflammation, especially in the acute phase, PGE2 may induce the apoptosis of NK cells. To the best of our knowledge, there is no information
available on the effect of PGE2 on the apoptosis of human or animal NK cells, and therefore this is the first report on this subject. All the findings indicate that, depending on the type of cells, PGE2 can produce either a proapoptotic (Föller et al., 2006; Huang et al., 2009; Kovarova and Koller, 2014; Nagano et al., 2014) or antiapoptotic effect on affected cells (Nishihara et al., 2003; Yamamoto et al., 2010; Coulombe et al., 2014). It was found that PGE2 increased apoptosis of normal lung fibroblasts in a dose-dependent manner (Huang et al., 2009). In other studies, it was demonstrated that PGE2 induced apoptosis of K562 human leukaemia cells (Föller et al., 2006), mast cells (Kovarova and Koller, 2014) and rat microglia (Nagano et al., 2014). On the other hand, it was found that PGE2 protected normal and transformed intestinal epithelial cells from apoptosis induced by diverse stimuli (Nishihara et al., 2003). Moreover, Coulombe et al. (2014) showed that during influenza A virus infection, PGE2 was upregulated, which led to the inhibition of apoptosis in macrophages. In the further stage of our research, it was determined which of the EP receptors mediated in the induction of the proapoptotic effect of PGE2 on the analyzed cells. The experiments did not demonstrate that the blockade of any of the receptors would prevent PGE2-induced apoptosis and depletion of NK cells. This does not seem to be due to an inadequate blockade of EP receptors, as it was demonstrated in another part of the research that exposure of PBMCs to the EP4 receptor
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antagonist prevented PGE2-induced inhibition of NK cell proliferation. Also, in another studies, we were able to determine that the selective blockade of EP4 receptor prevented PGE2-induced down-regulation of CD25 expression on T cells (Maślanka et al., 2014) and inhibition of IFN-γ production by WC1+ T cells (Przybysz et al., 2016). The fact that short-term exposure to PGE2 did not influence the apoptosis of NK cells, but that the proapoptotic effect of PGE2 emerged only after prolonged exposure seems to implicate that this effect may not be a result of the direct activation of EP1-EP4 receptors, but may involve complex cellular reactions. Hence, the results obtained thus far suggest that the proapoptotic effect of PGE2 on bovine NK cells is clearly of secondary character, and its induction is not mediated by EP1-EP4 receptors. This conclusion may be supported by research results showing that PGE2-induced apoptotic cell death in K562 human leukaemia cells was mediated by the Ca2 +-permeable transient receptor potential canonical 7 channel (Föller et al., 2006). The above conclusion as well as the current research data remain in accord with the results recently published by Nagano et al. (2014), who demonstrated that PGE2 induced apoptosis in cultured rat microglia, but this effect was not prevented by pretreatment with EP1–EP4 antagonists. Therefore, these investigators concluded that the above effect was unlikely to occur through EP1–EP4 receptors. Regarding the study on the proliferation of NK cells, it was observed that their exposure to the two higher PGE2 concentrations significantly reduced the percentage of proliferating cells in the total NK cell population. These results indicate that PGE2 may have an antiproliferative effect on NK cells. This conclusion, as well as the results of our investigation, can find support in our previous studies, which revealed that the inhibition of PGE2 synthesis mediated by meloxicam (a relatively selective COX-2 inhibitor) promoted proliferation of mitogenstimulated bovine NK cells (Maślanka, 2013a). Despite some claims that PGE2 inhibits proliferation of NK cells, to the best of our knowledge, no studies have been performed and reported that would provide direct evidence supporting the influence of this eicosanoid on proliferation of human or animal NK cells. NK cells rapidly proliferate during viral infections to provide an expanded pool of effector cells to suppress the infection (Nguyen et al., 2002; Andrews et al., 2003; Björkström et al., 2011). In most cases, a viral infection induces an inflammatory response, which leads to the production of various proinflammatory mediators, including PGE2. Therefore, it is highly possible that PGE2 can act as an inhibitor of bovine NK cell proliferation, but this occurs only in the presence of a factor that triggers this process and simultaneously induces an inflammatory response (e.g. viral infections). It needs to be noted that the current study demonstrated that the antiproliferatory effect of PGE2 appeared only at very high PGE2 concentrations, which suggests that under in vivo conditions such an effect might take place in the course of an illness with a strongly expressed inflammatory response. It should be underlined that any conclusion implicating that PGE2 can act as a physiological inhibitor of the proliferation of mature NK cells would be erroneous because in healthy animals these cells do not proliferate at a significant rate (Warren et al., 1996; Koka et al., 2003). The selective blockade of EP4 receptor, but not EP1, EP2 and EP3 receptors, prevented PGE2-induced inhibition of NK cell proliferation. These results suggest that antiproliferative action of PGE2 toward bovine NK cells is mediated via the EP4 receptor. This results is unsurprising as it has been evidenced that PGE2 supresses NK cell migration and INF-γ production by NK cells primarily through the EP4 receptor (Holt et al., 2011). Most clearly, then, in the case of NK cells the EP4 receptor serves as an inhibitory receptor. Our determinations also agree with the research results reported by Kvirkvelia et al. (2002), who found that PGE2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 (cytotoxic T-lymphocyte line) T cells. The current results implicate a possibility of preventing/inactivating the antiproliferative action of PGE2 toward bovine NK cells via a blockade of the EP4 receptor by its selective antagonist, or through the
inhibition of the production of PGE2. Considering the fact that the mechanism of action of NSAIDs involves blocking the synthesis of this eicosanoid, it is possible to claim that the research reported herein casts a new light on some undiscovered aspects of NSAIDs administration. These drugs are administered in treatment of various infections in order to alleviate the clinical signs of an illness, which arise from an inflammatory response that accompanies the infection. Our findings suggest that the use of NSAIDs may bring about an additional advantage in the treatment of cattle with infections caused by intracellular pathogens, such as giving protection to NK cells against the antiproliferative and proapoptotic effects of PGE2. However, it should be emphasized that at this moment the pharmacological protection of bovine NK cells achieved by blocking the effect of PGE2 on these cells can only be hypothesized as we lack firm knowledge whether PGE2 under in vivo conditions would actually inhibit the infection-induced proliferation of bovine NK cells and induce their apoptosis. These questions cannot be resolved without further research. In recapitulation, the results of the study reported herein suggest that PGE2 may produce a proapoptotic effect on NK cells in cattle, but this effect does not occur promptly. The proapoptotic effect of PGE2 on bovine NK cells is most clearly secondary in character, and its induction is not mediated by the EP1-EP4 receptors. Moreover, the results indicate that PGE2 can have an antiproliferative effect on bovine NK cells and this effect is mediated through the EP4 receptor. The proapoptotic and antiproliferative effects occurred at relatively high concentrations of PGE2, suggesting that under in vivo conditions such effects can be expected nearly exclusively in an illness accompanied by a very strongly expressed inflammatory response. The results also suggest an additional advantage to using NSAIDs in cows with infections caused by intracellular pathogens, such as the protection of NK cells against the antiproliferative and proapoptotic effects of PGE2. Conflict of interest statement The authors declare that they have no conflict of interests. Acknowledgments The study was supported by grant no. 15.610.008-300 from the University of Warmia and Mazury in Olsztyn. References Anderson, G.D., Hauser, S.D., McGarity, K.L., Bremer, M.E., Isakson, P.C., Gregory, S.A., 1996. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97, 2672–2679. Andrews, D.M., Scalzo, A.A., Yokoyama, W.M., Smyth, M.J., Degli-Esposti, M.A., 2003. Functional interactions between dendritic cells and NK cells during viral infection. Nat. Immunol. 4, 175–181. Bastos, R.G., Johnson, W.C., Mwangi, W., Brown, W.C., Goff, W.L., 2008. Bovine NK cells acquire cytotoxic activity and produce IFN-γ after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells. Vet. Immunol. Immunopathol. 124, 302–312. Biron, C.A., Nguyen, K.B., Pien, G.C., Cousens, L.P., Salazar-Mather, T.P., 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220. Björkström, N.K., Lindgren, T., Stoltz, M., Fauriat, C., Braun, M., Evander, M., Michaëlsson, J., Malmberg, K.J., Klingström, J., Ahlm, C., Ljunggren, H.G., 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208, 13–21. Boysen, P., Storset, A.K., 2009. Bovine natural killer cells. Vet. Immunol. Immunopathol. 130, 163–177. Cooper, M.A., Fehniger, T.A., Caligiuri, M.A., 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22, 633–640. Coulombe, F., Jaworska, J., Verway, M., Tzelepis, F., Massoud, A., Gillard, J., Wong, G., Kobinger, G., Xing, Z., Couture, C., Joubert, P., Fritz, J.H., Powell, W.S., Divangahi, M., 2014. Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages. Immunity 40, 554–568. Föller, M., Kasinathan, R.S., Duranton, C., Wieder, T., Huber, S.M., Lang, F., 2006. PGE2-induced apoptotic cell death in K562 human leukaemia cells. Cell. Physiol. Biochem. 17, 201–210. French, A.R., Yokoyama, W.M., 2003. Natural killer cells and viral infections. Curr. Opin. Immunol. 15, 45–51.
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Maślanka, T., Spodniewska, A., Barski, D., Jasiecka, A., Zuśka-Prot, M., Ziółkowski, H., Markiewicz, W., Jaroszewski, J.J., 2014. Prostaglandin E2 down-regulates the expression of CD25 on bovine T cells, and this effect is mediated through the EP4 receptor. Vet. Immunol. Immunopathol. 160, 192–200. Nagano, T., Kimura, S.H., Takemura, M., 2014. Prostaglandin E2 induces apoptosis in cultured rat microglia. Brain Res. 1568, 1–9. Nguyen, K.B., Salazar-Mather, T.P., Dalod, M.Y., Van Deusen, J.B., Wei, X.Q., Liew, F.Y., Caligiuri, M.A., Durbin, J.E., Biron, C.A., 2002. Coordinated and distinct roles for IFNalpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J. Immunol. 169, 4279–4287. Nishihara, H., Kizaka-Kondoh, S., Insel, P.A., Eckmann, L., 2003. Inhibition of apoptosis in normal and transformed intestinal epithelial cells by cAMP through induction of inhibitor of apoptosis protein (IAP)-2. Proc. Natl. Acad. Sci. U. S. A. 100, 8921–8926. Nygård, G., Larsson, A., Gerdin, B., Ejerblad, S., Berglindh, T., 1992. Viability, prostaglandin E2 production, and protein handling in normal and inflamed human colonic mucosa cultured for up to 48 h in vitro. Scand. J. Gastroenterol. 27, 303–310. Przybysz, J., Chrostowska, M., Ziółkowski, H., Jaroszewski, J.J., Maślanka, T., 2016. The influence of prostaglandin E2 on the production of IFN-γ by bovine CD4+, CD8+ and WC1+ T cells. Res. Vet. Sci. 105, 31–35. Taub, D.D., Sayers, T.J., Carter, C.R.D., Ortaldo, J.R., 1995. Alpha and beta chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155, 3877–3888. Warren, H.S., Kinnear, B.F., Kastelein, R.L., Lanier, L.L., 1996. Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells. J. Immunol. 156, 3254–3259. Wu, J., Lanier, L.L., 2003. Natural killer cells and cancer. Adv. Cancer Res. 90, 127–156. Yamamoto, E., Izawa, T., Juniantito, V., Kuwamura, M., Sugiura, K., Takeuchi, T., Yamate, J., 2010. Involvement of endogenous prostaglandin E2 in tubular epithelial regeneration through inhibition of apoptosis and epithelial-mesenchymal transition in cisplatininduced rat renal lesions. Histol. Histopathol. 25, 995–1007.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells Tomasz Maślanka a,⁎, Małgorzata Chrostowska b, Iwona Otrocka-Domagała c, Anna Snarska d, Mateusz Mikiewicz d, Monika Zuśka-Prot a, Agnieszka Jasiecka a, Hubert Ziółkowski a, Włodzimierz Markiewicz a, Jerzy J. Jaroszewski a a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 13, 10-719 Olsztyn, Poland Wipasz S.A., Wadąg 9, 10-373 Olsztyn, Poland c Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 13, 10-719 Olsztyn, Poland d Department of Internal Medicine, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego Street 14, 10-719 Olsztyn, Poland b
a r t i c l e
i n f o
Article history: Received 16 February 2016 Received in revised form 5 May 2016 Accepted 22 May 2016 Keywords: NK cells PGE2 EP receptor Apoptosis Proliferation Cattle
a b s t r a c t The aim of this research was to determine whether prostaglandin E2 (PGE2) affects bovine NK cells in respect of their counts, apoptosis and proliferation, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) these effects. We here report that long-term, but not short-term, exposure of bovine peripheral blood mononuclear cells to PGE2 at 10−5 M, 10−6 M and 10−7 M, but not at 10−8 M, caused a significant increase in the percentage of early apoptotic cells among NK cell subset. Moreover, PGE2 at 10−5 M and 10−6 M, but not at 10−7 M and 10−8 M, induced a considerable decrease in the absolute count of NK cells. The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors did not prevent the PGE2-induced apoptosis and depletion of NK cells. The results suggest that the proapoptotic effect of PGE2 is secondary in character and the induction of this effect is not mediated through EP receptors. Furthermore, the studies demonstrated that PGE2 at 10−5 M and 10−6 M, but not at 10−7 M and 10−8 M, highly significantly reduced the percentage of proliferating NK cells. The EP1, EP1/2 and EP3 receptor antagonists were unable to block this effect significantly, whereas the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of NK cells proliferation. These results indicate that PGE2 at certain concentrations may impair the proliferation of NK cells and this effect is mediated via the EP4 receptor. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction NK cells are effector lymphocytes of the innate immune system that represent a first line of defence against intracellular pathogens and tumors. NK cells perform two major functions. The first one is to recognize and lyse tumor cells and virally infected cells in the absence of previous sensitization (French and Yokoyama, 2003; Wu and Lanier, 2003); in addition to cytotoxicity, another important action of NK cells is to control intracellular pathogens by the production of large amounts of IFNγ (Goff et al., 2006; Bastos et al., 2008). The second function of NK cells is to regulate the innate and adaptive immune responses by secreting various chemokines (Taub et al., 1995; Inngjerdingen et al., 2001) or cytokines (Biron et al., 1999; Cooper et al., 2001). Several studies have indicated a role of NK cells in important infectious diseases of cattle (Boysen and Storset, 2009).
⁎ Corresponding author. E-mail address: [email protected] (T. Maślanka).
http://dx.doi.org/10.1016/j.rvsc.2016.05.009 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2) is typically perceived as a pro-inflammatory mediator. However, this eicosanoid actually shows a “Janus face” in terms of its effect on an inflammatory reaction because it exerts both pro- and anti-inflammatory effects (Konya et al., 2013). The effects of PGE2 are mediated via four distinct G protein-coupled E-prostanoid (EP) receptors, referred to as EP1, EP2, EP3 and EP4 receptors (Kalinski, 2012). It has been demonstrated that all these receptor subtypes are expressed by murine NK cells (Holt et al., 2011). Recently, we have found that prolonged exposure of bovine peripheral blood mononuclear cells (PBMCs) to meloxicam, i.e. a drug from the group of non-steroidal anti-inflammatory drugs (NSAIDs), raised the absolute count of NK cells and promoted the proliferation of mitogen-stimulated NK cells (Maślanka, 2013a). The major mechanism of action of NSAIDs consists in reducing the synthesis of PGE2 via the inhibition of cyclooxygenase-2 (COX-2), i.e. an enzyme that converts arachidonic acid into PGE2. Taking above into consideration, we have hypothesized that PGE2 compromises the proliferation of bovine NK cells in cattle. Our preliminary studies demonstrated that PGE2 at 10−6 M reduced the proliferation of NK cells derived from peripheral blood of cattle (Maślanka,
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2013b). This finding was a starting point for the current research. Whereas the role of NK cells in the immune system is gaining importance, we have concluded that broader research on the effect of this eicosanoid on bovine NK cells is recommendable. The primary objective of our study has been to determine at what concentrations PGE2 is capable of producing an inhibitory effect on the proliferation of NK cells in cattle and to identify which one(s) of EP receptor subtype(s) mediate(s) this effect. During our investigation, we observed that exposure of NK cells to PGE2 led to their depletion. This result implicated that PGE2 could induce the apoptosis of NK cells, hence the subsequent purpose of our study was to verify this hypothesis. As the experiments completed thus far have shown that PGE2 exerts the proapoptotic effect on NK cells, a study has been undertaken to identify which of the EP receptor subtype(s) mediate(s) this effect. While the results have a cognitive value, they can also contribute to certain practical solutions, because medications that inhibit production of PGE2 (i.e. NSAIDs) are used for the treatment of symptoms associated with various infections. 2. Materials and methods 2.1. Animals Blood was drawn from the jugular vein of clinically healthy HolsteinFriesian heifers from a farm at the Research Station of the University of Warmia and Mazury, located in Bałdy (Poland). The animals were housed and treated in accordance with the rules of the Local Ethics Commission for Animal Experiments in Olsztyn (affiliated to the National Ethics Commission for Animal Experimentation, Polish Ministry of Science and Higher Education). The study was approved by the above mentioned Local Ethics Commission. 2.2. Isolation of PBMCs and culture conditions Blood was collected into 10 mL heparinized sterile vacutainer tubes [Becton Dickinson (BD) Biosciences, San Jose, CA, USA]. PBMCs were isolated as previously described (Maślanka, 2013a; Maślanka et al., 2014), adjusted to a final concentration of 4 × 106 (evaluation of apoptosis) or 2 × 106 cells/mL (the remaining parameters) in complete medium [RPMI 1640, 10% FBS, 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate and 10 U/mL penicillin/streptomycin (all from Sigma-Aldrich)] and seeded in 24-well plates in 1 mL aliquots. In order to determine whether PGE2 affects the apoptosis and absolute count of NK cells, PBMCs were cultured with medium alone or in the presence of PGE2 (10−5 M, 10−6 M, 10−7 M and 10−8 M; Sigma-Aldrich) for 24 h and for 7 days. To evaluate the effect of PGE2 on the proliferation of NK cells, PBMCs were incubated without (medium) or with PGE2 (10−5 M, 10−6 M, 10−7 M and 10−8 M) for 24 h with concomitant stimulation with concanavalin A (Con A; 5 μg/mL; Sigma-Aldrich) in the presence of 5-bromo-2′-deoxyuridine (BrdU; APC BrdU Flow Kit, BD Biosciences) at a final concentration of 60 μM in cell culture medium during the last 8 h. Two experiments were performed, using 5 different animals in each one. Since PGE2 was dissolved in ethanol, an adequate volume of the solvent was added to control wells; all wells contained the same amount of ethanol. For the identification of EP receptor subtype(s) responsible for the PGE2-induced apoptosis and reduction in the number of NK cells, PBMCs were exposed only to PGE2 (10−6 M) or were pretreated with EP1 (SC-19220; Sigma-Aldrich) or EP1/2 (AH-6809; Sigma-Aldrich) or EP3 (L-798,106; Tocris Bioscience, Bristol, UK) or EP4 (L-161982; Tocris Bioscience) receptor antagonists (all at a final concentration of 10−5 M) for 1 h and then were exposed to PGE2 (10−6 M) for 7 days. PBMCs incubated in medium alone served as the control. For the identification of EP receptor subtype(s) responsible for the PGE2-induced inhibition of the proliferation of NK cells, PBMCs were exposed only to PGE2 (10−6 M) or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor
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antagonists (all at a final concentration of 10− 5 M) for 1 h and then were treated with PGE2 (10−6 M) for 24 h with concomitant stimulation with Con A (5 μg/mL) in the presence of BrdU at a final concentration of 60 μM in cell culture medium during the last 8 h. PBMCs incubated in medium alone served as the control. Moreover, to exclude the possibility that EP receptor antagonists alone could affect the evaluated parameters, PBMCs were cultured only in the presence of these agents. Two experiments were performed, using 5 different animals in each one. PGE2 and all antagonists of the EP receptors were dissolved in ethanol and DMSO, respectively. Therefore, the same amount of DMSO/ethanol and DMSO was added to control wells and wells treated only with PGE2, respectively. 2.3. Flow cytometry 2.3.1. Extracellular staining for NK cells Cells were removed from the wells by pipetting and rinsing with FACS buffer [FB; 1 × Dulbecco's PBS (Sigma-Aldrich) devoid of Ca2 + and Mg2+ with 2% (v/v) heat-inactivated FBS] and transferred into individual tubes and centrifuged. After additional washing in 2 mL FB, the cells were re-suspended in FB and stained with AF488-conjugated mouse anti-bovine CD335 mAb (1:20; NK cell marker; AKS1, IgG1; Serotec, Oxford, UK). After 45 min incubation (on ice and in the dark), the cells were washed in 2 mL FB. 2.3.2. Staining for apoptosis evaluation Cells stained with AF488-conjugated mouse anti-bovine CD335 mAb (as described above) were washed once in 1 mL of 1× Annexin V binding buffer (BD Biosciences). The supernatants were removed by centrifugation and the cells were suspended in 1× Annexin V binding buffer. A 5 μL of PE-conjugated Annexin V and 5 μL of 7-AAD (both from from BD Biosciences) were added to the cells. The cells were mixed gently and incubated for 15 min at RT in the dark, and then diluted with 400 μL of 1× Annexin V binding buffer and analyzed by flow cytometry within 1 h. 2.3.3. Staining for determination of proliferating cells PBMCs were labeled for NK cells (as described above), and thereafter stained for incorporated BrdU according to the manufacturer's procedure (APC BrdU Flow Kit, BD Biosciences). 2.3.4. FACS acquisition and analysis Flow cytometry analysis was performed using a FACSCanto II cytometer (BD Biosciences). The data were acquired by FACSDiva version 6.1.3 software (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc., Stanford, USA). The cytometry setup and tracking beads (BD Biosciences) were used to initialize PMT settings. Unstained and single fluorochrome-stained samples were used to set fluorochrome compensation levels. The entire volume of each sample was always acquired. The absolute counts of NK cells (i.e. number of NK cells per sample) were calculated using the dual platform method, i.e. the absolute cell count was determined by calculating the data obtained from cell counting chamber (a cell count per well) by the percentage of NK (i.e. CD335+ ) cells (the data from flow cytometric immunophenotyping). 2.4. Statistical analyses Results were the mean (± SD) of two independent experiments with 5 animals per experiment (overall n = 10). Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Differences were deemed significant when the P values were b0.05. SigmaPlot Software Version 12.0 (Systat Software Inc., San Jose, USA) was used for statistical analysis and plotting graphs.
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3. Results Exposure to PGE2 exerts proapoptotic effect on NK cells and induces their depletion
The effect of PGE2 on the apoptosis and absolute count of NK cells was assessed 24 h and 7 days after PBMCs exposure to this eicosanoid. No effect of PGE2 (at all used concentrations) on the apoptosis and absolute count of NK was demonstrated at the first time-point
Fig. 1. The effect of PGE2 on the apoptosis and absolute count of NK cells. PBMCs were cultured without (medium) or with PGE2 (10−5–10−8 M) for 7 days. The results are expressed as a percentage of early apoptotic (A) and late apoptotic/necrotic cells (B) within the NK cell population. The absolute count represents the number of NK cells per sample (C). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.05, **P b 0.01, ***P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). The dot plot of Annexin V versus 7-AAD was used for the assessment of apoptosis. Annexin V− 7-AAD− cells were considered viable; Annexin V+ 7-AAD− cells were deemed early-apoptotic; and double positive cells (Annexin V+ 7-AAD+) were considered to be a mixture of late apoptotic and primary necrotic cells (D). Examples of cytograms (density plots) showing percentages of early apoptotic, late apoptotic/necrotic and viable NK cells (E).
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Fig. 2. The effect of the blockade of PGE2 receptors (EP) on the apoptosis and absolute count of NK cells. To identify the EP receptor subtype(s) responsible for PGE2-induced apoptosis of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were exposed to PGE2 for 7 days. PBMCs incubated in medium alone served as the control. The results are expressed as a percentage of early apoptotic (A) and late apoptotic/necrotic cells (B) within the NK cell population. The absolute count represents the number of NK cells per sample (C). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Examples of cytograms (density plots) showing percentages of early apoptotic (Annexin V+ 7-AAD−), late apoptotic/necrotic (Annexin V+ 7AAD+) and viable (Annexin V− 7-AAD−) NK cells (D).
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(data not shown). It was found that 7-day-long exposure of PBMCs to PGE2 at 10− 5 M, 10− 6 M and 10− 7 M, but not at 10− 8 M, caused a significant increase in the percentage of early apoptotic (Fig. 1A and E) and of a mixed population of late apoptotic/necrotic cells among NK cells (Fig. 1B and E). PGE2 at 10−5 M and 10−6 M induced a considerable decrease in the absolute count of NK cells, whereas at lower concentrations it did not affect this parameter (Fig. 1C). The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors does not prevent PGE2-induced cell death To identify the EP receptor subtype(s) responsible for PGE2-induced apoptosis and depletion of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists and then were exposed to PGE2 for 7 days. The EP1, EP1/2, EP3 and EP4 antagonists were unable to abolish the PGE2-induced apoptosis and depletion of NK cells (Fig. 2). This is proven by the fact that the percentage of early apoptotic (Fig. 2A and D) and late apoptotic/necrotic NK cells (Fig. 2B and D) and the absolute number of NK cells (Fig. 2C) from samples pretreated with EP1, EP1/2, EP3 and EP4 antagonists were not different compared to those in samples exposed only to PGE2, while they were significantly higher (the percentage of early apoptotic and late apoptotic/necrotic NK cells) or lower (the absolute number of NK cells) than the control values. Thus, the blockade of any of the EP-receptor subtypes did not prevent the PGE2-induced apoptosis of NK cells. The percentage of early apoptotic and late apoptotic/necrotic NK cells, as well as the absolute number of NK cells from samples treated only with EP receptor antagonists did not differ substantially from the control values (data not shown). PGE2 impairs proliferation of NK cells The effect of PGE2 on the proliferation of NK cells was determined in PBMCs incubated with or without this eicosanoid for 24 h with the accompanying Con A stimulation in the presence of BrdU. These studies demonstrated that PGE2 at 10−5 M and 10− 6 M, but not at 10− 7 M
and 10−8 M, highly significantly reduced the percentage of proliferating NK cells (Fig. 3). The selective blockade of EP4 receptors prevents PGE2-induced inhibition of proliferation of NK cells To identify the EP receptor subtype(s) responsible for PGE2-induced inhibition of proliferation of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were treated with PGE2 for 24 h with concomitant stimulation with Con A in the presence of BrdU. The EP1, EP1/2 and EP3 antagonists were unable to block significantly the PGE2-induced inhibition of NK cell proliferation. This is proven by the fact that the percentage of proliferating NK cells from samples pretreated with EP1, EP1/2 and EP3 antagonists did not differ significantly from the values of these parameters obtained in samples exposed only to PGE2, while being considerably lower than the control values (Fig. 4). On the contrary, the percentage of proliferating NK cells from samples pretreated with the EP4 antagonist were higher than the values of this parameter obtained from samples exposed only to PGE2, but they did not differ significantly from the control values (Fig. 4). Thus, the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of proliferation of NK cells. The percentage of BrdU+ cells among NK cells in samples treated only with EP receptor antagonists showed no difference when compared with the control values (data not shown). 4. Discussion There are various levels of PGE2 attained at inflammatory sites, depending on the location of inflammation, type of disease as well as its course and stage. Literature indicates that PGE2 can reach concentrations of the order of 10−6–10−9 M (Nygård et al., 1992; Anderson et al., 1996; Hinson et al., 1996; Hirata et al., 2001). Thus, the concentrations of 10−6–10−8 M applied in our research mimic a real inflammatory levels of PGE2.
Fig. 3. The effect of PGE2 on the proliferation of NK cells. To evaluate the effect of PGE2 on proliferation NK cells, PBMCs were incubated without (medium) or with PGE2 (10−5–10−8 M) for 24 h with concomitant stimulation with Con A in the presence of 5-bromo-2′-deoxyuridine (BrdU) in cell culture medium during the last 8 h. The results are expressed as a percentage of BrdU-incorporating cells among the NK cell population (A). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Dot plot cytograms demonstrate the distribution of BrdU-positive and -negative cells within NK cells (B).
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Fig. 4. The effect of the blockade of PGE2 receptors (EP) on the proliferation of NK cells. To identify the EP receptor subtype(s) responsible for PGE2-induced inhibition of proliferation of NK cells, PBMCs were exposed only to PGE2 or were pretreated with EP1 or EP1/2 or EP3 or EP4 receptor antagonists for 1 h and then were treated with PGE2 for 24 h with concomitant stimulation with Con A in the presence of 5-bromo-2′-deoxyuridine (BrdU) in cell culture medium during the last 8 h. The results are expressed as a percentage of BrdU-incorporating cells among the NK cell population (A). Results are the mean (±SD) of two independent experiments with 5 animals per experiment (n = 10, *P b 0.001, one-way ANOVA with Bonferroni's multiple comparison test). Dot plot cytograms demonstrate the distribution of BrdU-positive and -negative cells within NK cells (B).
In the current experiments, average percentages of early apoptotic cells and a mixed population of late apoptotic/necrotic cells among NK cells in the control samples were 11.16% and 8.46%, respectively. These are normal levels because, under in vitro conditions, a certain pool of cells continually undergoes apoptosis, which is associated with the specific character of cell culture. Short-term treatment with PGE2 did not affect the apoptosis or count of NK cells, whereas 7-day exposure of PBMCs to this eiconasoid in the concentrations 10−5, 10−6 and 10−7 M, but not 10−8 M, induced a significant increase in the percentage of early apoptotic and mixed population of late apoptotic/necrotic cells within NK cells, but the intensity of this effect diminished as the concentration of PGE2 was lowered. This relates to the results of investigations into the effect of PGE2 on counts of NK cells, where it was revealed that exposure of PBMCs to PGE2 in the concentrations 10−5 and 10− 6 M led to a significant depletion of NK cells, with the effect being more distinctly expressed at a higher concentration of the eiconasoid. Such results implicate that PGE2 produces a proapoptotic effect on bovine NK cells, and (a) this effect does not appear promptly, (b) induction of this effect requires a relatively high concentration of PGE2, and (c) the intensity of this effect is dependent on the concentration of PGE2. Thus, the data obtained from this study suggest that in cattle with inflammation, especially in the acute phase, PGE2 may induce the apoptosis of NK cells. To the best of our knowledge, there is no information
available on the effect of PGE2 on the apoptosis of human or animal NK cells, and therefore this is the first report on this subject. All the findings indicate that, depending on the type of cells, PGE2 can produce either a proapoptotic (Föller et al., 2006; Huang et al., 2009; Kovarova and Koller, 2014; Nagano et al., 2014) or antiapoptotic effect on affected cells (Nishihara et al., 2003; Yamamoto et al., 2010; Coulombe et al., 2014). It was found that PGE2 increased apoptosis of normal lung fibroblasts in a dose-dependent manner (Huang et al., 2009). In other studies, it was demonstrated that PGE2 induced apoptosis of K562 human leukaemia cells (Föller et al., 2006), mast cells (Kovarova and Koller, 2014) and rat microglia (Nagano et al., 2014). On the other hand, it was found that PGE2 protected normal and transformed intestinal epithelial cells from apoptosis induced by diverse stimuli (Nishihara et al., 2003). Moreover, Coulombe et al. (2014) showed that during influenza A virus infection, PGE2 was upregulated, which led to the inhibition of apoptosis in macrophages. In the further stage of our research, it was determined which of the EP receptors mediated in the induction of the proapoptotic effect of PGE2 on the analyzed cells. The experiments did not demonstrate that the blockade of any of the receptors would prevent PGE2-induced apoptosis and depletion of NK cells. This does not seem to be due to an inadequate blockade of EP receptors, as it was demonstrated in another part of the research that exposure of PBMCs to the EP4 receptor
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antagonist prevented PGE2-induced inhibition of NK cell proliferation. Also, in another studies, we were able to determine that the selective blockade of EP4 receptor prevented PGE2-induced down-regulation of CD25 expression on T cells (Maślanka et al., 2014) and inhibition of IFN-γ production by WC1+ T cells (Przybysz et al., 2016). The fact that short-term exposure to PGE2 did not influence the apoptosis of NK cells, but that the proapoptotic effect of PGE2 emerged only after prolonged exposure seems to implicate that this effect may not be a result of the direct activation of EP1-EP4 receptors, but may involve complex cellular reactions. Hence, the results obtained thus far suggest that the proapoptotic effect of PGE2 on bovine NK cells is clearly of secondary character, and its induction is not mediated by EP1-EP4 receptors. This conclusion may be supported by research results showing that PGE2-induced apoptotic cell death in K562 human leukaemia cells was mediated by the Ca2 +-permeable transient receptor potential canonical 7 channel (Föller et al., 2006). The above conclusion as well as the current research data remain in accord with the results recently published by Nagano et al. (2014), who demonstrated that PGE2 induced apoptosis in cultured rat microglia, but this effect was not prevented by pretreatment with EP1–EP4 antagonists. Therefore, these investigators concluded that the above effect was unlikely to occur through EP1–EP4 receptors. Regarding the study on the proliferation of NK cells, it was observed that their exposure to the two higher PGE2 concentrations significantly reduced the percentage of proliferating cells in the total NK cell population. These results indicate that PGE2 may have an antiproliferative effect on NK cells. This conclusion, as well as the results of our investigation, can find support in our previous studies, which revealed that the inhibition of PGE2 synthesis mediated by meloxicam (a relatively selective COX-2 inhibitor) promoted proliferation of mitogenstimulated bovine NK cells (Maślanka, 2013a). Despite some claims that PGE2 inhibits proliferation of NK cells, to the best of our knowledge, no studies have been performed and reported that would provide direct evidence supporting the influence of this eicosanoid on proliferation of human or animal NK cells. NK cells rapidly proliferate during viral infections to provide an expanded pool of effector cells to suppress the infection (Nguyen et al., 2002; Andrews et al., 2003; Björkström et al., 2011). In most cases, a viral infection induces an inflammatory response, which leads to the production of various proinflammatory mediators, including PGE2. Therefore, it is highly possible that PGE2 can act as an inhibitor of bovine NK cell proliferation, but this occurs only in the presence of a factor that triggers this process and simultaneously induces an inflammatory response (e.g. viral infections). It needs to be noted that the current study demonstrated that the antiproliferatory effect of PGE2 appeared only at very high PGE2 concentrations, which suggests that under in vivo conditions such an effect might take place in the course of an illness with a strongly expressed inflammatory response. It should be underlined that any conclusion implicating that PGE2 can act as a physiological inhibitor of the proliferation of mature NK cells would be erroneous because in healthy animals these cells do not proliferate at a significant rate (Warren et al., 1996; Koka et al., 2003). The selective blockade of EP4 receptor, but not EP1, EP2 and EP3 receptors, prevented PGE2-induced inhibition of NK cell proliferation. These results suggest that antiproliferative action of PGE2 toward bovine NK cells is mediated via the EP4 receptor. This results is unsurprising as it has been evidenced that PGE2 supresses NK cell migration and INF-γ production by NK cells primarily through the EP4 receptor (Holt et al., 2011). Most clearly, then, in the case of NK cells the EP4 receptor serves as an inhibitory receptor. Our determinations also agree with the research results reported by Kvirkvelia et al. (2002), who found that PGE2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 (cytotoxic T-lymphocyte line) T cells. The current results implicate a possibility of preventing/inactivating the antiproliferative action of PGE2 toward bovine NK cells via a blockade of the EP4 receptor by its selective antagonist, or through the
inhibition of the production of PGE2. Considering the fact that the mechanism of action of NSAIDs involves blocking the synthesis of this eicosanoid, it is possible to claim that the research reported herein casts a new light on some undiscovered aspects of NSAIDs administration. These drugs are administered in treatment of various infections in order to alleviate the clinical signs of an illness, which arise from an inflammatory response that accompanies the infection. Our findings suggest that the use of NSAIDs may bring about an additional advantage in the treatment of cattle with infections caused by intracellular pathogens, such as giving protection to NK cells against the antiproliferative and proapoptotic effects of PGE2. However, it should be emphasized that at this moment the pharmacological protection of bovine NK cells achieved by blocking the effect of PGE2 on these cells can only be hypothesized as we lack firm knowledge whether PGE2 under in vivo conditions would actually inhibit the infection-induced proliferation of bovine NK cells and induce their apoptosis. These questions cannot be resolved without further research. In recapitulation, the results of the study reported herein suggest that PGE2 may produce a proapoptotic effect on NK cells in cattle, but this effect does not occur promptly. The proapoptotic effect of PGE2 on bovine NK cells is most clearly secondary in character, and its induction is not mediated by the EP1-EP4 receptors. Moreover, the results indicate that PGE2 can have an antiproliferative effect on bovine NK cells and this effect is mediated through the EP4 receptor. The proapoptotic and antiproliferative effects occurred at relatively high concentrations of PGE2, suggesting that under in vivo conditions such effects can be expected nearly exclusively in an illness accompanied by a very strongly expressed inflammatory response. The results also suggest an additional advantage to using NSAIDs in cows with infections caused by intracellular pathogens, such as the protection of NK cells against the antiproliferative and proapoptotic effects of PGE2. Conflict of interest statement The authors declare that they have no conflict of interests. Acknowledgments The study was supported by grant no. 15.610.008-300 from the University of Warmia and Mazury in Olsztyn. References Anderson, G.D., Hauser, S.D., McGarity, K.L., Bremer, M.E., Isakson, P.C., Gregory, S.A., 1996. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97, 2672–2679. Andrews, D.M., Scalzo, A.A., Yokoyama, W.M., Smyth, M.J., Degli-Esposti, M.A., 2003. Functional interactions between dendritic cells and NK cells during viral infection. Nat. Immunol. 4, 175–181. Bastos, R.G., Johnson, W.C., Mwangi, W., Brown, W.C., Goff, W.L., 2008. Bovine NK cells acquire cytotoxic activity and produce IFN-γ after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells. Vet. Immunol. Immunopathol. 124, 302–312. Biron, C.A., Nguyen, K.B., Pien, G.C., Cousens, L.P., Salazar-Mather, T.P., 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220. Björkström, N.K., Lindgren, T., Stoltz, M., Fauriat, C., Braun, M., Evander, M., Michaëlsson, J., Malmberg, K.J., Klingström, J., Ahlm, C., Ljunggren, H.G., 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208, 13–21. Boysen, P., Storset, A.K., 2009. Bovine natural killer cells. Vet. Immunol. Immunopathol. 130, 163–177. Cooper, M.A., Fehniger, T.A., Caligiuri, M.A., 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22, 633–640. Coulombe, F., Jaworska, J., Verway, M., Tzelepis, F., Massoud, A., Gillard, J., Wong, G., Kobinger, G., Xing, Z., Couture, C., Joubert, P., Fritz, J.H., Powell, W.S., Divangahi, M., 2014. Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages. Immunity 40, 554–568. Föller, M., Kasinathan, R.S., Duranton, C., Wieder, T., Huber, S.M., Lang, F., 2006. PGE2-induced apoptotic cell death in K562 human leukaemia cells. Cell. Physiol. Biochem. 17, 201–210. French, A.R., Yokoyama, W.M., 2003. Natural killer cells and viral infections. Curr. Opin. Immunol. 15, 45–51.
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