Effect of lipopolysaccharide and cytokines on synthesis and secretion of leukotrienes from endometrial epithelial cells of pigs
Introduction
Leukotriene (LT), prostaglandin (PG) and thromboxane synthesis is initiated by the release of arachidonic acid (AA) from cell membranes under the influence of phospholipase A2. In relation to the LT production, AA is converted in a two-step process into an unstable intermediate, LTA4, by a catalytic complex: 5-lipooxygenase (5-LO) and the 5-LO activating protein. This substance is subject to further transformation by the cytosolic LTA4 hydrolase (LTAH) to LTB4 or converted to the tripeptide glutathione under the influence of LTC4 synthase (LTCS) to generate LTC4. LTC4 and its derivatives, LTD4 and LTE4, are known as cysteinyl-LTs (Rinaldo-Matthis and Haeggström, 2010). The LTs are produced mainly in immune system cells and are of great importance for regulating innate immune and inflammatory responses. The role of LTs in the pathogenesis of many inflammatory diseases was previously documented (Liu and Yokomizo, 2015). The physiological LT effects refer to the modulation of many processes in the reproductive tract (Abu and Konje, 2000).
Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-4 and IL-10 are important for the many processes under physiological and pathological conditions. These factors are produced and released by immunological cells and other cell types. There is a presence of TNF-α in the endometrium and fetal membranes of pigs (Tayade et al., 2007, Linton et al., 2010, Khalaj et al., 2015). The TNF-α and its receptors are also present in the cattle (Okuda et al., 2010) and horse (Galvão et al., 2013) endometrium indicating a role of these factors in endometrial function. In pigs, IL-1β produced by peri-implantation embryos and the endometrium could affect conceptus-uterine communications during early pregnancy (Ross et al., 2003). This cytokine also activates uterine enzymes involved in the synthesis of PGE2 (Franczak et al., 2010). Moreover, Seo et al. (2012) described functional receptors for IL-1β in the endometrium of pigs. IL-4 and its receptor have been identified in utero-placental tissues indicating an immunobiological role of this cytokine during pregnancy (De Moraes-Pinto et al., 1997). Similarly, IL-10 was detected in the endometrium and trophoblast of pigs (Linton et al., 2010). Furthermore, the presence of IL-10 was described in the inflamed endometrium of mares (Woodward et al., 2013) while receptors for IL-10 are present in human adenomiotic tissues (Qin et al., 2012).
Uterine inflammation is the most frequent reproductive disorder in livestock, leading to both economic and animal welfare problems; and is caused mainly by bacteria. One of the factors initiating uterine inflammation is lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria), recognized by Toll-like receptor 4 and the CD14 complex present in immune and endometrial cells. As a consequence, mediators of inflammatory processes are synthesized and released (Cronin et al., 2012). Uterine inflammation in rats induced by LPS or Escherichia coli (E.coli) resulted in increased TNF-α and IL-1β concentrations in peripheral blood (Jana et al., 2005). Similarly, large amounts of these cytokines and IL-6 and IL-8 are synthesized and secreted during uterine inflammation in ruminants and mares; in the latter species, IL-10 gene expression was also increased (Gabler et al., 2009, Shao et al., 2012).
Little information is available about the synthesis and release of LTs from the uterus, nor about the mechanisms involved in controlling these processes. Studies on this subject have demonstrated that LTB4 and LTC4 synthesis occurs in the pig (Jana et al., 2014) and horse (Guzeloglu et al., 2013) uterus. Moreover, uterine inflammation in pigs (Jana et al., 2014) and cattle (Belluzzi et al., 2004, Barański et al., 2013) led to the increased synthesis and release of both LTs. LPS, TNF-α, IL-1β, IL-4 and IL-10 all stimulate increased relative abundance of LTAH and LTCS and secretion of LTB4 and LTC4 from endometrial strips of pigs following saline and E.coli intrauterine infusions (Czarzasta et al., 2014). Moreover, strong 5-LO, LTAH and LTCS immune responses were discovered in epithelial cells of these tissues (Jana et al., 2014). Therefore, it was hypothesized that LPS, TNF-α, IL-1β, IL-4 and IL-10 function locally to modulate LT synthesis and secretion from endometrial epithelial cells and influence cell viability. The aim of the present study was to examine the influence of LPS, TNF-α, IL-1β, IL-4 and IL-10 on the relative abundance of mRNA and protein of 5-LO, LTAH and LTCS in pig endometrial epithelial cells and on release of LTB4 and LTC4 from these cells. The viability of endometrial epithelial cells in response to these factors was also assessed.
Section snippets
Animals and uterine tissue collection
Uteri from six gilts were obtained from a local abattoir. Entire uteri were removed within 5 min after the gilts were slaughtered. A macroscopic examination of the gonads was conducted to identify the phase of estrous cycle (Akins and Morrissette, 1968). Uteri obtained on day 8 of the estrous cycle were used in the study. This day of the estrous cycle was chosen because the isolated cells easily become confluent in culture conditions when collected on this day of the estrous cycle. The uteri
Effect of LPS and cytokines on 5-LO, LTAH and LTCS mRNA
The relative abundance of 5-LO mRNA in the epithelial cells increased (P < 0.01) in response to 10 ng/ml of LPS, TNF-α and IL-10, or with 1 and 10 ng/ml treatments of IL-1β compared with the non-treated cells or after treatment with 100 ng/ml of LPS, 1 ng/ml of TNF-α and IL-10, or 10 ng/ml of IL-4 (Fig. 1A). In the epithelial cells, the relative abundance of LTAH mRNA was greater (P < 0.001) after treatment with the smaller doses of LPS and IL-4 and larger dose of IL-10 than in the control sample or in
Discussion
The current in vitro experiment focused on the effects of LPS, TNF-α, IL-1β, IL-4 and IL-10 on the relative abundance of 5-LO, LTAH and LTCS mRNA and proteins, and secretion of LTB4 and LTC4 from uterine epithelial cells of pigs. The influence of these factors on cell viability was also examined.
In the present study, there was an increased in the relative abundance (mRNA and/or protein) of 5-LO, LTAH and LTCS mRNA and protein in endometrial epithelial cells of pigs treated with LPS, TNF-α,
Conflict of interest
None.
Acknowledgments
This research was financed from a grant of the Polish Ministry of Scientific Research and Higher Education (NN308128339) as well as the statutory research funds of the Polish Academy of Sciences. We are very grateful to Professor Aneta Andronowska for providing us with knowledge of how to conduct epithelial cell cultures.
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