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Background: Mast cells cultured from human peripheral blood have been widely used to study human mast cell function. Prostanoids are the important regulators of mast cell activity, however, there were no reports about the class of prostanoid receptors expressed on such cultured cells.
Aims: The present study was to characterize pharmacologically the prostanoid receptors by investigating the effects of prostanoid receptor agonists on the immunoglobulin E (IgE)-mediated histamine release from the cultured mast cells.
Methods: Mast cells cultured from human progenitor cells in peripheral blood were sensitized with human myeloma IgE, and then challenged with anti-human IgE following pretreatment with diverse prostanoid receptor agonists. The histamine content in supernatants and cell pellets were measured by histamine auto-analyzer.
Results: Of the prostanoid receptor agonists tested, the prostaglandin E2 (PGE2) receptor (EP receptor) agonist PGE2 (10−7 to 10−11 M) produced concentration-related potentiation of IgE-mediated histamine release from the cultured mast cells. Sulprostone, an EP1/EP3 agonist, SC-46275, a selective EP3 agonist, and 11-deoxy-PGE1, a selective EP2/EP3/EP4 agonist also caused a significant increase in histamine release induced by anti-IgE. BW245C, fluprostone, cicaprost and U46619 for the prostaglandin D2, F2α, I2, and thromboxane A2 receptors respectively, and the EP2/EP4 receptor agonist butaprost had little effect on anti-IgE stimulated histamine release from mast cells.
Conclusions: The present results suggest that PGE2 potentiates the IgE-mediated histamine release from the cultured mast cell via EP3 and/or EP1 receptors.
Prostanoids consisting of prostaglandin (PG) D2, PGE2, PGF2α, PGI2 and thromboxane A2 (TXA2) are the primary products of cyclooxygenase metabolism of arachidonic acid (1). In addition to a wide range of physiological actions, these prostanoids also play a vital role in a number of pathological processes, especially in the inflammatory disorders (2, 3). Prostanoids have shown to be an important modulator of inflammatory cell activity during inflammatory responses (3).
Mast cells are known for their critical roles in asthma, allergic reactions and other inflammatory diseases attributed to their potent capability to produce multiple proinflammatory mediators after activation (4). A series of studies have demonstrated that prostanoids, particularly the PGD2 and PGE2, were able to regulate the synthesis and release of mast cell products. For instance, PGD2 effectively inhibited immunologically stimulated histamine release from rat mast cells (5). Prostaglandin E2 inhibited IgE-mediated histamine and leukotriene C4 release from human lung mast cells (6), but enhanced anti-IgE induced production of IL-6 by the rat peritoneal mast cells and murine bone marrow-derived mast cells (7).
Like other local hormones, prostanoids exert their actions through binding the five major specific receptors for PGD2 (DP receptor), PGE2 (EP receptor), PGF2α, (FP receptor), PGI2 (IP receptor) and TXA2 (TP receptor), which are linked to different transduction cascades in targeted cells (2, 3). It is, therefore, critical to identify the prostanoid receptor types to understand the modification of prostanoids on mast cell function. Although mast cells cultured from human peripheral blood have been widely used in different research fields relevant to mast cells, there were no reports about the characterization of prostanoid receptors expressed on such cultured cells. The present study was to characterize pharmacologically the prostanoid receptors involved in the regulation of IgE-mediated histamine release from the cultured mast cells.
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In the present study, BW245C, fluprostone, cicaprost, U46619 or PGE2 alone did not produce any effect on unstimulated mast cells (data not shown). After incubation of mast cells with agonists at concentrations between 10−8 and 10−6 M for 10 min, BW245C, fluprostone, cicaprost and U-46619 were all shown to have no effects on anti-IgE-induced histamine release from mast cells (Table 1). In contrast, PGE2 (10−9 to 10−7 M) significantly enhanced histamine release from anti-IgE activated mast cells in a dose-dependent manner with a maximal effect of 32.37 ± 4.58% at the highest tested concentrations of 10−7 M (Fig. 1).
Table 1. Effects of prostanoid receptor agonists on anti-immunoglobulin (IgE) stimulated histamine release (%) from cultured mast cells
|Group||n||Anti-IgE (%)||Anti-IgE + agonists (%)|
|10−8 M||10−7 M||10−6 M|
|Fluoprostone||5||30.34 ± 3.88||33.42 ± 4.90||33.10 ± 4.34||33.07 ± 3.47|
|BW245C||4||26.29 ± 3.65||30.04 ± 3.03||26.19 ± 3.12||27.46 ± 6.33|
|Cicaprost||4||26.29 ± 3.65||28.44 ± 5.69||28.92 ± 5.86||26.90 ± 6.23|
|U-46619||5||28.45 ± 3.65||28.30 ± 3.94||29.34 ± 4.20||29.33 ± 4.25|
Figure 1. Effect of prostaglandin (PG) E2 on the anti-immunoglobulin E (IgE)-induced histamine release from cultured mast cells. Sensitized mast cells were pretreated with PGE2 for 10 min and then challenged with anti-IgE for 30 min. Results were corrected for spontaneous histamine release, which was 8.71 ± 1.57% (n = 8). Values are expressed as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.005 when compared with anti-IgE alone induced histamine release.
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Prostaglandin E2 exerts its effects by activating four different EP receptor subtypes: EP1, EP2, EP3 and EP4 receptor. To further evaluate which EP receptor subtypes mediated the action of PGE2 in cultured mast cells, we investigated the effects of synthetic agonists with high affinity to one or more EP receptor subtypes on these mast cells. All tested EP receptor agonists by themselves did not produce any significant effects on mast cells without stimulation (data not shown). The highly selective EP3 agonist SC-46275 (10−10 to 10−6 M), the selective EP1/EP3 agonist sulprostone (10−8 to 10−6 M) and the selective EP2/EP3/EP4 agonist 11-deoxy-PGE1 (10−7 and 10−6 M) all enhanced significantly the anti-IgE-stimulated histamine release from cultured mast cells in a dose dependent manner (Fig. 2). The maximal enhancement of different agonists at 10−6 M concentration and EC25 value were evaluated as seen in Table 2. SC-46275 showed more potent enhancing effects on mast cells than other agonists. Butaprost had no significant effect on histamine release from anti-IgE-activated mast cells (Fig. 2).
Figure 2. Effect of EP receptor agonists on the anti-immonoglobulin (IgE) induced histamine release from cultured mast cells. Sensitized mast cells were pretreated with agonists for 10 min and then challenged with anti-IgE for 30 min. Results were corrected for spontaneous histamine release, which was 9.02 ± 1.53% (SC-46275, n = 5), 8.15 ± 1.62% (sulprostone, n = 5), 8.68 ± 1.97% (11-deoxy-PGE1, n = 4) and 8.26 ± 2.57% (butaprost, n = 4). Values are given as mean ± SE. *P < 0.05, **P < 0.01 when compared with anti-IgE alone induced histamine release.
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Table 2. The enhancing effects of EP receptor agonists on anti-immunoglobulin E (IgE)-induced histamine release from cultured mast cells
| ||n||EC25 (×10−8 M)||Potentiation at 10−6 M (%)|
|PGE2||8||5.87 ± 1.89||ND|
|SC-46275||5||0.34 ± 0.17||51.71 ± 7.82|
|Sulprostone||5||2.35 ± 1.83||45.30 ± 2.13|
|11-deoxy-PGE1||4||4.25 ± 1.65||36.35 ± 16.61|
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In the present study, the selective and potent DP receptor agonist BW245C, FP receptor agonist fluprostone, IP receptor agonist cicaprost and TP receptor agonist U46619, did not produce any effect on histamine release from immunologically activated mast cells, suggesting that those prostanoid receptors are unlikely to be present in the cultured mast cells. On the contrary, the result that PGE2 caused a dose-dependent enhancement of anti-IgE-induced histamine release shows that the EP receptors are predominant in these cells.
Prostaglandin E2 exerts its biological action by interacting with four subtypes of EP receptors, EP1, EP2, EP3 and EP4 receptors on target cells. EP2 and EP4 receptors activated adenylate cyclase leading to elevation of intracellular cAMP concentration and hence, mediated PGE2-induced inhibitory effects in different cells (10, 11). EP1 receptors activated phospholipase C and phosphatidyl inositol turnover and stimulated the release of intracellular Ca2+. EP3 receptors principally inhibited adenylate cyclase through a Gi-protein and also stimulated calcium release (2, 3). Contrary to the inhibitory actions of EP2 and EP4 receptors, EP1 and EP3 receptors mediated promoting effects induced by PGE2 in various mast cells (7, 12). The present observation that PGE2 produced a potentiating effect on anti-IgE-induced histamine release suggests that the excitatory EP1/EP3 subtypes, and not the inhibitory EP2/EP4 receptor subtypes, are present in the cultured mast cells.
SC-46275 was an highly selective and potent EP3 agonist in guinea pig and mouse in vivo (13). It has been shown to be about 106-fold more potent at EP3 receptor than at EP1 receptor in isolated guinea pig ileal smooth muscle and to activate EP1 receptors only at concentrations of up to 3 × 10−5 M (13). Although sulprostone was considered as a selective EP1/EP3 agonist, it has been shown to have higher affinity to the EP1 receptor than the EP3 receptor in Chinese hamster ovary cells (14). In isolated guinea pig ileum, in contrast to the weak activity of SC46275 at the EP1 receptor, sulprostone at 2 × 10−8 M was able to activate EP1 receptors (13). In the present study that both SC-46275 and sulprostone enhanced histamine release from anti-IgE activated mast cells with a similar efficacy, suggesting that EP1 and EP3 receptors both are very likely to exist on cultured mast cells. 11-deoxy-PGE1 showed affinities to EP2, EP3, and EP4 receptors with a specificity of EP2 > EP4 > EP3 > EP1 whereas butaprost showed affinity only to the EP2 receptor (2, 14). The present findings that anti-IgE-induced histamine release was enhanced by 11-deoxy-PGE1, but not affected by butaprost demonstrate the absence of EP2/EP4 receptors, and support the existence of EP3 receptors in cultured mast cells. Therefore, these data suggest the involvement of EP1 and/or EP3 receptors in PGE2-directed potentiation of histamine release from immunologically activated mast cells.
In our culture system, more than 90% of cultured mast cells expressed tryptase and only about 10% of cells contained both tryptase and chymase. These cultured mast cells therefore were the same phenotype as the mast cells dispersed from human lung tissues according to their protease contents (15), however, our results were opposite to the findings reported with human lung mast cells in which it has been reported that PGE2 inhibited immunologically stimulated histamine release (6). It shows that mast cells obtained under the artificial conditions are different from the normal human lung mast cells in some ways.