Result And Discussion

Estrogens have diverse effects on cell growth, differentiation and homeostatic functions. Estrogens decrease low-density lipoprotein and lipoprotein A, increase high-density lipoprotein levels, and promote vasodilatation and blood flow.

Figure 1. Effect of 17p -estradiol on immunoglobulin M production by splenocytes of C57BL/6N mice. Splenocytes were treated with 10-12 - 10-6 ME2 in the presence of 7.8 ^g/ml LPS for 24 h. Level of IgM was measured by ELISA. Data are means ± SD (n=3). Data with asterisk marks are significantly different from the values of the control group atp < 0.05*, 0.01** or 0.001***.

We first evaluated the effect of E2 on the production of IgM by splenocytes isolated from male C57BL/6N mice. As shown in Fig. 1, the production of IgM was obviously accelerated by E2, and enhanced by 2.0-fold at 10-10 M. This E2 concentration is equivalent to the circulating concentration of postmenopausal women treated hormone replacement therapy (Roger and Eastell, 1998).

Then, it was investigated whether ERs participate in the regulation of IgM production or not. Splenocytes were treated with E2 in the presence of ICI 182780, an ER antagonist, blocks forming ER dimmer and decomposes ERs (Wakeling and Bowler, 1992; Parisot et al., 1995). As shown in Fig.2, the enhancing effect of E2 on IgM production was

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IT

TT

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T

0 10-12 10-11 10-10 10-9 10-8 10-7 10-6 E2 concentration (M)

0 10-12 10-11 10-10 10-9 10-8 10-7 10-6 E2 concentration (M)

ICI182780

Figure 2. Effect of an ER antagonist ICI 182780 on 17P-eatradiol stimulated IgM production. Splenocytes were stimulated with 10-10 M E2 in the presence or absence of 10 M ICI 182780 for 24 h with 7.8 |ag/ml LPS. The cells were pre-incubated with ICI 182780 for 30 min before E2 application. Data are means ± SD (n=3). Letters that are different from each other denote significant difference at p < 0.05 (Scheffe's F test).

completely canceled by 10-5 M ICI 182780. This suggests that up-regulation of IgM production by E2 is mediated through ERs. ERs were classified into two kinds of subtype ERa and ERp. Thus, it was evaluated which kind of ERs is concerned with the regulation of IgM production by using agonists specific to ERa (PPT) and ERP (DPN). PPT is an ERa agonist, and possesses 410-fold selectivity for ERa over ERP (Harris et al., 2002).

As shown in Table 1, both PPT and DNP significantly enhanced IgM production, as well as E2. These results suggest that the up-regulation of IgM production by E2 is mediated by both ERa and ERp.

Table 1. Effect of ERa and ERP agonists on IgM production by mouse splenocytes.

Table 1. Effect of ERa and ERP agonists on IgM production by mouse splenocytes.

(M)

PPT

DPN

0

576.8 ± 41.5

406.7 ± 39.6

10-9

870.8 ± 105.9*

855.9 ± 48.8***

10-8

1004.1 ± 91.8**

826.4 ± 77.8***

10-7

1048.5 ± 117.6*

706.5 ± 84.8***

Splenocytes were stimulated with 10-9 - 10-7 M PPT (ERa agonist), or 10-9 -10-7 M DPN (ERP agonist) for 24 h in the presence with 7.8 |ag/ml LPS. Data are means ± SD (n = 3). Data containing asterisk marks are significantly different from the values in the control group at p < 0.05*, 0.01** or 0.001***.

Splcnocytc 0.8

E2-BSA concentration (M)

Figure 3. Effect of E2-conjugated BSA IgM on production by mouse splenocytes. Splenocytes were treated with 10-6 M E2-BSA-FITC for 1 h at room temperature. The cells were fixed with 3.7% formaldehyde for 5 min at room temperature. The specimens were analyzed with a Leica confocal laser scaning microscope unit. Splenocytes were treated with 10-11 - 10-8 M E2-BSA in presence of 7.8 |ag/ml LPS for 24 h. Data are presented as means ± SD (n=3).

It is well-known that ERs are located on plasma membranes as well as in nucleus (Chen et al., 1999). Then, we examined whether plasma membrane-associated ER participates in the regulation of IgM production or not. Though E2 can bind to ERs in both locations, E2-BSA can bind only to plasma membrane-associated ER. Splenocytes were treated with E2-BSA labeled with FITC. As shown in Fig. 3, plasma membrane-associated ER on splenocytes were stained by E2-BSA-FITC, however, IgM production of splenocytes was not stimulated by E2-BSA.

In this study, we demonstrated that E2 enhances the IgM production of splenocytes isolated from C57BL/6N mice. E2 up-regulates the IgM production through both of ERa and ERp.

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