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Alterations of T cell activation signalling and cytokine production by postmenopausal estrogen levels.

Ku LT, Gercel-Taylor C, Nakajima ST, Taylor DD - Immun Ageing (2009)

Bottom Line: Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E(2), JAK2 protein expression was increased 1.64-fold (p < 0.001) and JAK3 enhanced 1.79-fold (p < 0.001) compared to 4 pg/ml. mRNA levels for CD3-zeta, JAK2, and JAK3 were significantly increased following exposure to 40 pg/ml E(2) (2.39, 2.01, and 2.21 fold, respectively) versus 4 pg/ml.Jurkat T cells exposed to 4 pg/ml E(2) expressed significantly diminished activation signalling proteins, correlating with reduced IL-2 production.Lower signalling protein levels appear to result from decreased CD3-zeta, JAK2, and JAK3 gene expressions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Women's Health, University of Louisville School of Medicine, Louisville, Kentucky, USA. dr.lowellku@gmail.com

ABSTRACT

Background: Immunosenescence is an age-associated disorder occurring primarily in T cell compartments, including altered subset composition, functions, and activation. In women, evidence implicates diminished estrogen in the postmenopausal period as a contributing factor to diminished T cell responsiveness. Since hypoestrogenism is present in postmenopausal women, our objective focused on whether T cell activation, defined as signalling molecule expressions and activation, and function, identified as IL-2 production, were affected by low estrogen.

Methods: Using Jurkat 6.1 T cells, consequences of 4 pg/ml (corresponding to postmenopausal levels) or 40 pg/ml (premenopausal levels) of estradiol (E(2)) were analyzed on signalling proteins, CD3-zeta, JAK2, and JAK3, determined by Western immunoblotting. These consequences were correlated with corresponding gene expressions, quantified by real time-polymerase chain reaction. Tyrosine phosphorylation of CD3-zeta was defined by immunoprecipitation and western immunoblotting following activation by T cell receptor (TcR) cross-linking. CD3-zeta expression and modulation was also confirmed in T cells from pre- and postmenopausal women. To assess functional consequences, IL-2 production, induced by PMA and ionomycin, was determined using enzyme-linked immunosorbent spot assay (ELISpot).

Results: At 40 pg/ml E(2), the level of signalling protein CD3-zeta was elevated 1.57-fold, compared with cells exposed to 4 pg/ml E(2). The CD3-zeta proteins also exhibited altered levels of activation-induced phosphorylation in the presence of 40 pg/ml E(2) versus 4 pg/ml: 23 kD phosphorylated form increased 2.64-fold and the 21 kD form was elevated 2.95-fold. Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E(2), JAK2 protein expression was increased 1.64-fold (p < 0.001) and JAK3 enhanced 1.79-fold (p < 0.001) compared to 4 pg/ml. mRNA levels for CD3-zeta, JAK2, and JAK3 were significantly increased following exposure to 40 pg/ml E(2) (2.39, 2.01, and 2.21 fold, respectively) versus 4 pg/ml. These findings were confirmed in vivo, since T cells from postmenopausal women exhibited 7.2-fold diminished CD3-zeta expression, compared to pre-menopausal controls and this expression was elevated 3.8-fold by addition of 40 pg/ml E(2). Functionally, Jurkat cells exposed to 40 pg/ml E(2) and activated exhibited significantly elevated numbers of IL-2 producing colonies compared to 4 pg/ml (75.3 +/- 2.2 versus 55.7 +/- 2.1 colonies, p < 0.0001).

Conclusion: Jurkat T cells exposed to 4 pg/ml E(2) expressed significantly diminished activation signalling proteins, correlating with reduced IL-2 production. Lower signalling protein levels appear to result from decreased CD3-zeta, JAK2, and JAK3 gene expressions. These findings may provide a molecular basis for immunosenescence associated with the postmenopausal state.

No MeSH data available.


Related in: MedlinePlus

Suppression of CD3-zeta, JAK 2 and JAK 3 mRNA levels by physiologic estradiol concentrations. Suppression of mRNA levels for CD3-zeta, JAK 2, and JAK 3 by exposure to 4 pg/ml versus 40 pg/ml estradiol quantified by real-time PCR. Results are presented as a mean ± standard deviation of cDNA concentration, resulting from duplicate runs.
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Figure 6: Suppression of CD3-zeta, JAK 2 and JAK 3 mRNA levels by physiologic estradiol concentrations. Suppression of mRNA levels for CD3-zeta, JAK 2, and JAK 3 by exposure to 4 pg/ml versus 40 pg/ml estradiol quantified by real-time PCR. Results are presented as a mean ± standard deviation of cDNA concentration, resulting from duplicate runs.

Mentions: To identify whether reduced expression of signalling proteins, in the presence of 4 pg/ml versus 40 pg/ml estradiol, was the result of diminished gene expression, the levels of mRNA for these signalling molecules were quantified by real-time PCR. RT-PCR data demonstrated that expressions of CD3-zeta, JAK2, and JAK3 mRNAs were significantly decreased in Jurkat cells incubated in 4 pg/ml estradiol compared to 40 pg/ml (Figure 6). Expression of CD3-zeta was 2.39-fold lower in cells exposed to 4 pg/ml compared to cells incubated with 40 pg/ml (p < 0.001). There was also diminished levels of gene expression for Janus kinases, JAK2 (2.01-fold) and JAK3 (2.21-fold), in T cells incubated in 4 pg/ml compared to those incubated in 40 pg/ml (p < 0.001). Gene expressions for CD3-zeta and JAK 3, incubated in 4 pg/ml E2 were not significantly different than that observed in cells exposed to 0 pg/ml (p > 0.05); while JAK 2 expression was significantly lower in 0 pg/ml E2 compared to 4 pg/ml (p < 0.05).


Alterations of T cell activation signalling and cytokine production by postmenopausal estrogen levels.

Ku LT, Gercel-Taylor C, Nakajima ST, Taylor DD - Immun Ageing (2009)

Suppression of CD3-zeta, JAK 2 and JAK 3 mRNA levels by physiologic estradiol concentrations. Suppression of mRNA levels for CD3-zeta, JAK 2, and JAK 3 by exposure to 4 pg/ml versus 40 pg/ml estradiol quantified by real-time PCR. Results are presented as a mean ± standard deviation of cDNA concentration, resulting from duplicate runs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656459&req=5

Figure 6: Suppression of CD3-zeta, JAK 2 and JAK 3 mRNA levels by physiologic estradiol concentrations. Suppression of mRNA levels for CD3-zeta, JAK 2, and JAK 3 by exposure to 4 pg/ml versus 40 pg/ml estradiol quantified by real-time PCR. Results are presented as a mean ± standard deviation of cDNA concentration, resulting from duplicate runs.
Mentions: To identify whether reduced expression of signalling proteins, in the presence of 4 pg/ml versus 40 pg/ml estradiol, was the result of diminished gene expression, the levels of mRNA for these signalling molecules were quantified by real-time PCR. RT-PCR data demonstrated that expressions of CD3-zeta, JAK2, and JAK3 mRNAs were significantly decreased in Jurkat cells incubated in 4 pg/ml estradiol compared to 40 pg/ml (Figure 6). Expression of CD3-zeta was 2.39-fold lower in cells exposed to 4 pg/ml compared to cells incubated with 40 pg/ml (p < 0.001). There was also diminished levels of gene expression for Janus kinases, JAK2 (2.01-fold) and JAK3 (2.21-fold), in T cells incubated in 4 pg/ml compared to those incubated in 40 pg/ml (p < 0.001). Gene expressions for CD3-zeta and JAK 3, incubated in 4 pg/ml E2 were not significantly different than that observed in cells exposed to 0 pg/ml (p > 0.05); while JAK 2 expression was significantly lower in 0 pg/ml E2 compared to 4 pg/ml (p < 0.05).

Bottom Line: Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E(2), JAK2 protein expression was increased 1.64-fold (p < 0.001) and JAK3 enhanced 1.79-fold (p < 0.001) compared to 4 pg/ml. mRNA levels for CD3-zeta, JAK2, and JAK3 were significantly increased following exposure to 40 pg/ml E(2) (2.39, 2.01, and 2.21 fold, respectively) versus 4 pg/ml.Jurkat T cells exposed to 4 pg/ml E(2) expressed significantly diminished activation signalling proteins, correlating with reduced IL-2 production.Lower signalling protein levels appear to result from decreased CD3-zeta, JAK2, and JAK3 gene expressions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Women's Health, University of Louisville School of Medicine, Louisville, Kentucky, USA. dr.lowellku@gmail.com

ABSTRACT

Background: Immunosenescence is an age-associated disorder occurring primarily in T cell compartments, including altered subset composition, functions, and activation. In women, evidence implicates diminished estrogen in the postmenopausal period as a contributing factor to diminished T cell responsiveness. Since hypoestrogenism is present in postmenopausal women, our objective focused on whether T cell activation, defined as signalling molecule expressions and activation, and function, identified as IL-2 production, were affected by low estrogen.

Methods: Using Jurkat 6.1 T cells, consequences of 4 pg/ml (corresponding to postmenopausal levels) or 40 pg/ml (premenopausal levels) of estradiol (E(2)) were analyzed on signalling proteins, CD3-zeta, JAK2, and JAK3, determined by Western immunoblotting. These consequences were correlated with corresponding gene expressions, quantified by real time-polymerase chain reaction. Tyrosine phosphorylation of CD3-zeta was defined by immunoprecipitation and western immunoblotting following activation by T cell receptor (TcR) cross-linking. CD3-zeta expression and modulation was also confirmed in T cells from pre- and postmenopausal women. To assess functional consequences, IL-2 production, induced by PMA and ionomycin, was determined using enzyme-linked immunosorbent spot assay (ELISpot).

Results: At 40 pg/ml E(2), the level of signalling protein CD3-zeta was elevated 1.57-fold, compared with cells exposed to 4 pg/ml E(2). The CD3-zeta proteins also exhibited altered levels of activation-induced phosphorylation in the presence of 40 pg/ml E(2) versus 4 pg/ml: 23 kD phosphorylated form increased 2.64-fold and the 21 kD form was elevated 2.95-fold. Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E(2), JAK2 protein expression was increased 1.64-fold (p < 0.001) and JAK3 enhanced 1.79-fold (p < 0.001) compared to 4 pg/ml. mRNA levels for CD3-zeta, JAK2, and JAK3 were significantly increased following exposure to 40 pg/ml E(2) (2.39, 2.01, and 2.21 fold, respectively) versus 4 pg/ml. These findings were confirmed in vivo, since T cells from postmenopausal women exhibited 7.2-fold diminished CD3-zeta expression, compared to pre-menopausal controls and this expression was elevated 3.8-fold by addition of 40 pg/ml E(2). Functionally, Jurkat cells exposed to 40 pg/ml E(2) and activated exhibited significantly elevated numbers of IL-2 producing colonies compared to 4 pg/ml (75.3 +/- 2.2 versus 55.7 +/- 2.1 colonies, p < 0.0001).

Conclusion: Jurkat T cells exposed to 4 pg/ml E(2) expressed significantly diminished activation signalling proteins, correlating with reduced IL-2 production. Lower signalling protein levels appear to result from decreased CD3-zeta, JAK2, and JAK3 gene expressions. These findings may provide a molecular basis for immunosenescence associated with the postmenopausal state.

No MeSH data available.


Related in: MedlinePlus