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. 2009 Mar 5:6:1.
doi: 10.1186/1742-4933-6-1.

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

Lowell T Ku  1 , Cicek Gercel-Taylor, Steven T Nakajima, Douglas D Taylor
Affiliations

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

Lowell T Ku et al. Immun Ageing. .

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.

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Figures

Figure 1
Figure 1
Modulation of Jurkat cell CD3-zeta protein expression by physiologic estradiol concentrations. Expression of CD3-zeta protein expression by Jurkat cells incubated in the presence of either 4 pg/ml or 40 pg/ml estradiol for 48 hours defined by Western immunoblotting. The bar graph presents the quantitation of triplicate digitized gel images expressed as mean ± standard deviation. The insert shows a representative Western blot gel image.
Figure 2
Figure 2
Modulation of normal T cell CD3-zeta protein expression by physiologic estradiol concentrations. Expression of CD3-zeta protein expression by T cells isolated from either normal premenopausal or postmenopausal female volunteers, assayed immediately after isolation and following an incubation in the presence of either 4 pg/ml or 40 pg/ml estradiol for 48 hours defined by Western immunoblotting. The bar graph presents the quantitation of triplicate digitized gel images expressed as mean ± standard deviation. The insert shows a representative Western blot gel image.
Figure 3
Figure 3
Activation-induced phosphorylation of CD3-zeta at physiologic estradiol concentrations. Representative western immunoblotting results demonstrating the major phosphorylated CD3-zeta bands. Jurkat cells were incubated for 48 hours in 0, 4 or 40 pg/ml estradiol and then activated by cross-linking the TcR. Cellular CD3-zeta was immunoprecipitated and the phosphorylated bands were identified using anti-pCD3-zeta as the primary antibody.
Figure 4
Figure 4
Modulation of JAK 2 protein expression by physiologic estradiol concentrations. Modulation of JAK 2 protein expression in Jurkat cells incubated in the presence of either 4 pg/ml or 40 pg/ml estradiol for 48 hours defined by Western immunoblotting. The bar graph shows the quantitation of triplicate digitized gel images expressed as mean ± standard deviation. The insert shows a representative Western blot gel image.
Figure 5
Figure 5
Modulation of JAK 3 protein expression by physiologic estradiol concentrations. Modulation of JAK 3 protein expression in Jurkat cells incubated in the presence of either 4 pg/ml or 40 pg/ml estradiol for 48 hours defined by Western immunoblotting. The bar graph presents the quantitation of triplicate digitized gel images expressed as mean ± standard deviation. The insert shows a representative Western blot gel image.
Figure 6
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.
Figure 7
Figure 7
Interleukin-2 (IL-2) production following induction by PMA and ionomycin in physiologic estradiol concentrations. IL-2 production by Jurkat cells exposed to 4 pg/ml estradiol for 48 hours and activated with PMA and ionomycin for 24 hours compared to cells exposed to 40 pg/ml estradiol and activated. The number of IL-2 producing colonies was determined by ELISPOT assay and the values plotted are mean ± standard deviation. The inset images present representative assay plates for activated Jurkat cells in the presence of either 4.0 pg/ml E2 or 40.0 pg/ml E2.
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