This is the first study to investigate expression of the activation markers HLA-DR and CD38, and HIV-1 chemokine coreceptors on blood and cervical CD4+ T-cells in premenopausal and postmenopausal women. CD4+ T-cells in the cervix were found to have several characteristics that would support HIV-1 transmission including elevated immune activation compared to blood and high levels of expression of both CCR5 (median, 47%) and CXCR4 (median, 75%). Percentages of activated cells and CXCR4 expression did not differ substantially between premenopausal and postmenopausal women in either blood or the cervix. However, postmenopausal women had significantly higher expression of CCR5 in both blood and cervix. These findings suggest that postmenopausal women may be at greater biologic risk of R5 HIV-1 acquisition than premenopausal women.
Activated CD4+ T-cells have been linked to increased risk of HIV-1 infection,17–18
although mechanisms underlying this association are not fully understood. In the present study, we observed that percentages of DR+38+CD4+ T-cells were six-times higher in the cervix than in the blood of healthy HIV-1 seronegative women, consistent with two other small studies.22–23
Reasons for higher proportions of activated CD4+ T-cells in the cervix are unknown, although, higher percentages of memory T-cell populations are found in the cervix than blood.24–25
It has been proposed that elevated immune activation in the cervix is a result of antigenic stimulation.26
A strength of this study was that women were screened and excluded if they were found to have a vaginal infection, which can increase DR and 38 expression in the cervix.27
Nevertheless, antigens, from vaginal flora are present in all women, and may contribute to levels of immune activation and CCR5 expression. This is the first study to measure CCR5 and CXCR4 on DR+38+CD4+ T-cells in the cervix. A prior study evaluating 12 premenopausal women demonstrated higher percentages of CCR5+CD4+ cells in the cervix compared to blood.22
In addition, a study of Rhesus macaques demonstrated over 4-fold higher CCR5 expression in vaginal compared to blood CD4+ T-cells.26
Our results build on these prior studies, showing that the majority of activated CD4+ T-cells in the cervix expressed CCR5 and CXCR4. These data suggest that activated cervical CD4+ T-cells may be highly susceptible to HIV-1 infection due to high levels of expression of both HIV-1 coreceptors, which may account for the association of activated CD4+ T-cells with HIV-1 transmission.
The relationship between menopause and percentages of activated blood and cervical CD4+ T-cells was evaluated for the first time in this study. Declines in ovarian sex hormone production28
have been linked to increases in proinflammatory cytokines including, TNF-α and IL-6. Interestingly, we found no significant differences in percentages activated CD4+ T-cells between premenopausal and postmenopausal women in either the blood or the cervix. These findings are consistent with those of a previous study that reported no age-related differences in percentages of DR+38+CD4+ T-cells in blood of either healthy HIV-1 seronegative individuals or HIV-1-infected individuals,30
and further extends these observations to the female genital tract.
An important determinant of HIV-1 susceptibility is CCR5 expression on CD4+ T-cells. Levels of CCR5 expression correlate with cellular susceptibility, in vitro.31–33
Furthermore, individuals heterozygous for the CCR5 delta 32 mutation, which is associated with lower levels of CCR5 expression, may be less susceptible to HIV-1 acquisition.15
In the present study, substantially higher percentages of CCR5+CD4+ T-cells and CCR5+DR+38+CD4+ T-cells were observed in postmenopausal compared to premenopausal women in both blood and the cervix. Density of CCR5 molecules was also elevated on activated cervical CD4+ T-cells of postmenopausal women. The relative importance of percentages of CCR5+ cells and density of CCR5 molecules in HIV-1 transmission and replication is somewhat controversial. Several in vitro studies suggest concentrations of CCR5 molecules are the most important determinant of HIV-1 susceptibility,31–33
and one group reported that concentrations of CCR5 molecules on peripheral blood CD4+ T-cells correlate with viral load.34
Nonetheless, data from our lab35
suggest that CCR5 percentages and density are both important determinants; in lymph node cells from untreated R5-tropic HIV-1-infected individuals, percentages of CCR5+ cells and numbers of CCR5 molecules per cell predicted the amount of HIV-1 RNA levels within subsets of cells defined by DR and 38 expression. Extrapolation of existing data on the role of CCR5 in HIV-1 transmission and chronic infection suggests that elevated percentages of CCR5+ target cells and density of CCR5 in postmenopausal women may increase their vulnerability to HIV-1 acquisition as well as contribute to higher levels of virus replication following HIV-1 infection.
Mechanisms underlying differences in percentages of CCR5+CD4+ T-cells in postmenopausal and premenopausal women are unclear. Estrogen and progesterone receptors have been demonstrated on T-cells28, 36
and the CCR5 promoter contains hormone response elements, supporting transcriptional control of CCR5 by sex hormones.37
In oophorectomized mice38
receiving exogenous estrogen (blood levels 150–200 pg/mL) and in women receiving oral contraceptives,39
CCR5 expression on CD4+ T-cells was increased, opposite to the effect on CCR5 expression in women with physiologically low estrogen observed in the current study. Nevertheless, the effect of sex hormones on immune modulators may change over the lifespan40
and therefore it is difficult to directly extrapolate these studies to hormonal effects in menopause.
Alternatively, immune changes related to aging could account for heightened CCR5 expression in postmenopausal women. Prior studies have shown CCR5 RNA is higher in blood of older compared to younger mice41
and blood CD4+ T-cells of older compared to younger men and women.40
Although one group found that the percentage of CCR5+CD4+ T-cells was not significantly different between older and younger HIV-1-infected individuals,30
these results should be viewed with caution because some specimens were shipped overnight prior to analysis, a process known to downregulate CCR5 expression. Importantly, if age rather than sex hormones underlies the increased CCR5 expression observed in the present study, older women and men may be at increased risk of HIV-1 acquisition.
CXCR4 levels have not been associated with HIV-1 transmission, although they are linked to HIV-1 susceptibility in cell lines.42
In the present study, percentages of CXCR4+ were higher than CCR5+ cells on total and activated cervical CD4+ T-cells. It has been hypothesized that lower density of CXCR4 compared to CCR5 may account for preferential R5 virus transmission.42
Nevertheless, CXCR4 density was similar to or higher than CCR5 density on cervical lymphocytes in the present study. Thus, these findings support the hypothesis that there are multiple factors contributing to preferential R5 virus transmission13
and that coreceptor expression is not the only restriction factor. Intriguingly, recent studies suggest that seminal plasma induces increased CCR5 expression on CD4+ T-cells and thereby may promote R5 over X4-tropic HIV-1 transmission.43
One limitation of our study is that flow cytometry provides relative percentages, not absolute numbers of cells. HIV-1 susceptibility likely relates to the absolute number of available target cells in cervical tissue, not just relative percentage within CD4+ T-cells. Absolute numbers of blood CD4+ T-cells do decline after age 65 years,44
but women included here were younger. Further, it is unclear whether absolute CD4+ T-cell count from the blood translates to absolute numbers in mucosal tissue. Future research should be directed at evaluating absolute numbers of CCR5+ cells within cervical tissues of premenopausal compared to postmenopausal women. Another shortcoming was that the present study only evaluated CCR5 expression in the endocervix. Nevertheless, there are other sites in the female genital tract where CCR5+ T-cells are present and HIV-1 transmission may occur including the vagina,26, 45
and the endometrium.46
Further studies are needed to determine whether differences between premenopausal and postmenopausal women in CCR5 expression on CD4+ T-cells are also found in other areas of the female genital tract. Final limitations of the present study are that our observations are phenotypic and the sample size of cervical data from postmenopausal women is small. Further studies are needed to confirm our observations and demonstrate whether these differences in CCR5 expression result in true differences in how readily HIV-1 is transmitted to postmenopausal women. Importantly, a recent study demonstrated enhanced HIV-1 replication in ectocervical explants obtained from postmenopausal compared to those from premenopausal women, supporting the hypothesis that elevated CCR5 expression in postmenopausal women may result in differential HIV-1 susceptibility.47
Currently an estimated 50 million American women are postmenopausal48
and approximately 21 million more women will reach menopause in the next 10 years.49
These data suggest increasing numbers of postmenopausal women will be exposed to HIV-1 infection in the next decade. Indeed, new HIV-1 infections are already increasing in older women compared to younger women in the United States; from 1999 to 2004, numbers of new diagnoses increased by 28% in women over 40 years old whereas they decreased by 13% in women between the ages of 13 and 39.4
Importantly, our study suggests that postmenopausal women are likely to be at greater biologic risk of HIV-1 acquisition than reproductive-age women. One of the major priorities of the National HIV/AIDS Strategy is to lower the annual number of new infections by 2015.50
The present study underscores the urgent need to better understand the impact of sex hormones and aging on HIV-1 acquisition, and mechanisms underlying differential risk in order to design appropriate public health messages and effective prevention measures for this older population.