In the whole blood, there were significantly higher percentages of CCR5+CD4+ T cells (median: 22% vs. 13%) and CCR5+DR+38+CD4+ T cells (median: 76% vs. 62%) in postmenopausal compared with premenopausal women (Figs. 2A, B). In the cervix, there were even greater differences in percentages of CCR5+ cells between postmenopausal and premenopausal women on CD4+ T cells (median: 70% vs. 42%) and DR+38+CD4+ T cells (median: 80% vs. 57%) (Figs. 2E, F). There were no significant differences between premenopausal and postmenopausal women in the mean number of CCR5 molecules on whole blood CD4+ T cells or activated DR+38+CD4+ T cells (Figs. 2C, D). The concentration of CCR5 on cervical CD4+ T cells tended to be higher in postmenopausal (median: 3427 mol/cell) compared with premenopausal women (median: 2091 mol/cell) (Fig. 2G), and postmenopausal women had significantly more CCR5 molecules on activated cervical CD4+ T cells (median: 3176 mol/cell) than premenopausal women (median: 1776 mol/cell) (Fig. 2H).
In the blood, percentages of CXCR4+CD4+ T cells were slightly, but significantly, higher in postmenopausal (median: 88%; 95% CI: 81% to 90%) compared with premenopausal women (median: 81%; 95% CI: 72% to 85%; P = 0.04), but there were no differences in CXCR4 expression in the activated subset (median: 43% vs. 44%, respectively, P = 0.8). Postmenopausal women tended to have a higher percentage of cervical CXCR4+CD4+ T cells than premenopausal women (median: 87% vs. 67%; P = 0.2). Percentages of CXCR4+DR+38+CD4+ T cells in the cervix and density of CXCR4 on CD4+ T cells from the blood or cervix did not differ between postmenopausal and premenopausal women (data not shown).
To further investigate differences in HIV-1 coreceptor expression between premenopausal and postmenopausal women, data were analyzed to determine the relationship between age and CCR5 or CXCR4 expression. There was a linear relationship between age and percentages of CCR5+CD4+ T cells and CCR5+DR+38+CD4+ T cells in both whole blood and cervix (Fig. 3). Using an adjusted model, it could not be determined if the linear relationship between percent CCR5 expression and age was due to menopause or the aging process. There were no significant linear relationship between age and percentages of CXCR4+ cells or density of CXCR4 expression on total or activated CD4+ T cells in blood or cervix (data not shown).
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 with 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.
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 and aging29 have been linked to increases in proinflammatory cytokines including, TNF-α and IL-6. Interestingly, we found no significant differences in percentages of activated of 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 individuals30 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 with 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 laboratory35 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 and contribute to higher levels of virus replication after 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 cells,28,36 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 which is 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 with younger mice41 and blood CD4+ T cells of older compared with 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 before 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, both 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 with 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 65,44 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 with 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 the ectocervix,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 with those from premenopausal women, supporting the hypothesis that elevated CCR5 expression in postmenopausal women may result in differential HIV-1 susceptibility.47
We thank Kristina Carroll, Lauren Tobin, and Chelsea Bergman for their technical assistance.
2. Comparison of female to male and male to female transmission of HIV in 563 stable couples. European study group on heterosexual transmission of HIV. BMJ. 1992;304:809–813.
3. Lindau ST, Leitsch SA, Lundberg KL, et al.. Older women's attitudes, behavior, and communication about sex and HIV: a community-based study. J Womens Health (Larchmt). 2006;15:747–753.
4. Espinoza L, Hall HI, Hardnett F, et al.. Characteristics of persons with heterosexually acquired HIV infection, United States 1999-2004. Am J Public Health. 2007;97:144–149.
5. Smith SM, Baskin GB, Marx PA. Estrogen protects against vaginal transmission of simian immunodeficiency virus. J Infect Dis. 2000;182:708–715.
6. Smith SM, Mefford M, Sodora D, et al.. Topical estrogen protects against SIV vaginal transmission without evidence of systemic effect. AIDS. 2004;18:1637–1643.
7. Linsk NL. HIV among older adults: age-specific issues in prevention and treatment. AIDS Read. 2000;10:430–440.
8. Miller CJ, Li Q, Abel K, et al.. Propagation and dissemination of infection after vaginal transmission of Simian immunodeficiency virus. J Virol. 2005;79:9217–9227.
9. Zhang Z, Schuler T, Zupancic M, et al.. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science. 1999;286:1353–1357.
10. Gupta P, Collins KB, Ratner D, et al.. Memory CD4(+) T cells are the earliest detectable human immunodeficiency virus type 1 (HIV-1)-infected cells in the female genital mucosal tissue during HIV-1 transmission in an organ culture system. J Virol. 2002;76:9868–9876.
11. Hladik F, Sakchalathorn P, Ballweber L, et al.. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity. 2007;26:257–270.
12. Hirbod T, Kaldensjo T, Broliden K. In situ distribution of HIV-binding CCR5 and C-Type lectin receptors in the human endocervical mucosa. PLoS One. 2011;6:e25551.
13. Grivel JC, Shattock RJ, Margolis LB. Selective transmission of R5 HIV-1 variants: where is the gatekeeper? J Infect Dis. 2010:9:(suppl 1):S1–S17.
14. Hogan CM, Hammer SM. Host determinants in HIV infection and disease. Part 2: genetic factors and implications for antiretroviral therapeutics. Ann Intern Med. 2001;134:978–996.
15. Marmor M, Sheppard HW, Donnell D, et al.. Homozygous and heterozygous CCR5-Delta32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr. 2001;27:472–481.
16. Reeves JD, Gallo SA, Ahmad N, et al.. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci U S A. 2002;99:16249–16254.
17. Begaud E, Chartier L, Marechal V, et al.. Reduced CD4 T cell activation and in vitro susceptibility to HIV-1 infection in exposed uninfected Central Africans. Retrovirology. 2006;3:35.
18. Koning FA, Otto SA, Hazenberg MD, et al.. Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection. J Immunol. 2005;175:6117–6122.
19. Reichelderfer PS, Coombs RW, Wright DJ, et al.. Effect of menstrual cycle on HIV-1 levels in the peripheral blood and genital tract. WHS 001 Study Team. AIDS. 2000;14:2101–2107.
20. Meditz AL, Schlichtemeier R, Folkvord JM, et al.. SDF-1alpha is a potent inducer of HIV-1-Specific CD8+ T-cell chemotaxis, but migration of CD8+ T cells is impaired at high viral loads. AIDS Res Hum Retroviruses. 2008;24:977–985.
21. de Roda Husman AM, Koot M, Cornelissen M, et al.. Association between CCR5 genotype and the clinical course of HIV-1 infection. Ann Intern Med. 1997;127:882–890.
22. Prakash M, Kapembwa MS, Gotch F, et al.. Higher levels of activation markers and chemokine receptors on T lymphocytes in the cervix than peripheral blood of normal healthy women. J Reprod Immunol. 2001;52:101–111.
23. Quayle AJ, Kourtis AP, Cu-Uvin S, et al.. T-lymphocyte profile and total and virus-specific immunoglobulin concentrations in the cervix of HIV-1-infected women. J Acquir Immune Defic Syndr. 2007;44:292–298.
24. Saba E, Grivel JC, Vanpouille C, et al.. HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model. Mucosal Immunol. 2010;3:280–290.
25. Hladik F, Lentz G, Delpit E, et al.. Coexpression of CCR5 and IL-2 in human genital but not blood T cells: implications for the ontogeny of the CCR5+ Th1 phenotype. J Immunol. 1999;163:2306–2313.
26. Veazey RS, Marx PA, Lackner AA. Vaginal CD4+ T cells express high levels of CCR5 and are rapidly depleted in Simian immunodeficiency virus infection. J Infect Dis. 2003;187:769–776.
27. Nkengasong JN, Kestens L, Ghys PD, et al.. Human immunodeficiency virus type 1 (HIV-1) plasma virus load and markers of immune activation among HIV-infected female sex workers with sexually transmitted diseases in Abidjan, Cote d'Ivoire. J Infect Dis. 2001;183:1405–1408.
28. Pfeilschifter J, Koditz R, Pfohl M, et al.. Changes in proinflammatory cytokine activity after menopause. Endocr Rev. 2002;23:90–119.
29. Bruunsgaard H, Pedersen M, Pedersen BK. Aging and proinflammatory cytokines. Curr Opin Hematol. 2001;8:131–136.
30. Kalayjian RC, Landay A, Pollard RB, et al.. Age-related immune dysfunction in health and in human immunodeficiency virus (HIV) disease: association of age and HIV infection with Naive CD8+ Cell depletion, reduced expression of CD28 on CD8+ Cells, and reduced thymic volumes. J Infect Dis. 2003;187:1924–1933.
31. Lee B, Sharron M, Montaner LJ, et al.. Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1999;96:5215–5220.
32. Platt EJ, Wehrly K, Kuhmann SE, et al.. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol. 1998;72:2855–2864.
33. Reeves JD, Gallo SA, Ahmad N, et al.. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci U S A. 2002;99:16249–16254.
34. Reynes J, Portales P, Segondy M, et al.. CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression. AIDS. 2001;15:1627–1634.
35. Meditz AL, Haas MK, Folkvord JM, et al.. HLA-DR+CD38+CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo. J Virol. 2011;85:10189–10200.
36. Asin SN, Heimberg AM, Eszterhas SK, et al.. Estradiol and progesterone regulate HIV type 1 replication in peripheral blood cells. AIDS Res Hum Retroviruses. 2008;24:701–716.
37. Moriuchi H, Moriuchi M, Fauci AS. Nuclear factor-kappa B potently up-regulates the promoter activity of RANTES, a chemokine that blocks HIV infection. J Immunol. 1997;158:3483–3491.
38. Mo R, Chen J, Grolleau-Julius A, et al.. Estrogen regulates CCR gene expression and function in T lymphocytes. J Immunol. 2005;174:6023–6029.
39. Prakash M, Kapembwa MS, Gotch F, et al.. Oral contraceptive use induces upregulation of the CCR5 chemokine receptor on CD4(+) T cells in the cervical epithelium of healthy women. J Reprod Immunol. 2002;54:117–131.
40. Yung RL, Mo R. Aging is associated with increased human T cell CC chemokine receptor gene expression. J Interferon Cytokine Res. 2003;23:575–582.
41. Mo R, Chen J, Han Y, et al.. T cell chemokine receptor expression in aging. J Immunol. 2003;170:895–904.
42. Fiser AL, Vincent T, Brieu N, et al.. High CD4+ T-cell surface CXCR4 density as a risk factor for R5 to X4 switch in the course of HIV-1 infection. J Acquir Immune Defic Syndr. 2010;55:529–535.
43. Balandya E, Sheth S, Sanders K, et al.. Semen protects CD4+ target cells from HIV infection but promotes the preferential transmission of R5 tropic HIV. J Immunol. 2010;185:7596–7604.
44. Huppert FA, Solomou W, O'Connor S, et al.. Aging and lymphocyte subpopulations: whole-blood analysis of immune markers in a large population sample of healthy elderly individuals. Exp Gerontol. 1998;33:593–600.
45. Pudney J, Quayle AJ, Anderson DJ. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol Reprod. 2005;73:1253–1263.
46. Kaldensjo T, Petersson P, Tolf A, et al.. Detection of intraepithelial and stromal langerin and CCR5 positive cells in the human endometrium: potential targets for HIV infection. PLoS One. 2011;6:e21344.
47. Rollenhagen C, Asin SN. Enhanced HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women correlates with increased inflammatory responses. Mucosal Immunol. 2011;4:671–681.