Ex vivo analysis of HIV-1 co-receptors at the endocervical mucosa of women using oral contraceptives
Dr M. S. Kapembwa, Department of GU/HIV Medicine, Imperial College of Science, Technology and Medicine, Northwick Park and St Mark's Hospitals, Watford Road, Harrow, Middlesex,UK.
Combined oral contraceptives may alter the microenvironment of the female genital tract and, thus, influence susceptibility of endocervical cells to HIV-1 transmission. The mechanism for this effect is unknown but might involve combined oral contraceptive up-regulation of chemokine receptors on CD4+ endocervical cells. We measured chemokine co-receptor (CCR5 and CXCR4) expression on cervical intraepithelial CD4+ T lymphocytes, macrophages and dendritic cells using flow cytometry in 32 healthy women, 16 of whom were combined oral contraceptive users and 16 non-users. All women tested negative for sexually transmitted infections. Combined oral contraceptive users showed a higher proportion of CCR5+ CD4+ T lymphocytes compared with combined oral contraceptive non-users (P < 0.05). However, expression of both co-receptors on cervical intraepithelial macrophages and dendritic cells was no different between the two groups. Up-regulation of CCR5 on cervical intraepithelial CD4+ T lymphocytes offers a potential explanation by which women receiving combined oral contraceptives may be at increased risk of HIV transmission.
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The endocervical epithelium contains numerous CD4+ cells that are important in mucosal immune defence against exogenous pathogens. They also play a decisive role in the initial selection of HIV-1 phenotypic variants. Following sexual contact, infection appears to be initiated by non-syncytium-inducing HIV variants that utilise the CCR5 chemokine receptor, the main co-receptor for M-tropic viruses expressed primarily on macrophages and dendritic cells. In contrast, syncytium-inducing HIV variants found predominantly on T lymphocytes in late stage disease use the CXCR4 co-receptor. Possible explanations for the preferential transmission of CCR5-utilising HIV variants include a higher density of CD4-bearing antigen-presenting cells at the mucosal surface combined with the presence of stromal derived factor-1, a ligand for the CXCR4 chemokine co-receptor.
Previous work suggests hormonal contraceptives may influence the susceptibility of cervical mucosal cells to HIV-1 infection. In particular, use of combined oral contraceptives and depot medroxyprogesterone acetate have been associated with increased HIV-1 DNA shedding from the cervix.1,2 Potential mechanisms for this observation include increased area of cervical ectopy among oral contraceptive users, direct immunosuppressive effect of combined oral contraceptives and increased rates of chlamydial infection. In this study, we sought to explore other mechanisms by which combined oral contraceptives might potentiate HIV transmission by investigating CCR5 and CXCR4 chemokine co-receptor expression on CD4+ cervical intraepithelial cells.
Normal, healthy women underwent a standardised speculum examination that included cervical inspection for the presence of inflammation. Women with evidence of cervical ectopy, erythema, oedema or mucopus at the os were excluded from the study.
Samples of endocervical secretions were obtained using sterile cotton-tipped swabs for screening of Chlamydia trachomatis using direct immunofluorescence (Syva MicroTrac, UK), Neisseria gonorrhoea and herpes simplex were identified following culture. Wet high vaginal smears were examined microscopically for Trichomonas vaginalis, bacterial vaginosis and Candida spp; in addition, Gram-stained smear preparations were made to quantify the number of inflammatory cells in the cervix. Only women without sexually transmitted infections (Syphilis, C. trachomatis, N. gonorrhoea, bacterial vaginosis, Candida spp, herpes simplex virus) were studied.
Cervical intraepithelial samples were obtained using the cytobrush technique from 32 women, 16 of whom were receiving combined oral contraceptives (containing 20–50 μg of oestradiol) and the remaining half were not. All cytobrush samples were negative for blood contamination as assessed by labelling with the red cell marker anti-glycophorin A. To further minimise any cellular variability due to hormonal cycling, women were sampled during mid-cycle (median 12 days, range 8th and 15th day of the menstrual cycle). The study was approved by the hospital ethics committee.
Cervical intraepithelial leucocytes were surface-stained using monoclonal antibodies for T-lymphocyte subsets (CD3+, CD4+, CD8+) and macrophage/monocytes (CD14+) as previously described.3 Because no specific marker for dendritic cells exists, these cells were identified by the expression of MHC class II molecules and the absence of markers for other cell types (i.e. CD3, CD14, CD16, CD19, CD56-negative and HDL-DR+). Cell preparations were labelled in parallel with appropriate isotype control antibodies and data were analysed using three-colour FACS Calibur software (Becton Dickinson, UK). Chemokine receptors were studied on all CD4+ cell types with a fourth colour, allophycocyanin: anti-CCR5 and anti-CXCR4. Mean values were compared using the Student's t test with a P value of <0.05 considered significant.
Polymorphonuclear leucocytes were counted in five non-adjacent oil immersion fields (×1000) and the mean polymorphonuclear leucocytes per high power field (phf) was computed. Women showing 0–40 polymorphonuclear leucocytes/hpf were considered normal. Counts greater than 40 polymorphonuclear leucocytes/hpf were considered indicative of cervicitis and women with such counts were excluded from the study.
Table 1 shows the clinical and socio-demographic features of the cohort.
Table 1. Demographic features of women receiving combined oral contraceptives (n= 16) compared with those not receiving combined oral contraceptives (n= 16). Age and parity are expressed as mean (SEM).
|No. of subjects||16||16|
|Age||31 (2)||25 (1)|
|Previous sexually transmitted infection||6||7|
|Coitus within 72 hours||3||3|
|Parity||1 (1)||1 (2)|
Most subjects were Caucasian with mean age 31 years for combined oral contraceptive users and 25 years for those receiving no oral contraceptives. Both chemokine receptors were expressed on CD4+ lymphocytes, macrophages and dendritic cells in cervical intraepithelial samples (Table 2), although women receiving combined oral contraceptives generally showed higher fluorescent intensity than non-users. In particular, expression of CCR5 on cervical intraepithelial CD4+ T lymphocytes was significantly different between combined oral contraceptive users and non-users (P < 0.05), whereas that of CXCR4 was not. In contrast, analysis of CCR5 and CXCR4 expression on cervical intraepithelial macrophages and dendritic cells showed no differences between the two groups of women.
Table 2. Percentage of cervical intraepithelial CD4+ T cells, macrophages and dendritic cells expressing CCR5 and CXCR4 chemokine receptors among healthy women receiving combined oral contraceptives (n= 16) and those not receiving combined oral contraceptives (n= 16). Data are presented as mean (SEM). Significance was determined by a two-tailed parametric Student's t test.
|CCR5||CD4+ T lymphocytes||48 (4)||66 (7)*|
|Macrophages||22 (5)||31 (8)|
|Dendritic cells||5 (1)||12 (5)|
|CXCR4||CD4+ T lymphocytes||66 (5)||70 (5)|
|Macrophages||31 (4)||44 (8)|
|Dendritic cells||15 (4)||22 (6)|
Our study shows that the use of combined oral contraceptives is associated with increased expression of chemoreceptor co-receptors CCR5 on CD4+ T lymphocytes in healthy women. Such up-regulation may be a direct effect of the progesterone component on these cells and is relevant to pathogenesis of HIV infection. For example, CCR5 density on CD4+ T lymphocytes is pivotal to infectability of these cells including in vivo virus production. Furthermore, this finding suggests a mechanism by which mucosal CD4+ T lymphocytes may be rendered susceptible to infection by dual tropic HIV variants in vivo. However, while this may be one factor in the observed increased risk HIV transmission in women taking combined oral contraceptives, there may be other important considerations such as cellular activation and recruitment. We have previously shown that the immunological microenvironment within cervical mucosa differs from that of peripheral blood.4 Thus, in the absence of any local infection, cervical intraepithelial lymphocytes appear highly activated with coincidentally, significantly elevated CCR5 co-receptor expression, a finding in keeping with increased cellular exposure to antigen or altered cytokine expression at this site. Intraepithelial T lymphocytes are located at the mucosal surface of the endocervix. However, the close proximity and continual movement of these cells increases the likelihood of localised conjugates forming and, thus, of productive HIV infection. More recently, we have reported increased numbers of CD4+ T lymphocytes and macrophages in cervical epithelium of healthy volunteers following sexual intercourse.5 These observations suggest combined oral contraceptives and coitus may enhance HIV transmission across the mucosa via the mechanisms of cellular recruitment, activation and up-regulation of HIV co-receptors, in particular, of CCR5 on T lymphocytes.
There are clearly several limitations to our study. Firstly, CCR5 expression can vary among individuals and the extent of inter-individual variability was not determined. It is, therefore, unclear to what extent the observed differences in CCR5 expression are due to natural variability or the effect of combined oral contraceptives. Secondly, data on compliance of combined oral contraceptive use and duration were not collected. It is, therefore, possible that some combined oral contraceptive users were not actually taking their combined oral contraceptives at the time of sampling and the effects of combined oral contraceptives on cervical immune cells may not be apparent if used for a short time.
Despite the above confounders, our data illustrate the potential importance of combined oral contraceptives in modulating cervical mucosal immunity. The change in CCR5 co-receptor expression on cervical intraepithelial CD4+ T lymphocytes may be a consequence of heightened cell mediated immunity in women using combined oral contraceptives. This finding, if confirmed by larger prospective studies, offers a potential explanation for the apparent increase in HIV transmission in women using combined oral contraceptives and may have important implications for family planning and public health.