Repertoire of chemokine receptor expression in the female genital tract: implications for human immunodeficiency virus transmission

Abstract

Sexually transmitted diseases, genital ulcer disease, and progesterone therapy increase susceptibility to lentivirus transmission. Infection of cells by human immunodeficiency virus (HIV) is dependent on expression of specific chemokine receptors known to function as HIV co-receptors. Quantitative kinetic reverse transcription-polymerase chain reaction was developed to determine the in vivo expression levels of CCR5, CXCR4, CCR3, CCR2b, and the cytomegalovirus-encoded US28 in peripheral blood mononuclear cells and cervical biopsies from 12 women with and without sexually transmitted diseases, genital ulcer disease, and progesterone-predominant conditions. Our data indicate that CCR5 is the major HIV co-receptor expressed in the female genital tract, and CXCR4 is the predominantly expressed HIV co-receptor in peripheral blood. CCR5 mRNA expression in the ectocervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In peripheral blood, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in cervical tissue despite expression in peripheral blood mononuclear cells from five individuals. CCR5 was significantly increased (p < 0.02) in biopsies from women with sexually transmitted diseases and others who were progesterone predominant. In vitro studies demonstrate that progesterone increases CCR5, CXCR4, and CCR3 expression and decreases CCR2b expression in lymphocytes and monocytes/macrophages. Characterization of chemokine receptors at the tissue level provides important information in identifying host determinants of HIV-1 transmission.

Figure 1.

Chemokine receptor mRNA quantification is linear over a range of at least 10⁵ copies. Threshold cycle number (C_t) refers to the cycle number at which the reporter signal exceeds background. Results were based on triplicate determinations. The correlation coefficient for all curves was 0.99.

Figure 2.

Comparison of chemokine receptor expression in PBMCs and uterine cervix. CCR5 mRNA expression in the cervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In PBMCs, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in any of the cervical biopsies despite expression in PBMCs from five individuals.

Figure 3.

Digital microscopic images of chemokine receptor expression in an immunohistochemically stained cervical biopsy from HIV-1-seropositive patient 12 (Tables 1 and 2) ▶ ▶ . Chemokine receptor-expressing cells appear brown (arrows) in sections counterstained with hematoxylin (blue). A: CCR5-expressing cells are clustered beneath the surface epithelium. B: CXCR4-expressing cells from the same biopsy have a similar localization as the CCR5-expressing cells. Increased numbers of CCR5-expressing cells were found compared with CXCR4-expressing cells. Magnification, ×350.

Figure 4.

Localization of cells co-expressing CD4 and CCR5 (yellow) using double-label immunofluorescence and laser confocal image analysis (patient 6). Cells expressing only CD4 appeared red (arrowheads), and cells expressing only CCR5 were not identified. The epithelium-submucosa junction is denoted by arrows.

Figure 5.

Immunophenotypic characterization of cell types known to express CCR5 in representative cervical biopsies from patients listed in Table 1 ▶ . Activated/memory T lymphocytes (CD45RO), macrophages (CD68), and Langerhans’ cells (S-100) were visualized and quantified using image analysis. Positive cells appear brown (arrowheads) in sections counterstained with hematoxylin (blue). A lymphoid follicle was identified (arrow) in one of the biopsies. Magnification, ×200.

Figure 6.

Assisted computerized image analysis photomicrograph of cervical biopsies from patients 8 (A) and 9 (B) stained by immunohistochemistry for IL-2. IL-2-producing cells were identified by a juxtanuclear focal staining pattern (arrows) surrounded by extracellular immune reactivity caused by adherence of cytokines to matrix proteins. Tissue sections were counterstained with hematoxylin (blue). Magnification, ×400.

Figure 7.

A: Quantification of progesterone-induced chemokine receptor mRNA up-regulation after 3 days of PBMC culture in the presence of 50 ng/ml progesterone. CCR5, CXCR4, and CCR3 mRNA were significantly increased after progesterone treatment, whereas CCR2b mRNA decreased after treatment. B: Representative histograms from immunophenotyping/flow cytometry experiments. Untreated (top) and progesterone-treated (bottom) cells were double labeled with CD14-fluorescein isothiocyanate and CCR5-PE and gated based on CD14 expression and side scatter. Progesterone up-regulated CCR5 protein expression fivefold in CD14⁺ monocytes. The increase in CCR5/CXCR4 expression peaked at 3 days and remained elevated or slightly diminished by day 6.