Mucosal delivery of CpG oligodeoxynucleotides expands functional dendritic cells and macrophages in the vagina

Abstract

Antigen-presenting cells (APC) are specialized sentinel cells that sense pathogens within tissues and then activate appropriate immune effector cells in lymphoid organs. Recent evidence, however, suggests that APC can also induce effector cells in non-lymphoid organs. The purpose of this study was to determine the effect of intravaginal (IVAG) delivery of CpG-oligodeoxynucleotide (ODN) on expansion of resident genital APC. Our results show that delivery of CpG-ODN to the murine genital tract induced a rapid and significant, but transient expansion of genital APC in situ. As early as 12 hr post CpG-ODN delivery, we observed an enhanced level of F4/80+ major histocompatibility complex (MHC) class II-negative macrophages in the genital tissue. This was followed by increased levels of F4/80/MHC class II double-positive cells, as well as MHC class II, CD11c and CD86 triple-positive dendritic cells (DC) at 48 hr. Expanded APC levels at 48 hr post CpG-ODN resulted in increased ability of genital cells to induce an allogenic mixed leucocyte reaction. By 72 hr after CpG-ODN treatment, APC levels were not distinguishable from naive levels. Therefore, these results clearly show that administration of CpG-ODN to the genital tract induced a marked but transient enhancement of APC within the genital tissue, and that these APC appear to possess functional capacity. Furthermore, these results indicate that IVAG-CpG-ODN may be an important factor for the enhancement of local antigen presentation in the genital tract through increased DC numbers.

Figure 1

(a) Effects of vaginal CpG-ODN delivery on inflammatory cell numbers in the vagina. Groups of mice were treated with a single injection of Depo-Provera. On day 4 after Depo-Provera treatment, groups of mice were either killed immediately or killed at 12, 28, 48 or 72 hr following vaginal delivery of CpG-ODN. For each time point, vaginal tissues (including cervix but not the uterus) from at least five mice were pooled, digested and counted as described in Materials and methods). Cell counts are expressed as the number of cells/mouse. Error bars indicate the SEM of at least four separate experiments for all time-points except the 72 hr time-point, which was carried out twice. (b) Flow cytometric analysis was performed on differentially treated genital tissues as described in Materials and methods. White histogram plots indicate the staining obtained by an isotype-control antibody, while the dark histograms represent the staining of the specified antibody. The numbers indicated percentage of positive cells within the leukocyte subpopulation. Antibody-specific values are deemed positive if the expression levels are higher than 5% of the isotype control antibody.

Figure 2

Infiltration kinetics of APC into vaginal tissue following vaginal CpG-ODN delivery. Groups of mice were treated with a single injection of Depo-Provera. On day 4 after Depo-Provera treatment, groups of mice were either killed immediately or killed at 12, 28, 48 and 72 hr following vaginal delivery of CpG-ODN. Immediately thereafter, vaginal tissues (including cervix but not the uterus) from at least five mice were digested, stained for flow cytometry and analysed with WINLIST software as described in Materials and methods. White histogram plots indicate the staining obtained by an isotype-control antibody, while the dark histograms represent the staining of the specified antibody. The numbers indicated percent of positive cells within the leucocyte subpopulation. Antibody specific values are deemed positive if the expression levels are higher than 5% of the isotype-control antibody.

Figure 3

Subset of APCs infiltrating the vaginal tissue following IVAG CpG-ODN delivery. Four days following Depo-Provera treatment, groups of mice were either killed immediately or killed at 12, 48 and 72 hr following vaginal delivery of CpG-ODN. Immediately thereafter, vaginal tissues (including cervix but not the uterus) from at least five mice were digested, stained for flow cytometry and analysed with WINLIST software as described in Materials and methods. The numbers shown are representative of cells that coexpress both of the markers in the dotplot and indicate the percent of positive cells within the leukocyte subpopulation.

Figure 4

Distribution/localization of APCs within the vaginal tract following IVAG CpG-ODN delivery. Four days following Depo-Provera treatment, groups of mice were either killed immediately or killed at 12, 28 or 48 hr following vaginal delivery of CpG-ODN. Immediately thereafter, vaginal tissues were collected, placed in OCT and frozen in liquid nitrogen. Immunohistochemical analysis of MHC class II, CD11b and F4/80 was performed on 8 µm cryostat sections using the immunoperoxidase technique. Images were captured using a 4× objective lens. Average sections, representative of at least three independent experiments using at least three mice/group are shown.

Figure 5

Infiltration kinetics of CD11c-positive cells into the vaginal tissue following vaginal CpG-ODN delivery. (a) Four days following Depo-Provera treatment, groups of mice were either killed immediately or killed at 12, 28 or 48 hr following vaginal delivery of CpG-ODN. Immediately thereafter, vaginal tissues (including cervix but not the uterus) from at least five mice were digested, stained for flow cytometry and analysed with WINMDI software as described in Materials and methods. During the analysis of the acquired data we first gated the leucocyte population on CD86⁺ cells, and then analysed for the expression of CD11c and MHC class II cell surface markers. Percent of gate values indicate the percentage of the cells positive for each marker among the CD86⁺ cells, while the percent values indicate the percentage of CD11c/MHC class II/CD86 triple-positive cells among the total cell population. (b) Four days following Depo-Provera treatment, groups of mice were either killed immediately or killed at 48 hr following vaginal delivery of CpG-ODN. Immediately thereafter, vaginal tissues were collected, placed in OCT and frozen in liquid nitrogen. Immunohistochemical analysis of CD11c was performed on 8 µm cryostat sections using the immunoperoxidase technique. Images were captured using either a 4× objective lens or a 20× objective lens (inset). Average sections, representative of at least three independent experiments using at least three mice/group are shown.

Figure 6

Functional capacity of CpG-induced APCs in the genital tract. Four days following Depo-Provera treatment, groups of mice were either killed immediately (□) or killed 48 hr following vaginal delivery of CpG-ODN (▪). Immediately thereafter, vaginal tissues (including cervix but not the uterus) from at least five mice were digested and plated on 96-well plates in indicated numbers. Subsequently, 8 × 10⁵ splenocytes from autologous BALB/c mice were administered to each well. Proliferation of T cells was measured by means of ³H thymidine incorporation after 5 days of coculture. Results are mean c.p.m. ± SD of triplicate cultures