Protection against ascending infection of the genital tract by Chlamydia trachomatis is associated with recruitment of major histocompatibility complex class II antigen-presenting cells into uterine tissue

Abstract

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts' immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45(+) major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.

FIG. 1

Mouse strain variation in uterine CD45⁺ MC infiltrate. Each symbol represents the results of an individual mouse. Uteri were obtained 7, 10, 21, and 24 days postinfection with 4.5 × 10⁵ IFU of C. trachomatis NI1 or with administration of vehicle alone. MC were obtained, and the percentages of CD45 staining were determined by flow cytometry. CD45⁺ MC numbers were determined as follows: number of MC recovered times the proportion of CD45⁺ cells. The increase in CD45⁺ MC numbers in infected animals was obtained by subtracting a mean value (n = 2) for uninfected mice of the appropriate strain. The results were indistinguishable at days 7 and 10 and also at days 21 and 24, and so the results were pooled for an early and late time point. The lines represent the mean values for each group.

FIG. 2

Proliferative responses (A) and cytokine production (B) by pooled draining lymph node cells taken from groups of 5 mice 17 days post-i.vag. infection with 4.5 × 10⁵ IFU. Symbols (panel A): •, BALB/c EB infected; ■, C3H EB infected; ○, BALB/c sham infected; □, C3H sham infected. Error bars encompass the standard deviation of triplicate cultures. In panel B, the LNC from EB-infected mice cultured with EB (+) or medium alone (−) are as indicated. The cytokines detected were IFN-γ (□) and IL-10 (▨). No IL-4 was detected. Error bars encompass the standard deviation of duplicate wells. No cytokines were detected in the cultures of cells from uninfected mice. Results representative of two experiments are shown.

FIG. 3

Intracellular cytokine staining of LNC from mice infected with 3.5 × 10⁵ to 4.5 × 10⁵ IFU of C. trachomatis NI1 and restimulated in vitro with 0.5 μg of inactivated EB per ml. LNC were obtained 13 to 17 days postinfection. Gates for analysis were set on large cells on the basis of light scatter. (A) Double labeling with anti-αβTCR antibody. (B) Double labeling with anti-CD4 and anti-CD8. (C) Specificity controls for labeling of intracellular TNF-α (blocking with excess TNF-α and the need for permeabilization). Cells were from BALB/c mice and the control TNF-α labeling is shown in panel A. Each graph shows the percent positive cells in the upper right quadrant following subtraction of the isotype control staining results.

FIG. 4

Mouse strain differences in uterine CD45⁺ MC infiltrates at 7 to 10 days post-i.vag. infection with 4.5 × 10⁵ IFU of C. trachomatis NI1. Plots represent light scatter properties of infiltrates isolated from three individual mice from each strain. Samples were gated on CD45⁺ cells. All tissues were PCR positive for C. trachomatis and contained at least fivefold more CD45⁺ MC than did the control tissues. The numbers represent the ratio of large (R2) to small (R1) cells, and these values differed significantly between strains: BALB/c versus C3H, P < 0.01; and BALB/c nu/nu versus BALB/c, P < 0.001.

FIG. 5

Phenotypic variation in the composition of uterine CD45⁺ MC infiltrate at 7 days postinfection with 3.5 × 10⁵ IFU of C. trachomatis NI1. Uterine tissue was pooled from five individual mice from each strain, and the MC were prepared. Analysis was performed on gated CD45⁺ MC and, because of different levels of autofluorescence and isotype binding, large (R2) and small (R1) cells were analyzed separately and the results then combined. The percentage of cells binding isotype-matched control antibodies has been subtracted in all cases. Columns: ▨, CD4⁺; ⊠, CD8β⁺; □, CD4⁻ CD8β⁻.

FIG. 6

Phenotypic characterization of large (R2) MC from BALB/c uteri. Tissues were pooled from 10 animals at 8 days postinfection with 3.5 × 10⁵ IFU of C. trachomatis NI1, the MC were isolated, and the cells were gated for flow cytometry analysis on the basis of light scatter properties and expression of CD45. Histograms display the results for R2 CD45⁺ MC. Solid histograms show labeling with the antibody noted in the figure; open histograms show binding of an isotype-matched control. The figure summarizes the results of three separate experiments.

FIG. 7

Functional characterization of large (R2) MC from BALB/c uteri. Allogeneic (B10) T cells were stimulated by uterine MC obtained from a pool of 10 mice at 9 days postinfection with 3 × 10⁵ IFU of C. trachomatis N1 (A) or mature spleen DC isolated from uninfected BALB/c mice (B). Stimulator cells were irradiated (20 Gy) before addition to hanging-drop cultures. Each point represents the mean proliferation of triplicate cultures.