Differential regulation of CD4 lymphocyte recruitment between the upper and lower regions of the genital tract during Chlamydia trachomatis infection

Abstract

Genital infection with Chlamydia trachomatis results in both the local recruitment of protective immune responses and an inflammatory infiltrate that may also participate in tubal pathology. As a beginning to understanding the etiology of immune system-mediated tubal pathology, we evaluated the regional recruitment of lymphocyte subsets to different areas of the female genital tract (GT) over the course of a murine infection with the mouse pneumonitis agent of Chlamydia trachomatis (MoPn). Using flow cytometric techniques we found that the CD4 lymphocyte subset was preferentially recruited to the upper GT (oviduct and uterine horn) over the lower GT (cervical-vaginal region) throughout the course of MoPn infection. The influx of CD4 cells also correlated with the expression of endothelial cell adhesion molecules (ECAMs) and in vitro lymphocyte adherence in the upper GT. Interestingly, the expression of ECAMs in the lower GT was not maintained longer than 7 days after infection, even in the presence of viable chlamydiae. Taken together, these data suggest that regulatory mechanisms of lymphocyte recruitment differ between the upper and lower regions of the GT and may influence the clearance of chlamydiae and the development of tubal pathology.

FIG. 1

CD4 lymphocyte recruitment to different regions of the GT during infection with MoPn. GTs were harvested on various days throughout the course of a MoPn infection. They were dissected into three regions: the CV region, the UH, and the OD. Pooled tissue from five mice were stained for anti-CD4 and analyzed by flow cytometry. The percentage of CD4 cells present in each pool (unshaded histogram) after subtracting the value for the irrelevant control antibody (shaded histogram) is shown.

FIG. 2

Expression of VCAM-1 in different regions of the GT following MoPn infection. Frozen sections of GTs prepared from uninfected (top panels) and infected mice (bottom panels) were stained with an anti-VCAM-1 monoclonal antibody and visualized using immunoperoxidase histochemistry. Venules staining positive for VCAM-1 can be seen in the CV region 7 days after infection (arrow in bottom left panel), in the UH on day 14 (arrow in bottom middle panel), and in the OD by day 21 (arrow in bottom right panel).

FIG. 3

Quantitation of VCAM-1-expressing venules and viable chlamydiae in different regions of the GT throughout the course of MoPn infection. (A) The CV, UH, and OD regions from individual mice were homogenized and cultured for the isolation of chlamydiae. Each data point represents the mean ± standard error of the mean (SEM) of six values. (B) Venules expressing VCAM-1 from each GT region of two to six individual mice harvested on various days throughout MoPn infection were counted. Data are expressed as the means ± SEM. ∗, P < 0.05 (CV region on day 7 and UH on day 21) by ANOVA and Dunn's post-hoc test.

FIG. 4

Correlation of CD4 cells and VCAM-1 expression in the upper versus the lower GT. GTs from individual mice were divided into the upper tract (UH plus OD regions) and the lower tract (CV region) and stained for anti-CD4. The number of CD4 cells was paired with the number of VCAM-1-positive venules per square millimeter for days 0, 7, 14, 21, 35, and 49. Correlation coefficients were obtained using the Spearman rank order test; P < 0.04, n = 37 for the upper GT, and P = 0.156, n = 18 for the lower GT.

FIG. 5

Lymphocyte adherence on GT sections throughout MoPn infection. MLN cells from mice infected with MoPn were harvested on day 7. Single cell suspensions (2 × 10⁶ cells) were applied to freshly cut frozen GT sections harvested at various days during the course of MoPn infection. Adherent lymphocytes from 10 to 15 venules per section were counted. Cells from multiple sections for each time point from 2 to 4 mice were counted. Each data point represents the mean ± standard error of the mean (SEM) of the number of adherent lymphocytes from 30 to 120 individual venules. ∗, P < 0.05 for the CV region on day 7 and for both the CV region and UH plus OD on day 21 compared to adherence on venules in the UH and OD at day 0, when ECAMs are not expressed (ANOVA and Dunn's post-hoc test).

FIG. 6

Blocking of lymphocyte adherence in the upper and lower regions of the GT. MLN cells from mice infected with MoPn were harvested on day 7. Monoclonal antibodies against murine VCAM-1, ICAM-1, MAdCAM-1, and an isotype-matched control antibody were incubated on freshly cut GT frozen sections for 30 min at room temperature. Single cell suspensions (2 × 10⁶ cells) were applied to freshly cut frozen GT sections from the CV region on day 7 (A) or the UH plus OD regions on day 14 (B). Adherent lymphocytes from 10 to 15 venules per section were counted. Cells from multiple sections for each time point were counted. Each data point represents the mean ± standard error of the mean (SEM) of the number of adherent lymphocytes from 90 to 139 individual venules. ∗, P < 0.05 by ANOVA and Dunnett's (A) and Dunn's (B) post-hoc test.