Laser capture microdissection and cDNA array analysis of endometrium identify CCL16 and CCL21 as epithelial-derived inflammatory mediators associated with endometriosis

Abstract

Background: Understanding the pathophysiology of chemokine secretion in endometriosis may offer a novel area of therapeutic intervention. This study aimed to identify chemokines differentially expressed in epithelial glands in eutopic endometrium from normal women and those with endometriosis, and to establish the expression profiles of key chemokines in endometriotic lesions.

Methods: Laser capture microdissection isolated epithelial glands from endometrial eutopic tissue from women with and without endometriosis in the mid-secretory phase of their menstrual cycles. Gene profiling of the excised glands used a human chemokine and receptor cDNA array. Selected chemokines were further examined using real-time PCR and immunohistochemistry.

Results: 22 chemokine/receptor genes were upregulated and two downregulated in pooled endometrial epithelium of women with endometriosis compared with controls. CCL16 and CCL21 mRNA was confirmed as elevated in some women with endometriosis compared to controls on individual samples. Immunoreactive CCL16 and CCL21 were predominantly confined to glands in eutopic and ectopic endometrium: leukocytes also stained. Immunoreactive CCL16 was overall higher in glands in ectopic vs. eutopic endometrium from the same woman (P < 0.05). Staining for CCL16 and CCL21 was highly correlated in individual tissues.

Conclusion: This study provides novel candidate molecules and suggests a potential local role for CCL16 and CCL21 as mediators contributing to the inflammatory events associated with endometriosis.

Figure 1

Laser capture microdissection. (A) Frozen section of eutopic endometrial tissue from an endometriosis patient (mid-secretory phase) stained with HistoGene™. The epithelial glands were isolated with laser capture microdissection (LCM). (B) Tissue section following LCM demonstrating the precision in dissection and cell capture. (C) RNA yields from microdissected glands expressed as total RNA (ng) per number of gland from each tissue sample. Microdissected glandular RNA samples from control (C, n = 4) and endometriosis (E, n = 4) tissues were separately, reverse transcribed and a uterine epithelial cell-specific gene (HtrA3) was amplified using PCR to assess RNA quality (insert).

Figure 2

Real-time PCR quantitation of CCL16 and CCL21 mRNA expression in control and endometriosis subjects. (A) mRNA expression of CCL16 and (B) CCL21 was measured in glandular RNA samples from individuals in control (■) and endometriosis cohorts (■, ●, ▲ representing the 3 patients' individual data). Data is presented as relative units where chemokine expression has been normalized to 18S RNA expression for each sample (n = 3 per group, RT-PCR performed in duplicates). The average value for each subject group is indicated with a horizontal line. * denotes a statistically significant difference between the 2 groups (P = 0.049, Mann-Whitney U-test).

Figure 3

Immunostaining for CCL16 and CCL21 in eutopic and ectopic tissue from control and endometriosis subjects. Staining intensity for (A) CCL16 and (B) CCL21 in glandular epithelium of normal mid secretory endometrium (n = 12) (■) and in glandular epithelium in paired eutopic (▲) and ectopic (▼) endometrium from women with endometriosis (n = 10), also in the mid-secretory phase of the menstrual cycle. Open symbols represent subjects whose samples were used in the gene array study. Staining intensity was scored on a scale of 0 (no stain) to 4 (intense stain). Scores are shown for individual samples. Bars represent mean values. * denotes a statistical difference between 2 samples from the same patients (P = 0.047, Wilcoxon Matched-Pairs Signed-Ranks Test).

Figure 4

Immunostaining for CCL16 and CCL21. Representative tissues immunostained for CCL16 (A, C, E) and CCL21 (B, D, F). CCL16 and CCL21 staining, in control eutopic endometrial tissue (A, B), eutopic tissue (C, D), and matched ectopic lesions (E, F) from the same endometriosis patient. Leukocytes stained for CCL21 are shown in the insert in (F). (G, H) Endometrial sections with non-immune IgG substituted for the primary antibodies, as negative controls. Scale bars represent 200 μm.