Project description:Endometriosis is an inflammatory disease and bone marrow-derived cells are abundant in endometriotic lesions and in the peritoneal fluid of women with the disease. This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication contributes to the pathology of endometriosis. Changes in gene expression elicited by exposure to factors secreted by the opposing cell type were measured by DNA microarray to test this hypothesis. 716 named genes were differentially expressed in cultured endometrial stromal cells in response to factors secreted by macrophages. Genes that were up-regulated included IL8/CXCL8, MMP3, phospholamban, CYR61/CCN1, CTGF/CCN2, tenascin C, and NNMT, whereas integrin alpha 6 was down-regulated. In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by cultured endometrial stromal cells. The data document reciprocal communication between macrophages and endometrial stromal cells and suggest that interaction with macrophages stimulates the expression of genes in endometrial stromal cells that contribute to migration, adhesion, invasion, neovascularization and mitosis of endometrial cells that may support the establishment of endometriosis. Experiment Design: Goal of the experiment: To examine reciprocal communication between a telomerase-immortalized human endometrial stromal cell line (THESC, ATCC #CRL-4003) and U-937 macrophages (ATCC #CRL-1593.2 treated with phorbol ester to stimulate differentiation), and also to assess the response of THESC cells to estrogen plus progesterone. Keywords: human, cultured endometrial stromal cell line, macrophage Experimental factors: hormone treatment and treatment with macrophage-conditioned medium of endometrial stromal cells, treatment of macrophages with endometrial stromal cell conditioned medium Experimental design: T HESC cells were grown in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 10% charcoal-dextran treated fetal bovine serum albumin, 1% pen/strep, and 500 ng/ml puromycin. THESC cells were treated for 8 days with vehicle (0.1% ethanol) or estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate). On day 6, serum was removed from the culture medium. On day 7, half of the cells were treated with U-937 macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM phorbol myristate acetate (PMA) for 48 h to stimulate differentiation into macrophages. The U-937 macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. The experiment was repeated with three different passages of cells (passages 7, 9, and 12) to yield an experimental n of 3. The two experimental groups were macrophages treated with non-conditioned control medium and macrophages treated with THESC-conditioned medium. Quality control steps: The cRNA that was synthesized from each cultured cell passage and treatment was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: The samples were telomerase-immortalized human endometrial stromal cells and human U-937 monocytic cell line differentiation into macrophages by treatment with phorbol ester. The cell lines were obtained from American Type Culture Collection (#CRL-4003 and #CRL-1593.2). Manipulations of biological samples and protocols used: T HESC cells were grown in culture in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 2% charcoal/dextran treated fetal bovine serum, 1% penicillin/streptomycin, and 500 ng/ml puromycin. The cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + macrophage-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + macrophage-conditioned medium. On day 6, serum was removed from the culture medium of all cells. On day 7, half of the cells were treated with macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. To prepare macrophage-conditioned medium, U-937 (ATCC #CRL-1593.2) cells were grown in RPMI-1640 medium with 10% fetal bovine serum until the cells covered 80% of the flask surface. These U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. Differentiated macrophages were maintained in phenol red-free, fetal bovine serum-free DMEM/F12 medium supplemented with 1% penicillin/streptomycin for 48 hours. The cell suspension was centrifuged and the conditioned medium was collected, filtered, and stored at -80C. Before use, macrophage-conditioned medium was supplemented with 1% ITS+ and puromycin. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. The differentiated macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. Experimental factor: hormone treatment, treatment with macrophage-conditioned medium, and treatment with T-HESC cell conditioned medium Technical protocols: The cells were rinsed and TRI reagent (MRC) was added to the culture flasks. The cells were scraped into centrifuge tubes, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 1.0 microgram of T-HESC cell total RNA or 2 micrograms of macrophage total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

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