ABSTRACT: Cytokines are implicated in the development of inflammatory diseases such as endometriosis. This project was designed to test the hypothesis that specific cytokines that are secreted by macrophages, such as tumor necrosis factor ? (TNF?) and interleukin 1 beta (IL1?), cause gene expression changes in endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with TNF? (5ng/ml) ± IL1? (1ng/ml). DNA microarray and real time RT-PCR were used to study the gene expression changes in T-HESC cells. Two hundred and nineteen genes featuring in various gene ontologies were found to be differentially expressed in T-HESC cells treated with TNF? ± ILI?. The gene ontologies included functions expected to be associated with the development of endometriosis such as peptidases, cell adhesion, cell death/apoptosis, cell cycle, growth factors, cytoskeletal organization, defense/immune system, signal transduction, and transcriptional regulation. The differential expression of 4 genes (interleukin 8 (IL8), interleukin 6 (IL6), IL1? and matrix metalloproteinase (MMP3) was confirmed by real time RT-PCR. All four genes were up-regulated in response to TNF? ± ILI? in T-HESC cells. The effect of TNF? ± ILI? on migration and invasion of T-HESC cells, as measured with Boyden chambers, was not affected by treatment with these cytokines. Goal of the experiment: To examine differential gene expression in telomerase-immortalized human endometrial stromal cells (T-HESC) in response to treatment with tumor necrosis factor alpha (TNF?) ± interleukin 1? (IL1?). Keywords: cytokines, endometrial stromal cells, endometriosis Experimental factors: cytokine treatment Experimental design: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were treated with vehicle or with the cytokines, TNF? (5ng/ml) ± IL1? (1ng/ml). Three different passages of cells were collected for each treatment group to provide biological replicates. Quality control steps: The cRNA that was synthesized from each cell culture sample was used for hybridization to a single CodeLink (Applied Microarrays, Tempe, AZ) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were cultured cells of the telomerase-immortalized human endometrial stromal cell (T-HESC) line obtained from ATCC (CRL-4003). Manipulations of biological samples and protocols used: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO) with 10% fetal bovine serum albumin (FBS, Hyclone, South Logan, UT), insulin, selenious acid, transferrin, bovine serum albumin and linoleic acid (ITS+ Premix, BD Biosciences, Bedford, MA), puromycin (Cellgro, Manassas, VA) and penicillin/ streptomycin (Hyclone, South Logan, UT). After the cells reached 80% confluence they were starved for 24 hours in T-HESC starvation medium (DMEM+ ITS+ puromycin+ penicillin/streptomycin) before treatment. The cells were treated with vehicle or with TNF? (5ng/ml) alone, IL1? (1ng/ml) alone, or with the two cytokines combined at the same concentrations for 48 hours. Total RNA was then isolated from the cell cultures for use in the DNA microarray experiments. Each treatment was carried out with 3 different passages of cells. Technical protocols: The cultured endometrial cells were rinsed with PBS and TRI reagent (MRC) was added. The cells were scraped into tubes and centrifuged in the presence of bromochloropropane and sodium acetate. 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 was evaluated with the RNA 6000 NanoLabChip in an Agilent Bioanalyzer. Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total T-HESC cell 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 columns. 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 columns. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94°C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37°C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46°C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa 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 each and once in 0.05% Tween 20 for 20 sec. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
