Yukiko Tagashira, M. D. , Fuminori Taniguchi, M. D. , Tasuku Harada, M

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Interleukin-10 attenuates TNF-α–induced interleukin-6 production in endometriotic stromal cells Yukiko Tagashira, M.D., Fuminori Taniguchi, M.D., Tasuku Harada, M.D., Ayako Ikeda, M.D., Ayako Watanabe, M.D., Naoki Terakawa, M.D. Fertility and Sterility Volume 91, Issue 5, Pages 2185-2192 (May 2009) DOI: 10.1016/j.fertnstert.2008.04.052 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Endometriotic stromal cells exhibit repressed interleukin (IL)-10 gene expression. Gene expression of IL-10 in the endometriotic stromal cells was significantly lower than in the endometrial stromal cells. ∗P<.05. Data from real-time reverse-transcriptase polymerase chain reaction (mean ± SE) for IL-10 gene expression in endometrial or endometriotic stromal cells are shown. (B) Endometriotic stromal cells expressed IL-10 and its receptors protein. Immunocytochemical staining revealed expression of IL-10, IL-10 receptor (IL-10R) 1, and IL-10R2 proteins in the cytoplasm of endometriotic stromal cells. Fertility and Sterility 2009 91, 2185-2192DOI: (10.1016/j.fertnstert.2008.04.052) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of exogenous interleukin (IL)-10 on IL-6 gene expression and protein production in endometriotic stromal cells. Gene expression was measured by real-time reverse-transcriptase polymerase chain reaction. Concentrations of IL-6 protein in supernatants were measured by ELISA. Adding tumor necrosis factor (TNF)-α (0.1 ng/mL) significantly increased IL-6 gene expression and protein production. Results are expressed as percentage of control values. Pretreatment with IL-10 (10 ng/mL) significantly reduced the levels of TNF-α–induced IL-6 gene expression and protein production. Bars represent the mean ± SE. The mean and SE were calculated from each patient's endometriotic stromal cell culture (n = 12). The cells were used for experiments during one- to two-cell passages. ∗P<.05. Fertility and Sterility 2009 91, 2185-2192DOI: (10.1016/j.fertnstert.2008.04.052) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Effect of IL-10 on phosphorylation of ERK1/2, JNK1/2, and IκB proteins in endometriotic stromal cells. The subconfluent endometriotic stromal cells were pretreated with IL-10 (10 ng/mL) within 1 h, and then tumor necrosis factor (TNF)-α (0.1 ng/mL) was added for 10 min. Proteins were separated by electrophoresis on a 4%-to-10% gradient polyacrylamide gel and immunoblotted using antiphospho-p44/42 MAP kinase (Thr202/Tyr204) antibody, antiphospho-SAPK/JNK (Thr183/Tyr185) antibody, and antiphospho-IκBα(Ser32) antibody. TNF-α enhanced the expression of phosphorylation of these signal transduction molecules. Exogenous IL-10 attenuated these phosphorylated protein expressions. (B) Effect of IL-10 on phosphorylation of STAT1, STAT3, and STAT5 proteins in endometriotic stromal cells. Subconfluent endometriotic stromal cells were exposed to IL-10 (10 ng/mL) for 0, 1, 5, 10, 15, 30, or 60 min. Phospho-STAT3 was enhanced by exogenous IL-10. In contrast, IL-10 did not change the expression of phospho-STAT1 and phospho-STAT5 (not shown). Fertility and Sterility 2009 91, 2185-2192DOI: (10.1016/j.fertnstert.2008.04.052) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Effect of the JAK inhibitor on tumor necrosis factor (TNF)-α–induced interleukin (IL)-6 gene expression. Subconfluent endometriotic stromal cells were preincubated for 24 h with AG490 (30 μmol/L) and incubated in the presence of TNF-α (0.1 ng/mL) for 4 h. Interleukin-10 was exposed for an hour before adding TNF-α. The IL-6 gene expression was assessed using reverse-transcriptase polymerase chain reaction. (B) Effect of IL-10 on translocation of intranuclear p65. The concentration of intranuclear p65 protein was determined using NF-κB transcription factor ELISA. TNF-α (0.1 ng/mL) markedly increased intranuclear p65 concentration. Adding IL-10 (10 ng/mL) significantly reduced p65 concentration. ∗P<.05. Fertility and Sterility 2009 91, 2185-2192DOI: (10.1016/j.fertnstert.2008.04.052) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions