Possible involvement of signal transducer and activator of transcription-3 in cell–cell interactions of peritoneal macrophages and endometrial stromal.

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Possible involvement of signal transducer and activator of transcription-3 in cell–cell interactions of peritoneal macrophages and endometrial stromal cells in human endometriosis Fumiko Itoh, M.D., Yoshihiro Komohara, M.D., Ph.D., Kiyomi Takaishi, M.D., Ph.D., Rituo Honda, M.D., Ph.D., Hironori Tashiro, M.D., Ph.D., Satoru Kyo, M.D., Ph.D., Hidetaka Katabuchi, M.D., Ph.D., Motohiro Takeya, M.D., Ph.D. Fertility and Sterility Volume 99, Issue 6, Pages 1705-1713.e1 (May 2013) DOI: 10.1016/j.fertnstert.2013.01.133 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Phenotypes of macrophages in ascites from patients with and without endometriosis. (A) Cytospin specimens of ascites from patients with endometriosis contained many CD68+ and CD163+ macrophages. The total number of macrophages was calculated from the number of living cells in the ascites and the percentage of CD68+ or CD163+ cells in the living cells. (B) CT = control group; EM = endometriosis group. (C) CD163 expression was scored, and the difference between the endometriosis and control groups was evaluated. Data were analyzed via the Mann-Whitney U test. Fertility and Sterility 2013 99, 1705-1713.e1DOI: (10.1016/j.fertnstert.2013.01.133) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Proliferation of ESCs cocultured with M2 macrophages (Mac). (A) Immortalized ESCs or pESCs were cocultured with and without M2 macrophages. Specific CD204 immunostaining distinguished macrophages from ESCs in cocultures. (B, C) Immortalized ESCs were cocultured with and without M2 macrophages. Endometrial stromal cell proliferation was evaluated via BrdU labeling. Ratios (%) of BrdU+ ESCs were counted after double immunostaining for BrdU and CD204. In addition, BrdU incorporation into ESCs was evaluated using ELISA. (D) After culture with CM from cultured macrophages (mCM) or cocultured cells (cCM), iESC proliferation was evaluated via WST assays. Data were analyzed using Student’s t test. ∗P< .05. Fertility and Sterility 2013 99, 1705-1713.e1DOI: (10.1016/j.fertnstert.2013.01.133) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Stat3 activation in ESCs cocultured with M2 macrophages (Mac). (A) Cocultured cells and control iESCs were double-immunostained for pStat3 and CD10 (an ESC marker). (B) Immortalized ESCs were stimulated with CM from cultured macrophages (mCM) or cocultured cells (cCM), and Stat3 phosphorylation was analyzed using Western blotting. (C) Following Stat3, protein was silenced in iESC, coculture was started, and total numbers of iESC were evaluated. (D) The inhibitory effect of a Stat3 inhibitor, corosolic acid (CA; final concentration, 10 μM), on ESC proliferation was investigated using WST assays. Data were analyzed using Student’s t test. ∗P< .05. Fertility and Sterility 2013 99, 1705-1713.e1DOI: (10.1016/j.fertnstert.2013.01.133) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunostaining of pStat3 and CD163 in human endometriotic lesions and normal endometrium. (A) Tissue sections of pelvic endometriotic lesions were stained with anti-pStat3 and anti-CD163 antibodies. (B) Sections of normal endometrium from patients with uterine fibromas were stained with anti-pStat3 antibody. Data were analyzed using Student’s t test. Fertility and Sterility 2013 99, 1705-1713.e1DOI: (10.1016/j.fertnstert.2013.01.133) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Cytokine secretion from cocultured cells and control ESCs. (A) Cytokine secretion was evaluated with the cytokine array kit. Granulocyte macrophage–CSF, RANTES, IL-1RA, and MCP-1 production showed greater up-regulation in CM from cocultured cells (cCM) compared with cultured iESC medium (eCM). (B) Expressions of CCR2, GM-CSF, and GM-CSFR mRNAs were analyzed by PCR. Macrophages and iESCs evidenced GM-CSF and GM-CSFR mRNAs. (C) Immortalized ESCs were cultured with or without GM-CSF, and the percentage of total cells was evaluated with WST assays. Mac = macrophage. Data were analyzed using Student’s t test. ∗P< .05. Fertility and Sterility 2013 99, 1705-1713.e1DOI: (10.1016/j.fertnstert.2013.01.133) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions