Human Reproduction vol.7 no.9 pp. 1326-1328, 1992
Expression of aminopeptidase N and neutral endopeptidase on the endometrial stromal cells in endometriosis and adenomyosis
(Nichirei Co., Tokyo, Japan), My7/CD13 (Coulter Immunology, Hialeah, FL, USA), and LeuM3/CD14 (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA).
Department of Gynaecology and Obstetrics, Faculty of Medicine, and 2Chest Disease Research Institute, Kyoto University, Sakyo-ku, Kyoto 606, Japan
Results
'To whom correspondence should be addressed
Indirect immunofluorescence staining revealed that endometrial stromal cells (ESC) in the ectopic endometrium of patients with endometriosis or adenomyosis expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CDIO antigen, both of which are expressed on ESC in the normal endometrium throughout the menstrual cycle. Thus, ESC hi the ectopic endometrium resembled ESC in the normal endometrium not only morphologically but also antigenkally. Both peptidase antigens may be useful markers for the histologkal diagnosis of endometriosis and adenomyosis. Key words: adenomyosis/aminopeptidase N/endometriosis/ imrnunohistology/neutral endopeptidase
Introduction Recently we reported that human endometrial stromal cells (ESC) expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CDIO antigen, and suggested their usefulness as cell surface markers of ESC (Imai et al., 1992). In this study, we examined the ectopic endometrium immunohistologicaUy to determine whether or not ectopic ESC express these two peptidase antigens. Materials and methods Adenomyotic tissue samples from 12 women aged between 29 and 56 years old, tissue samples of ovarian chocolate cysts from four women between 36 and 47 years old, and small peritoneal tissue samples with blueberry spots from two 48-year-old women were examined. A definitive diagnosis of the disease was made based on the morphological evaluation of the haematoxylin/eosinstained tissue preparations. Informed consent was obtained from all the patients. Frozen tissue sections were prepared and stained by an indirect immunofluorescence method as described elsewhere (Imai et al., 1992). Antibodies used were MCS2/CD13, NU-N1/CD10, NU-Ter/CD2, NU-B2/CD20 and Bear- I/CD lib 1326
As shown in Figures 1 and 2, both aminopeptidase N/CD13 antigen and neutral endopeptidase/CDIO antigen were detected on the ESC of the ectopic endometria in all of the adenomyosis, ovarian endometriosis and peritoneal endometriosis cases examined. ESC of adenomyosis expressed CD 13 antigen very weakly in four out of 12 patients. Endometrial glandular cells in the ectopic endometrium expressed neither the CD 13 antigen nor the CD10 antigen. In the endometriotic foci, there were many macrophages, neutrophils, lymphocytes, and plasma cells. Some of these cells bore the aminopeptidase N/CD13 antigen and/or neutral endopeptidase/CDIO antigen, but they could be distinguished from ESC by using a combination of CDllb, CD14, CD2, and CD20 antibodies. In normal ovarian tissue, peritoneum, and myometrial layer, few CD 13- and CDlO-positive cells were detected except theca cells, which were strongly positive for the CD 13 antigen as reported previously (Fujiwara et al., 1992). Many cells expressing CD1 lb and/or CD14 antigens, probably monocytes/macrophages, were observed in and around the endometriotic foci (Figure 2c), but few in the adenomyotic foci (Figure lc). Few CD2-bearing cells (T-lymphocytes) and CD20-bearing cells (B-lymphocytes) were observed in and around the ectopic endometria (data not shown). Discussion The present study showed that ESC in the ectopic endometrium expressed both aminopeptidase N/CD13 antigen and neutral endopeptidase/CDIO antigen, as did ESC in the normal endometrium (Imai et al., 1992). However, aminopeptidase N expression on ESC was sometimes very weak in adenomyosis. This suggests that ectopic ESC resembles ESC in the normal endometrium, but some difference may exist between them. The diagnosis of endometriosis on microscopic examination is based on the presence of ectopic endometrial epithelium associated with endometrial stroma (Clement, 1987). The stroma constitutes an important element in endometriosis. Although the endometrial stromal cells have a characteristic morphology, other spindle cells such as those of the ovarian stroma bear some morphological resemblance to endometrial stroma (Ramzy, 1989). Haney (1987) reported that the endometrial stroma is © Oxford University Press
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
Kimitoshi Imai1, Hideharu Kanzaki, Hiroshi Fujiwara, Masatoshi Kariya, Norihiko Okamoto, Kenji Takakura, Mkhiyuki Maeda2 and Takahide Mori
Peptidase antigen expression in ectopic endometrtum
Fig. 2. Indirect immunofluorescence staining of ectopic endometrium in the ovary with monoclonal antibodies. Tissues were stained with: (a) MY7/CD13, (b) NU-N1/CD10, (c) LeuM3/CD14. Haematoxylin and eosin staining is shown in (d). Endometrial stromal cells (ESQ, but not endometrial gland (G), in the ectopic endometrium were positively stained with CD13 (a) and CD10 antibodies (b). Ovarian stroma (OS) was negative for CD13 and CD10 (a,b). CD14-positive cells, possibly tissue macrophages, were occasionally noticed (c). ( a - d ) Magnification X126.
1327
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
Fig. 1. Indirect immunofluorescence staining of ectopic endometrium in the myometrial layer with monoclonal antibodies. Tissues were stained with: (a) MCS2/CD13, (b) NU-N1/CD10, (c) LeuM3/CD14. Haematoxylin and eosin staining is shown in (d). Endometrial stromal cells (ESQ, but not endometrial gland (G), in ectopic endometrium were positively stained with CD 13 (a) and CD 10 antibodies (b). Myometrium (M) was negative for CD13 and CD10 (a,b). CD14-positive cells, possibly tissue macrophages, were rarely seen (c). ( a - d ) Magnification X126.
K.Imal et at.
Acknowledgements This work was partially supported by Grants-in-Aid for Scientific Research (no. 03454396) from the Ministry of Education, Sciences, and Culture of Japan and by the Shimizu Foundation Research Grant for 1990. References Bancroft.K., Williams.C.A.V. and Elstein.M. (1989) Minimal/mild endometriosis and infertility. A review. Br. J. Obstet. Gynaecol., 96, 454-460. Clement.P.B. (1987) Endometriosis, lesion of the secondary mullerian system, and pelvic mesothelial proliferations. In Kurman,R.J. (ed.), Blaustein 's Pathology of the Female Genital Tract. Springer- Verlag, New York, pp. 516-536. Dorken.B., MSller.P., Pezzutto.A., Schuwarz-Albiez,R. and Moldenhauer.G. (1989) B-cell antigens: CD10. In Knapp.W., Dorken.B., Gilks.W.R., Rieber.E.P., Schmidt.RlE., Stein,H. and von dem Bome,A.E.G.Kr. (eds), Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo, pp. 3 3 - 3 4 . Erdos.E.G. and Skidgel.R.A. (1989) Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J., 3, 145-151. Fujiwara.H., Maeda.M., Imai.K., Fukuoka.M., Yasuda,K., Horie,K., Takakura.K., Taii.S. and Mori.T. (1992) Differential expression of aminopeptidase N on human ovarian granulosa cells and thecal cells. J. Clin. Endocrinol. Metab., 74, 9 1 - 9 5 . Gadd.S. (1989) Cluster report: CD13. In Knapp,W., Dorken.B., Gilks.W.R., Rieber.E.P., Schmidt.R.E., Stein.H. and von dem Bome.A.E.G.Kr. (eds), Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo, pp. 782-784. Haney.A.F. (1987) Endometriosis: pathogenesis and pathophysiology. In Wilson.E.A. (ed.), Endometriosis. Alan R.Liss, Inc., New York, pp. 2 3 - 5 1 . 1328
Imai.K., Maeda.M., Fujiwara.H., Okamoto.N., Kariya.M., Emi,N., Takakura.K., Kanzaki.H. and Mori.T. (1992) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol. Reprod., 46, 328-334. Ramzy.I. (1989) Pathology. In Schenken.R.S. (ed.), Endometriosis. Contemporary Concepts in Clinical Management. J.B.Lippincott Company, Philadelphia, USA, pp- 4 9 - 8 1 . Sidorowicz,W., Zownir.O. and Behal.F.J. (1981) Action of human pancreas alanine aminopeptidase on biologically active peptides: kinin converting activity. Clin. Chem. Acta, 111, 6 9 - 7 9 . Received on 27 April 1992; accepted on July 9, 1992
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
particularly difficult to identify, because the individual stromal cells are frequently indistinguishable from the surrounding fibroblasts. Furthermore, endometriosis may be associated with and should be distinguished from endosalpingiosis, a lesion characterized by glands lined by benign endosalpingeal epithelium (Clement, 1987). Endosalpingiosis lacks endometrial stroma and the typical inflammatory response (Clement, 1987). In this study, immunohistological examination wim CD13 and CD10 antibodies in combination with other antibodies proved useful for the detection of ectopic ESC and for the differential diagnosis of endometriosis. Aminopeptidase N(EC3.4.11.2)/CD13 antigen and neutral endopeptidase (EC3.4.21.1 l)/CD10 antigen are membrane-bound peptidases, expressed on a variety of cells including some haemopoietk lineages of cells (Gadd, 1989; Dorken et al., 1989). They degrade many kinds of biologically active peptides (Sidorowicz et al, 1981; Erdos et al., 1989). The peptidases on the ectopic ESC might degrade biologically active peptides in inappropriate sites, which might lead to clinical manifestations such as decreased fertility even in mild or minimal endometriosis (Bancroft et al., 1989). Further studies on these peptidase enzymes in ectopic as well as in the normal endometrium may help clarify the pathophysiology of endometriosis and adenomyosis.
Expression of aminopeptidase N and neutral endopeptidase on the endometrial stromal cells in endometriosis and adenomyosis
(Nichirei Co., Tokyo, Japan), My7/CD13 (Coulter Immunology, Hialeah, FL, USA), and LeuM3/CD14 (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA).
Department of Gynaecology and Obstetrics, Faculty of Medicine, and 2Chest Disease Research Institute, Kyoto University, Sakyo-ku, Kyoto 606, Japan
Results
'To whom correspondence should be addressed
Indirect immunofluorescence staining revealed that endometrial stromal cells (ESC) in the ectopic endometrium of patients with endometriosis or adenomyosis expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CDIO antigen, both of which are expressed on ESC in the normal endometrium throughout the menstrual cycle. Thus, ESC hi the ectopic endometrium resembled ESC in the normal endometrium not only morphologically but also antigenkally. Both peptidase antigens may be useful markers for the histologkal diagnosis of endometriosis and adenomyosis. Key words: adenomyosis/aminopeptidase N/endometriosis/ imrnunohistology/neutral endopeptidase
Introduction Recently we reported that human endometrial stromal cells (ESC) expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CDIO antigen, and suggested their usefulness as cell surface markers of ESC (Imai et al., 1992). In this study, we examined the ectopic endometrium immunohistologicaUy to determine whether or not ectopic ESC express these two peptidase antigens. Materials and methods Adenomyotic tissue samples from 12 women aged between 29 and 56 years old, tissue samples of ovarian chocolate cysts from four women between 36 and 47 years old, and small peritoneal tissue samples with blueberry spots from two 48-year-old women were examined. A definitive diagnosis of the disease was made based on the morphological evaluation of the haematoxylin/eosinstained tissue preparations. Informed consent was obtained from all the patients. Frozen tissue sections were prepared and stained by an indirect immunofluorescence method as described elsewhere (Imai et al., 1992). Antibodies used were MCS2/CD13, NU-N1/CD10, NU-Ter/CD2, NU-B2/CD20 and Bear- I/CD lib 1326
As shown in Figures 1 and 2, both aminopeptidase N/CD13 antigen and neutral endopeptidase/CDIO antigen were detected on the ESC of the ectopic endometria in all of the adenomyosis, ovarian endometriosis and peritoneal endometriosis cases examined. ESC of adenomyosis expressed CD 13 antigen very weakly in four out of 12 patients. Endometrial glandular cells in the ectopic endometrium expressed neither the CD 13 antigen nor the CD10 antigen. In the endometriotic foci, there were many macrophages, neutrophils, lymphocytes, and plasma cells. Some of these cells bore the aminopeptidase N/CD13 antigen and/or neutral endopeptidase/CDIO antigen, but they could be distinguished from ESC by using a combination of CDllb, CD14, CD2, and CD20 antibodies. In normal ovarian tissue, peritoneum, and myometrial layer, few CD 13- and CDlO-positive cells were detected except theca cells, which were strongly positive for the CD 13 antigen as reported previously (Fujiwara et al., 1992). Many cells expressing CD1 lb and/or CD14 antigens, probably monocytes/macrophages, were observed in and around the endometriotic foci (Figure 2c), but few in the adenomyotic foci (Figure lc). Few CD2-bearing cells (T-lymphocytes) and CD20-bearing cells (B-lymphocytes) were observed in and around the ectopic endometria (data not shown). Discussion The present study showed that ESC in the ectopic endometrium expressed both aminopeptidase N/CD13 antigen and neutral endopeptidase/CDIO antigen, as did ESC in the normal endometrium (Imai et al., 1992). However, aminopeptidase N expression on ESC was sometimes very weak in adenomyosis. This suggests that ectopic ESC resembles ESC in the normal endometrium, but some difference may exist between them. The diagnosis of endometriosis on microscopic examination is based on the presence of ectopic endometrial epithelium associated with endometrial stroma (Clement, 1987). The stroma constitutes an important element in endometriosis. Although the endometrial stromal cells have a characteristic morphology, other spindle cells such as those of the ovarian stroma bear some morphological resemblance to endometrial stroma (Ramzy, 1989). Haney (1987) reported that the endometrial stroma is © Oxford University Press
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
Kimitoshi Imai1, Hideharu Kanzaki, Hiroshi Fujiwara, Masatoshi Kariya, Norihiko Okamoto, Kenji Takakura, Mkhiyuki Maeda2 and Takahide Mori
Peptidase antigen expression in ectopic endometrtum
Fig. 2. Indirect immunofluorescence staining of ectopic endometrium in the ovary with monoclonal antibodies. Tissues were stained with: (a) MY7/CD13, (b) NU-N1/CD10, (c) LeuM3/CD14. Haematoxylin and eosin staining is shown in (d). Endometrial stromal cells (ESQ, but not endometrial gland (G), in the ectopic endometrium were positively stained with CD13 (a) and CD10 antibodies (b). Ovarian stroma (OS) was negative for CD13 and CD10 (a,b). CD14-positive cells, possibly tissue macrophages, were occasionally noticed (c). ( a - d ) Magnification X126.
1327
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
Fig. 1. Indirect immunofluorescence staining of ectopic endometrium in the myometrial layer with monoclonal antibodies. Tissues were stained with: (a) MCS2/CD13, (b) NU-N1/CD10, (c) LeuM3/CD14. Haematoxylin and eosin staining is shown in (d). Endometrial stromal cells (ESQ, but not endometrial gland (G), in ectopic endometrium were positively stained with CD 13 (a) and CD 10 antibodies (b). Myometrium (M) was negative for CD13 and CD10 (a,b). CD14-positive cells, possibly tissue macrophages, were rarely seen (c). ( a - d ) Magnification X126.
K.Imal et at.
Acknowledgements This work was partially supported by Grants-in-Aid for Scientific Research (no. 03454396) from the Ministry of Education, Sciences, and Culture of Japan and by the Shimizu Foundation Research Grant for 1990. References Bancroft.K., Williams.C.A.V. and Elstein.M. (1989) Minimal/mild endometriosis and infertility. A review. Br. J. Obstet. Gynaecol., 96, 454-460. Clement.P.B. (1987) Endometriosis, lesion of the secondary mullerian system, and pelvic mesothelial proliferations. In Kurman,R.J. (ed.), Blaustein 's Pathology of the Female Genital Tract. Springer- Verlag, New York, pp. 516-536. Dorken.B., MSller.P., Pezzutto.A., Schuwarz-Albiez,R. and Moldenhauer.G. (1989) B-cell antigens: CD10. In Knapp.W., Dorken.B., Gilks.W.R., Rieber.E.P., Schmidt.RlE., Stein,H. and von dem Bome,A.E.G.Kr. (eds), Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo, pp. 3 3 - 3 4 . Erdos.E.G. and Skidgel.R.A. (1989) Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J., 3, 145-151. Fujiwara.H., Maeda.M., Imai.K., Fukuoka.M., Yasuda,K., Horie,K., Takakura.K., Taii.S. and Mori.T. (1992) Differential expression of aminopeptidase N on human ovarian granulosa cells and thecal cells. J. Clin. Endocrinol. Metab., 74, 9 1 - 9 5 . Gadd.S. (1989) Cluster report: CD13. In Knapp,W., Dorken.B., Gilks.W.R., Rieber.E.P., Schmidt.R.E., Stein.H. and von dem Bome.A.E.G.Kr. (eds), Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo, pp. 782-784. Haney.A.F. (1987) Endometriosis: pathogenesis and pathophysiology. In Wilson.E.A. (ed.), Endometriosis. Alan R.Liss, Inc., New York, pp. 2 3 - 5 1 . 1328
Imai.K., Maeda.M., Fujiwara.H., Okamoto.N., Kariya.M., Emi,N., Takakura.K., Kanzaki.H. and Mori.T. (1992) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol. Reprod., 46, 328-334. Ramzy.I. (1989) Pathology. In Schenken.R.S. (ed.), Endometriosis. Contemporary Concepts in Clinical Management. J.B.Lippincott Company, Philadelphia, USA, pp- 4 9 - 8 1 . Sidorowicz,W., Zownir.O. and Behal.F.J. (1981) Action of human pancreas alanine aminopeptidase on biologically active peptides: kinin converting activity. Clin. Chem. Acta, 111, 6 9 - 7 9 . Received on 27 April 1992; accepted on July 9, 1992
Downloaded from https://academic.oup.com/humrep/article-abstract/7/9/1326/622511 by University of Western Sydney Library user on 13 January 2019
particularly difficult to identify, because the individual stromal cells are frequently indistinguishable from the surrounding fibroblasts. Furthermore, endometriosis may be associated with and should be distinguished from endosalpingiosis, a lesion characterized by glands lined by benign endosalpingeal epithelium (Clement, 1987). Endosalpingiosis lacks endometrial stroma and the typical inflammatory response (Clement, 1987). In this study, immunohistological examination wim CD13 and CD10 antibodies in combination with other antibodies proved useful for the detection of ectopic ESC and for the differential diagnosis of endometriosis. Aminopeptidase N(EC3.4.11.2)/CD13 antigen and neutral endopeptidase (EC3.4.21.1 l)/CD10 antigen are membrane-bound peptidases, expressed on a variety of cells including some haemopoietk lineages of cells (Gadd, 1989; Dorken et al., 1989). They degrade many kinds of biologically active peptides (Sidorowicz et al, 1981; Erdos et al., 1989). The peptidases on the ectopic ESC might degrade biologically active peptides in inappropriate sites, which might lead to clinical manifestations such as decreased fertility even in mild or minimal endometriosis (Bancroft et al., 1989). Further studies on these peptidase enzymes in ectopic as well as in the normal endometrium may help clarify the pathophysiology of endometriosis and adenomyosis.
