Stanczyk et al.

I I [3-hydroxyandrostenedione by human adrenal homog­enates. J Clin Endocrinol Metab 1972;34:215-24.

14. Chang L, Stanczyk FZ, Cassidenti DL, Putz Z, Lobo RA. Is the ovary a source of 11[3-hydroxyandrostenedione among hyperandrogenic women? [Abstract 435]. Pre­sented at the thirty-eighth annual meeting of the Society

December 1991 Am J Obstet Gynecol

for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991.

IS. Carmina E, Levin JH, Malizia G, Lobo RA. Ovine corti­cotropin-releasing factor and dexamethasone responses in hyperandrogenic women. Fertil Steril 1990;54:245-50.

Effects of peritoneal macrophages from patients with endometriosis on the proliferation of endometrial carcinoma cell line ECC-l

Ren-jie Zhang, MD,. Robert A. Wild, MD,. Diane Medders, BS,. and

Subba Rao Gunupudi, PhDb

Oklahoma City, Oklahoma, and Sunnyvale, California

Endometriosis has been shown to be associated with increased number and activity of peritoneal macrophages. The peritoneal macrophage-conditioned media from 33 women with or without endometriosis were studied for their effects on an endometrial carcinoma cell line, ECC-1. The media from six of six stage III/IV cases demonstrated a mitogenic effect, which was blocked by an antibody to epidermal growth factor receptor. However, the conditioned media from seven of nine stage 1111 cases and 14 of 18 normal women did not show a mitogenic effect. The difference between stage III/IV and the other two groups was significant (p < 0.01). The incorporation of tritium-thymidine was three times higher with the media from stage III/IV cases, as compared with that of controls. When purified cytokines were tested in the tritium-thymidine uptake assay, only epidermal growth factor-transforming growth factor-a was mitogenic on ECC-1, whereas tumor necrosis factor, interleukin-1, and platelet-derived growth factor had no effect. Thus peritoneal macrophages in patients with endometriosis may play an important role in the progression of endometriosis, and the noted effects could be mediated by epidermal growth factor or a related growth factor. (AM J OBSTET GYNECOL 1991 ;165:1842-6.)

Key words: Cytokine, epidermal growth factor, endometriosis, endometrial carcinoma cell line

Endometriosis is a common disease in reproductive­aged women and is often associated with infertility. Re­cent studies l

.s have shown that women with this disease

have an increased number and activity of peritoneal macrophages. Growth factors (cytokines) from these

macro phages or peritoneal fluid could decrease fertility by damaging gametes or embryos.6, 7 However, whether

the macrophages produce a cytokine(s) to promote the

From the Section of Reproductive Endocrinology and Infertility, De­partment of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center," and Adeza Biomedical.' Presented in part at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. Reprint requests: Robert A. Wild, MD, OUHSC, Department ofOb­stetrics and Gynecology, PO Box 26901, 4SP720, Oklahoma City, OK 73190. 616133550

1842

ectopic growth of endometrial cells or create a hostile environment by inhibiting these cells as a nonspecific

inflammatory response is not clear. Stromal cell prolif­eration is stimulated by peritoneal fluid,S so it is spec­

ulated that the development of endometriosis may be

stimulated by a growth factor(s).

To determine whether a peritoneal macrophage-de­rived cytokine(s) from women with endometriosis has

growth-promoting activity on endometrial epithelial cells, an endometrial epithelial cell line, ECC-I,g was used for an in vitro proliferation study. To determine which cytokine has mitogenic activity on ECC-I, several

commercial preparations of cytokines were compared. These preparations were tumor necrosis factor (TNF­

a), interleukin-l (IL-I), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF)-trans­

forming growth factor-a (TGF-a).

Volume 165 Number 6, Part 1

Material and methods

The collection and use of human tissue was approved by the Institutional Review Board of the University of Oklahoma Health Science Center. Peritoneal fluid was obtained from 15 patients with laparoscopically staged endometriosis (Revised American Fertility Society clas­sification, 1985), Nine were stage II II and 6 were stage III/IV Of 9 with stage I1II, 7 were infertile; of 6 with stage IIIIIV, 4 were infertile. Of 18 fertile women un­dergoing sterilization without endometriosis, 5 had adhesions and 13 had a normal pelvis discovered at surgery; 8 were in the follicular phase and 10 were in the luteal phase of the cycle. Of 9 with stage II II, 4 were in the follicular phase and 5 were in the luteal phase. Of 6 stage IIIIIV endometriosis patients, 3 were in the follicular phase and 3 were in the luteal phase.

Macrophages were isolated from peritoneal fluid by layering 1: 1 diluted samples over Ficoll-Paque (Phar­macia LKB, Piscataway, N.J.) gradients and centrifuged at 400g at 18° to 20° C for 30 minutes. The macrophage fractions were washed twice with Hanks' balanced salt solution, spun at 60 to 100g for 10 minutes, and then resuspended in Dulbecco's modified Eagle's-high-glu­cose medium (Hazleton, Denver, Pa.) with 0.2% lact­albumin hydrolyzate (Gibco, Grand Island, N.Y.). The suspension was plated at 7.5 X 105 cells per well in the 1.5 em diameter tissue culture wells (Falcon, Los An­geles) for 1 hour. At the end of preincubation >90% of the cells were adherent. There was no difference in adherence between macrophage suspensions. The non­adherent cells were removed, and the media were then replaced with 1.5 ml of control medium, Dulbecco's modified Eagle's medium-nutrient mixture F-12 Ham with 1 % fetal bovine serum (Sigma, St. Louis), and in­cubated as an adherent monolayer for 24 hours in 5% carbon dioxide at 37° C in a humidified incubator. Cell viability was assessed by microscopic visualization of cell morphologic types and adherence. Macrophages rep­resented >90% of all the cells stained by Wright­Giemsa. The macrophage-conditioned media were col­lected, spun at 1500 rpm at room temperature for 10 minutes, and stored at - 20° C until use.

A clonal continuous monolayer of the human en­dometrial carcinoma cell line ECC-l, which was derived from the well-differentiated estrogen-sensitive tumor EnCa-l0l grown in nude mice, was used for this study because of the close similarity between the endometrial epithelial cells and ECC-l cells.9

-11 The ECC-l cells were

plated in the 96-well tissue culture plates (Corning Glass Works, Corning, N.Y.) at a density of 1.5 x 105 per well and grown to 70% confluence in nutrient mixture F-12 Ham with 10% fetal bovine serum. The nutrient mixture F-12 Ham was supplemented with glucose 0.4 mg/ml (Sigma), insulin 0.2 U Iml (Collaborative Re-

Peritoneal macrophages and proliferation of endometriosis 1843

s ;J

800 ;a v E 700 '0 .... +'

600 ~ 0 tJ '- 500 0

~ v 400

..'<: Ol +' 0.. 300 ;J

<lJ ~ 200 ;a § ~

:>, 100 ..•.. ..c: -' ... I &0:-. ...

::r: 0 C'J Control I/II III/IV

Endometriosis

Fig. 1. Effect of macrophage-conditioned media from patients with and without endometriosis on tritium-thymidine incor­poration of ECC-l cells. Cells were harvested 24 hours after exposure to macrophage-conditioned media or control media. Difference of DNA synthesis between treatment with macro­phages from patients with stage Ill/IV endometriosis versus other two groups is highly significant (p < 0.01).

search Inc., Bedford, Mass.), human transferrin 25 jJ.g/ml (Sigma), cholera toxin 2.5 ng/ml (Sigma), estra­diol 0.2 ng/ml (Sigma), 1 % Pen/Strep mixture (Gibco), and L-glutamine 143 jJ.g/ml (Gibco). Two days after plating, the serum-enriched media were replaced by control media (Dulbecco's modified Eagle's medium­nutrient mixture F-12 Ham, 1 % fetal bovine serum) for 18 hours before addition of the macrophage­conditioned media or media supplemented with var­ious concentrations of recombinant human TNF-a (Amgen, Thousand Oaks, Calif.), IL-l (Accurate Chemicals, Westbury, N.Y.), PDGF (PDGF-BB, Gen­zyme, Boston), EGF (Genzyme), orTGF-a(Gibco). The cells were then continuously incubated for another 24 hours.

In another set of experiments ECC-l cells were pre incubated with a monoclonal antihuman EGF re­ceptor antibody (10 jJ.g/ml, Genzyme) for 1 hour. Then control medium, EGF 10 ng/ml in control medium, or macropahge-conditioned medium from five patients with stage IIIIIV endometriosis was added to ECC-l cells, and the cells wre incubated for 24 hours. Appro­priate controls were run without pretreatment with the monoclonal antibody.

Tritium-thymidine (ICN, Irvine, Calif., 0.1 jJ.Ci per well, 10 Ci/mmollL) was added 6 hours before the cells were harvested. The cell cultures were terminated with a semiautomatic cell harvester (Cambridge Technology, Watertown, Mass.). Incorporation of the radioactivity

1844 Zhang et al. December 1991 Am J Obstet Gynecol

100000,------------------------------------------

l -~ ...... E c. ~

~ as -c. 10000 :s Q)

.5 :2 E ;>. .c -I I

CO")

EGF (ng/mJ)

-B- TGF- Q'(ng/mJ)

--*- IL-l (u/mJ)

-+- PDGF (ng/ml

-b- TNF- Q' (u/mJ)

1000L-~~~~--~JJ~ll--L-LLULlli--~LLLUlll_~I_LI~I~I~!I~!11

o 10 100 1000 10000

Growth Factor Added

Fig. 2. Dose-dependent effect of cytokines on tritium-thymidine uptake by ECC-1 cells. EGF - TGF­a promoted tritium-thymidine uptake in a dose-dependent manner, but IL-1, PDGF, and TNF-a did not.

into cellular deoxyribonucleic acid (DNA) was mea­sured by liquid scintillation counting.

To evaluate the effect of several cytokines on cell viability, total cell number, and cellular protein synthe­sis, ECC-l cells were cultured in 1.5 em diameter tissue culture plates at a concentration of 3 x 105 per wei!. Again, after the cells reached 70% confluence, they were subsequently incubated in control medium for 18 hours. The media were replaced with media supple­mented with EGF (10 ng/ml), TNF-a (10 Vlml), IL-l (10 Vlml), or PDGF (50 ng/ml) and incubated for 24 hours. Cell viability was then tested by try pan blue ex­clusion. Total cell numbers were counted on a hemo­cytometer, and protein concentrations were deter­mined by protein assay (Bio-Rad, Richmond, Calif.).

The Wilcoxon rank-sum test was used for compari­son of the mitogenic activities by macro phages from different patient groups.

Results

Macrophage-conditioned media from 6 of 6 patients with stage III/IV endometriosis demonstrated mito­genic activity to the target cell, ECC-l. Seven of 9 with stage II II and 14 of 18 without the disease did not have the mitogenic effect (Fig. 1). The difference between stage IIIIIV and the other two groups was highly sig­nificant (p < 0.01). The incorporation of tritium-thy­midine by the target cells was threefold higher with the medium from the stage IIIIIV patients as compared with that from the controls. There was no significant difference in mitogenic activity between the macro­phage-conditioned media from endometriosis stage 1111 and controls.

The mitogenic effect of EGF - TGF-a on target cell

DNA synthesis was dose dependent at I to 1000 ng/m!. In comparison, TNF-a (1 to 1000 Vlml), IL-l (1 to 1000 V Iml), or PDGF (1 to 1000 ng/ml) showed no effect (Fig. 2). Viability of cells was >95% after the incubation period with the cytokines. Increases in total cell count and protein content were found only with the EGF (10 ng/ml) treatment, not with IL-l (10 Vlml), TNF-a (10 Vlml), or PDGF (50 ng/ml) (data not shown).

Monoclonal anti-EGF-receptor antibody (10 fLg/ml) caused >50% inhibition of the mitogenic activity of EGF (10 ng/ml, Fig. 3, B) and of macrophage-condi­tioned media from five of five patients with stage III/IV endometriosis (Fig. 3, C to G). The monoclonal antibody by itself had no significant effect on mitogenic activity (Fig. 3, A).

Comment

The association between endometriosis and an in­crease in the number and activity of peritoneal mac­rophages has led investigators to question whether these macrophages play an important role in the patho­genesis of the disease. Cytokines derived from perito­neal macrophages of patients with endometriosis pro­duce different effects on various target cells. They have a proliferative effect on lymphocytes, lymphoma cells, and 3T3 fibroblasts; the mediator is thought to be IL­l, PDGF, or fibroblast growth factorY·!4 But cytokines have a cytolytic effect on L929 fibroblasts and WEHI-164 fibrosarcoma cells, which are TNF-a-sensitized target cells.!s, !6 Peritoneal fluid from patients with en­dometriosis has been shown to stimulate the prolifer­ation of human endometrial stromal cells."

To study the effect of macrophage-derived cyto-

Volume 165 Number 6, Part 1

Peritoneal macrophages and proliferation of endometriosis 1845

_ Control media I22Ll mAb to EGF-R (10 ug/ml) added &S:'J EGF 10 ng/ml

0 o macrophage conditioned medium Stage III/IV 0

20 c: ..--<

X 18

S 16

0, 14 u OJ 12 ~ (1j

10 -' 0, ;:i 8 OJ ~ 6

"0

S 4 >.

2 .c -'

! 0 ::r: C'J A B c D G E F

Fig. 3. Neutralization of monoclonal anti-EGF-receptor antibody (mAB to EGF-R) on ECC-l cells. mAB to EGFR was added to control medium (A), EGF 10 ng/ml (B), or macrophage-conditioned media from patients with stage IIIIIV endometriosis (C to G). Antibody caused >50% inhibition of mitogenic activity of commercially obtained EGF and of macrophage-derived media from five pa­tients with endometriosis. Antibody by itself had no significant effect on mitogenic activity (A).

kine(s) from patients with endometriosis on endome­trial epithelial cells, the ECC-I cell model was used, because an adequate number of purified epithelial cells from human endometrial tissue could not be grown. The ECC-l cells are well-differentiated homogeneous epithelial cells that are cytokeratin positive (epithelial cell marker) and vimentin negative (stroma cell marker) by immunohistochemical methods.9 We have shown that antiendometrial antibodies from patients with en­dometriosis react with both endometrial glandular ep­ithelial cells and ECC-l cells but not with endometrial stroma cells.1O The close physiologic similarity between endometrial epithelial cells and ECC-l cells is observed in their response to estrogen, progesterone, and inter­feron gamma.9.!!

Macrophage-conditioned medium from patients with stage IIIIIV endometriosis stimulated ECC-l cell proliferation. Furthermore, a monoclonal antibody to EGF receptor blocked the mitogenic effect of the mac­rophage-conditioned medium. Because the macro­phages were cultured in the absence of exogenous stim­ulants in vitro, these macropahges were likely to have been stimulated in the peritoneal cavity of the patients before they were studied. Therefore it is reasonable to speculate that, in the normal pelvis or in milder stages of endometriosis, the activity and secretion of macro­phages might not favor the growth of ectopic endo­metrial epithelial cells. However, during the progres­sion of endometriosis, as the disease becomes more se­vere, macrophages might receive sufficient stimuli to

be altered and reach certain levels of maturation. The macro phages might then secrete growth-stimulating substances into the peritoneal cavity, to which the ec­topic cells of endometrial origin exhibit stimulated growth activity. One of the mediators might be related to EGF - TGF-a or a cytokine that reacts with the EGF receptor.

Among the tested commercial cytokines, EGF - TGF­a was found to enhance proliferation, but TNF-a, IL­l, and PDGF did not. EGF and TGF-a are significantly homologous!7.!8 because they bind the same receptor.!9 Both are potent mitogens for a variety of cultured cells. In vitro, the proliferation of mouse uterine epithelial cells is enhanced by EGF but not by PDGF, fibroblast growth factor, or nerve growth factor.·o The receptor for EGF has been found in the rat uterus·! and in stromal and glandular epithelial cells of the human uterus. EGF is thought to be essential for growth of normal and neoplastic endometrium!· Induction of transcription and secretion of TGF-a by activated hu­man monocytes has been reported recently!' However, it is not known whether macrophages produce EGF.

Peritoneal macrophage-derived cytokine(s) from pa­tients with advanced stages of endometriosis and com­mercial EGF - TGF-a proliferated the endometrial ep­ithelial cell line ECC-l in vitro. These results suggested that the mediator in macrophage-conditioned media might be related to EGF - TGF-a. However, the mito­genic effect from the macrophage-conditioned me­dium, which was blocked by antibody to EGF receptor,

1846 Zhang et al.

could be due to other factors that work in synergy with EGF or to another factor that binds to EGF. Identifi­cation of these specific mediator(s) is currently under investigation.

We thank Dr. P. G. Satyaswaroop for the generous gift of the ECC-I cell line. We thank Dr. Reichlin, Mr. J. M. Ojago, and Dr. M. Hill for technical advice.

REFERENCES

1. Halme ], Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macro phages in infertile women with mild endometriosis. AM] OBSTET GYNECOL 1983;145:333-7.

2. Haney AF, Misukonis MA, Weinberg ]B. Macrophages and infertility. Oviductal macrophages as potential me­diators of infertility. Ferti! Steril 1983;39:310-5.

3. Zeller ]M, Henig I, Radwanska E, Dmowski WP. En­hancement of human monocyte and peritoneal macro­phage chemiluminescence activities in women with en­dometriosis. Am] Reprod Immunol 1987;13:78-83.

4. Olive DL, Weinberg ]B, Haney AF. Peritoneal macro­phages and infertility: the association between cell num­ber and pelvic pathology. Ferti! Steril 1985;44:772-7.

5. Hill]A, Faris HMP, Schiff I, Anderson DJ. Characteriza­tion of leukocyte subpopulation in the peritoneal fluid of women with endometriosis. Ferti! Steril 1988;50:216-22.

6. Chacho K], Chacho MS, Andresen P], Scommegna A. Peritoneal fluid patients with and without endometriosis prostanoids and macrophages and their effect on the spermatozoa penetration assay. AM ] OBSTET GYNECOL 1986;154:1290-9.

7. Morcos RN, Gibbons WE, Findley WE. Effect of perito­neal fluid in vitro, cleavage of 2-cell mouse embryos: pos­sible role in infertility associated with endometriosis. Fertil Steril 1985;44:678-83.

8. Surrey ES, Halme ]. Effect of peritoneal fluid from en­dometriosis patients on endometrial stromal cell prolif­eration in vitro. Obstet Gynecol 1990;76:792-7.

9. Satyaswaroop PC, Sivagarajah A, Zaino RJ, MordeI R. Hormonal control of growth of human endometrial car­cinoma in the nude mouse model. In: Bresciani F, King RGF, Lippman ME, Raynaud ]P, eds. Progress in cancer research and therapy. New York: Raven Press, 1988;35:430-5.

10. Wild RA, Satyawaroop PG, Shiver AC. Epitheliallocaliza-

December 1991 Am J Obstet Gynecol

tion of antiendometrial antibodies associated with endo­metriosis. Am] Reprod ImmunoI1987;13:62-5.

11. Tabibzadeh SS, Satyaswaroop PG, Rao PN. Antiprolifer­ative effect of interferon-r in human endometrial epithe­lial cells in vitro: potential local growth modulatory role in endometrium. ] Clin Endocrinol Metab 1988;67: 131-8.

12. Hill] A, Anderson D]. Lymphocyte activity in the presence of peritoneal fluid from fertile women and infertile women with and without endometriosis. AM ] OBSTET GYNECOL 1989;161:8614.

13. Fakih H, Baggett B, Holtz G,Tsang KY, Lee ]C, William­son HO. Interleukin-l: a possible role in the infertility associated with endometriosis. Ferti! Steril 1987;47: 213-7.

14. Halme], White C, Kauma S, Estes], Haskill S. Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. ] Clin Endocrinol Metab 1988;66: 1044-9.

15. Halme J. Release of tumor necrosis factor-a by human peritoneal macro phages in vivo and in vitro. AM] OBSTET GYNECOL 1989;161:1718.

16. Eisermann], Odem RR, Gast M], Collins ]L, Pineda J. Tumor necrosis factor in peritoneal fluid of women un­dergoing laparoscopic surgery. Ferti! Steril 1988;50: 573-9.

17. Lee DC, Rose TM, Webb NR, Todaro GJ. Cloning and sequence analysis of a cDNA for rat transforming growth factor-a. Nature 1985;313:489-91.

18. Marquardt H, Hunkapiller MW, Hood LE, Todaro GJ. Rat transforming growth factor type I: structure and re­lation to epidermal growth factor. Science 1984;223: 1079-82.

19. Cohen S, Ushiro H, Stoscheck C, Chinkers MA. A native 170,000 epidermal growth factor receptor-kinase com­plex from shed membrane vesicles. ] Bioi Chern 1982;257: 1523-31.

20. Tomooka Y, Diaugustine RP, Mciachlan]A. Proliferation of mouse uterine epithelial cells in vitro. Endocrinology 1986; 118: 1011-8.

21. Mukku VR, Stancel GM. Receptors for epidermal growth factor in the rat uterus. Endocrinology 1985;117:149-54.

22. Chegini N, Rao CV, Wakim N, Sanfilippo J. Binding of 125I-epidermal growth factor in human uterus. Cell Tissue Res 1986;246:543-8.

23. Madtes DK, Malden LT, Raines EW, Ross R. Induction of transcription and secretion ofTGF-a by activated human monocytes. Chest 1991;99(suppl 3):79S.