Immuuoloav
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 24:45-50 (C) 1990 MUNKSGAARD
,
5
Danazol Suppresses the Production of Interleukin-1 ~ and Tumor Necrosis Factor by Human Monocytes HIDEHIRO MORl, MIKI NAKAGAWA, NAOKI ITOH, KEISUKE WADA, AND TERUHIKO TAMAYA Department of Obstetrics and Gynecology. Gifu University School of Medicine. Gifu, Japan ABSTRACT: The effects of estradiol (E 2 ) , progesterone (P), and danazol on the production of interleukin-IB (IL-II3) and tumor necrosis factor (TNF) by OK-432 (a streptococcal preparationj-stimulated monocytes were examined. E 2 and P at physiologic concentrations enhanced IL-113 and TNF production by monocytes from donors with lower control levels (without steroids added) of IL-113 and TNF. However, E 2 and P at physiologic concentrations did not affect IL-113 and TNF production by monocytes from donors with higher control levels of IL-113 and TNF. Danazol inhibited IL-113 and TNF production by monocytes in a dose-dependent manner from not only donors with lower control levels of IL-113 and TNF but also donors with higher control levels oflL-113 and TNF. Danazol at a concentration of 10- 6 M significantly suppressed IL-113 and TNF production in the presence of E 2 and/or P at concentrations giving peak responses of IL-113 production. These findings suggest possible new mechanisms of action for danazol in the treatment of endometriosis and infertility associated with immune abnormalities. (Am J Reprod Immunol. 1990; 24:45-50) Key words: Estradiol, progesterone INTRODUCTION
Danazol, an isoxazole derivative of the synthetic steroid 17a-ethinyl testosterone, has been the mainstay of the medical management of endometriosis for over a decade. The hypoestrogenic and hyperandrogenic environment created by danazol is extremely hostile to the growth of endometriotic tissue. Danazol has also been used to treat such autoimmune diseases as idiopathic thrombocytopenic purpura,' systemic lupus erythematosis.r and angioneurotic edema.:' Substantial evidence has indicated that gonadal steroids such as estradiol (E z) and progesterone (P) influence immune function." Although there is considerable evidence indicating in vivo effects of danazol and gonadal steroids, the mechanisms of their actions on the immune response have not been fully clarified. Monocytes and macrophages play a crucial role in the regulation of the cellular and humoral immune responses. Some aspects of this regulatory function are mediated by one of its soluble products, interleukin-1 OL-1l, which acts as an activator ofT and B cells." IL-1 also has stimulatory and regulatory effects on the growth and differentiation of numerous cell types, and is a mediator of inflammatory responses." Two distinct Submitted for publication -January 24, 1990; accepted June 12. 1990. Address reprint requests to Dr. Hidehiro Mori, Department of Obstetrics and Gynecology. Gifu University School of Medicine, 40 Tsukusa-machi, Gifu 500, .Inpan.
genes that produce IL-1 activit{' have been identified, referred to as IL-1a and IL-ll3." IL-ll3 is a prominent product of monocytes and macrophages." In addition to IL-1, activated monocytes and macrophages release tumor necrosis factor (TNF), also known as cachectin." Although TNF is distinct from IL-1 in its amino acid sequence and receptor recognition, TNF and IL-1 share a variety of biological activities, including lymphocyteactivating properties.P-?" OK-432, a streptococcal preparation, has been used as a biological response modifier," and has been shown to activate human monocytes and macrophas-es and promote IL-1 and TNF production by them.' ,11 The present study was undertaken to investigate the effects ofE 2 , P, and danazol on the in vitro production ofIL-ll3 and TNF by OK-432-stimulated human monocytes. MATERIALS AND METHODS
Reagents Danazol was a generous gift from Tokyo Tanabe Co. Ltd., Tokyo, Japan. E z and P were obtained from Sigma Chemical Co., St. Louis, MO. These steroids were dissolved in absolute ethanol and diluted to the desired concentration with a commercial serum-free medium, ASF-301 (Ajinomoto Co. Ltd., Tokyo, Japan) just before use. The steroid preparations were endotoxin-free as assessed by the Limulus test (sensitivity limit: 60 pg/ ml, Funakoshi Pharmaceutical Co. Ltd., Tokyo, Japan). The final concentration of ethanol in the cultures did not exceed 0.1 %, and this level of ethanol had no effect on the production oflL-ll3 and TNF by monocytes. OK-432, which is a lyophilized preparation of the avirulent Su strain, group A Streptococcus pyogenes, was kindly supplied by Chugai Pharmaceutical Co. Ltd., Tokyo, Japan. The "Klinische Einheit (KE)" unit was used to express the strength of the preparation. One KE of OK-432 is equivalent to 0.1 mg of dried Streptococci. Isolation of Human Monocytes Heparinized peripheral blood (250~400 ml) was collected from healthy volunteers (five male and seven female medical students, mean age ±SD: 24.1±2.2 years). None of the blood donors had any history of recent infection or illness or was malnourished. Endotoxin was not detected in all the blood samples. All the female donors had regular menstrual cycles and were not using oral contraceptives. Female blood samples were obtained during the follicular phase of the cycle. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradient centrifugation. PBMCs at the interface were washed twice with Hanks' balanced salt solution (HBSS) and suspended to
46
MaRl ET AL.
a concentration of 2 x lOG cells/ml in ASF-301 medium. Monocytes were isolated by adherence on fetal calf serum-coated plastic dish. The PBMC suspension was incubated for 60 min at 37°C in a plastic Petri dish (#3003; Falcon, Oxnard, CAl. After nonadherent cells were thoroughly washed off, adherent cells were recovered by exposure for 15 min to 1 mM EDTA in cold phosphate-buffered saline. After washing three times with HBSS, cell viability was evaluated by the trypan blue exclusion test, and the cells were adjusted to 2 x lOG viable cells/ml with ASF-301 medium. Under these conditions, more than 90 c!c, of the adherent cells belonged to the monocyte-macrophage lineage as assessed by phase contrast microscopy and non-specific esterase staining. Contaminating cells were almost exclusively lymphocytes. Induction of IL-1 and TNF The monocytes were seeded at 5 x 10 5 cells/250 f.ll! well in 24-well culture plates (#25820; Corning Glass Works, Corning, NY), and 250 f.ll of ASF-301 medium alone (control) or ASF-301 medium containing various concentrations of E 2 , P, and/or danazol were added to the cells. Six h later, 20 u.l of OK-432 solution was added at a final concentration of 0.2 KE/ml, and the cells were incubated for 24 h at 37°C in humidified air with 5% CO 2 , All cultures were performed in triplicate. After incubation, the supernatant was collected, passed through a 0.22 urn filter (Millipore, Bedford, MAl and stored at -20°C until use for assay of IL-1f3 and TNF. Cell viability was also assessed by microscopic visualization of cell morphology and adherence, and the trypan blue exclusion test. Assays of IL-1f3 and TNF in Supernatant The IL-1f3 level was measured using an 112 5 radioimmunoassay kit (Medgenix, Belgium). The minimum detection sensitivity of IL-1f3 by this method was 0.2 ng/ ml. The TNF level was determined using an enzymelinked immunosorbent assay kit (T cell sciences, Cambridge, MAl. The minimum detection sensitivity of TNF by this method was 10 pg/ml. Statistical Analysis Correlation coefficient (Fig. 1) was determined by Spearman's rank test. Dose response data (Figs. 2-4) were tested by Bartlett's test for homogeneity of variance, however they were frequently found to be heterocedastic. Therefore, these data were analysed using Friedman's nonparametric two-factor analysis of variance by ranks. The significance of difference between two groups (Table I) was evaluated by Student's paired t test. A result was considered significant when P was less than 0.05. RESULTS
IL-1f3 and TNF Production by OK-432-stimulated Monocytes Maximal production of both IL-1f3 and TNF were obtained at a final concentration of 0.2 KE/ml of OK-432 (Fig. 5Al. The production of IL-1f3 and TNF by OK432-stimulated monocytes reached their plateaus at 24 h, whereas unstimulated monocytes did not produce detectable levels of IL-1f3 and TNF (Fig. 5B).
0.5
•
0
r =
0.34
0.4 0
•
c 0.3 E
•
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 24:45-50 (C) 1990 MUNKSGAARD
,
5
Danazol Suppresses the Production of Interleukin-1 ~ and Tumor Necrosis Factor by Human Monocytes HIDEHIRO MORl, MIKI NAKAGAWA, NAOKI ITOH, KEISUKE WADA, AND TERUHIKO TAMAYA Department of Obstetrics and Gynecology. Gifu University School of Medicine. Gifu, Japan ABSTRACT: The effects of estradiol (E 2 ) , progesterone (P), and danazol on the production of interleukin-IB (IL-II3) and tumor necrosis factor (TNF) by OK-432 (a streptococcal preparationj-stimulated monocytes were examined. E 2 and P at physiologic concentrations enhanced IL-113 and TNF production by monocytes from donors with lower control levels (without steroids added) of IL-113 and TNF. However, E 2 and P at physiologic concentrations did not affect IL-113 and TNF production by monocytes from donors with higher control levels of IL-113 and TNF. Danazol inhibited IL-113 and TNF production by monocytes in a dose-dependent manner from not only donors with lower control levels of IL-113 and TNF but also donors with higher control levels oflL-113 and TNF. Danazol at a concentration of 10- 6 M significantly suppressed IL-113 and TNF production in the presence of E 2 and/or P at concentrations giving peak responses of IL-113 production. These findings suggest possible new mechanisms of action for danazol in the treatment of endometriosis and infertility associated with immune abnormalities. (Am J Reprod Immunol. 1990; 24:45-50) Key words: Estradiol, progesterone INTRODUCTION
Danazol, an isoxazole derivative of the synthetic steroid 17a-ethinyl testosterone, has been the mainstay of the medical management of endometriosis for over a decade. The hypoestrogenic and hyperandrogenic environment created by danazol is extremely hostile to the growth of endometriotic tissue. Danazol has also been used to treat such autoimmune diseases as idiopathic thrombocytopenic purpura,' systemic lupus erythematosis.r and angioneurotic edema.:' Substantial evidence has indicated that gonadal steroids such as estradiol (E z) and progesterone (P) influence immune function." Although there is considerable evidence indicating in vivo effects of danazol and gonadal steroids, the mechanisms of their actions on the immune response have not been fully clarified. Monocytes and macrophages play a crucial role in the regulation of the cellular and humoral immune responses. Some aspects of this regulatory function are mediated by one of its soluble products, interleukin-1 OL-1l, which acts as an activator ofT and B cells." IL-1 also has stimulatory and regulatory effects on the growth and differentiation of numerous cell types, and is a mediator of inflammatory responses." Two distinct Submitted for publication -January 24, 1990; accepted June 12. 1990. Address reprint requests to Dr. Hidehiro Mori, Department of Obstetrics and Gynecology. Gifu University School of Medicine, 40 Tsukusa-machi, Gifu 500, .Inpan.
genes that produce IL-1 activit{' have been identified, referred to as IL-1a and IL-ll3." IL-ll3 is a prominent product of monocytes and macrophages." In addition to IL-1, activated monocytes and macrophages release tumor necrosis factor (TNF), also known as cachectin." Although TNF is distinct from IL-1 in its amino acid sequence and receptor recognition, TNF and IL-1 share a variety of biological activities, including lymphocyteactivating properties.P-?" OK-432, a streptococcal preparation, has been used as a biological response modifier," and has been shown to activate human monocytes and macrophas-es and promote IL-1 and TNF production by them.' ,11 The present study was undertaken to investigate the effects ofE 2 , P, and danazol on the in vitro production ofIL-ll3 and TNF by OK-432-stimulated human monocytes. MATERIALS AND METHODS
Reagents Danazol was a generous gift from Tokyo Tanabe Co. Ltd., Tokyo, Japan. E z and P were obtained from Sigma Chemical Co., St. Louis, MO. These steroids were dissolved in absolute ethanol and diluted to the desired concentration with a commercial serum-free medium, ASF-301 (Ajinomoto Co. Ltd., Tokyo, Japan) just before use. The steroid preparations were endotoxin-free as assessed by the Limulus test (sensitivity limit: 60 pg/ ml, Funakoshi Pharmaceutical Co. Ltd., Tokyo, Japan). The final concentration of ethanol in the cultures did not exceed 0.1 %, and this level of ethanol had no effect on the production oflL-ll3 and TNF by monocytes. OK-432, which is a lyophilized preparation of the avirulent Su strain, group A Streptococcus pyogenes, was kindly supplied by Chugai Pharmaceutical Co. Ltd., Tokyo, Japan. The "Klinische Einheit (KE)" unit was used to express the strength of the preparation. One KE of OK-432 is equivalent to 0.1 mg of dried Streptococci. Isolation of Human Monocytes Heparinized peripheral blood (250~400 ml) was collected from healthy volunteers (five male and seven female medical students, mean age ±SD: 24.1±2.2 years). None of the blood donors had any history of recent infection or illness or was malnourished. Endotoxin was not detected in all the blood samples. All the female donors had regular menstrual cycles and were not using oral contraceptives. Female blood samples were obtained during the follicular phase of the cycle. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradient centrifugation. PBMCs at the interface were washed twice with Hanks' balanced salt solution (HBSS) and suspended to
46
MaRl ET AL.
a concentration of 2 x lOG cells/ml in ASF-301 medium. Monocytes were isolated by adherence on fetal calf serum-coated plastic dish. The PBMC suspension was incubated for 60 min at 37°C in a plastic Petri dish (#3003; Falcon, Oxnard, CAl. After nonadherent cells were thoroughly washed off, adherent cells were recovered by exposure for 15 min to 1 mM EDTA in cold phosphate-buffered saline. After washing three times with HBSS, cell viability was evaluated by the trypan blue exclusion test, and the cells were adjusted to 2 x lOG viable cells/ml with ASF-301 medium. Under these conditions, more than 90 c!c, of the adherent cells belonged to the monocyte-macrophage lineage as assessed by phase contrast microscopy and non-specific esterase staining. Contaminating cells were almost exclusively lymphocytes. Induction of IL-1 and TNF The monocytes were seeded at 5 x 10 5 cells/250 f.ll! well in 24-well culture plates (#25820; Corning Glass Works, Corning, NY), and 250 f.ll of ASF-301 medium alone (control) or ASF-301 medium containing various concentrations of E 2 , P, and/or danazol were added to the cells. Six h later, 20 u.l of OK-432 solution was added at a final concentration of 0.2 KE/ml, and the cells were incubated for 24 h at 37°C in humidified air with 5% CO 2 , All cultures were performed in triplicate. After incubation, the supernatant was collected, passed through a 0.22 urn filter (Millipore, Bedford, MAl and stored at -20°C until use for assay of IL-1f3 and TNF. Cell viability was also assessed by microscopic visualization of cell morphology and adherence, and the trypan blue exclusion test. Assays of IL-1f3 and TNF in Supernatant The IL-1f3 level was measured using an 112 5 radioimmunoassay kit (Medgenix, Belgium). The minimum detection sensitivity of IL-1f3 by this method was 0.2 ng/ ml. The TNF level was determined using an enzymelinked immunosorbent assay kit (T cell sciences, Cambridge, MAl. The minimum detection sensitivity of TNF by this method was 10 pg/ml. Statistical Analysis Correlation coefficient (Fig. 1) was determined by Spearman's rank test. Dose response data (Figs. 2-4) were tested by Bartlett's test for homogeneity of variance, however they were frequently found to be heterocedastic. Therefore, these data were analysed using Friedman's nonparametric two-factor analysis of variance by ranks. The significance of difference between two groups (Table I) was evaluated by Student's paired t test. A result was considered significant when P was less than 0.05. RESULTS
IL-1f3 and TNF Production by OK-432-stimulated Monocytes Maximal production of both IL-1f3 and TNF were obtained at a final concentration of 0.2 KE/ml of OK-432 (Fig. 5Al. The production of IL-1f3 and TNF by OK432-stimulated monocytes reached their plateaus at 24 h, whereas unstimulated monocytes did not produce detectable levels of IL-1f3 and TNF (Fig. 5B).
0.5
•
0
r =
0.34
0.4 0
•
c 0.3 E
•
