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Possible role of mast cells in the mechanism of action of intrauterine contraceptive devices
Possible role of mast cells in the mechanism of action of intrauterine contraceptive devices A. Tursi, A. Mastrorilli, D. Ribatti, L. Loiudice, R. Contino, and J. Claudatus
Bari, Italy The presence and number of mast cells and their relationship to other inflammatory cells were investigated in smears from intrauterine contraceptive devices. The results obtained suggest a possible role of mast cells in the mechanism of action of intrauterine contraceptive devices, on the basis of the peculiar known functions of mast cells. (AM. J. 0BSTET. GYNECOL. 148:1064, 1984.)
The presence of mast cells in the human uterus was first described by von Numers in 1942. Their biologic role has not yet been completely elucidated, but, on the basis of the current knowledge of mast cell functions, 1 it is possible that they may be involved in the physiology and derangement of menstruation, as well as in the dynamic alterations that take place in pregnancy and parturition. An increase in mast cells has been reported in the uterus and in the cellular film taken from removed intrauterine contraceptive devices. 2 - 4 Mast cells are generally increased in chronic inflammation, no matter where this process takes place, 5 • 6 and intrauterine contraceptive devices are capable of inducing a chronic sterile inflammatory process. 7 • 8 The purpose of our study was to evaluate the morphologic and quantitative aspects of mast cells in smears from intrauterine contraceptive devices in relationship to the clinical findings, in order to investigate the possible role of mast cells in the mechanism of action of intrauterine contraceptive devices. Light microscopy does not appear to be completely reliable in establishing quantitative changes in the mast cell population. 2 Possibly, a number of mast cells that are completely or partially degranulated may be overlooked because of scarce or absent metachromatic staining with toluidine blue. Therefore, in this study, we also used electron microscopy on the cellular films present on the surface of removed intrauterine contraceptive devices, to identify mast cells and study their morphologic changes and relationships to other inflammatory cells.
Material and methods Light microscopy. Triplicate slide smears were obtained from intrauterine contraceptive devices removed from 5 to 25 months after insertion in 100
From Cattedra di Immunologia Clinica and Istituto di Clinica Ostetrica e Ginecologica II, University of Bari Medical School. Supported in part by grants from Consiglio Nazionale delle Ricerche and Ministero Pubblica Istruzione, Rome, Italy. Received for publication April 29, 1983; revised September 18, 1983; accepted November 2, 1983. Reprint requests: Praf. A. Tursi, Cattedra di Immunologia Clinica, Policlinico, 70124 Bari, Italy.
1064
healthy women (age range, 24 to 40 years). Normal endometrial smears were obtained from 30 women (age range, 28 to 41 years). The slides were stained by Papanicolaou, toluidine blue, and Giemsa methods, and cellular populations were identified and counted, with particular attention being given to the mast cells. Electron microscopy. Twenty-two samples were studied by electron microscopy. The cellular layers adherent to the intrauterine contraceptive devices were fixed in glutaraldehyde (25% in Millonig buffer 0.13M, pH 7 .4) for 20 minutes, washed, postfixed in 1% osmium tetroxide solution for 1 hour, dehydrated and embedded in Araldite. Semi thin sections were stained with 1% toluidine blue aqueous solution or by the Giemsa method, mounted with dibutylphthalate-xylene medium, and observed under the light microscope, to preliminarily identify the areas to be subsequently examined by electron microscopy. Ultrathin sections were cut on a Reichert OMH2 ultramicrotome, stained with 5% uranyl acetate and 0.1% lead citrate, and observed with a TEM Hitachi HS8 electron microscope.
Results Light microscopy. Neutrophils, lymphocytes, endometrial cells, and macrophages were observed. The latter cells predominated, and they had hyperchromatic nuclei with distinct nucleoli, and vacuolated cytoplasm with or without phagocytosed material. No giant cells were seen, but red blood cells, degenerated spermatozoa, and cellular debris were present. Mast cells were observed in all smears examined, with a range of from 120 to 180 cells/10 microscopic fields at 100x for each slide, as compared with smears of endometrium obtained from 30 normal women, which had only 10 to 50 cells. Their cytoplasm stained metachromatically with toluidine blue, because of cytoplasmic granules, whereas other mast cells were typically degranulated. (Fig. 1, d). Pelvic pain, spotting, and other manifestations of pathologic conditions due to use of intrauterine contraceptive devices were observed in most cases with
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Fig. 1. Smear from an intrauterine contraceptive device. a, Electron micrograph of a mast cell showing semilunar features of the granules. (Original magnification x95,000.) b, Electron micrograph of a mast cell with a macrophage-like aspect. (Original magnification X 12,000.) c, Electron micrograph of an endometrial cell showing numerous and pronounced microvilli. (Original magnification x 12,000.) d, Optical microscope micrograph of cytologic composition of smear from an intrauterine contraceptive device, stained with toluidine blue, showing typical mast cells (arrows) with specific metachromasia. (Original magnification X 1,000.)
highly increased numbers of mast cells, whereas no clinical manifestations were present in a few cases, although the number of mast cells was similarly elevated. 9 Endometrial cells were present, free or in clumps, and did not show dysplastic or neoplastic alterations. Electron microscopy. A conspicuous number of mast cells was intermingled with the other inflammatory cells (macrophages, neutrophils, and lymphocytes) and endometrial cells, thus confirming the aspects observed with light microscopy. Most mast cells presented the semilunar features of granules characteristic of a slow but progressive degranulation in response to lowlevel but constant stimulation 10 (Fig. 1, a). Mast cells were in close physical contact with the other inflamma-
tory cells. Some mast cells had peculiar morphologic aspects-some had a macrophage-like appearance, and others had a lymphoid-like appearance (Fig. 1, b). Spermatozoa were present in some macrophagic cells, and endometrial cells showed aspects peculiar to inflammatory stimulation (numerous and pronounced microvilli, well-developed cytoplasmic canalicular system, etc.) (Fig. 1, c).
Comment The precise roles of mast cells in inflammatory and immunologic phenomena, other than the classic reaginic primary type reaction, are largely unknown; however, there is reason to believe, on both biochem-
1066 Tursi et al. Am.
ical and morphologic grounds, that these roles are complex. 5 The list of mediators of inflammation that are synthesized or newly generated by mast cells is long and continues to grow. Their established or suggested effects include smooth muscle and vascular endothelial contraction, vascular dilatation and increased vascular permeability with subsequent effects on local blood flow and shifts of fluid and solutes from intravascular to extravascular compartments, attraction of eosinophils and neutrophils, activation of platelets, activation or inhibition of clotting mechanism, promotion of fibrinolysis, generation of kallikrein activity, and interactions with fibronectin that may influence cell adherence. Apart from the classic IgE-mediated mast cell degranulation, several physical, chemical, and biologic (immunologic and nonimmunologic) factors are able to stimulate degranulation of the mast cell. These include cold, some metals, radiocontrast dyes, lectins, polymers (48/80), ionophores, lysosomal polypeptides, insect and venom polypeptides, anaphylatoxins (C3a and C5a), anti-IgE antibody, aggregated IgG, T-cell products, 5 • 11 etc. The diverse mediators, once released, collectively have an almost limitless potential to influence the progression of an individual immunologic reaction. The specific consequences of mast cell participation probably varies in different hypersensitivity phenomena, and these cells may have multiple roles even in a single immunologic reaction. A long-standing belief has been that mast cells normally function by regulating properties of the local microenvironment, particularly vascular tone and permeability. A relatively slow sustained release of mediators could occur during the course of late or delayed, as opposed to immediate, hypersensitivity reactions. At times, mast cell mediators may function to dampen rather than enhance inflammatory processes, as could be the case with our users of intrauterine contraceptive devices who showed increased numbers of mast cells without clinically adverse manifestations. On the basis of the above-presented data and our findings, we suggest that mast cells are involved in the process of decapacitation of spermatozoa (antifertilizing action) and/or inhibition of implantation of the blastocyst (microabortive action). An unknown moderate but persistent stimulus, certainly correlated with the presence of the intrauterine contraceptive device, could produce a slow but sustained release of mediators, as demonstrated by the semilunar appearance of the granules of most of the mast cells. Therefore, mast cells can act either directly on the spermatozoa through their enzymatic mediators or indirectly by stimulating
J.
April 15, 1984 Obstet. Gynecol.
phagocytic and/or enzymatic activity of the other inflammatory cells. Furthermore, the vasoactive mediators of mast cells could cause alterations in the environment (increased vascular permeability, smooth muscle contraction, and secretion of mucus; involvement of coagulation and fibrinolysis system), thus contributing to the creation of conditions unfavorable to the implantation of the blastocyst. Finally, the mast cells are involved in the "coral" process of the chronic inflammatory reaction induced by intrauterine contraceptive devices, which is hostile to conception, by means of several mechanisms, such as direct action of inflammatory cells, cytotoxic action of chemical mediators, modification of the microenvironment, and other as yet unidentified mechanisms. In conclusion, it is highly possible that mast cells, stimulated by nonimmunologic or immunologic factors, could primarily cause or participate in a process of hypersensitivity reaction of the late or delayed type, triggered by the "physical" presence of the intrauterine contraceptive device. We wish to thank Mr. F. Di Pietro for his skillful technical assistance.
REFERENCES
J., and Edwards, A. M., editors: The Mast Cell, London, 1979, Pitman Medical. Sheppard, B. L., and Bohmar, J.: Mast cells in the human uterus, in Pepys, J., and Edwards, A. M., editors: The Mast Cell, London, 1979, Pitman Medical, p. 142. Kobayashi, T. K., Okamoto, M., Marami, K., and Yuasa, M.: The presence of the mast cells in IUD smear, Acta Cytol. (Baltimore) 23:3, 1980. Ragni, N., Rugiati, S., Rossato, P., Venturini, P. L., Foglia, G., and Capitano, G. L.: Histological and ultrastructural changes of the endometrium in women using inert and copper coiled IUD's, Acta Eur. Fertil. 8:193, 1977. Galli, S. J., and Dvorak, H. F.: Basophils and mast cells: Structure, function, and role in hypersensitivity, in Good, R. A., and Day, S. B., editors: Comprehensive Immunology, series 6, New York, 1979, Plenum Publishing Corp.,
1. Pepys,
2. 3.
4.
5.
p. 1.
6. Tursi, A., Colotta, F., Ribatti, D., and DiPietro, F.: Mastcellule e sinovite reumatoide, Reumatismo 33:237, 1981. 7. Mishell, D. R., Jr., Bell, J. H., Good, R. G., and Moyer, D. L.: The intrauterine device: A bacteriological study of the endometrial cavity, AM. j. 0BSTET. GYNECOL. 96:ll9, 1966.
8. Sagiroglu, N., and Sagiroglu, E.: Biological mode of action of the Lippes loop in intrauterine contraception, AM. j. 0BSTET. GYNECOL. 106:506, 1970. 9. Loiudice, L., Orsini, G., Mastrorilli, A., and Schonauer, S.: Le mast cells nella citologia endometriale di donne portatrici di IUD, Contracc. Fertil. Steril. 3:235, 1982. 10. Selye, H.: The Mast Cells, Washington, D. C., 1965, Butterworth Publishers. 11. Askenase, P. W.: Effector cells in late and delayed hypersensitivity reactions that are dependent on antibodies or T cells, Prog. Immunol. 4:829, 1980.
Bari, Italy The presence and number of mast cells and their relationship to other inflammatory cells were investigated in smears from intrauterine contraceptive devices. The results obtained suggest a possible role of mast cells in the mechanism of action of intrauterine contraceptive devices, on the basis of the peculiar known functions of mast cells. (AM. J. 0BSTET. GYNECOL. 148:1064, 1984.)
The presence of mast cells in the human uterus was first described by von Numers in 1942. Their biologic role has not yet been completely elucidated, but, on the basis of the current knowledge of mast cell functions, 1 it is possible that they may be involved in the physiology and derangement of menstruation, as well as in the dynamic alterations that take place in pregnancy and parturition. An increase in mast cells has been reported in the uterus and in the cellular film taken from removed intrauterine contraceptive devices. 2 - 4 Mast cells are generally increased in chronic inflammation, no matter where this process takes place, 5 • 6 and intrauterine contraceptive devices are capable of inducing a chronic sterile inflammatory process. 7 • 8 The purpose of our study was to evaluate the morphologic and quantitative aspects of mast cells in smears from intrauterine contraceptive devices in relationship to the clinical findings, in order to investigate the possible role of mast cells in the mechanism of action of intrauterine contraceptive devices. Light microscopy does not appear to be completely reliable in establishing quantitative changes in the mast cell population. 2 Possibly, a number of mast cells that are completely or partially degranulated may be overlooked because of scarce or absent metachromatic staining with toluidine blue. Therefore, in this study, we also used electron microscopy on the cellular films present on the surface of removed intrauterine contraceptive devices, to identify mast cells and study their morphologic changes and relationships to other inflammatory cells.
Material and methods Light microscopy. Triplicate slide smears were obtained from intrauterine contraceptive devices removed from 5 to 25 months after insertion in 100
From Cattedra di Immunologia Clinica and Istituto di Clinica Ostetrica e Ginecologica II, University of Bari Medical School. Supported in part by grants from Consiglio Nazionale delle Ricerche and Ministero Pubblica Istruzione, Rome, Italy. Received for publication April 29, 1983; revised September 18, 1983; accepted November 2, 1983. Reprint requests: Praf. A. Tursi, Cattedra di Immunologia Clinica, Policlinico, 70124 Bari, Italy.
1064
healthy women (age range, 24 to 40 years). Normal endometrial smears were obtained from 30 women (age range, 28 to 41 years). The slides were stained by Papanicolaou, toluidine blue, and Giemsa methods, and cellular populations were identified and counted, with particular attention being given to the mast cells. Electron microscopy. Twenty-two samples were studied by electron microscopy. The cellular layers adherent to the intrauterine contraceptive devices were fixed in glutaraldehyde (25% in Millonig buffer 0.13M, pH 7 .4) for 20 minutes, washed, postfixed in 1% osmium tetroxide solution for 1 hour, dehydrated and embedded in Araldite. Semi thin sections were stained with 1% toluidine blue aqueous solution or by the Giemsa method, mounted with dibutylphthalate-xylene medium, and observed under the light microscope, to preliminarily identify the areas to be subsequently examined by electron microscopy. Ultrathin sections were cut on a Reichert OMH2 ultramicrotome, stained with 5% uranyl acetate and 0.1% lead citrate, and observed with a TEM Hitachi HS8 electron microscope.
Results Light microscopy. Neutrophils, lymphocytes, endometrial cells, and macrophages were observed. The latter cells predominated, and they had hyperchromatic nuclei with distinct nucleoli, and vacuolated cytoplasm with or without phagocytosed material. No giant cells were seen, but red blood cells, degenerated spermatozoa, and cellular debris were present. Mast cells were observed in all smears examined, with a range of from 120 to 180 cells/10 microscopic fields at 100x for each slide, as compared with smears of endometrium obtained from 30 normal women, which had only 10 to 50 cells. Their cytoplasm stained metachromatically with toluidine blue, because of cytoplasmic granules, whereas other mast cells were typically degranulated. (Fig. 1, d). Pelvic pain, spotting, and other manifestations of pathologic conditions due to use of intrauterine contraceptive devices were observed in most cases with
Mast cells in action of IUDs
Volume 148 Number 8
1065
Fig. 1. Smear from an intrauterine contraceptive device. a, Electron micrograph of a mast cell showing semilunar features of the granules. (Original magnification x95,000.) b, Electron micrograph of a mast cell with a macrophage-like aspect. (Original magnification X 12,000.) c, Electron micrograph of an endometrial cell showing numerous and pronounced microvilli. (Original magnification x 12,000.) d, Optical microscope micrograph of cytologic composition of smear from an intrauterine contraceptive device, stained with toluidine blue, showing typical mast cells (arrows) with specific metachromasia. (Original magnification X 1,000.)
highly increased numbers of mast cells, whereas no clinical manifestations were present in a few cases, although the number of mast cells was similarly elevated. 9 Endometrial cells were present, free or in clumps, and did not show dysplastic or neoplastic alterations. Electron microscopy. A conspicuous number of mast cells was intermingled with the other inflammatory cells (macrophages, neutrophils, and lymphocytes) and endometrial cells, thus confirming the aspects observed with light microscopy. Most mast cells presented the semilunar features of granules characteristic of a slow but progressive degranulation in response to lowlevel but constant stimulation 10 (Fig. 1, a). Mast cells were in close physical contact with the other inflamma-
tory cells. Some mast cells had peculiar morphologic aspects-some had a macrophage-like appearance, and others had a lymphoid-like appearance (Fig. 1, b). Spermatozoa were present in some macrophagic cells, and endometrial cells showed aspects peculiar to inflammatory stimulation (numerous and pronounced microvilli, well-developed cytoplasmic canalicular system, etc.) (Fig. 1, c).
Comment The precise roles of mast cells in inflammatory and immunologic phenomena, other than the classic reaginic primary type reaction, are largely unknown; however, there is reason to believe, on both biochem-
1066 Tursi et al. Am.
ical and morphologic grounds, that these roles are complex. 5 The list of mediators of inflammation that are synthesized or newly generated by mast cells is long and continues to grow. Their established or suggested effects include smooth muscle and vascular endothelial contraction, vascular dilatation and increased vascular permeability with subsequent effects on local blood flow and shifts of fluid and solutes from intravascular to extravascular compartments, attraction of eosinophils and neutrophils, activation of platelets, activation or inhibition of clotting mechanism, promotion of fibrinolysis, generation of kallikrein activity, and interactions with fibronectin that may influence cell adherence. Apart from the classic IgE-mediated mast cell degranulation, several physical, chemical, and biologic (immunologic and nonimmunologic) factors are able to stimulate degranulation of the mast cell. These include cold, some metals, radiocontrast dyes, lectins, polymers (48/80), ionophores, lysosomal polypeptides, insect and venom polypeptides, anaphylatoxins (C3a and C5a), anti-IgE antibody, aggregated IgG, T-cell products, 5 • 11 etc. The diverse mediators, once released, collectively have an almost limitless potential to influence the progression of an individual immunologic reaction. The specific consequences of mast cell participation probably varies in different hypersensitivity phenomena, and these cells may have multiple roles even in a single immunologic reaction. A long-standing belief has been that mast cells normally function by regulating properties of the local microenvironment, particularly vascular tone and permeability. A relatively slow sustained release of mediators could occur during the course of late or delayed, as opposed to immediate, hypersensitivity reactions. At times, mast cell mediators may function to dampen rather than enhance inflammatory processes, as could be the case with our users of intrauterine contraceptive devices who showed increased numbers of mast cells without clinically adverse manifestations. On the basis of the above-presented data and our findings, we suggest that mast cells are involved in the process of decapacitation of spermatozoa (antifertilizing action) and/or inhibition of implantation of the blastocyst (microabortive action). An unknown moderate but persistent stimulus, certainly correlated with the presence of the intrauterine contraceptive device, could produce a slow but sustained release of mediators, as demonstrated by the semilunar appearance of the granules of most of the mast cells. Therefore, mast cells can act either directly on the spermatozoa through their enzymatic mediators or indirectly by stimulating
J.
April 15, 1984 Obstet. Gynecol.
phagocytic and/or enzymatic activity of the other inflammatory cells. Furthermore, the vasoactive mediators of mast cells could cause alterations in the environment (increased vascular permeability, smooth muscle contraction, and secretion of mucus; involvement of coagulation and fibrinolysis system), thus contributing to the creation of conditions unfavorable to the implantation of the blastocyst. Finally, the mast cells are involved in the "coral" process of the chronic inflammatory reaction induced by intrauterine contraceptive devices, which is hostile to conception, by means of several mechanisms, such as direct action of inflammatory cells, cytotoxic action of chemical mediators, modification of the microenvironment, and other as yet unidentified mechanisms. In conclusion, it is highly possible that mast cells, stimulated by nonimmunologic or immunologic factors, could primarily cause or participate in a process of hypersensitivity reaction of the late or delayed type, triggered by the "physical" presence of the intrauterine contraceptive device. We wish to thank Mr. F. Di Pietro for his skillful technical assistance.
REFERENCES
J., and Edwards, A. M., editors: The Mast Cell, London, 1979, Pitman Medical. Sheppard, B. L., and Bohmar, J.: Mast cells in the human uterus, in Pepys, J., and Edwards, A. M., editors: The Mast Cell, London, 1979, Pitman Medical, p. 142. Kobayashi, T. K., Okamoto, M., Marami, K., and Yuasa, M.: The presence of the mast cells in IUD smear, Acta Cytol. (Baltimore) 23:3, 1980. Ragni, N., Rugiati, S., Rossato, P., Venturini, P. L., Foglia, G., and Capitano, G. L.: Histological and ultrastructural changes of the endometrium in women using inert and copper coiled IUD's, Acta Eur. Fertil. 8:193, 1977. Galli, S. J., and Dvorak, H. F.: Basophils and mast cells: Structure, function, and role in hypersensitivity, in Good, R. A., and Day, S. B., editors: Comprehensive Immunology, series 6, New York, 1979, Plenum Publishing Corp.,
1. Pepys,
2. 3.
4.
5.
p. 1.
6. Tursi, A., Colotta, F., Ribatti, D., and DiPietro, F.: Mastcellule e sinovite reumatoide, Reumatismo 33:237, 1981. 7. Mishell, D. R., Jr., Bell, J. H., Good, R. G., and Moyer, D. L.: The intrauterine device: A bacteriological study of the endometrial cavity, AM. j. 0BSTET. GYNECOL. 96:ll9, 1966.
8. Sagiroglu, N., and Sagiroglu, E.: Biological mode of action of the Lippes loop in intrauterine contraception, AM. j. 0BSTET. GYNECOL. 106:506, 1970. 9. Loiudice, L., Orsini, G., Mastrorilli, A., and Schonauer, S.: Le mast cells nella citologia endometriale di donne portatrici di IUD, Contracc. Fertil. Steril. 3:235, 1982. 10. Selye, H.: The Mast Cells, Washington, D. C., 1965, Butterworth Publishers. 11. Askenase, P. W.: Effector cells in late and delayed hypersensitivity reactions that are dependent on antibodies or T cells, Prog. Immunol. 4:829, 1980.