Human endometrium: an active site of cytokine production and action.

0163-769X/91/1203-0272$03.00/0 Endocrine Reviews Copyright © 1991 by The Endocrine Society

Vol. 12, No. 3 Printed in U.S.A.

Human Endometrium: An Active Site of Cytokine Production and Action* S. TABIBZADEH Department of Pathology, University of South Florida Health Sciences Center, Tampa, Florida 33612

I. II. III. IV. V. VI. VII. VIII. IX. X. XL XII. XIII.

XIV.

XV.

XVI. XVII.

Introduction Cytokine Network Interactive Cellular Network in Human Endometrium Analogy of Human Menstruation to Inflammatory Processes IFN7 IL-1 IL-6 TNFa TGFa/EGF TGF0 CSF-1 IGFs Potential Functions of Cytokines in Human Endometrium A. Cytokines and proliferation B. Cytokines and lymphoid infiltration C. Cytokines and endometrial microenvironments D. Cytokines and menstruation E. Cytokines and uteroplacental functions F. Cytokine-cytokine interaction Interactions of Cytokines with Gonadal Steroid Hormones A. Are cytokines mediators of gonadal steroid actions? Influence of Cytokines on Endometrial Functions by Interaction with Hypothalamic-Pituitary-Gonadal Axis. A. Systemic Effects of Cytokines Model Systems for Studying the Cytokine Actions in Human Endometrium Perspectives

I. Introduction Complex interactions of a network of cells comprised of epithelial, stromal, endothelial, and lymphoid cells in human endometrium are displayed as orchestrated phases of proliferation, differentiation, and menstrual shedding. It is clear that these dynamic cell-to-cell interactions require a well developed array of intercellular Send reprint requests and correspondence to: S. Tabibzadeh, M.D., Department of Pathology, University of South Florida Health Sciences Center, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612. "This work is supported by Public Health Research Grant CA46866.

communication signals. Many of the changes that occur in human endometrium are reminiscent of those associated with the inflammatory and reparative processes. Thus, it is not surprising that emerging evidence suggests the involvement of proinflammatory cytokines in endometrial reactions. Expression of the receptors for cytokines, production of these factors by endometrial cells, and regulation of endometrial functions by these factors all indicate the potential of cytokines to serve at autocrine, paracrine, and endocrine levels in human endometrium. In the succeeding sections, the emerging evidence for the role of cytokines in human endometrium is presented. In this context, the complex network of cells able to release cytokines in human endometrium is addressed first. From the long list of cytokines, those cytokines whose potential participations in endometrial functions have been suggested are reviewed. In each section, some salient features of cytokines, available information on the production of each specific cytokine in human endometrium, and when available, cytokine receptor expression are discussed. In addition, those functions of cytokines that are relevant to endometrium and interactions of cytokines with steroid hormones are addressed. At present, data regarding the secretion or the potential roles of some cytokines are only available in nonhuman endometria, and similar information regarding human endometrium are lacking. The extrapolation of these data to humans should be entertained with caution and with the understanding that the role of cytokines may differ in different species. Similarly, the data on gestational endometrium should be interpreted with caution as major differences exist in the anatomical, morphological, biochemical, and physiological features of gestational and nongestational endometria. Functions of cytokines that may be mediated through their interaction with the hypothalamic-pituitary-gonadal axis and indirectly affect endometrium are highlighted. Some model systems for unraveling the role of cytokines in human endometrium are discussed and finally the intriguing 272

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CYTOKINE PRODUCTION IN ENDOMETRIUM

questions regarding the role of cytokines in human endometrium that remain to be answered are presented in the perspective section.

II. Cytokine Network It has become apparent that cytokines, in addition to functioning as immunoregulatory proteins, also serve as paracrine, autocrine, and endocrine factors and modulate an array of cell functions ranging from proliferation to differentiation (1-17). It has also become clear that in addition to T cells and monocytes, other cells including epithelial, endothelial, and mesenchymal cells produce overlapping as well as distinct sets of cytokines. These factors are all polypeptides and act on a variety of cell types in a non-antigen-specific manner. Originally, each cytokine was narrowly defined for a specific action, but it has now become apparent that all cytokines are pleiotropic and that the release of any set of these factors initiates a cascade of local events with ensuing release of other cytokines and activation of surrounding cells. Autocrine and paracrine actions of cytokines may be directed to modulate cell proliferation and differentiation, to induce expression of new antigenic epitopes and adhesion molecules, to alter cell morphology, and to induce chemotaxis (1-17). Among the distant and systemic effects are major changes in acute phase proteins and fever (5,18,19). In view of the distant, systemic, and hormonelike actions of cytokines, it has been suggested that these factors be named homeokines (17). Originally described as a separate group, growth factors are now included in the cytokine family of molecules (1). The number of factors in the cytokine family is constantly increasing and include the interleukins (IL-1 to IL-11), colony stimulating factors (M-CSF, G-CSF, GM-CSF), tumor necrosis factors (TNFa and /?), and interferons (IFN«, j9, and 7). Also included in this category are transforming growth factor-a and 0 (TGF/3 1-5) families, activin, inhibin, chemotactic factors and epidermal, fibroblast, insulin-like, nerve, and platelet-derived growth factors (1-17, 20, 21).

III. Interactive Cellular Network in Human Endometrium Human endometrium consists of glandular and surface epithelia and a surrounding stroma (22). Vascular supply of endometrium consists of endothelial cells mantled by basal lamina and tethered by a smooth muscle layer in the basalis. This layer is gradually lost close to the surface epithelium (23). Epithelium of human endometrium consists of a distinct group of cells. Lumen of endometrial cavity is covered by a surface epithelium that is contiguous with the glandular compartment. The glandular cells are comprised of those residing adjacent

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to myometrium, the so-called basalis, and those in the upper layer dubbed functionalis. This latter layer may arbitrarily be divided into upper and lower compartments (24-26). These compartments exhibit distinct differences in regard to proliferation and HLA-DR expression. We reported that in the proliferative phase, the proliferative activity is highest in the epithelium in the upper functional layer while it is negligible in the basalis (27). A similar observation has been reported by others in human and in primate endometria (24-26, 28). In contrast, differences in the expression of the class II proteins of the major histocompatibility complex, the so called HLADR molecules, in different layers of endometrial epithelium are observed. In the proliferative phase, the HLADR molecules are expressed markedly in the basalis epithelium while the expression of these antigens is markedly reduced in the lower and particularly the upper functional layers (29). Glandular epithelium undergoes sequential phases of proliferation, differentiation, and shedding throughout the menstrual cycle. It is suggested that these changes are driven by the coincident variations of estrogen and progesterone levels in the peripheral circulation. However, the mechanism by which the steroids induce these changes in endometrium are poorly understood, and it is unclear at present what mechanism (s) are responsible for the induction of these distinct differences in the proliferation and HLA-DR expression in different layers of endometrium. The heterogeneity of the cells in the human endometrial stroma is made evident by the presence of diverse groups of lymphoid as well as nonlymphoid cells. At present, the heterogeneous nature of the lymphoid cells of endometrial stroma may be defined on the basis of the reactivity of these cells with various monoclonal antibodies. However, markers that specifically characterize stromal cells have not been described, and thus far identification of these cells may only be achieved by exclusion. Despite homogeneous appearance of nonlymphoid stromal cells in hematoxylin and eosin-stained sections of human endometria, heterogeneous development of predecidual reaction in stromal cells in the secretory phase of the menstrual cycle suggests the existence of various populations of stromal cells poised to respond differently to regulatory signals. Stromal cells similar to the endometrial epithelium exhibit proliferative activity only in the proliferative phase (27). The proliferative activity observed in the endometrial stroma and originally ascribed by Ferenczy et al. (28) to the stromal cells in the secretory phase is shown to be limited to the lymphoid cells (27). Presence of aggregates of lymphoid cells, polymorphonuclear leukocytes, mast cells, and stromal granulocytes in endometrial stroma has been known for some time (22). However, with the advent of monoclonal antibodies


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that react with specific types of cells, the diversity of the lymphoid cells of endometrial stroma has become more apparent (29-36). Immunostaining for leukocyte common antigen has demonstrated that lymphoid cells are a major cell population in human endometrium (29-36). This group of cells is scattered as single cells in the endometrial stroma, or within endometrial glands, or are present as aggregates primarily adjacent to the endometrial glands in the basalis. T lymphocytes both with helper/inducer and cytotoxic/suppressor phenotypes and monocytes/macrophages constitute the majority of the lymphoid cells, whereas B cells, dendritic cells, and NK cells form a smaller population of lymphoid cells in human endometria (29-36). It is likely that during each menstruation, along with other cells of endometrium, a substantial number of lymphoid cells are lost. Proliferative activity of the endometrial lymphoid cells including T cells and monocytes/macrophages, however, ensures that after each menstrual shedding the lymphoid cell pool within endometrium is reconstituted (27). Recently, a group of molecules has been described, the expression of which may be responsible for the homotypic and heterotypic binding of lymphoid cells. Intercellular adhesion molecule-1 (ICAM-1) interacts with lymphocyte function-associated-1 (LFA-1) and serves as an adhesion molecule (33). The expression of ICAM-1 on endometrial epithelium, stroma, and lymphoid cells and sole expression of LFA-1 on the lymphoid cells may allow the interaction of lymphoid with other endometrial cells based on the reciprocal nature of LFA-l/ICAM-1 interaction and may provide a means of aggregation of lymphoid cells or their scattering in the endometrial stroma (33). Expression of steroid receptors in lymphoid cells of human endometrium concurs with the view that similar to other target cells in endometrium, the function of lymphoid cells is regulated by gonadal steroids (34).

IV. Analogy of Human Menstruation to Inflammatory Processes The inflammatory response is initiated by tissue damage due to chemical, physical, and infectious insults. This is followed by formation of thrombi, recruitment of polymorphonuclear and later on mononuclear cells, fibroblast proliferation, angiogenesis, and deposition of collagen. The granulation tissue thus formed is gradually replaced by a scar, and concomitantly the epithelial surfaces are repaired. Although the etiology of the tissue damage during menstruation is not induced by an exogenous factor, the menstrual phase of the cycle bears strong resemblance to the events occurring during the inflammatory response. During the secretory phase of the menstrual cycle, the endometrial epithelial cells undergo secretory changes. Some view this as a prelethal

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stage of cellular state (37). Apparently, the sudden fall in the serum level of estrogen and progesterone on the postovulatory days 25-26 of the cycle coincides with the loss of integrity of the lysosomes and the release of acid hydrolases. The cytoplasm, plasma membrane, and desmosomal junctions undergo autodigestion. The injury to the endothelial cells promotes platelet aggregation, release of prostaglandin F2a, thrombosis, and contraction of vessels (38). Ischemic necrosis initiated by occlusion of vessels on postovulatory day 25 reaches maximum levels during menses. The infiltration of the endometrial tissue by the so-called stromal granulocytes that starts in the late secretory phase reaches a peak in the menstrual phase. From first to third day of the cycle, the damage to endometrium becomes more apparent. The functionalis, including the epithelial and surrounding mantle of stroma, is gradually detached from the underlying basalis. Detachment is initiated in the fundus and slowly extends to the isthmus causing a thin denuded endometrium that retains the basalis and part of the surface epithelium particularly at the lower uterine segment and isthmus of the uterus. On day 3 of the cycle, for about 6-12 h, spreading and migration of epithelial cells are the principal mechanisms of restoration of the surface of the denuded endometrium. Subsequently, a wave of proliferative activity in the surface epithelial cells and in stumps of the basalis glands restores the integrity of endometrium on day 5 of the cycle. Concomitant with the repair of the denuded surface by proliferating epithelium, endometrial endothelium also exhibits regenerative features in the latter phase of the menstrual cycle (28, 39-42). As described, menstruation carries a striking similarity to the processes of inflammation and wound repair with the exception that formation of granulation tissue or scar does not contribute to the remodeling and repair of the denuded endometrium. From this analogy, it is not surprising to find that the cytokines known for their proinflammatory roles are shown and will be shown to modulate a variety of functions in human endometrium.

V. IFN7 IFN7, originally defined for its antiviral activity, may be differentiated from other interferons by its sensitivity to extreme temperature and pH (1). Activated T cells are the primary source of this factor; however, some studies indicate that NK cells may also produce this cytokine (43). IFN7 is a homodimer consisting of 166 amino acid residues, a signal sequence of 23 amino acids, and two sites for N-linked glycosylation. The sequences of IFN7 are conserved which is probably responsible for the lack of cross-species activity of IFN7 (1). The first clue as to the potential role of IFN7 in human


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endometrium originated from our observation that the expression of HLA-DR molecules is markedly enhanced in endometrial epithelial cells that are adjacent to lymphoid aggregates (29). The majority of cells within these aggregates are T cells and express markers indicative of their activation. These markers include HLA-DR, HLADP, HLA-DQ, and the recently described molecule belonging to the integrin family of molecules, the very late activation antigen-1 or VLA-1 (23, 29-32). In addition, it is shown that cells isolated from endometrial cavity release IFN7 (44). IFN7 induces HLA-DR molecules and inhibits the proliferation of human endometrial epithelium (30, 32, 45, 46). These data, coupled with the evidence that the local administration of IFN7 only focally induces HLA-DR expression in epithelial cells, support the concept of the paracrine effect of IFN7 in human endometrium (47-49). By localizing IFN7 receptor on the endometrial epithelium, we could demonstrate that this epithelium fulfills one essential requirement for responding to IFN7. This expression is consistent throughout the menstrual cycle (23). Thus, the variability of HLA-DR expression in endometrial epithelial cells as seen throughout the menstrual cycle is not related to the variation of IFN7 receptor (23, 29). It may be related to the variation of a factor encoded by chromosome 21, which is required to confer to cells biological sensitivity to IFN7 (50). Alternatively, it may be related to the variation of IFN7 produced by T cells or alteration of the sensitivity of the endometrial epithelium to this factor. IFN7 differentially induces the expression of HLA-DR as compared to HLADP and HLA-DQ molecules in human endometrial epithelial cells in vitro (32). Similarly, human endometrial epithelium primarily expresses HLA-DR rather than HLA-DP and HLA-DQ in vivo (32). Taken together, it is likely that endometrial T cells are activated and produce IFN7 and that at least one manifestation of such in vivo production may be the induction of expression of HLA-DR molecules on endometrial epithelium. We have also demonstrated that IFN7 exerts antiproliferative effect on endometrial epithelial cells in vitro (45). In view of the strategic localization of activated T cells in lymphoid aggregates adjacent to the basalis glands, we have suggested that the low proliferative activity of the basalis epithelium may also be due to the local effect of this cytokine (45). The in vivo and in vitro findings that are in accord with the hypothesis of IFN7 production and its local effects in human endometrium are summarized in Table 1. VI. IL-1 IL-1 consists of two distinct but related molecules, termed IL-la and IL-1/3. The polypeptide chains are only

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TABLE 1. In vivo and in vitro evidence for the focal effect of IFN7 in human endometrium Presence of lymphoid aggregates in the basalis Presence of large number of T cells in lymphoid aggregates Expression of markers on lymphoid aggregate T cells that signify cell activation (HLA-DR, HLA-DP, HLA-DQ, VLA-1) Focal enhancement of expression of HLA-DR in human endometrial epithelium adjacent to T cells within lymphoid aggregates Focal induction of HLA-DR expression in endometrial epithelium by the local administration of IFN7 in vivo Demonstration of IFN7 production by endometrial T cells Low proliferative activity of the epithelium in the basalis Demonstration of antiproliferative activity of IFN7 on endometrial epithelium [Compiled from Refs. 22, 23, 27, 29, 31, 44, 45, 47, and 48.]

26% homologous, and have distinct isoelectric points (pi 5 and 7, respectively). However, both molecules bind to the same receptor and mediate similar actions (1-3, 5158). An unusual structural feature of these molecules is the lack of the conventional leader sequence that allows the proteolytic cleavage of the protein into extracellular space. It is unclear how the IL-1 without the hydrophobic sequence is translocated within the cells. Since there is no evidence for the presence of processed IL-1 molecules in macrophages, the mechanism by which cells process the IL-1 precursor to the biologically active secreted product is unclear (59). It has been suggested that the processing of the IL-1 precursor occurs at the plasma membrane or in the extracellular space (60). Alternatively, it is suggested that stored IL-1 is released from cells only after a significant cellular insult (61). Regardless of the mechanism of the IL-1 secretion, one consequence of the lack of the leader sequence seems to be the presence of a biologically active form of the molecule on the plasma membranes (3). In situ hybridization has revealed the presence of ILla and IL-1/? messenger RNA in the mouse uterus in cells with morphological characteristics of macrophages (62). The expression of IL-1/? mRNA has also been shown in human endometrium in the late secretory phase of the menstrual cycle (63). In parallel with this observation, human serum levels of IL-1 were shown to be variable during the menstrual cycle and to reach maximal levels in the secretory phase (64). By using D-10 bioassay, which is based on the proliferative response of a mouse thymocyte cell line to IL-1, it has been shown recently that decidual cells release IL-1 into culture medium, and neutralization experiments demonstrate that this activity may be ascribed to IL-1 rather than other cytokines (65). One caveat to the interpretation of these data is the presence of a significant number of cells of monocyte/ macrophage lineage in the decidua and the difficulty of excluding these cells from preparations of decidual cells. Monocytes and macrophages are known to secrete enor-


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mous amounts of IL-1 in response to lipopolysaccharide (LPS) that commonly contaminates the media, sera, and labwares used in the tissue culture. Thus, further proof is required to demonstrate decidual cells as the source of the IL-1 in decidual cell cultures. Amniotic fluid is also shown to contain both IL-la and IL-1/3 species (66). Both endometrial epithelial as well as stromal cells exhibit IL-1 binding sites with characteristic specificity and affinity. The dissociation constants of these bindings are similar to those observed on other cells including monocytes and fibroblasts (67, 68). It is thus clear that both the gene and the gene product of IL-1 as well as IL1 binding sites exist in human endometrium suggesting that this molecule may potentially serve a variety of functions in this tissue.

VII. IL-6 From various names assigned to IL-6, it is clear that this molecule mediates a host of different responses and regulates functions of many cell types. Originally, presence of a second prominent mRNA (IFN/32) induced by virus-infected fibroblasts defined the molecule (5). Based on the complementary DNA sequence, it was predicted that the gene for IFNj82 encodes a protein of 26 kilodaltons (kD), thus the name, 26-kD protein (69). Independently described for its effects on induction of proliferation of B cells and plasmacytoma, the molecule was named, respectively, as B cell growth factor-2 (BSF-2) or hybridoma/plasmacytoma growth factor (70). Later, it became clear that all these biological activities are attributable to the product derived from the same mRNA transcript (5, 71). In humans, the translation product of IL-6 consists of 212 amino acids which in contrast to the IL-1 possesses a signal peptide of 28 amino acids that contains many hydrophobic residues (72, 73). The location of N-terminus of the secreted product varies somewhat resulting in a secretory product of 183 to 184 amino acids (72). Phosphorylation and glycosylation of the secreted IL-6 result in a group of differentially modified phosphoglycoproteins in the size range of 23-30 kD (74). We showed that endometrial stromal cells free of epithelial, endothelial, and lymphoid cells secrete IL-6 into culture medium (75). A number of inflammation-associated cytokines (IL-la, IL-lft TNFa, and IFN7) induce stromal cells to secrete IL-6. IL-la, however, is by far the most potent inducer of IL-6 production in endometrial stromal cells. In contrast, LPS is the most potent stimulus of IL-6 production in macrophages and fibroblasts (76). Endometrial stromal cells are insensitive to the bacterial LPS and do not secrete IL-6 with amounts of up to 1 tig/ml of LPS in the culture medium (75). Endometrial stromal IL-6 is heterogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel elec-

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trophoresis and consists of isoforms with mobilities in the range of 23-30 kD. These isoforms are phosphorylated and glycosylated and are similar to those produced by the induced human fibroblasts and monocytes. We have confirmed the presence of immunoreactive IL-6 in human endometrium by localizing this cytokine by immunoperoxidase staining. Both scattered stromal cells as well as endometrial epithelium express IL-6 immunoreactivity throughout the menstrual cycle (77). However, it seems that other sources of IL-6 are present in gestational tissues. Human trophoblasts seem to have IL-6 receptor and also produce IL-6 (78). In view of the ability of IL-6 to induce human chorionic gonadotropin (hCG) in trophoblast cultures, it is suggested that IL-6 may regulate the release of hCG from trophoblasts by an autocrine mechanism (78). Although the uterus was not one of the organs studied, a recent study demonstrated the expression of mRNA for IL-6 in various organs of normal individuals suggesting that, in vivo, this factor may be involved in normal physiological functions (79, 80).

VIII. TNFa The protein variously described as Coley's toxin, cachectin, TNF, or TNFa became known in the late 1800s for its ability to induce hemorrhagic necrosis in tumors (6, 7). The identity of this factor was established in 1975, when Carswell et al. reported that a serum factor from animals sensitized with Bacillus Calmette-Guerin and injected 2 weeks later with LPS (endotoxin) causes hemorrhagic necrosis of certain transplanted mouse tumors (6, 7). It became known, however, that TNFa is a pleiotropic factor that exerts a variety of effects ranging from proinflammatory and cytotoxic, to growth and immunomodulatory on a host of different cells (3). TNFa initially synthesized as a membrane-bound 26 kD prohormone is proteolytically cleaved to yield a mature 17 kD protein. A 14 kD prosequence polypeptide remains associated with the membrane (81). Apparently, several cytokines including IL-1, TGFa, and CSF-1 are secreted by a similar mechanism leaving a membrane-bound form of the cytokine associated with the cell. Consequently, this pattern of secretion of cytokines is different from the common mode of secretion which involves cleavage of the nascent protein by signal peptidase during the translocation of the nascent polypeptide chain from the endoplasmic reticulum (81). Despite a large body of data regarding the effects of TNFa, limited knowledge exists concerning the in vivo sources of this cytokine. TNFa was originally thought to be exclusively a product of macrophages. We are beginning to realize, however, that alternative sources of TNFa exist in human tissues (82, 83). Since TNFa


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mRNA is constitutively present and synthesis of this factor is primarily controlled at the posttranscriptional level, the cells expressing the TNFa mRNA may not necessarily translate the message to the product (84, 85). This limits the contribution of such studies as in situ hybridization and Northern blotting. Our understanding of the potential sources of TNFa in vivo has evolved largely through recent use of monoclonal and polyclonal antibodies to TNFa and immunohistochemical procedures (86-88). Identification of biologically active TNFa in human decidua, and in amniotic fluids and supernatants of placental and decidual tissues, and demonstration of TNF receptors on placental cells and TNFa mRNA in uterine cells have provided the first clues that TNFa may be a key cytokine in gestational and endometrial responses (89-92). The physiological mechanism that signals the release of TNFa from cells is largely unknown. Furthermore, the signals for the release of the cytoplasmic TNFa from various cells in human endometrium remain to be determined. However, it has recently been demonstrated that interaction of cells with molecules including the LFA-3, CD44, and CD45 results in the release of cellular TNFa (93). CD45-positive cells are ubiquitous in human endometrium, and we have observed LFA-3 on endometrial epithelium suggesting the presence of alternative pathways that may contribute to the release of TNFa in this tissue (32). The release of both the epithelial as well as decidual TNFa may be essential to the presence of this factor in the amniotic fluid and in supernatants of decidual explants (89, 90). IX. TGFa/EGF Epidermal growth factor (EGF) is a heat-stable polypeptide hormone that was originally isolated from extracts of submaxillary glands of male mice. Human EGF was identified and isolated from urine in 1975. In the same year, the sequence of an inhibitor of gastric acid secretion, /?-urogastrone, was published (9-12). Later, it was found that the /?-urogastrone elicits the same mitogenic action of EGF, and it is now accepted that 0urogastrone is the human form of EGF (9-12). The identification of TGFs is the outcome of the initial observation that transformation of cells by sarcoma viruses produces a specific and dramatic decrease in the [125I]EGF binding capacity (11). It was later shown that the two sets of molecules termed TGFa and TGF/? are required for the anchorage-independent growth of normal cells and that EGF can substitute for TGFa in this regard (11). In humans as well as other species, TGFa shows homology in amino acid sequence to EGF and binds to a receptor that shares homology with the V-erbB oncogene protein sequences (94). Both EGF and TGFa

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are synthesized as larger precursor molecules. EGF is probably synthesized as a transmembrane pre-pro-EGF molecule consisting of 1217 amino acids. EGF is derived from the action of two esterases from the intermediate factor called pro-EGF. EGF may be found in virtually all body fluids including amniotic fluid (12). The expression of mRNA for EGF and EGF protein has been shown in the uteri of estrogenstimulated immature mice whereas the receptor for EGF has been described both in rat and in human uteri (95103). Most studies indicate that EGF receptors are ubiquitously present in human endometrium and that endometrial epithelium, stroma, and endothelium uniformly express the receptors throughout the menstrual cycle (98-102). However, one report indicates presence of the receptor only in endometrial epithelium. Apparently, the endometrial epithelium in the proliferative phase exhibits a linear cell surface expression, and enhanced cytoplasmic staining in glandular epithelium is observed during gestation (103). Membrane fractions from normal human endometrium exhibit a dissociation constant of 0.64 nmol/liter, and affinity cross-linking studies reveal the mol wt of the receptor to be in the range of 150 to 170K (104). TGFa mRNA has been localized both in the mouse decidua and in the nongestational mouse uterus (105). X. TGF0 TGF/3 is now known to consist of five different isoforms, each coded by a separate gene on different chromosomes. These factors are homodimers with a mol wt of 25,000. Each monomer unit consists of 112 amino acids with unique sequences unrelated to any other previously known polypeptides. It has become clear that the biological activities of TGF/3s exceed the context of their original description. These molecules affect cell functions such as cell morphology, cell differentiation, cell proliferation, and morphogenesis (13-15, 106-115). Although it is clear that TGF/3s along with other cytokines are involved in inflammatory and repair processes, their participation in endometrial functions and implantation only recently has been suggested. A recent study has shown by in situ hybridization that the message for TGF/3i is expressed in endometrial epithelium of the mouse uterus during the preimplantation period and in the mouse decidua (116). This is not unexpected, as TGF/3 mRNA appears soon after fertilization and thereafter the gene is probably transcriptionally active to some extent in all tissues with high amounts of the TGF/? mRNA being expressed in placenta (108-115). One caveat to the interpretation of the in situ hybridization data is that the immunolocalization of TGF/3 has been discordant with the sites that were demonstrated to


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express the mRNA (108). Immunohistochemical staining has confirmed that the gene product of TGF/3 is expressed in the epithelium on day 1-4 of pregnancy and also appears in the extracellular spaces among the stromal cells of the mouse endometrium (116). However, the immunolocalization data must be interpreted with caution as it has been suggested that immunolocalization reveals sites of mature TGF/3 rather than the site of its synthesis (108). After the formation of decidua, the decidual cells also express immunoreactivity for the TGF/3. An immunosuppressive factor has been identified in the pregnant mice uteri as well as in the amniotic fluid and in view of the neutralization of its bioactivity by the antibody to TGF/32 and not with a TGF/3rspecific antiserum, it is suggested that this molecule is identical or is closely related to TGF/32 (117, 118). The expression of TGF/3 mRNA in human endometrium has only recently been described (63). Four distinct cell surface proteins have been described that bind TGF/3 with high affinity and specificity expected from physiologically relevant receptors (type IIV) (110). The expression of the type IV receptor is limited to pituitary cells. The expression of TGF/3 receptors has not yet been described for the endometrium; however, in view of the widespread expression of type IIII receptors, it is likely that these receptors are expressed in this tissue as well. The hypothesis that one receptor mediates multiple functions prevails over the opinion that each set of functions is displayed through interaction of TGF/3 with different receptors (110). TGF/3 is secreted in a latent form and must be activated to become biologically functional. In view of the ubiquitous expression of the mRNA, protein, and receptor, it is suggested that the stringent control over the action of TGF/3 may be through a tight control of activation of this protein (111-113). Although the signals that cause the in vivo activation of TGF/? are not known, it has been shown that proteases such as plasmin, cathepsin D, alkaline and acid phosphatases, as well as I F N Y can activate the latent form of TGF/3 (112,114). XL CSF-1 The human CSF-1 (M-CSF) gene is transcribed into a group of differentially spliced mRNAs that encode different forms of the growth factor. A 554 amino acid precursor is translated from the 4 kilobase mRNA into a transmembrane glycoprotein. This precursor is glycosylated, dimerized, and then cleaved in the secretory component of the cell to yield a soluble 90 kD protein that is secreted from the cell (1,16). Other differentially spliced mRNA species differ from the 4 kilobase mRNA in their 3'-untranslated regions and yield proteins identical to those derived from the larger transcript. An

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exception is a 1.6 kilobase mRNA that encodes a protein that, in view of the lack of recognition site for the proteases, is expressed in plasma membrane and retains biological activity (1, 16). Originally, CSF-1 was described for its activity as a factor promoting the growth of bone marrow precursors. However, it has become clear that the expression of this protein is not limited to the bone marrow elements and that CSF-1 is also expressed preferentially in the glandular epithelium of pregnant mice (119). CSF-1 and its receptor exhibit specific temporal and spatial patterns of expression in the human and mouse uteroplacental units (120-122). CSF-1 mRNA first becomes detectable in the mouse uterine epithelium 8.5 days postcoitum and persists throughout the gestation. This expression is elevated at least 100-fold on days 14-16 of pregnancy. Murine uterine CSF-1 mRNA consists of a major 2.3 and a minor 4.5 kilobase species (120). A recent study has failed to demonstrate the CSF-1 mRNA in nonpregnant human endometria, and CSF-1 message was only demonstrated in gestational endometria (123). The actions of CSF-1 is mediated by a class of high affinity receptor that is encoded by the c-fms protooncogene. This cell surface glycoprotein is a member of the family of growth factor receptors that exhibits an intracellular tyrosine kinase activity. In addition, the receptor possesses a membrane-spanning domain and an extracellular ligandbinding domain (124). c-fms expression initially appears in murine decidua on day 7.5, apparently in decidual cells and then in the trophoblasts on day 9.5 (122). A 3.7 kilobase mRNA of CSF-1 receptor has been shown both in the proliferative and first trimester human endometria; however, the cells that express the message for the receptor have not yet been defined (120). The expression of mRNA of CSF-1 receptor has also been shown in the first trimester placenta, and in situ hybridization has localized the message to the syncytial trophoblasts (120). CSF-1 is also shown in the amniotic fluid of mouse and human. In mouse, the level of CSF-1 in the amniotic fluid reaches a peak of about 6-fold of that observed in the serum on day 14 of pregnancy. Similarly, in human amniotic fluid, levels reported in the third trimester are 3-fold those reported in the first trimester (120).

XII. Insulin-like Growth Factors (IGFs) IGFs or somatomedins are polypeptides that are similar in structure to proinsulin and relaxin (125). In contrast to insulin, however, these factors are widely expressed (126, 127). Two IGFs are called, respectively, somatomedin A (IGF-I) and somatomedin B (IGF-II). It has been suggested that IGF-II functions as a fetal growth factor in rodents (127). IGF-I has growth pro-


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moting effect in the hypophysectomized rat, whereas IGF-II elicits an insignificant response (128, 129). A low level expression of IGF-II mRNA has been shown in the adult rat uterus (127). In contrast, abundant expression of mRNA of IGF-I can be demonstrated in the adult rat uterus (126). This expression shows slight variation in the cycling rats reaching to maximum levels in late diestrus and early proestrus (129). The expression of IGF-I mRNA species as well as the immunoreactive IGF-I can be coordinately increased after administration of estrogen to immature and ovariectomized rats (129132). Similarly, the abundance of IGF-II mRNA message is increased 2- to 5-fold after estrogen administration (127). In addition, specific IGF-I binding sites have been reported in rat uterine membranes similar to those reported for other tissues (133). Autoradiography of the [125I]IGF-I bound to the uterus, however, showed myometrium as the prime site of the binding (134). Estrogen modestly increases the IGF-I binding sites in the uteri of immature rats (133). One characteristic feature of IGFs, unlike other growth factors, is that these molecules are present in the biological fluids and tissue extracts bound to the IGF binding proteins (134). It is suggested that these binding proteins may, by limiting the availability of the IGF molecules, modulate their functions; however, there is a report indicating that the binding protein may enhance the action of the polypeptide (135). The message of a binding protein called IGFBP-1 as well as its respective immunoreactive molecule were localized to luminal epithelium and stroma in rat (134). In humans, the IGFBP-1 is localized to the stromal cells, and increased immunoreactivity of this protein is found in the secretory phase as well as in gestational endometria (136,137).

XIII. Potential Functions of Cytokines in Human Endometrium We presented data that demonstrate the expression of cytokines at the gene or protein levels in endometrium and show secretion of these cytokines by endometrial cell constituents in animals or in in vitro systems. However, further data are required to confirm the role of cytokines in human endometrium in vivo. The following is a summary of the potential functions of cytokines in endometrium. A. Cytokines and proliferation Both EGF and TGFa exhibit mitogenic and angiogenic potential (138, 139). EGF was found to be mitogenic on epithelial cells of the mouse uterus. In the same study, other growth factors, including fibroblast and plateletderived growth factors, and somatomedin-C were found to be inactive (140). In view of the enhancement of EGF

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receptor production by estradiol, it has been suggested that the mitogenic effect of estrogen may be mediated through EGF (140, 141). It has been shown that IGF-I, at relatively low concentrations, can stimulate the DNA synthesis in the perimyometrial stroma in the presence of estrogen in organ cultures or in tissues from estrogenprimed animals suggesting that this factor may contribute to the proliferative activity of endometrium (133, 142). Whereas several cytokines that enhance proliferative responses in endometrial epithelial cells have been described, only IFN7 has been described to inhibit the proliferation of this tissue (45). Based on the inhibition of proliferation of human endometrial epithelial cells by IFN7, we have suggested that this factor may regulate the endometrial epithelial proliferation in the basalis adjacent to the lymphoid aggregates (45). B. Cytokines and lymphoid infiltration It is conceivable that cytokines, in view of their ability to induce leukocyte-endothelial adhesion and chemotactic potential, promote infiltration of endometrium by lymphoid cells. Several cytokines including TGF/3, IL-1, TNF, and IFN7 are known for their chemotactic effects (14, 143-149). The chemotactic activity of IL-1 on polymorphonuclear leukocytes was recently demonstrated in vivo by injection of recombinant IL-1/3 into the testicular microcirculation of rat (150). IFN7 may also serve a similar function as local administration of IFN7 results in focal infiltration of the mouse and human tissues by a lympho-histiocytic infiltrate (47-49). Aggregates of lymphoid cells in human endometrium are characteristically present adjacent to HLA-DR- and ICAM-1-positive epithelium (33). Both HLA-DR and ICAM-1 have been reported as adhesion molecules and considering that IFN7 induces both proteins, we have speculated that this cytokine may facilitate lymphoepithelial interactions and binding (33, 151, 152). C. Cytokines and endometrial microenvironments As described previously, it is evident that human endometrium consists of a variety of cells. Distinct patterns of proliferation and expression of different molecules occur in endometrial glands and stromal cells in different layers of human endometrium. Lymphoid cells are distributed as scattered cells in the endometrial stroma and as aggregates specifically in the basalis adjacent to endometrial glands. Predecidual reaction develops characteristically around vessels and under surface epithelium and then involves all stromal cells in the upper functional layer (153). The distinct patterns of distribution of cells and different proliferative and differentiative potential of cells in various layers of endometrium suggest the existence of specific microenvironments in endometrium.


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It is conceivable that these microenvironments may be induced due to local effects of cytokines. For example, we have suggested that the focal HLA-DR expression in the basalis epithelium and the low proliferative activity of this compartment may be due to local production of IFN7 by activated T cells of human endometrium (23). The hypothesis that cytokines may induce local microenvironments is supported by the observations that local injection of cytokines induce only focal effects and that cytokines may be expressed on the plasma membrane of cells. For example, after injection of IL-1 and IFN7, tissues are locally infiltrated by the inflammatory cells (47-49,150). After administration of IFN7, HLA-DR is focally expressed on thyroid epithelium, and implants that slowly release TGF/3 cause local inhibition of development of breast ducts in neonatal mouse (47, 48, 154). Many of the cytokines including IL-1, TNFa, EGF, TGFa, and CSF-1 are present as a functionally active, plasma membrane-anchored molecule (1, 3,16,18, 105). The local effect of cytokines and the existence of the plasma-membrane bound cytokines may be important in creating distinct microenvironments in which a mantle of cytokine-responsive cells surrounds the cytokine-expressing cells. D. Cytokines and menstruation Many manifestations of the menstrual process, including the infiltration of the endometrium by mononuclear cells and repair and remodeling of the damaged endometrium, may be orchestrated by cytokines. Many of the actions of TNFa are associated with inflammation. TNFa is chemotactic to polymorphonuclear leukocytes and causes activation and degranulation of these cells (3, 155). Induction of angiogenesis and procoagulant activity on endothelial cells, mitogenic action on fibroblasts, pyrogenic action, and induction of acute phase proteins are just a few of the diverse actions of TNFa that are directed at target cells in the inflammatory focus or on distant sites (3, 156). In this context, it is interesting to note that the administration of TNFa induces injury in the endothelial cells of the uterus and causes multifocal hemorrhage and inflammatory cell accumulation in the mouse uteri reminiscent of those observed during menstruation (157). IL-1 is one of the proinflammatory cytokines, and it induces production of PGE2, collagenase, and phospholipase A2 (1, 5, 158). We have recently demonstrated that IL-1 is able to increase prostaglandin E2 production by human endometrial epithelial cells (67). This is in line with the observation of increased prostaglandin E2 levels in human endometrium in the secretory phase of the menstrual cycle (159, 160). It is also of interest to note that coincident with this increase, serum levels of IL-1 and IL-1 mRNA in human endometrium

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are increased during the secretory phase (63, 64). In addition, induction of HLA-DR in an epithelial cell line by IL-1 suggests that this cytokine may contribute to the observed expression of HLA-DR on human endometrial epithelium in vivo (161). It is thought that some of the IL-1 effects may be mediated through elaboration of other cytokines including IL-6 (1, 5). We have recently demonstrated that IL-1 induces synthesis and release of IL-6 by human endometrial stromal cells suggesting that some of the IL-1-mediated effect(s) in human endometrium occurs through elaboration of the IL-6 by stromal cells (75). TGF/3 mRNA is expressed after vascular insult as well as during the proliferation and repair of the damaged epithelium (107, 162). TGF/3 can modulate epithelial proliferation, enhances gland formation, is chemotactic, and is associated with recruitment of monocytes and polymorphonuclear leukocytes into the damaged tissue. It also promotes angiogenesis, activation and proliferation of fibroblasts, and the deposition of extracellular matrices including collagen, fibronectin, proteoglycan, and tenascin (13-15, 106-115). In this regard, it is of interest to note that deposition of all extracellular matrices including collagen, fibronectin, and tenascin have been described in human endometrium (163, 164). In view of the demonstration of cytokine production in endometrium along with the multitude of functions that cytokines exert in inflammatory processes, it is reasonable to assume that IL-1, IL-6, TNFa, and TGFjS may be important in changes in endometrium that are linked to the inflammatory events. E. Cytokines and uteroplacental functions A large body of data indicates that cytokines are involved in diverse reactions in the uteroplacental unit. Presence of TNFa in amniotic fluid, the presence of TNFa receptor on the cells from placenta, and the demonstration of growth inhibition of trophoblasts by TNFa suggest that this factor may be important in placental development (89-92,165). Presence of TNFa in amniotic fluid of women with intraamniotic infection and preterm labor has been described, and it has been shown that the administration of endotoxin to pregnant animals induces abortion or preterm labor (166-168). Thus, it has been suggested that TNFa, along with other cytokines, may be significant in the onset of parturition (166-168). Based on the coexpression of CSF-1 on endometrial epithelium along with the expression of CSF-1 receptor mRNA in trophoblasts and decidual cells, it has been suggested that CSF-1 is involved in placental development (119-122). Indirect evidence supporting this view may be the increased resorption and reduction of successful implantation by the administration of CSF-1 (120). In addition, it is shown that CSF-1 regulates hCG


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and PRL production by cultured human cytotrophoblasts suggesting alternative roles of CSF-1 in placental functions (120). The suggestion that TGF0 also functions in uteroplacental unit is based on the production of a unique immunosuppressive molecule related to the TGF/32 in decidua as well as the temporal and spatial distribution of TGF/3i message and its respective product in pregnant mouse uteri (116-118). Based on these findings as well as the demonstration of TGFa mRNA in decidua, EGF, and IGF-I and their receptors in specific cells of mouse endometria during pregnancy, and presence of multiple cytokines including CSF-1, IL-1, EGF, and TNFa in amniotic fluid, it is likely that cytokines are involved in the development, growth, and differentiation of cells in the uteroplacental unit and placental functions. However, the distinct functions that these cytokines exert in the gestational endometrium or placenta remain to be shown. F. Cytokine-cytokine interactions Many cytokines result in the production of their own species or other cytokines. For example, IL-1 increases production of IL-1, IL-6, IFN7, and TNFa. IL-6 induces production of other cytokines such as TNFa and GMCSF. In other instances, cytokines may exhibit agonistic or antagonistic effects in the target tissues (1-17). It is unclear why similar functions are mediated by multiple cytokines. However, it is suggested that many, if not all, cytokines are integrated into a vast and intricate network that operates by a coordinated regulation of expression of various cytokines, their effector genes and in some cases, modulation of expression of their high affinity receptors. The individual peptides of the cytokine family may be equivalent to the characters of alphabet or code. Thus, the information transmitted to cells may be the outcome and net effect of the set of regulatory peptide molecules rather than be dependent on the effect of the individual cytokine. It seems that this redundancy may assure error-free transmission of information to cells in the presence of noise (169). A summary of the functions of the cytokines relevant to human endometrium is provided in Table 2.

XIV. Interactions of Cytokines with Gonadal Steroid Hormones Gonadal steroid hormones potentially influence cytokines at three levels. Steroids may alter the expression of the cytokine at the transcriptional or posttranscriptional level, modulate the expression of the cytokine receptor, or alter the net effect of the cytokine in the target cells. In regard to the influence of gonadal steroid hormones on cytokine secretion, there is evidence that when chal-

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TABLE 2. Functions of cytokines relevant to human endometrium Modulation of proliferation (IFN7, IL-1, IL-6, TNFa, EGF, TGFa, TGF/3, CSF-1) Induction of HLA-DR and ICAM-1 (IFN7) Alteration of morphology (IFN7, IL-1, TNFa, TGF/3) Gland formation (TGF/3) Induction of adhesion molecules (IFN7, IL-1, TNFa, TGF/3) Induction of cytokines (IFN7, IL-1, TNFa, IL-6, CSF-1, TGFa) Chemotaxis and induction of lymphoid infiltration (IFN7, IL-1, TNFa, TGF0) Edema (IL-1, TNFa) Deposition of extracellular matrices (TGF/8) Induction of PGE2 (IL-1, TNFa) Activation of T cells (IL-1, IL-6) Activation of polymorphonuclear leukocytes (TNFa) Injury to uterine vessels (TNFa) Angiogenesis (TGF/3) Elevation of body temperature (IL-1, IL-6, TNFa) [Compiled from Ref. 1-19, 30, 32, 45-49, 67,106-115,133,138-152, 155-158, and 162.]

lenged with purified protein derivative of mycobacteria, female mice infected with the mycobacterium Bacillus Calmette-Guerin produce significantly more IFN7 than male mice (170). Furthermore, some data also demonstrate that the IFN7 promoter may be positively regulated by estrogen (171). There is also evidence that ovarian gonadal steroid treatment blocks the release of IL-1 as well as TNFa by human blood monocytes (172174). TGFa mRNA is induced in the mouse uterus after treatment with estrogen, and stimulation by diethylstilbestrol results in the accumulation of high levels (50-70 ng/ml) of mature TGFa in the mouse uterine fluid (105). In addition, progestin increases secretion of TGF0 in T47D, a breast carcinoma cell line, apparently by a posttranscriptional mechanism (175). In contrast, estrogen inhibits and antiestrogen increases TGF/3 secretion in the same cell line (176). Production of IL-6 by human endometrial stromal cells may be down-regulated by the physiological level of estrogen (75). As stated previously, the expression of immunoreactive IGF-I and its respective mRNA can be significantly increased in the mouse uterus by gonadal steroid treatment (129-132). The significant elevation in the mouse uterine CSF-1 during pregnancy suggests that the expression of this protein may also be linked to the hormonal influences. Indeed, the concentration of the uterine CSF-1 can be regulated by both estrogen and progesterone (119). Induction of pseudopregnancy by the administration of oral gonadal steroid hormones to women induces the expression of CSF-1 mRNA in their endometria (123). The uterine expression of EGF can also be modulated by the administration of estrogen to immature mice (177). These findings suggest that gonadal steroid hormones are involved in the regulation of cytokine secretion from gonadal steroid-sensitive tissues and cells.


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Limited information is available regarding modulation of cytokine receptor expression by steroids in human tissues. However, there is evidence that glucocorticoids up-regulate the high affinity receptors of IL-6 on human epithelial cells, offering an explanation for the frequently observed synergy between IL-6 and glucocorticoids (178). It is also shown that the administration of estrogen to immature female rats induces a 3-fold increase in the binding of [125I]EGF to uterine membranes. This increase is not associated with any change in the affinity of uterine membrane receptors for EGF, raising the possibility that modulation of receptor expression of EGF is involved in the estrogen-stimulated growth (97). The expression of EGF receptor within rat ovary has also been shown to be modulated by gonadal steroid hormones (179). Estrogen also induces an increase in the level of the mRNA of the CSF-1 receptor in human endometrium (123). In respect to the modulation of cytokine function by steroids, we have been able to demonstrate that estradiol 17/3, at physiological level, can down-regulate the expression of HLA-DR molecules induced by IL-1 in human endometrial epithelial cells (161). Taken together, these data show that gonadal steroid hormones influence cytokine functions at three different levels; cytokine secretion, cytokine receptor expression, and cellular responses. Thus, one important question is whether cytokines are mediators of gonadal steroid action. This issue is further discussed in the section below. A. Are cytokines mediators of gonadal steroid actions? In regard to endometrial functions, it is now undisputed that gonadal steroid hormones, estrogen, and progesterone profoundly affect diverse actions in human endometrium which include alterations, in morphology, proliferation, secretion, prevention of cell death, as well as modulation of estrogen and progesterone receptor levels, and various enzyme activities (180-189). However, the precise cellular and molecular mechanisms of estrogen actions are not known, and in certain instances it is not clear whether steroids directly affect target tissues or indirectly influence cells through elaboration of intermediate factors released within endometrium. One area of controversy is the mitogenic effect of estrogens. Estrogens have typically been associated with the increased proliferative activity of the endometrial epithelium (190, 191). However, estrogen binding (i.e. receptors) has not always correlated with the induction of DNA synthesis in endometrial epithelial cells (192, 193). The receptors for estrogen are uniformly distributed in the human endometrial epithelium of the functionalis and the basalis whereas the proliferative activity of the functionalis epithelium far exceeds that seen in the basalis (194).

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Thus, it has been suggested that estrogen-induced responses in the endometrial epithelium may be mediated by affecting the underlying stromal cells which contain estrogen receptors (195, 196). Also, it has been reported that estrogen fails to enhance the proliferation of primary cultures of purified epithelial cells isolated from humans and rodents (196-198), but enhances proliferation when epithelial and stromal cells are cocultured (192, 198). One model proposes that growth factors induced by estrogen in stromal cells are responsible for the estrogen-induced proliferation observed in these experiments (199). Alternatively, it has been suggested that estrogens enhance epithelial cell proliferation by blocking the synthesis of inhibitors of cell proliferation secreted by stromal cells (200). Based on experiments performed in mouse uteri, three stages of response to estrogen have been determined (201): 1) A lag phase of 10-14 h when there is little or no effect of estrogen on DNA synthesis whereas metabolic activities are on the rise. 2) The effect of estrogen on DNA synthesis becomes marked 18-48 h after its administration. 3) Around day 3, an inhibitory effect of estrogen may be observed when the DNA synthesis is reduced to control values or lower. Based on this last observation, it has been speculated that late events in estrogen action may be due to accumulation of products that inhibit DNA synthesis (201). Considering the mitogenic and antiproliferative impacts of a diverse group of cytokines in regulation of growth of endometrial epithelial cells, coupled with the data that suggest cytokine production may be regulated by gonadal steroid hormones, it may be speculated that cytokines are the local mediators of estrogen action in tissues.

XV. Influence of Cytokines on Endometrial Functions by Interaction with HypothalamicPituitary-Gonadal Axis A. Systemic effects of cytokines Emerging evidence suggests that various cytokines may alter endometrial functions as a result of their actions on hypothalamus, pituitary, or gonads (Table 3). At the level of the hypothalamus, IL-1 is shown to inhibit the release of hypothalamic LHRH in rats (202). Recently, it was demonstrated that TGF« (2-100 ng/ml) also elicits a dose-related increase in LHRH by the median eminence of the juvenile rats in vitro (203). Several cytokines exert their functions on the pituitary. IL-1 inhibits the ovarian steroid-induced LH surge in rats and TNFa similar to GnRH causes rapid increase in the release of LH, PRL, and ACTH from rat pituitary cells in vitro (202, 204). IL-6 has been shown to be produced by anterior mouse pituitary cells, and it has been suggested that this factor may function as an intrapituitary releasing factor (205, 206). The release of


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TABLE 3. Evidence that links the cytokines with the hypothalamicpituitary-gonadal axis IL-1 mediated inhibition of hypothalamic LHRH TGFa-mediated release of LHRH IL-1-mediated release of FSH, LH, and PRL release by pituitary cells IL-6 mediated release of FSH, LH, and PRL release by pituitary cells TNF-mediated release of LH, PRL, and ACTH from pituitary cells Activin-mediated stimulation of FSH release from pituitary Inhibin-mediated inhibition of FSH release from pituitary IL-1-mediated inhibition of steroid-induced LH surge IL-1-mediated modulation of sex steroid secretion by the ovary IL-1-mediated inhibition of lutenization by granulosa cells of the ovary TNF-mediated modulation of steroidogenesis by ovarian follicles IL-1-mediated modulation of Leydig cell steroidogenesis EGF-mediated modulation of Leydig cell steroidogenesis Gonadotropin and FSH-mediated control of EGF receptor by ovary [Compiled from Refs. 202-221.]

IL-6 by rat pituitary cells, which is enhanced by IL-1/3 in vitro, can be inhibited by a rabbit anti-IL-1/3 antiserum (207). In an in vitro study, both IL-1/3 and IL-6 induced, within 3 h, significant releases of FSH, LH, and PRL from rat pituitary cells comparable to those induced by GnRH and TRH (207). From the members of the TGF/3 family, activin and inhibin are also known, respectively, for their stimulatory and inhibitory functions on the release of FSH from pituitary (208). Thus, it is becoming clear that IL-1, IL-6, and members of the TGF family of molecules alter the secretion of pituitary hormones and consequently affect endometrial functions. Gonads are also emerging as sites of cytokine action. According to one report, IL-1 at the level of ovary interferes with steroidogenesis in immature female rats and causes significant inhibition of both estradiol and progesterone secretion (209). Another report, however, indicates that IL-1 increases progesterone production from preovulatory follicles in cyclic hamsters (210). Apparently, IL-1 also inhibits lutenization of porcine granulosa cells in the ovary (211). Granulosa cells of the ovary have been shown to be the source of TNFa as well as target cells for this cytokine (212). In view of the significant overlap in the biological functions of IL-la and TNFa, it is not surprising to find that similar effects of TNFa on steroidogenesis by the follicles have been reported. TNFa increases the steroidogenesis from rat ovarian follicles and theca cells whereas it significantly inhibits the basal and FSH-stimulated progesterone production from the granulosa cells of preovulatory follicles (213). The effect of IL-1 on rat Leydig cell steroidogenesis has also been controversial with some studies indicating stimulation while others suggested an inhibition of steroidogenesis by these cells (214, 215). It has been suggested that in rats, the luteotropic action of the PRL on ovary is at least in part mediated by an immunoreactive TGF/?-like substance (216). Stimulation of progesterone

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production by rat and mouse Leydig cells by EGF has been shown, and it has been suggested that the strong inhibitory effect of EGF on androgen production may in part be due to the loss of the steroidogenic potential of Leydig cells after prolonged culture (217). Modulation of secretion of human gonadotropic hormone by EGF from the first trimester placenta indicates that multiple cytokines influence secretion of systemic hormones (218). It is also being realized that pituitary hormones may also influence the cytokine action by alteration of their secretion or binding sites. It has been shown that FSH maintains and increases the number of EGF receptors and LH/hCG reduces the binding sites for EGF on immature rat granulosa cells, suggesting a regulatory role for growth and selection of ovarian follicles by this growth factor (219). In an independent study, it was found that in hypophysectomized or intact rats, FSH or hCG increases EGF binding to ovaries while PRL, estrogen, or progesterone does not (220). These data as well as the demonstration of an immunoreactive LHRH in splenic lymphocytes (221) demonstrate that the functions of the hypothalamic-pituitarygonadal axis are controlled by various sets of cytokines and enforce the view that a tight association exists between cytokines and cells of the immune and the endocrine systems. One of the characteristic and consistent findings during the menstrual cycle is a 0.2 to 0.6 C elevation of body temperature immediately after ovulation. As occurs with fever, this rise in the body temperature appears to be associated with an upward shift of the thermoregulatory set point (64). The underlying mechanism for this temperature rise has remained unclear. In view of the pyrogenic activity ascribed to the proinflammatory cytokines, IL-1, IL-6, and TNFa, it may be speculated that these cytokines contribute to the postovulatory rise in body temperature (3, 5, 18, 19). TNFa induces a rapid onset of increased body temperature indistinguishable from that produced by IL-1. Whereas administration of IL-6 iv or ip has no effect on body temperature, intracerebroventricular injection of IL-6 results in a significant increase in core temperature (18, 19). The demonstration of regulation of IL-1 and TNFa activity by gonadal steroid hormones gives further credibility to the hypothesis that cytokines may be important in the postovulatory regulation of body temperature (172-174). Although the increased serum levels of IL-6 and TNFa in the secretory phase of the menstrual cycle in women have not been described, an increase in the serum levels of IL-1 during the secretory phase of the cycle is reported (64). This increased body temperature induced by cytokines may have functional relevance as both IL-1 and TNFa as well as IFN7 are more active at an elevated temperature (3, 222). Among effects of hyperthermia on


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cytokines, enhancement of IFN7 synthesis has been reported (223).

XVI. Model Systems for Studying the Cytokine Actions in Human Endometrium Several model systems exist that are suitable for studying the cytokine actions in endometrium. These include use of explant cultures, isolated single cell or gland cultures, cell lines, endometrium transplanted to nude mice, and perfused uteri (46, 224-237). Explants have been used for studying a variety of endometrial functions (224, 225). In explants, the three dimensional architecture of endometrium remains intact, and cell responses are more akin to that observed in normal tissue. However, these tissues rapidly undergo degenerative changes, and we have observed necrosis within 48 h of the initiation of these in vitro cultures, making them unsuitable for long-term studies. Both stromal cells and glands may be obtained to highest purity from minced endometrial fragments (225227). Cultures of epithelial cells initiated from isolated glands derived from normal human endometria allow studying the effects of cytokines in vitro. Using this model, we have been able to provide evidence that IFN7, a product of activated T cells, induces HLA-DR molecules in endometrial epithelium and inhibits their proliferation (30, 32, 45). However, in this system, glandular epithelium loses its estrogen and progesterone receptors in vitro and does not respond to estrogen in regard to estrogen-mediated growth and progesterone mediated down-regulation of estrogen receptor (228). Using the gland cultures has several other drawbacks. These studies should take into account the variation that may be observed among different individuals. In addition, in view of the changes that occur in endometria as they transit through the menstrual cycle, obtaining endometria in the same phase of the menstrual cycle represents additional difficulty. Furthermore, glands isolated from the same endometrium are not homogeneous in view of the region-to-region and gland-to-gland variations. Finally, the end product of the isolation procedure is fragmented glands rather than single cell suspensions; thus cell counts at the time of initial plating are unobtainable. These primary cultures have a finite lifespan and do not lend themselves to more than merely one passage. In addition, the epithelial cells lose the three-dimensional glandular appearance and polarization and form a monolayer on the substratum. Apparently the loss of polarization is associated with the loss of functional integrity. These latter problems may be obviated by growing the glands on matrigel, a basement membrane-like material that confers upon cells both morphological and functional polarization (229-231).

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Several cell lines of both epithelial and stromal cell lineages may be utilized in studies of cytokines in endometrium (46, 232-234). However, in order to establish the concomitant effects of steroids and cytokines, cells should be utilized that respond to both types of signals in vitro. We have demonstrated that ECCl cell line fulfills these requirements and shares with the glandular epithelium of endometrium certain characteristics including the epithelial lineage, expression of gonadal steroid hormone, and cytokine receptors. Furthermore, the functionality of the receptors is demonstrated by the effect of cytokines on individual cell parameters including the expression of class II molecules and changes in morphology and proliferation (46). These findings suggest that this cell line is a useful model for simultaneously studying the effects of cytokines and steroids on epithelial cells of endometrium. Athymic mouse has been used as a host to the human endometrium (235, 236). The transplanted endometrium remains inactive; however, it retains the ability to respond to steroid hormones (235, 236). The transplanted endometria have the advantages of retaining the hormone responsiveness, maintaining the three-dimensional architecture, and being in an in vivo environment. The distinct disadvantages of this system however, include the abnormal localization of endometrium and the aberrant host. Recently it has been shown that the viability of the human uterus may be maintained in vitro for a period of about 12 h by perfusion of its vasculature (237, 238). Notwithstanding the changes that the deprivation from peripheral blood circulation imposes, this system has unique characteristics that allow demonstration of the role of cytokines in an in i>iuo-like condition.

XVII. Perspectives Although the expression of cytokines in endometrium has begun to be illustrated, the identity of the cells that express the cytokines or their receptors and target cells for the cytokine actions remains to be defined. Clearly, the functions ascribed to various cytokines may explain some aspects of the endometrial physiology and embrace several aspects of the menstrual process. However, many questions remain to be answered. The cytokine network elaborated by various cells in human endometrium remains to be defined. The contribution of cytokines to epithelial, stromal, endothelial, and lymphoid cell proliferation, differentiation, activation, and organization remains to be illustrated, and the cytokine(s) associated with initiation of menstrual process and those mediating tissue repair should be clarified. One intriguing question is what are the mechanisms that promote the creation of distinct microenvironments in human endometrium.



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Equally important is the determination of the driving forces that contribute to the aggregation of lymphoid cells in the basalis or their distribution as single cells in the endometrial stroma and in epithelium. The regulation of the synthesis, receptor expression, and functions of the cytokines by steroids not only reveals how the systemic signals are translated into local effects in endometrium, they provide a basis for understanding the observed differences in gender-specific responses. Many more cytokine interactive processes will continue to be unraveled; however, a difficult task will be to delineate how these multiple interactive processes are integrated to create the characteristic phases of the menstrual cycle. It is clear that our knowledge of the involvement of the cytokines in endometrial physiology is at its inception; however, the presented data provide a strong foundation for further studies of this ever-expanding area of research.

Acknowledgment The author thanks Dr. P. G. Satyaswaroop for review of this manuscript.

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